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Arabidopsis Mutants Define a Central Role for the Xanthophyll Cycle in the Regulation of Photosynthetic Energy Conversion
Abstract
A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.
... Each component is defined by an underlying mechanism and rate of formation and relaxation (Jahns & Holzwarth, 2012). The most rapid responding form of NPQ is qE (energy-dependent quenching) which is activated by acidification of lumen (the ΔpH component of pmf) via protonation of photosystem II subunit S (PsbS) (Li et al., 2004;Niyogi et al., 2005) as well as activation of violaxanthin de-epoxidase (VDE), which catalyzes the conversion of violaxanthin (Vx) to antheraxanthin and then zeaxanthin (Zx) (Niyogi et al., 1998). There are additionally, more slowly responding forms of NPQ, including sustained quenching (qH) (Malnoë, Schultink, et al., 2018), photoinhibition (qI) which is related to the photodamage of PSII centres and subsequent repair (Andersson & Aro, 2004;Murata et al., 2007;Nawrocki et al., 2021), zeaxanthin-dependent quenching (qZ) which involves the accumulation of Zx but not the PsbS (Dall'Osto et al., 2005;Demmig-Adams & Adams, 1996;Li et al., 2004;Müller et al., 2001;Nilkens et al., 2010), and finally, qT, which involves antenna state transitions (where LHCII migrates to PSI) (Quick & Stitt, 1989). ...
... There are several forms of NPQ, defined by their rates of activation and deactivation (Pinnola & Bassi, 2018). These forms include the most rapid form, energy-dependent quenching (qE) (Li et al., 2004;Niyogi et al., 1998Niyogi et al., , 2005, state-transition quenching (qT) (Quick & Stitt, 1989), Zx-dependent quenching (qZ) (Demmig-Adams & Adams, 1996;Li et al., 2004;Müller et al., 2001;Nilkens et al., 2010), sustained quenching (qH) (Malnoë, Schultink, et al., 2018), and photoinhibitory quenching (qI) (Andersson & Aro, 2004;Murata et al., 2007;Nawrocki et al., 2021). In the following subsections, we will discuss the essential role of redox regulation on each NPQ form. ...
... It is triggered by the acidification of the lumen (proton gradient (ΔpH) component of pmf which results in the protonation of PsbS as well as the activation of VDE, which catalyzes the conversion of violaxanthin (Vx) to zeaxanthin (Zx). Zx subsequently dissipates excessive light energy and induces qE along with PsbS (Niyogi et al., 1998;Brooks et al., 2014). ...
Photosynthesis is the foundation of life on Earth. However, if not well regulated, it can also generate excessive reactive oxygen species (ROS), which can cause photodamage. Regulation of photosynthesis is highly dynamic, responding to both environmental and metabolic cues, and occurs at many levels, from light capture to energy storage and metabolic processes. One general mechanism of regulation involves the reversible oxidation and reduction of protein thiol groups, which can affect the activity of enzymes and the stability of proteins. Such redox regulation has been well studied in stromal enzymes, but more recently, evidence has emerged of redox control of thylakoid lumenal enzymes. This review/hypothesis paper summarizes the latest research and discusses several open questions and challenges to achieving effective redox control in the lumen, focusing on the distinct environments and regulatory components of the thylakoid lumen, including the need to transport electrons across the thylakoid membrane, the effects of pH changes by the proton motive force ( pmf ) in the stromal and lumenal compartments, and the observed differences in redox states. These constraints suggest that activated oxygen species are likely to be major regulatory contributors to lumenal thiol redox regulation, with key components and processes yet to be discovered.
... However, photosynthetic efficiency has been supposed to be limited by the kinetics of qE induction and relaxation, particularly under fluctuating light conditions, which frequently occur under natural conditions on partly cloudy days (Zhu et al. 2004, Murchie andNiyogi 2011). Since high amounts of Zx accelerate the induction of qE, but decelerate the relaxation of qE (Niyogi et al. 1998), acceleration of Zx synthesis and reconversion might be a promising target to improve photosynthetic efficiency. Consequently, bioengineering of the dynamics of NPQ induction and relaxation has been applied to improve photosynthesis under fluctuating light conditions (Kromdijk et al. 2016, Garcia-Molina and Leister 2020, Lehretz et al. 2022. ...
... The only general trend visible in both species was the 10-20% lower VAZ pool size found in VPZ plants compared to WT plants. The impact of overexpression of only either VDE or ZEP on the VAZ pool size has not been studied in detail so far, but it is known from Arabidopsis that knock-out of VDE (npq1 mutant) leads to a moderately increased (up to 1.5-fold) (Niyogi et al. 1998) or an unchanged (Kalituho et al. 2007) VAZ pool size compared to WT plants. In contrast, knock-out of ZEP (npq2 mutant) induces a much more pronounced increase (3-to 4fold) in the VAZ pool size (Niyogi et al. 1998, Kalituho et al. 2007). ...
... The impact of overexpression of only either VDE or ZEP on the VAZ pool size has not been studied in detail so far, but it is known from Arabidopsis that knock-out of VDE (npq1 mutant) leads to a moderately increased (up to 1.5-fold) (Niyogi et al. 1998) or an unchanged (Kalituho et al. 2007) VAZ pool size compared to WT plants. In contrast, knock-out of ZEP (npq2 mutant) induces a much more pronounced increase (3-to 4fold) in the VAZ pool size (Niyogi et al. 1998, Kalituho et al. 2007). Possibly, the amount of xanthophyll cycle enzymes, most importantly the ZEP amount, may have an impact on the VAZ pool size. ...
The generation of VPZ lines, which simultaneously overexpress violaxanthin de-epoxidase (VDE), PsbS and zeaxanthin epoxidase (ZEP), has been successfully used to accelerate the kinetics of the induction and relaxation of non-photochemical quenching (NPQ). Here, we studied the impact of the over-expression of VDE and ZEP on the conversion of the xanthophyll cycle pigments in VPZ lines of Arabidopsis thaliana and Nicotiana tabacum. The protein amount of both VDE and ZEP was determined to be increased to about 3-5fold levels of wild-type (WT) plants for both species. Compared to WT plants, the conversion of violaxanthin (Vx) to zeaxanthin (Zx), and hence VDE activity, was only marginally accelerated in VPZ lines, whereas the conversion of Zx to Vx, and thus ZEP activity, was strongly increased in VPZ lines. This indicates, that the amount of ZEP but not the amount of VDE is a critical determinant of the equilibrium of the de-epoxidation state of the xanthophyll cycle pigments under saturating light conditions. Comparing the two steps of epoxidation, particularly the second step (antheraxanthin (Ax) to Vx) was found to be accelerated in VPZ lines, implying that the intermediate Ax is released into the membrane during epoxidation by ZEP.
... Energy-dependent NPQ (qE) is the main and most rapid NPQ component in plants. The qE responds to the proton concentration of the lumen (Briantais et al., 1979;Niyogi, 1998;Li et al., 2000Li et al., , 2004 and deactivates within seconds (Demmig-Adams et al., 1996). Upon protonation, the PsbS protein catalyzes a rearrangement of the photosystem (PS) II supercomplex, which is required for qE activation (Johnson et al., 2011). ...
... Zeaxanthin is synthesized from the epoxidated xanthophyll violaxanthin (Vx) by the lumen-localized violaxanthin de-epoxidase (VDE) that is also activated by protonation (Li et al., 2004;Arnoux et al., 2009). The Zx levels respond to lumen pH on the minute timescale (Hager, 1969;Demmig et al., 1987;Hartel et al., 1996;Niyogi, 1998). Recent data point toward a function of Zx in regulating qE dynamics rather than the dissipation process itself (Holt et al., 2005;Johnson et al., 2009;Nilkens et al., 2010;Xu et al., 2015;Sacharz et al., 2017;Tutkus et al., 2019). ...
... After 3 wk of pre-cultivation in a phytotron (120 lmol photons m À2 s À1 , climate conditions as described above), plants were grown in the polytunnel from 21 March to 28 March 2019 (ND 1 ) or 4 April to 14 April 2019 (ND 2 , Figs 1a, S1a). Measurements were performed on Days 7-8 (ND 1 ) or 8-9 (ND 2 ) after transfer and on mature phytotrongrown plants (FD L : 5-6 wk, FD M : 4-5 wk FD H : 3-4 wk, DD S : 3-4 wk, DD F : 5-6 wk, DD SF : 4-5 wk) 1-2 wk before bolting of wild-type Columbia-0 (WT) and mutants: vccn1-1: Salk 103612; vccn1-2: Gabi-Kat 796C09, kea3-1: Gabi-Kat 170G09; kea3-2: Sail 556_E12, npq2: Salk_059469, npq2 kea3 (Armbruster et al., 2014;Duan et al., 2016;Herdean et al., 2016) and npq4 (Niyogi, 1998). npq2 and npq2 kea3-1 together with controls were grown under elevated humidity using a dome. ...
Understanding photosynthesis in natural, dynamic light environments requires knowledge of long‐term acclimation, short‐term responses, and their mechanistic interactions. To approach the latter, we systematically determined and characterized light‐environmental effects on thylakoid ion transport‐mediated short‐term responses during light fluctuations.
For this, Arabidopsis thaliana wild‐type and mutants of the Cl⁻ channel VCCN1 and the K⁺ exchange antiporter KEA3 were grown under eight different light environments and characterized for photosynthesis‐associated parameters and factors in steady state and during light fluctuations. For a detailed characterization of selected light conditions, we monitored ion flux dynamics at unprecedented high temporal resolution by a modified spectroscopy approach.
Our analyses reveal that daily light intensity sculpts photosynthetic capacity as a main acclimatory driver with positive and negative effects on the function of KEA3 and VCCN1 during high‐light phases, respectively. Fluctuations in light intensity boost the accumulation of the photoprotective pigment zeaxanthin (Zx). We show that KEA3 suppresses Zx accumulation during the day, which together with its direct proton transport activity accelerates photosynthetic transition to lower light intensities.
In summary, both light‐environment factors, intensity and variability, modulate the function of thylakoid ion transport in dynamic photosynthesis with distinct effects on lumen pH, Zx accumulation, photoprotection, and photosynthetic efficiency.
... Currently, there are numerous studies investigating the role of VDEs in resistance to high light stress. For instance, Arabidopsis VDE mutants (Atnpq1) have demonstrated significant suppression of NPQ and leaf developmentdependent photosynthetic defect damage (Havaux et al. 2000;Niyogi et al. 1998). In tobacco, the overexpression of LeVDE has been found to reduce its susceptibility to high light inhibition (Guan et al. 2014). ...
... Previous studies have indicated that the conversion of violaxanthin to zeaxanthin in plants is limited. The deletion or suppression of VDE in Arabidopsis (Atnpq1) and tobacco has been found to result in a reduction in NPQ (Niyogi et al. 1998;Chang et al. 2000). Reduced NPQ is found in both Arabidopsis Atvdr mutants and cucumber CsVDR silencing lines, which lead us to speculate on the functional redundancy of VDE and VDR in plants. ...
The xanthophyll cycle plays a pivotal role in protecting plants and algae against photodamage. Although the resistance of the violaxanthin de-epoxidase enzyme (VDE) to high light stress in the xanthophyll cycle has been extensively studied, there is limited knowledge about VDE-related (VDR) proteins, which exhibit a close homologous relationship with VDEs. In this study, we preliminary investigated VDR protein, focusing on basic bioinformatics, spatiotemporal gene expression patterns, and high light stress treatment. VDR exhibited a significant homology with VDE, and the CsVDR protein was localized in the chloroplasts. CsVDR was expressed in all tissues of Arabidopsis and cucumber, with the highest expression level observed in mature leaves cultivated for 20 days in cucumber. Interestingly, both CsVDR and AtVDR were identified as high light response genes. Under high light stress, the non-photochemical quenching and Fv/Fm exhibited a decrease in both the Atvdr mutants and TRSV::CsVDR lines compared to the WT. Additionally, the de-epoxidation ratio (A + Z)/(A + Z + V) of the Atvdr mutants was significantly reduced. This suggested that the xanthophyll cycle in Atvdr mutants and TRSV::CsVDR lines were less effective and more susceptible to photoinhibition of PSII under high light stress. Our findings provide compelling evidence for the involvement of VDR proteins in regulating plant response to high light, thereby offering a theoretical basis for further investigation into plant photoprotective pathways.
... This enzyme is widely distributed across photosynthetic eukaryotes, encoded by single or multiple gene copies [21]. In plants and green algae that are believed to harbor a single ZEP-encoded gene, ZEP disruption leads to constant accumulation of zeaxanthin and lack of violaxanthin yet just slight changes of growth [22][23][24][25][26]. By contrast, N. oceanica is proposed to contain two putative ZEP genes; one has been functionally examined through transient expression in tobacco leaves [27]. ...
... Plants contain a single ZEP-encoded gene and the ZEP disruption mutants (e.g., npq2 for Arabidopsis and aba2 for tobacco) constitutively accumulate zeaxanthin yet lacking its epoxidated derivatives [22,23]. Similarly, the green algae C. reinhardtii and Dunaliella salina defective in the single ZEP gene contain zeaxanthin but no violaxanthin [24,25]. ...
Background
The marine alga Nannochloropsis oceanica, an emerging model belonging to Heterokont, is considered as a promising light-driven eukaryotic chassis for transforming carbon dioxide to various compounds including carotenoids. Nevertheless, the carotenogenic genes and their roles in the alga remain less understood and to be further explored.
Results
Here, two phylogenetically distant zeaxanthin epoxidase (ZEP) genes from N. oceanica (NoZEP1 and NoZEP2) were functionally characterized. Subcellular localization experiment demonstrated that both NoZEP1 and NoZEP2 reside in the chloroplast yet with differential distribution patterns. Overexpression of NoZEP1 or NoZEP2 led to increases of violaxanthin and its downstream carotenoids at the expense of zeaxanthin in N. oceanica, with the extent of changes mediated by NoZEP1 overexpression being greater as compared to NoZEP2 overexpression. Suppression of NoZEP1 or NoZEP2, on the other hand, caused decreases of violaxanthin and its downstream carotenoids as well as increases of zeaxanthin; similarly, the extent of changes mediated by NoZEP1 suppression was larger than that by NoZEP2 suppression. Interestingly, chlorophyll a dropped following violaxanthin decrease in a well-correlated manner in response to NoZEP suppression. The thylakoid membrane lipids including monogalactosyldiacylglycerol also correlated with the violaxanthin decreases. Accordingly, NoZEP1 suppression resulted in more attenuated algal growth than NoZEP2 suppression did under either normal light or high light stage.
Conclusions
The results together support that both NoZEP1 and NoZEP2, localized in the chloroplast, have overlapping roles in epoxidating zeaxanthin to violaxanthin for the light-dependent growth, yet with NoZEP1 being more functional than NoZEP2 in N. oceanica. Our study provides implications into the understanding of carotenoid biosynthesis and future manipulation of N. oceanica for carotenoid production.
... It has been demonstrated that NPQ is indeed essential both for the optimal growth and survival of plants under natural conditions (Külheim et al. 2002). However, many studies have also shown that NPQ has a marginal effect on the photoprotection of PSII, particularly under constant or gradually changing incident irradiance (Niyogi et al. 1998, Havaux and Niyogi 1999, Li et al. 2000, Niyogi et al. 2001, Sarvikas et al. 2006, Murata et al. 2012). As such, there exists no general consensus on the photoprotective role of NPQ. ...
... On the surface, this observation suggests that Y(NPQ), although a highly dynamic and reversible quenching component, does not protect PSII significantly in field plant species. The result is consistent with the observation that NPQ-deficient plants do not show symptoms of chronic photoinhibition when grown under low to moderate light in a chamber (Niyogi et al. 1998, Havaux and Niyogi 1999, Li et al. 2000, Niyogi et al. 2001. Furthermore, it was demonstrated that NPQ offers only 0-25% higher photoprotection for wild-type Arabidopsis, compared with the npq 4 mutant (which lacks a functional PsbS protein) and npq 1 (which lacks violaxanthin de-epoxidase) (Sarvikas et al. 2006). ...
Non-photochemical quenching (NPQ) has been regarded as a safety valve to dissipate excess absorbed light energy not used for photochemistry. However, there exists no general consensus on the photo-protective role of NPQ. In the present study, we quantified the Photosystem II (PSII) photo-susceptibilities (mpi) in the presence of lincomycin, under red light given to five shade-acclimated tree species grown in the field. Photosynthetic energy partitioning theory was applied to investigate the relationships between mpi and each of the regulatory light-induced non-photochemical quenching [Y(NPQ)], the quantum yield of the constitutive non-regulatory non-photochemical quenching [Y(NO)], and the PSII photochemical yield in the light-adapted state [Y(PSII)] under different red irradiances. It was found that in the low to moderate irradiance range (50-800 μmol m-2 s-1) when the fraction of open reaction centers (qP) exceeded 0.4, mpi exhibited no association with Y(NPQ), Y(NO), and Y(PSII) across species. However, when qP < 0.4 (1,500 μmol m-2 s-1), there existed positive relationships between mpi and Y(NPQ) or Y(NO), but a negative relationship between mpi and Y(PSII). It is postulated that both Y(NPQ) and Y(NO) contain protective and damage components, and that using only Y(NPQ) or Y(NO) metrics to identify the photo-susceptibility of a species is a risk. It seems that qP regulates the balance of the two components for each of Y(NPQ) and Y(NO). Under strong irradiance, when both protective Y(NPQ) and Y(NO) are saturated/depressed, the forward electron flow [i.e., Y(PSII)] acts as the last defense to resist photoinhibition.
... One possible explanation is that TDO can rapidly introduce a second oxidation at the 5-carbon position of 4-hydroxymesotrione, causing this intermediate to be undetectable. After the second oxidation of mesotrione, oxy-mesotrione is generated 16,42 Mesotrione inhibits HPPD and affects the function of PDS through depletion of PQ. The image in (A) was taken 5 days after mesotrione treatment at the V1 growth stage. ...
... This is most likely the result of xanthophyll cyclemediated rebalancing, which involves the npq1-like genes among others. 42 Mature leaves of conventional soybean plants are slow to respond to mesotrione treatment due to the minimal expression of PDS genes in those leaves in combination with the slow formation of noninhibitory 4-hydroxy-mesotrione and 5-hydroxymesotrione, 23,27,28 a relatively high level reserve of xanthophylls, and the rebalance of xanthophylls. ...
BACKGROUND
Herbicide‐resistant weeds pose a challenge to agriculture and food production. New herbicide tolerance traits in crops will provide farmers with more options to effectively manage weeds. Mesotrione, a selective pre‐ and post‐emergent triketone herbicide used in corn production, controls broadleaf and some annual grass weeds via hydroxyphenylpyruvate dioxygenase (HPPD) inhibition. Recently, the rice HIS1 gene, responsible for native tolerance to the selective triketone herbicide benzobicyclon, was identified. Expression of HIS1 also confers a modest level of mesotrione resistance in rice. Here we report the use of the HIS1 gene to develop a mesotrione tolerance trait in soybean.
RESULTS
Conventional soybean is highly sensitive to mesotrione. Ectopic expression of a codon‐optimized version of the rice HIS1 gene (TDO) in soybean confers a commercial level of mesotrione tolerance. In TDO transgenic soybean plants, mesotrione is rapidly and locally oxidized into noninhibitory metabolites in leaf tissues directly exposed to the herbicide. These metabolites are further converted into compounds similar to known classes of plant secondary metabolites. This rapid metabolism prevents movement of mesotrione from treated leaves into vulnerable emerging leaves. Minimizing the accumulation of the herbicide in vulnerable emerging leaves protects the function of HPPD and carotenoid biosynthesis more generally while providing tolerance to mesotrione.
CONCLUSIONS
Mesotrione has a favorable environmental and toxicological profile. The TDO‐mediated soybean mesotrione tolerance trait described here provides farmers with a new option to effectively manage difficult‐to‐control weeds using familiar herbicide chemistry. This trait can also be adapted to other mesotrione‐sensitive crops (e.g. cotton) for effective weed management. © 2022 Bayer Crop Science. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
... In high-light conditions, several processes are activated in the photosynthetic thylakoid membrane to prevent over excitation, including nonphotochemical quenching (NPQ), which causes dissipation of singlet chlorophyll (Chl) excited-state energies in the PSII antenna as heat (2). Two processes contribute to NPQ on a second to minute timescale, both controlled by the thylakoid lumen pH: rapid (1-3 min) induced quenching termed as qE through activation of the proteins photosystem II subunit S and light-harvesting complex stress-related protein (LHCSR), and zeaxanthin (Zea)-dependent quenching, known as qZ that is activated by the xanthophyll cycle, which reversibly converts the carotenoid violaxanthin (Vio) into Zea via antheraxanthin (3)(4)(5). Molecular sites involved in qE and qZ are integrated in the light-harvesting antenna complexes of PSII, of which the most abundant ones are the Chl a/b binding protein complexes (light-harvesting complex II [LHCII], see Fig. 1). ...
... Many studies on the effects of Zea make use of carotenoid-less mutants, of which the most well-studied ones are those of the npq2 strains (4,13,14). The npq2 mutants, which contain a defect in the enzyme zeaxanthin deepoxidase and bind constitutively Zea in the V1 binding pocket, lack the carotenoid neoxanthin (Neo). ...
The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the Light-Harvesting Complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from ¹³C enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared to the trimeric, wild type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein-bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.
... The precise mechanism of how zeaxanthin mediates NPQ is not clear. However, 36 its level is determined by the balance between the activity of two enzymes of the xanthophyll 37 cycle: violaxanthin de-epoxidase (VDE, also known as NPQ1) that converts violaxanthin to 38 zeaxanthin, and zeaxanthin epoxidase (ZEP or NPQ2), which mediates the opposite reaction 39 (Niyogi et al., 1998;Jahns and Holzwarth, 2012). PsbS (also known as NPQ4) is a thylakoid 40 membrane protein that does not bind pigments (Li et al., 2000;Li et al., 2002). ...
Non-photochemical quenching (NPQ), the dissipation of excess light energy as heat, has been long recognized as a major protective mechanism that minimizes the potential for oxidative damage to photosystem II (PSII) reaction centers. Two major positive contributors to NPQ are the carotenoid zeaxanthin, generated from violaxanthin by the enzyme violaxanthin de-epoxidase (VDE or NPQ1), and the thylakoid protein PsbS (NPQ4). The involvement of the lumenal Deg proteases in the repair of PSII from photoinhibition prompted us to further explore their possible role in other responses of Arabidopsis thaliana to high light. Here we show that upon exposure to high light, the single deg1 and the triple deg158 mutants display different levels and kinetics of NPQ, compared to the deg58 mutant and WT that behave alike. In response to high light, the two genotypes lacking Deg1 over-accumulate NPQ1 and NPQ4. After temporal inhibition of protein translation in vivo, the level of these two proteins in deg1 is higher than in WT. Together, the results suggest that Deg1 represents a new level of regulation of the NPQ process through adjusting the quantity of NPQ1 and NPQ4 proteins, probably through their proteolysis.
... Some protective mechanisms to ameliorate light stress include the dissipation of excess light energy, antioxidant systems and photooxidative damage repair (Niyogi et al. 1998). The PS II activity loss is determined by the balance of the competing photodamage and repair rate (Kodru et al. 2020). ...
Pyropia haitanensis was cultured under two CO2 (410 (LC), 1000 (HC) μL L⁻¹) concentrations and six chloramphenicol (CAP)-methanol solutions (0, 0+methanol, 10, 50, 100, 250 μg mL⁻¹) to investigate the effects of elevated CO2 and CAP on its growth, photosynthesis and biochemical characteristics. HC had no obvious effects on the growth rate (RGR) with CAP in the range of 10 to 100 μg mL⁻¹, but the decrease of RGR by HC was statistically significant with the CAP dosage at 250 μg mL⁻¹. HC had no significant effect on net photosynthetic rates (Pn) in the present of CAP (10-250 μg mL⁻¹). CAP greatly reduced net photosynthesis as well as the maximal photochemical yield (Fv/Fm) and photosynthetic efficiency (αETR). In contrast, the maximum relative electron transport rates (rETRm) were almost constant with the CAP dosage from 10 to 100 μg mL⁻¹. HC significantly increased the energy fluxes (per RC) for absorption (ABS/RC), trapping (TRo/RC) and transport fluxes (ETo/RC) with the dosage of CAP at 250 μg mL⁻¹. Principal component analysis (PCA) indicated that CAP was positively correlated with the synthesis of free amino acids (FAA), contents of umami-, sweet- and essential AA were significantly enhanced with the interaction of HC and higher CAP dosage at 100 μg mL⁻¹, which led to the variation of flavor in algae. Furthermore, phycobiliproteins and soluble protein (SP) contents were remarkably reduced by CAP. Contents of chlorophyll a (Chl a), carotenoids (Car), soluble carbohydrates (SC) and C/N ratios were almost unchanged among treatments. The study indicates that future ocean acidification has no obvious effects on the biomass productivity of P. haitanensis, maintained steady photosynthetic activities with the CAP (within 100 μg mL⁻¹) and induces better flavor. The data obtained have important theoretical relevance for in-depth understanding of algal responses to global changes and oceanic contamination.
... These two reactions form the xanthophyll cycle, which is an important photoprotection process because zeaxanthin is involved in the nonphotochemical quenching (NPQ) of excess light energy in PS antennas through deactivation of excited singlet chlorophyll and elimination of oxygen radicals (Jahns and Holzwarth, 2012). Analysis of a mutant defective in the xanthophyll cycle, npq1, showed that zeaxanthin synthesis is necessary for approximately 70% of the total NPQ in Arabidopsis (Niyogi et al., 1998). Another study found that the accumulation of H 2 O 2 in vivo induced the inactivation of ZEP activity under long-lasting light stress (Holzmann et al., 2022). ...
... NPQ consists of several components, the main one being qE, which is characterized by fast induction and relaxation kinetics within a few minutes after light irradiation and returning to dark conditions, respectively (Nilkens et al. 2010;Ruban 2016). qE is induced by the protonation of PSII subunit PsbS and the conversion of pigments in the xanthophyll cycle (Li et al. 2000;Niyogi et al. 1998). In the xanthophyll cycle, violaxanthin is converted to zeaxanthin via antheraxanthin by violaxanthin de-epoxidase, which is activated by lumen acidification (Szabo et al. 2005). ...
Thioredoxin (Trx) is a small redox mediator protein involved in the regulation of various chloroplast functions by modulating the redox state of Trx target proteins in ever-changing light environments. Using reducing equivalents produced by the photosynthetic electron transport chain, Trx reduces the disulfide bonds on target proteins and generally turns on their activities. While the details of the protein-reduction mechanism by Trx have been well investigated, the oxidation mechanism that counteracts it has long been unclear. We have recently demonstrated that Trx-like proteins such as Trx-like2 and atypical Cys His-rich Trx (ACHT) can function as protein oxidation factors in chloroplasts. Our latest study on transgenic Arabidopsis plants indicated that the ACHT isoform ACHT2 is involved in regulating the thermal dissipation of light energy. To understand the role of ACHT2 in vivo, we characterized phenotypic changes specifically caused by ACHT2 overexpression in Arabidopsis. ACHT2-overexpressing plants showed growth defects, especially under high light conditions. This growth phenotype was accompanied with the impaired reductive activation of Calvin–Benson cycle enzymes, enhanced thermal dissipation of light energy, and decreased photosystem II activity. Overall, ACHT2 overexpression promoted protein oxidation that led to the inadequate activation of Calvin–Benson cycle enzymes in light and consequently induced negative feedback control of the photosynthetic electron transport chain. This study highlights the importance of the balance between protein reduction and oxidation in chloroplasts for optimal photosynthetic performance and plant growth.
... To more specifically address how a change in thylakoid lipid composition might influence NPQ, we focused on an analysis of the xanthophyll cycle (XC), which contributes substantially to the dissipation of excess light energy as heat. Acidification of the thylakoid lumen by the establishment of the light-driven proton gradient activates the enzyme Vx to Ax and further to Zx, thereby dissipating excess excitation energy (Niyogi et al. 1998). In addition to Vx de-epoxidation, the low luminal pH leads to protonation of the PSII subunit S (PsbS), which belongs to the LHC family of proteins. ...
Monogalactosyldiacylglycerol (MGDG) is the main lipid constituent of thylakoids and a structural component of photosystems and photosynthesis-related proteo-lipid complexes in green tissues. Previously reported changes in MGDG abundance upon stress-treatments are hypothesized to reflect mobilization of MGDG-based polyunsaturated lipid intermediates to maintain extraplastidial membrane integrity. While exchange of lipid intermediates between compartmental membranes is well documented, physiological consequences of mobilizing an essential thylakoid lipid, such as MGDG, for an alternative purpose are not well understood.Arabidopsis seedlings exposed to mild (50 mM) salt-treatment displayed significantly increased abundance of both MGDG and the extraplastidial lipid, phosphatidylcholine (PC). Interestingly, similar increases in MGDG and PC were observed in Arabidopsis fad3 mutant seedlings defective in ER-localized linolenic acid formation, in which compensatory plastid-to-ER-directed mobilization of linolenic acid-containing intermediates takes place. The postulated (salt) or evident ( fad3 ) plastid-ER-exchange of intermediates concurred with altered thylakoid function according to parameters of photosynthetic performance. While salt-treatment of wild type seedlings inhibited photosynthetic parameters in a dose-dependent manner, interestingly the fad3 mutant did not show overall reduced photosynthetic quantum yield. By contrast, we observed a reduction specifically of non-photochemical quenching (NPQ) under high light, representing only part of observed salt effects. The decreased NPQ in the fad3 mutant was accompanied by reduced activity of the xanthophyll cycle, leading to a reduced concentration of the NPQ-effective pigment zeaxanthin. The findings suggest that altered ER-located fatty acid unsaturation and ensuing inter-organellar compensation impacts on aspects of thylakoids related to the function of specific enzymes, rather than globally affecting thylakoid function.
Subject Areas
(2) Environmental and stress responses
(7) Membrane and transport
... Recently, zeaxanthin epoxidase (ZEP), which catalyzes the conversion of zeaxanthin to violaxanthin, was suggested to be redox-regulated by the Trx system because Arabidopsis mutants defective in NTRC or Trx m showed increased accumulation of zeaxanthin and high non-photochemical quenching (NPQ) of the chlorophyll fluorescence phenotype under low light conditions (Naranjo et al. 2016, Da et al. 2018. Zeaxanthin is synthesized under high light conditions and is required for NPQ induction (Niyogi et al. 1998). In contrast, ZEP was reported to be regulated by hydrogen peroxide under high light conditions (Holzmann et al. 2022). ...
... qE reacts within one-tenth of a second (Muller et al. 2001)-and is, therefore, the mechanism that responds most rapidly to sudden increases in light intensity-whereas qZ has a slower relaxation time of 10 to 15 minutes. Several proteins involved in NPQ have been identified through forward genetic screens, including PHOTOSYSTEM II SUBUNIT S (PSBS, also named NPQ4 based on the corresponding mutant; Li et al. (2000)), VIOLAXANTHIN DE-EPOXIDASE 1 (VDE1; or NPQ1), and ZEAXANTHIN EPOXIDASE (ZEP; or NPQ2) (Niyogi et al. 1998). ...
Photosynthetic organisms must cope with rapid fluctuations in light intensity. Nonphotochemical quenching (NPQ) enables the dissipation of excess light energy as heat under high light conditions, whereas its relaxation under low light maximizes photosynthetic productivity. We quantified variation in NPQ kinetics across a large sorghum (Sorghum bicolor) association panel in four environments, uncovering significant genetic control for NPQ. A genome-wide association study (GWAS) identified 20 unique regions in the sorghum genome associated with NPQ. We detected strong signals from the sorghum ortholog of Arabidopsis thaliana SUPPRESSOR OF VARIEGATION3 (SVR3) involved in plastid nucleus signaling and tolerance to cold. By integrating GWAS results for NPQ across maize (Zea mays) and sorghum association panels, we identified a second gene, NON-YELLOWING 1 (NYE1), originally identified by Gregor Mendel in pea (Pisum sativum) and involved in the degradation of photosynthetic pigments in light-harvesting complexes, along with OUTER ENVELOPE PROTEIN 37 (OEP37), that encodes a transporter in chloroplast envelope. Analysis of nye1 insertion alleles in A. thaliana confirmed the effect of this gene on NPQ kinetics across monocots and eudicots. We extended our comparative genomics GWAS framework across the entire maize and sorghum genomes, identifying four additional loci involved in NPQ kinetics. These results provide a baseline for engineering crops with improved NPQ kinetics and increasing the accuracy and speed of candidate gene identification for GWAS in species with high linkage disequilibrium.
... An increase of the qE capacity during long-term acclimation to HL conditions is accompanied by a reduction of photoinhibition, supporting the photoprotective function of this NPQ mechanism. The qE capacity is predominantly regulated by the amount of the PsbS protein (Li et al., 2000(Li et al., , 2002 and the amount of Zx (Kalituho et al., 2007;Nilkens et al., 2010;Niyogi et al., 1998). ...
Proper short‐ and long‐term acclimation to different growth light intensities is essential for the survival and competitiveness of plants in the field. High light exposure is known to induce the down‐regulation and photoinhibition of photosystem II (PSII) activity to reduce photo‐oxidative stress. The xanthophyll zeaxanthin (Zx) serves central photoprotective functions in these processes. We have shown in recent work with different plant species (Arabidopsis, tobacco, spinach and pea) that photoinhibition of PSII and degradation of the PSII reaction center protein D1 is accompanied by the inactivation and degradation of zeaxanthin epoxidase (ZEP), which catalyzes the reconversion of Zx to violaxanthin. Different high light sensitivity of the above‐mentioned species correlated with differential down‐regulation of both PSII and ZEP activity. Applying light and electron microscopy, chlorophyll fluorescence, and protein and pigment analyses, we investigated the acclimation properties of these species to different growth light intensities with respect to the ability to adjust their photoprotective strategies. We show that the species differ in phenotypic plasticity in response to short‐ and long‐term high light conditions at different morphological and physiological levels. However, the close co‐regulation of PSII and ZEP activity remains a common feature in all species and under all conditions. This work supports species‐specific acclimation strategies and properties in response to high light stress and underlines the central role of the xanthophyll Zx in photoprotection.
... In contrast to the response times obtained from activation during a dark-to-light induction, for example, in Wehner et al. (2006) and Nilkens et al. (2010), the limits of the NPQ operation range in oscillating light depend on an interplay of the activation and deactivation processes that both contribute to plant response to fluctuating light. Toward this goal, we explored the responses to oscillating light of the wild-type, the npq1 mutant, which cannot convert violaxanthin into zeaxanthin by VDE (Niyogi et al., 1998), and of the npq4 mutant that lacks the PsbS protein (Li et al., 2000). ...
In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light.
Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild‐types Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de‐epoxidase, the npq4 mutant lacking PsbS protein, and the mutants crr2‐2, and pgrl1ab impaired in different pathways of the cyclic electron transport.
The fastest was the PsbS‐regulation responding to oscillation periods longer than 10 s. Processes involving violaxanthin de‐epoxidase dampened changes in chlorophyll fluorescence in oscillation periods of 2 min or longer. Knocking out the PGR5/PGRL1 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH‐like pathway only slightly changed the photosynthetic dynamics.
Our observations are consistent with the hypothesis that nonphotochemical quenching in slow light oscillations involves violaxanthin de‐epoxidase to produce, presumably, a largely stationary level of zeaxanthin. We interpret the observed dynamics of photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.
... La lumière absorbée en excès peut conduire à la photoinhibition, c'est à dire une baisse dans l'efficacité photosynthétique pouvant aller jusqu'à altération des photosystèmes. Cette photoinhibition provient de la production de dérivé réactif de l'oxygène (ROS) (Asada, 1999;Niyogi et al., 1998) endommageant l'appareil photosynthétique comme le PSII (Powles, 1984). Cet excès de lumière doit donc être limité pour éviter ces conséquences. ...
Depuis les années 2000, sur le continuum Estuaire - Baie de la Seine les apports en phosphores (P) ont été réduit contrairement à ceux en azote (N) qui sont restés élevés. Cette réduction entraîne donc un déséquilibre de la stoechiométrie N:P affectant les communautés phytoplanctoniques. L'écart à l'équilibre optimal de Redfield (N:P = 16) est appelé dystrophie. Pour qualifier et quantifier l'effet de la dystrophie sur le phytoplancton les méthodes de fluorimétrie variable et de cytométrie en flux sont employées pour étudier les production primaire, la structure des communautés et leurs interactions. Dans l'estuaire, au-delà de la limitation en lumière les communautés phytoplanctoniques sont affectées par la distribution des sels nutritifs, les déséquilibres N:Si et P:Si provoquent la succession des communautés. Une dynamique complexe de la production primaire a également été mis en évidence avec un rôle important du temps de résidence mais également l'influence de certains facteurs encore mal identifiés. Les expériences en milieux contrôlés et l'étude d'une efflorescence dans la baie montrent que la baie est co-limitée par le N et le P en fonction du rapport N:P, rendant nécessaire l'établissement d'un nouveau calcul "d'efficacité d'utilisation de la ressource (RUE)" adapté à la dystrophie, le RUE NP. La dystrophie va affecter négativement les paramètres photosynthétiques et la production primaire, et accroître des indicateurs de stress physiologiques [activité de la phosphatase alcaline et les excrétions de carbone (TEP)]. Cette dystrophie influence aussi la structure des communautés en entraînant une baisse de la diversité fonctionnelle et modification de la taille des communautés. La prise est compte de l’effet de la dystrophie sur le phytoplancton associé à celui du changement climatique est ainsi essentiel pour la gestion des écosystèmes côtiers et la scénarisation de trajectoires dans le continuum dans un contexte de gestion de l’eau est donc majeure.
... In the β-branch, β-carotene was converted by CHYB to generate zeaxanthin through β-cryptoxanthin intermediate. Zeaxanthin can be further epoxidized in two steps to form violaxanthin by zeaxanthin epoxidase (ZEP), while the removal of the epoxy groups of violaxanthin catalyzed by violaxanthin de-epoxidase (VDE) (Niyogi et al., 1998). The conversion of violaxanthin to neoxanthin can be catalyzed by neoxanthin synthase (NSY) (Al-Babili et al., 2000). ...
Carotenoids, a group of essential pigments for petal coloration in many species, could be generally categorized into linear and cyclic groups. The cyclization of the linear substrate lycopene at both end groups by β- and ε-cyclases (LCYBs and LCYEs, respectively) is a key branching point of the biosynthesis of cyclic carotenoids in flowers. Common zinnia (Zinnia elegans L.), an Asteraceae family plant that has been widely used in landscape greening, is best known for its brilliant flower colors. However, the mechanism of its petal coloration is not well studied, especially with respect to carotenoid pigmentation. In this study, total carotenoid content and carotenoid compositions of petals were analyzed in two common zinnia cultivars, 'Dreamland Red’ (DRE) and 'Dreamland Yellow’ (DY). Despite the lighter color of DY petals, the total carotenoid content in DY flowers was significantly higher than that in DRE. Metabolomic analysis revealed that the major carotenoids of both cultivars are cyclic carotenoids, with β-carotene being the most abundant. In addition, one LCYB, LCYE, and capsanthin/capsorubin synthase (CCS) gene were identified from Z. elegans by homology-based search in transcriptome. After cloning and sequencing, full-length open reading frame of each gene is exactly the same between two cultivars. Bacterial pigment complementation experiments revealed that ZeLCYB and ZeCCS can cyclize lycopene at both ends to produce β-carotene. Unlike most LCYEs characterized from higher plants, ZeLCYE produced predominantly ε-carotene in E. coli system. However, ZeLCYE is likely to have a lower affinity for lycopene or lower activity compared with ZeLCYB. Gene expression analysis showed that DY has higher expression levels of ZeLCYB and ZeCCS than DRE at specific developmental stages, consistent with its higher total carotenoid content. This work would lay a solid foundation for future studies on carotenoid accumulation in Z. elegans.
... The most rapid component of NPQ is qE which is activated when there's a decrease in thylakoid lumen pH. NPQ1 of plants, which cannot synthesize zeaxanthin, shows NPQ reduction [12,13]. In this process, zeaxanthin synthesis increases the qE and qZ components. ...
The localization of carotenoids and macromolecular organization of thylakoid supercomplexes have not been reported yet in Chlamydomonas reinhardtii WT and cyclic electron transport mutants (pgrl1 and pgr5) under high light. Here, the various pigments, protein composition, and pigment-protein interactions were analyzed from the cells, thylakoids, and sucrose density gradient (SDG) fractions. Also, the supercomplexes of thylakoids were separated from BN-PAGE and SDG. The abundance of light-harvesting complex (LHC) II trimer complexes and pigment-pigment interaction were changed slightly under high light, shown by circular dichroism. However, a drastic change was seen in photosystem (PS)I-LHCI complexes than PSII complexes, especially in pgrl1 and pgr5. The lutein and β-carotene increased under high light in LHCII trimers compared to other supercomplexes, indicating that these pigments protected the LHCII trimers against high light. However, the presence of xanthophylls, lutein, and β-carotene was less in PSI-LHCI, indicating that pigment-protein complexes altered in high light. Even the real-time PCR data shows that the pgr5 mutant does not accumulate zeaxanthin dependent genes under high light, which shows that violaxanthin is not converting into zeaxanthin under high light. Also, the protein data confirms that the LHCSR3 expression is absent in PSI-LHCI and PSII complexes, and some of the core proteins were aggregated in pgr5 under high light.
... In this study we used WT Arabidopsis plants, ecotype Columbia (Col-0), transgenic lines 35S::HA-GI (David et al. 2006) and mutant lines aba1-6 (Niyogi et al. 1998), gi-2 (Fowler et al. 1999, gi-100 (Huq et al. 2000), fkf1 line SALK_059480 (Riboni et al. 2013) andco-10 (Laubinger et al. 2006). Seeds were germinated and plants were grown in a controlled environment at a temperature of 21-23 ∘ C and 65% relative humidity, either under LD (16 h light/8 h dark) or SD (8 h light/16 h dark) photocycles. ...
Transcriptional reprogramming plays a key role in drought stress responses, preceding the onset of morphological and physiological acclimation. The best-characterised signal regulating gene expression in response to drought is the phytohormone abscisic acid (ABA). ABA-regulated gene expression, biosynthesis and signalling are highly organised in a diurnal cycle, so that ABA-regulated physiological traits occur at the appropriate time of the day. The mechanisms that underpin such diel oscillations in ABA signals are poorly characterised. Here we uncover GIGANTEA (GI) as a key gatekeeper of ABA-regulated transcriptional and physiological responses. Time-resolved gene expression profiling by RNA sequencing under different irrigation scenarios indicates that gi mutants produce an exaggerated ABA response, despite accumulating wild-type levels of ABA. Comparisons with ABA-deficient mutants confirm the role of GI in controlling ABA-regulated genes and the analysis of leaf temperature, a read-out for transpiration, supports a role for GI in the control of ABA-regulated physiological processes. Promoter regions of GI/ABA-regulated transcripts are directly targeted by different classes of transcription factors, especially PHYTOCHROME-INTERACTING FACTORs, and (ABRE)-BINDING FACTOR, together with GI itself. We propose a model whereby diel changes in GI control oscillations in ABA responses. Peak GI accumulation at midday contributes to establishing a phase of reduced ABA sensitivity and related physiological responses, by gating DNA binding or function of different classes of transcription factors that cooperate or compete with GI at target regions.
... Transgenic and/or mutant plants with impaired biosynthetic capacity have been instrumental in identifying the functional role of specific metabolites. For instance, plants with impaired carotenoid biosynthesis have been used to demonstrate the role of carotenoids in photosynthesis and photoprotection under varying intensities of photosynthetic light (Niyogi et al., 1998;Li et al., 2009;Cazzaniga et al., 2012). Similarly, tocopherol biosynthesis enzymes have been identified, and it has been shown that tocopherol biosynthesis enzymes (vte) are crucial for the synthesis of tocopherol in plants (Havaux et al., 2005). ...
Tocopherols are lipophilic antioxidants in plants. Their potential role in protecting UV-B exposed plants has been alluded to, but not shown. Here we have determined UV-B induced accumulation of tocopherol in the model plant Arabidopsis thaliana and asked 1) whether induction relies on UV-B application duration, 2) if UV (UVR8) or blue light (phototropins or cryptochrome) photoreceptors mediate the accumulation of tocopherol and, 3) whether tocopherol contributes to UV-B photoprotection. Results show that UV-B induces accumulation of tocopherols in plants, with the strongest induction of α-tocopherol. The induction is dependent on exposure duration but not individual photoreceptors. Plants with impaired tocopherol biosynthesis show no UV-B induction of tocopherol and are found to be UV-sensitive. Overall, the results show for the first time that tocopherol accumulation is a component of UV-B acclimation and that impeding their responses leads to increased UV-susceptibility of the photosynthetic machinery.
... However, this possible constitutive quenching mechanism had a minor role in C. reinhardtii bkt5 because 77 K fluorescence measurements on dark or light-treated whole cells demonstrated that PSII in bkt5 cells is not in a constitutive quenching state (Additional file 1: Figure S8). The reduction of NPQ was more probably due to the changes in carotenoids compositions; mutants with altered xanthophylls and carotenes composition showed a reduction of NPQ both in microalgae and land plant [59,60]. However, independently from the reason for its reduction, the NPQ mechanisms are not involved in the increased resistance to strong irradiances observed in bkt5. ...
Background
Astaxanthin is a highly valuable ketocarotenoid with strong antioxidative activity and is natively accumulated upon environmental stress exposure in selected microorganisms. Green microalgae are photosynthetic, unicellular organisms cultivated in artificial systems to produce biomass and industrially relevant bioproducts. While light is required for photosynthesis, fueling carbon fixation processes, application of high irradiance causes photoinhibition and limits biomass productivity.
Results
Here, we demonstrate that engineered astaxanthin accumulation in the green alga Chlamydomonas reinhardtii conferred high light tolerance, reduced photoinhibition and improved biomass productivity at high irradiances, likely due to strong antioxidant properties of constitutively accumulating astaxanthin. In competitive co-cultivation experiments, astaxanthin-rich Chlamydomonas reinhardtii outcompeted its corresponding parental background strain and even the fast-growing green alga Chlorella vulgaris .
Conclusions
Metabolic engineering inducing astaxanthin and ketocarotenoids accumulation caused improved high light tolerance and increased biomass productivity in the model species for microalgae Chlamydomonas reinhardtii . Thus, engineering microalgal pigment composition represents a powerful strategy to improve biomass productivities in customized photobioreactors setups. Moreover, engineered astaxanthin accumulation in selected strains could be proposed as a novel strategy to outperform growth of other competing microalgal strains.
... Regarding the use of analytical techniques to determine photosynthetic pigments, high-performance liquid chromatography (HPLC) has been particularly widespread in studies aiming to understand the biochemical mechanisms associated with NPQ, and its major and fastest component called qE (Havaux and Kloppstech, 2001;Kopsell et al., 2012;Niyogi et al., 1998;Ruban, 2018). Despite there is no complete consensus about the site and underlying mechanism of qE, this energy dissipation mechanism is associated with protonation of PSII subunit S (PsbS) and the interconversion between violaxanthin and zeaxanthin, which is catalysed by the enzymes violaxanthin depoxidase and zeaxanthin epoxidase, respectively (Pascal et al., 2005;Pinnola and Bassi, 2018;Ruban et al., 2012). ...
Photosynthesis is an essential metabolic pathway for plants, contributing to growth and biomass production. Environmental adverse conditions have a negative impact on photosynthetic activity, reducing crop yield and productivity, a situation that has been worsen due to the actual global climate change scenario. Plants have different mechanisms to cope with this changing environment, ranging from photo-protective mechanisms to adaptive processes aiming acclimation. To understand these processes, and in the search for resistant varieties of crops, plant scientists have been assessing photosynthetic activity under different conditions and in different plant species. There are multiple methodologies to study photosynthesis; however, not all of them are suitable for every working condition or plant species. In this review, we offer an overview of the available methodologies to assess photosynthesis, from the most conventional to other less well known. We highlight the strength and weakness of each technique, and discuss how photosynthetic measurements can be linked and integrated to other methodologies (e.g. omics). Moreover, we address how photosynthesis is affected by intra-plant and inter-plants factors, as well as environmental variables. The analysis of the photosynthetic process from a wider and systemic perspective will lead to a closer understanding of plant physiology, ultimately improving crop yield and land use efficiency.
... Here we also show that cold and drought resulted in faster NPQ induction and relaxation, but this was highly variable among ecotypes and not exclusive to high elevation ones, potentially because cold and drought can happen at different elevations in different regions. NPQ can remove over 75% of absorbed light energy (Niyogi et al., 1998), thus its fast induction in response to stress can play an essential role for rapid defense against photoinhibition, preventing photodamage of the photosynthetic machinery by reducing ROS formation (Malnoë, 2018). Fast relaxation of NPQ (particularly evident in two Central Asian highland ecotypes) can also be vital because it allows more energy to be photochemically quenched under fluctuating light conditions, potentially promoting plant growth (Kromdijk et al., 2016). ...
Arabidopsis thaliana has a wide elevational range and much of its diversity may be associated with local adaptation to elevation. We took a multi-regional view of the genomics and physiology of elevational adaptation in Arabidopsis, with >200 ecotypes, including 17 newly collected from Africa. We measured plant responses to potential high elevation stressors: low pCO2, high light, and night freezing and conducted genome-wide association studies (GWAS). We found evidence of an adaptive cline in the western Mediterranean with low δ 13 C/early flowering at low elevations to high δ 13 C/late flowering at high elevations. By contrast, central Asian high elevation ecotypes flowered earlier. Antioxidants and pigmentation under high light and freezing showed regional differentiation but not elevational clines and may be associated with maladaptive plasticity. We found natural variation in non-photochemical quenching (NPQ) kinetics in response to chilling and fluctuating light, though with an unclear role in local adaptation. There were several candidate genetic loci mapped, including the ascorbate transporter PHT4;4 (associated with antioxidants) that influences the xanthophyll cycle, and may be involved in local adaptation to Morocco. Our study shows how the ecological strategies and genetic loci causing local adaptation to elevation change across regions and contribute to diversity in Arabidopsis.
... It has been shown in rice that genetic modification of the PsbS proteins resulted in a change in NPQ levels, which significantly affected dynamic photosynthesis and biomass production under fluctuating light conditions (Hubbart et al., 2012(Hubbart et al., , 2018. In addition, the qE is modulated by the conversion between violaxanthin and zeaxanthin catalysed by violaxanthin deepoxidase (VDE) and zeaxanthin epoxidase (ZEP) in the xanthophyll cycle depending on the oxidation level of the thylakoid lumen (Niyogi et al., 1998). In tobacco, overexpression of PsbS, VDE, and ZEP resulted in faster A recovery during the post-illumination phase due to faster NPQ relaxation compared with wild-type plants, yielding higher biomass production under field environments (Kromdijk et al., 2016). ...
Under field environments, fluctuating light conditions induce dynamic photosynthesis, which affects carbon gain by crop plants. Elucidating the natural genetic variations among untapped germplasm resources and their underlying mechanisms can provide an effective strategy to improve dynamic photosynthesis and ultimately, improve crop yields through molecular breeding approaches. In this review, we first overview two processes affecting dynamic photosynthesis, namely (1) biochemical processes associated with to CO2 fixation and photoprotection and (2) gas diffusion processes from the atmosphere to the chloroplast stroma. Next, we review the intra- and interspecific variations in dynamic photosynthesis in relation to each of these two processes. It is suggested that plant adaptations to different hydrological environments underlie natural genetic variation explained by gas diffusion through stomata. This emphasizes the importance of the coordination of photosynthetic and stomatal dynamics to optimize the balance between carbon gain and water use efficiency under field environments. Finally, we discuss future challenges in improving dynamic photosynthesis by utilising natural genetic variation. The forward genetic approach supported by high-throughput phenotyping should be introduced to evaluate the effects of genetic and environmental factors and their interactions on the natural variation in dynamic photosynthesis.
... It is induced by low values of pH in the thylakoid lumen (Müller et al. 2001, Ruban 2016. Arabidopsis thaliana mutants, npq1 (unable to convert violaxanthin to zeaxanthin; Niyogi et al. 1998) and lut2 (does not synthesize lutein or α-carotene; Pogson et al. 1996), are characterized by diminished levels of qE , Pogson et al. 1998). In addition, it has been proposed that lutein also might have a direct role in qE formation (Niyogi et al. 1997, Lokstein et al. 2002. ...
Alterations in photosynthetic performance of lutein-deficient mutant lut2 and wild type (wt) of Arabidopsis thaliana were followed after treatment with low temperature and high light for 6 d. The obtained results indicated lower electrolyte leakage, lower excitation pressure, and higher actual photochemical efficiency of PSII in lut2 plants exposed to combined stress compared to wt plants. This implies that lut2 is less susceptible to the applied stress conditions. The observed lower values of quantum efficiency of nonphotochemical quenching and energy-dependent component of nonphotochemical quenching in lut2 suggest that nonphotochemical quenching mechanism(s) localized within LHCII could not be involved in the acquisition of higher stress tolerance of lut2 and alternatives to nonphotochemical quenching mechanisms are involved for dissipation of excess absorbed light. We suggest that the observed enhanced capacity for cyclic electron flow and the higher oxidation state of P700 (P700+), which suggests PSI-dependent energy quenching in lut2 plants may serve as efficient photoprotective mechanisms, thus explaining the lower susceptibility of lut2 to the combined stress treatments.
... These results show that the PsbS protein plays an important role in HL stress amelioration during short-term HL treatments, but that PsbS is not an essential factor during long-term HL treatments. The major component of NPQ, energy-dependent quenching (qE) includes the accumulation of the xanthophyll cycle pigment zeaxanthin associated with the development of the pH gradient across the thylakoid membrane and the PsbS protein [7,25]. We compared the rise in kinetics of de-epoxidation state of the xanthophyll cycle pigments in WT and npq4-1 mutant plants. ...
The photosystem II PsbS protein of thylakoid membranes is responsible for regulating the energy-dependent, non-photochemical quenching of excess chlorophyll excited states as a short-term mechanism for protection against high light (HL) stress. However, the role of PsbS protein in long-term HL acclimation processes remains poorly understood. Here we investigate the role of PsbS protein during long-term HL acclimation processes in wild-type (WT) and npq4-1 mutants of Arabidopsis which lack the PsbS protein. During long-term HL illumination, photosystem II photo-chemical efficiency initially dropped, followed by a recovery of electron transport and photochem-ical quenching (qL) in WT, but not in npq4-1 mutants. In addition, we observed a reduction in light-harvesting antenna size during HL treatment that ceased after HL treatment in WT, but not in npq4-1 mutants. When plants were adapted to HL, more reactive oxygen species (ROS) were accumulated in npq4-1 mutants compared to WT. Gene expression studies indicated that npq4-1 mutants failed to express genes involved in plastoquinone biosynthesis. These results suggest that the PsbS protein regulates recovery processes such as electron transport and qL during long-term HL acclimation by maintaining plastoquinone biosynthetic gene expression and enhancing ROS homeostasis.
... Frontiers in Plant Science | www.frontiersin.org A common feature of zep mutant leaves is the increased accumulation of zeaxanthin and decreased production of antheraxanthin and violaxanthin (Rock and Zeevaart, 1991;Marin et al., 1996;Niyogi et al., 1998;Liu et al., 2020b). As expected, in our study, the amount of zeaxanthin increased about 11.2-fold in DY-bulk than that in Y-bulk, and violaxanthin decreased about 3.6-fold in DY-bulk. ...
Flower color is an important trait in Brassica species. However, genes responsible for the dark yellow flower trait in Chinese cabbage have not been reported. In this study, we identified a dark-yellow-flowered Chinese cabbage line SD369. Genetic analysis indicated that the dark yellow flower trait in SD369 was controlled by a single recessive locus, Br-dyp1 (dark yellow petal color 1 in Brassica rapa). Using bulked segregant RNA sequencing and kompetitive allele-specific PCR assays, Br-dyp1 was fine-mapped to an interval of 53.6 kb on chromosome A09. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra037130 (BraA09.ZEP), which encodes a zeaxanthin epoxidase, was the most likely candidate gene for Br-dyp1. Carotenoid profile analysis suggested that Bra037130 (BraA09.ZEP) might participate in the epoxidation from zeaxanthin to violaxanthin. The 679 bp insertion in dark yellow petal caused premature stop codon, thus caused the loss-of-function of the enzyme zeaxanthin epoxidase (ZEP), which disturbed the carotenoid metabolism, and caused the increased accumulation of total carotenoid, and finally converted the flower color from yellow to dark yellow. Comparative transcriptome analysis also showed that the “carotenoid biosynthesis” pathway was significantly enriched, and genes involved in carotenoid degradation and abscisic acid biosynthesis and metabolism were significantly downregulated. Furthermore, we developed and validated the functional marker Br-dyp1-InDel for Br-dyp1. Overall, these results provide insight into the molecular basis of carotenoid-based flower coloration in B. rapa and reveal valuable information for marker-assisted selection breeding in Chinese cabbage.
... The extent of thermal dissipation is measured by an energy-dependent (qE) component of nonphotochemical quenching (NPQ) of chlorophyll fluorescence (Ruban, 2016;Ruban and Wilson, 2021). In the Arabidopsis (Arabidopsis thaliana) mutants defective in the qE induction, the efficiency of light energy utilization by PSII fY(II)g was not upregulated (Niyogi et al., 1998), suggesting that the induction of qE does not limit the rate of electron transport under the constant HL. Under fluctuating light intensity, however, the qE induced at HL intensity may decrease the rate of electron transport through PSII by dissipating the limited light energy in the subsequent LL period. ...
The proton concentration gradient (ΔpH) and membrane potential (Δψ) formed across the thylakoid membrane contribute to ATP synthesis in chloroplasts. Additionally, ΔpH downregulates photosynthetic electron transport via the acidification of the thylakoid lumen. K+ exchange antiporter 3 (KEA3) relaxes this downregulation by substituting ΔpH with Δψ in response to fluctuation of light intensity. In the Arabidopsis (Arabidopsis thaliana) line overexpressing KEA3 (KEA3ox), the rate of electron transport is elevated by accelerating the relaxation of ΔpH after a shift from high light (HL) to low light (LL). However, the plant cannot control electron transport toward photosystem I (PSI), resulting in PSI photodamage. In this study, we crossed the KEA3ox line with the line (Flv) expressing the Flavodiiron proteins of Physcomitrium patens. In the double transgenic line (Flv-KEA3ox), electrons overloading toward PSI were pumped out by Flavodiiron proteins. Consequently, photodamage of PSI was alleviated to the wild-type level. The rate of CO2 fixation was enhanced in Flv and Flv-KEA3ox lines during HL periods of fluctuating light, although CO2 fixation was unaffected in any transgenic lines in constant HL. Upregulation of CO2 fixation was accompanied by elevated stomatal conductance in fluctuating light. Consistent with the results of gas exchange experiments, the growth of Flv and Flv-KEA3ox plants was better than that of WT and KEA3ox plants under fluctuating light.
... In contrast to the response times obtained from activation during a dark-to-light 132 induction, e.g., in (Wehner et al., 2004;Nilkens et al., 2010), the limits of the NPQ operation 133 range in oscillating light depend on an interplay of the activation and deactivation processes that 134 both contribute to plant response to fluctuating light. Towards this goal, we explored the 135 responses to oscillating light of the wild-type, the npq1 mutant, which cannot convert 136 violaxanthin into zeaxanthin by VDE (Niyogi et al., 1998), and of the npq4 mutant that lacks the 137 PsbS protein (Li et al., 2000). 138 The second objective was to discriminate between the roles played in an oscillating light by the 1B. ...
In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light.
Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild-type Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de-epoxidase, the npq4 mutant lacking PsbS-protein, and the mutants crr2-2 , and pgrl1ab impaired in different pathways of the cyclic electron transport.
The fastest was the PsbS-regulation responding to oscillation periods longer than 10s. Processes involving violaxanthin de-epoxidase dampened changes of chlorophyll fluorescence in oscillation periods of 2min or longer. Knocking out the PGRL1-PGR5 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH-like pathway only slightly changed the photosynthetic dynamics.
Our observations are consistent with the assumption that non-photochemical quenching in slow light oscillations involves violaxanthin de-epoxidase to produce, presumably, a stationary, non-oscillating level of zeaxanthin. We interpret the observed dynamics of Photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.
... Xanthophyll biosynthesis Arabidopsis mutants lacking zeaxanthin revealed reduced, albeit not complete, disappearance of NPQ. 121,133 However, it was discovered that normal NPQ could be restored with enhanced levels of ΔpH in isolated chloroplasts lacking zeaxanthin. 49,134,135 This suggests that even if zeaxanthin was an NPQ quencher, it was not the major one. ...
Photosystem II (PSII) uses light energy to split water into protons, electrons, and oxygen, ultimately sustaining heterotrophic life on Earth. The major light harvesting complex in plants (LHCII) is packed with chlorophylls and carotenoids and is the main supplier of excitation energy to PSII reaction centers. The protein scaffold acts as a programmed solvent for the pigments in LHCII, tuning their orientations while at the same time impeding concentration quenching to ensure efficient storage of excitation energy by chlorophylls. However, under stress, the very fuel of PSII, solar photons, can damage its delicate inner components and hamper photosynthesis. In a crucial regulatory strategy in plants, LHCII evolved a flexible design that allows it to switch between light-harvesting and dissipative conformations, thereby safely releasing the excess energy that is absorbed into heat. Several mechanisms have been proposed to explain chlorophyll de-excitation pathways in LHCII, such as chlorophyll-chlorophyll charge transfer states, resonance energy transfer from chlorophylls to a carotenoid S1 state, and chlorophyll-carotenoid reductive energy transfer. This Perspective critically assesses the listed proposals, addressing both the physical mechanism of quenching and the nature of the quenching pigment. These hypotheses are then discussed in the context of state-of-the-art biochemical, physiological, and genetic knowledge to scrutinize their likeliness to occur in the native thylakoid membranes.
Non-photochemical quenching (NPQ) mechanisms are crucial for protecting photosynthesis from photoinhibition in plants, algae, and cyanobacteria, and their modulation is a long-standing goal for improving photosynthesis and crop yields. The current work demonstrates that Chlorella ohadii , a green micro-alga that thrives in the desert under high light intensities which are fatal to many photosynthetic organisms, does not perform nor require NPQ to protect photosynthesis under constant high light. Instead of dissipating excess energy, it minimizes its uptake by eliminating the photosynthetic antenna of photosystem II, in addition to accumulating antioxidants that neutralize harmful reactive oxygen species (ROS) and ramping up cyclic electron flow around PSI. This NPQ-independent response proved efficient in preventing ROS accumulation and reducing oxidative damage to proteins in high-light-grown cells. This work contributes to the understanding of photoprotection under extreme high light intensities and provides potential targets for improving photoprotection.
Improving the efficiency of crop photosynthesis has the potential to increase yields. Genetic manipulation showed photosynthesis can be improved by speeding up relaxation of photoprotective mechanisms during sun to shade transitions. However, it is unclear if natural variation in relaxation of non-photochemical quenching (NPQ) can be exploited in crop breeding programs. To address this issue, we measured six NPQ parameters in the 40 founder lines and common parent of a Soybean Nested Association Mapping (SoyNAM) panel over two field seasons in Illinois. NPQ parameters did not show consistently variable trends throughout development, and variation between sampling days suggests environmental impacts on NPQ which last more than 24 hours. 17 genotypes were found to show small but consistent differences in NPQ relaxation kinetics relative to a reference line providing a basis for future mapping studies. Finally, a soybean canopy model predicted available phenotypic variation could result in a 1.6% difference in carbon assimilation when comparing fastest and slowest relaxing NPQ values.
Significance Statement
Evidence suggests increasing the rate of relaxation of photoprotection can lead to improved biomass and yield. We compare photoprotection relaxation rates in 41 diverse soybean genotypes grown in the field, identifying lines with faster rates of relaxation, and predict a potential 1.6% difference in daily carbon assimilation which could contribute to improving soybean performance.
Arabidopsis thaliana Mitogen-activated protein Kinase Phosphatase 1 (MKP1) negatively balances production of reactive oxygen species (ROS) triggered by Microbe-Associated Molecular Patterns (MAMPs) through uncharacterized mechanisms. Accordingly, ROS production is enhanced in mkp1 mutant after MAMP treatment. Moreover, mkp1 plants show a constitutive activation of immune responses and enhanced disease resistance to pathogens with distinct colonization styles, like the bacterium Pseudomonas syringae pv. tomato DC3000, the oomycete Hyaloperonospora arabidopsidis Noco2 and the necrotrophic fungus Plectosphaerella cucumerina BMM. The molecular basis of this ROS production and broad-spectrum disease resistance controlled by MKP1 have not been determined. Here, we show that the enhanced ROS production in mkp1 is not due to a direct interaction of MKP1 with the NADPH oxidase RBOHD, nor is it the result of the catalytic activity of MKP1 on RBHOD phosphorylation sites targeted by BOTRYTIS INDUCED KINASE 1 (BIK1) protein, a positive regulator of RBOHD-dependent ROS production. The analysis of bik1 mkp1 double mutant phenotypes suggested that MKP1 and BIK1 targets are different. Additionally, we showed that phosphorylation residues stabilizing MKP1 are essential for its functionality in immunity. To further decipher the molecular basis of disease resistance responses controlled by MKP1, we generated combinatory lines of mkp1-1 with plants impaired in defensive pathways required for disease resistance to pathogen: cyp79B2 cyp79B3 double mutant defective in synthesis of tryptophan-derived metabolites, NahG transgenic plant that does not accumulate salicylic acid, aba1-6 mutant impaired in abscisic acid (ABA) biosynthesis, and abi1 abi2 hab1 triple mutant impaired in proteins described as ROS sensors and that is hypersensitive to ABA. The analysis of these lines revealed that the enhanced resistance displayed by mkp1-1 is altered in distinct mutant combinations: mkp1-1 cyp79B2 cyp79B3 fully blocked mkp1-1 resistance to P. cucumerina, whereas mkp1-1 NahG displays partial susceptibility to H. arabidopsidis, and mkp1-1 NahG, mkp1-1 aba1-6 and mkp1-1 cyp79B2 cyp79B3 showed compromised resistance to P. syringae. These results suggest that MKP1 is a component of immune responses that does not directly interact with RBOHD but rather regulates the status of distinct defensive pathways required for disease resistance to pathogens with different lifestyles.
This article comments on:
Sands E, Davies S, Puxty RJ, Verge V, Bouget F-Y, Scanlan DJ, Carre IA. 2023. Genetic and physiological responses to light quality in a deep ocean ecotype of Ostreococcus, an ecologically important photosynthetic picoeukaryote. Journal of Experimental Botany 74, 6773–6789.
Main conclusion
Simultaneous genome editing of the two homeologousLCYeandZEPgenes ofNicotiana benthamianaresults in plants in which all xanthophylls are replaced by zeaxanthin.
Abstract
Plant carotenoids act both as photoreceptors and photoprotectants in photosynthesis and as precursors of apocarotenoids, which include signaling molecules such as abscisic acid (ABA). As dietary components, the xanthophylls lutein and zeaxanthin have photoprotective functions in the human macula. We developed transient and stable combinatorial genome editing methods, followed by direct LC–MS screening for zeaxanthin accumulation, for the simultaneous genome editing of the two homeologous Lycopene Epsilon Cyclase (LCYe) and the two Zeaxanthin Epoxidase (ZEP) genes present in the allopolyploid Nicotiana benthamiana genome. Editing of the four genes resulted in plants in which all leaf xanthophylls were substituted by zeaxanthin, but with different ABA levels and growth habits, depending on the severity of the ZEP1 mutation. In high-zeaxanthin lines, the abundance of the major photosystem II antenna LHCII was reduced with respect to wild-type plants and the LHCII trimeric state became unstable upon thylakoid solubilization. Consistent with the depletion in LHCII, edited plants underwent a compensatory increase in PSII/PSI ratios and a loss of the large-size PSII supercomplexes, while the level of PSI-LHCI supercomplex was unaffected. Reduced activity of the photoprotective mechanism NPQ was shown in high-zeaxanthin plants, while PSII photoinhibition was similar for all genotypes upon exposure to excess light, consistent with the antioxidant and photoprotective role of zeaxanthin in vivo.
Main Conclusion
WHIRLY1 deficient barley plants surviving growth at high irradiance displayed increased non-radiative energy dissipation, enhanced contents of zeaxanthin and the flavonoid lutonarin, but no changes in α-tocopherol nor glutathione.
Abstract
Plants are able to acclimate to environmental conditions to optimize their functions. With the exception of obligate shade plants, they can adjust their photosynthetic apparatus and the morphology and anatomy of their leaves to irradiance. Barley ( Hordeum vulgare L., cv. Golden Promise) plants with reduced abundance of the protein WHIRLY1 were recently shown to be unable to acclimatise important components of the photosynthetic apparatus to high light. Nevertheless, these plants did not show symptoms of photoinhibition. High-light (HL) grown WHIRLY1 knockdown plants showed clear signs of exposure to excessive irradiance such as a low epoxidation state of the violaxanthin cycle pigments and an early light saturation of electron transport. These responses were underlined by a very large xanthophyll cycle pool size and by an increased number of plastoglobules. Whereas zeaxanthin increased with HL stress, α -tocopherol, which is another lipophilic antioxidant, showed no response to excessive light. Also the content of the hydrophilic antioxidant glutathione showed no increase in W1 plants as compared to the wild type, whereas the flavone lutonarin was induced in W1 plants. HPLC analysis of removed epidermal tissue indicated that the largest part of lutonarin was presumably located in the mesophyll. Since lutonarin is a better antioxidant than saponarin, the major flavone present in barley leaves, it is concluded that lutonarin accumulated as a response to oxidative stress. It is also concluded that zeaxanthin and lutonarin may have served as antioxidants in the WHIRLY1 knockdown plants, contributing to their survival in HL despite their restricted HL acclimation.
Plants cope with sudden increases in light intensity through various photoprotective mechanisms. Redox regulation by thioredoxin (Trx) systems also contributes to this process. Whereas the functions of f- and m-type Trxs in response to such fluctuating light conditions have been extensively investigated, those of x- and y-type Trxs are largely unknown. Here, we analyzed the trx x single, trx y1 trx y2 double, and trx x trx y1 trx y2 triple mutants in Arabidopsis (Arabidopsis thaliana). A detailed analysis of photosynthesis revealed changes in photosystem I (PSI) parameters under low light in trx x and trx x trx y1 trx y2. The electron acceptor side of PSI was more reduced in these mutants than in the wild type. This mutant phenotype was more pronounced under fluctuating light conditions. During both low- and high-light phases, the PSI acceptor side was largely limited in trx x and trx x trx y1 trx y2. After fluctuating light treatment, we observed more severe PSI photoinhibition in trx x and trx x trx y1 trx y2 than in the wild type. Furthermore, when grown under fluctuating light conditions, trx x and trx x trx y1 trx y2 plants showed impaired growth and decreased level of PSI subunits. These results suggest that Trx x and Trx y prevent redox imbalance on the PSI acceptor side, which is required to protect PSI from photoinhibition, especially under fluctuating light. We also propose that Trx x and Trx y contribute to maintaining the redox balance even under constant low-light conditions to prepare for sudden increases in light intensity.
Key message
A mutation of CsARC6 not only causes white fruit color in cucumber, but also affects plant growth and fruit quality.
Abstract
Fruit color of cucumber is a very important agronomic trait, but most of the genes affecting cucumber white fruit color are still unknow, and no further studies were reported on the effect of cucumber fruit quality caused by white fruit color genes. Here, we obtained a white fruit mutant em41 in cucumber by EMS mutagenesis. The mutant gene was mapped to a 548 kb region of chromosome 2. Through mutation site analysis, it was found to be a null allele of CsARC6 (CsaV3_2G029290). The Csarc6 mutant has a typical phenotype of arc6 mutant that mesophyll cells contained only one or two giant chloroplasts. ARC6 protein was not detected in em41, and the level of FtsZ1 and FtsZ2 was also reduced. In addition, FtsZ2 could not form FtsZ ring-like structures in em41. Although these are typical arc6 mutant phenotypes, some special phenotypes occur in Csarc6 mutant, such as dwarfness with shortened internodes, enlarged fruit epidermal cells, decreased carotenoid contents, smaller fruits, and increased fruit nutrient contents. This study discovered a new gene, CsARC6, which not only controls the white fruit color, but also affects plant growth and fruit quality in cucumber.
Ribulose–1,5–bisphosphate carboxylase/oxygenase (Rubisco) is the rate–limiting enzyme for photosynthesis. Rubisco activase (RCA) can regulate the Rubisco activation state, influencing Rubisco activity and photosynthetic rate. We obtained transgenic maize plants that overproduced rice RCA (OsRCAOE) and evaluated photosynthesis in these plants by measuring gas exchange, energy conversion efficiencies in photosystem (PS) I and PSII, and Rubisco activity and activation state. The OsRCAOE lines showed significantly higher initial Rubisco activity and activation state, net photosynthetic rate, and PSII photochemical quantum yield than wild–type plants. These results suggest that OsRCA overexpression can promote maize photosynthesis by increasing the Rubisco activation state.
Cold stress is a key environmental constraint that dramatically affects the growth, productivity, and quality of tomato (Solanum lycopersicum); however, the underlying molecular mechanisms of cold tolerance remain poorly understood. In this study, we identified REDUCED CHLOROPLAST COVERAGE 2 (SlREC2) encoding a tetratricopeptide repeat (TPR) protein that positively regulates tomato cold tolerance. Disruption of SlREC2 largely reduced abscisic acid (ABA) levels, photoprotection and the expression of C-REPEAT BINDING FACTOR (CBF) pathway genes in tomato plants under cold stress. ABA deficiency in the notabilis (not) mutant, which carries a mutation in 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (SlNCED1), strongly inhibited the cold tolerance of SlREC2-silenced plants and empty vector control plants and resulted in a similar phenotype. In addition, foliar application of ABA rescued the cold tolerance of SlREC2-silenced plants, which confirms that SlNCED1-mediated ABA accumulation is required for SlREC2-regulated cold tolerance. Strikingly, SlREC2 physically interacted with β-RING CAROTENE HYDROXYLASE 1b (SlBCH1b), a key regulatory enzyme in the xanthophyll cycle. Disruption of SlBCH1b severely impaired photoprotection, ABA accumulation and CBF-pathway gene expression in tomato plants under cold stress. Taken together, this study reveals that SlREC2 interacts with SlBCH1b to enhance cold tolerance in tomato via integration of SlNCED1-mediated ABA accumulation, photoprotection, and the CBF-pathway, thus providing further genetic knowledge for breeding cold-resistant tomato varieties.
UV-B and UV-A radiation are natural components of solar radiation that can cause plant stress, as well as induce a range of acclimatory responses mediated by photoreceptors. UV-mediated accumulation of flavonoids and glucosinolates is well documented, but much less is known about UV effects on carotenoid content. Carotenoids are involved in a range of plant physiological processes, including photoprotection of the photosynthetic machinery. UV-induced changes in carotenoid profile were quantified in plants (Arabidopsis thaliana) exposed for up to ten days to supplemental UV radiation under growth chamber conditions. UV induces specific changes in carotenoid profile, including increases in antheraxanthin, neoxanthin, violaxanthin and lutein contents in leaves. The extent of induction was dependent on exposure duration. No individual UV-B (UVR8) or UV-A (Cryptochrome or Phototropin) photoreceptor was found to mediate this induction. Remarkably, UV-induced accumulation of violaxanthin could not be linked to protection of the photosynthetic machinery from UV damage, questioning the functional relevance of this UV response. Here, it is argued that plants exploit UV radiation as a proxy for other stressors. Thus, it is speculated that the function of UV-induced alterations in carotenoid profile is not UV protection, but rather protection against other environmental stressors such as high intensity visible light that will normally accompany UV radiation.
Mitochondrial alternative oxidase is an important protein involved in maintaining cellular metabolic and energy balance, especially under stress conditions. AOX-genes knockout is aimed at revealing the functions of AOX genes. Under unfavorable conditions, AOX-suppressed plants (mainly based on Arabidopsis AOX1a-knockout lines) usually experience strong oxidative stress. However, a compensation effect, which consists in the absence of AOX1a leading to an increase in defense response mechanisms concomitant with a decrease in reactive oxygen species contents has also been demonstrated. This review briefly describes the possible mechanisms underlying the compensation effect upon the suppression of AOX1a. Information about mitochondrial retrograde regulation of AOX is given. The importance of reactive oxygen species and mitochondrial membrane potential in triggering the signal transmitting from mitochondria in the absence of AOX or disturbance of mitochondrial electron transport chain functions is indicated. The few available data on the response of the cell in response to the absence of AOX at the level of changes in the hormonal balance and the reactions of chloroplasts are presented. The decrease in the relative amount of the reduced ascorbate at stable reactive-oxygen-species levels as a result of compensation in AOX1a-suppressed plants is proposed to be a sign of stress development. Obtaining direct evidence on the mechanisms and signaling pathways involved in AOX modulation in the genome should facilitate a deeper understanding of the role of AOX in the integration of cellular signaling pathways.
Plants employ an array of photoprotection mechanisms to alleviate the harmful effects of high light intensity. The violaxanthin cycle, which is associated with non-photochemical quenching (NPQ), involves violaxanthin de-epoxidase (VDE), and zeaxanthin epoxidase (ZEP) and is one of the most rapid and efficient mechanisms protecting plants under high light intensity. Woody bamboo is a class of economically and ecologically important evergreen grass species widely distributed in tropical and subtropical areas. However, the function of VDE in bamboo has not yet been elucidated. In this study, we found that high light intensity increased NPQ and stimulated the de-epoxidation of violaxanthin cycle components in moso bamboo (Phyllostachys edulis), whereas, samples treated with the VDE inhibitor (dithiothreitol) exhibited lower NPQ capacity, suggesting that violaxanthin cycle plays an important role in the photoprotection of bamboo. Further analysis showed that not only high light intensity but also extreme temperatures (4 and 42°C) and drought stress upregulated the expression of PeVDE in bamboo leaves, indicating that PeVDE is induced by multiple abiotic stresses. Overexpression of PeVDE under the control of the CaMV 35S promoter in Arabidopsis mutant npq1 mutant could rescue its NPQ, indicating that PeVDE functions in dissipating the excess absorbed light energy as thermal energy in bamboo. Moreover, compared with wild-type (Col-0) plants, the transgenic plants overexpressing PeVDE displayed enhanced photoprotection ability, higher NPQ capacity, slower decline in the maximum quantum yield of photosystem II (Fv/Fm) under high light intensity, and faster recovery under optimal conditions. These results suggest that PeVDE positively regulates the response to high light intensity in bamboo plants growing in the natural environment, which could improve their photoprotection ability through the violaxanthin cycle and NPQ.
The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants.
Carotenoids comprise the most widely distributed natural pigments. In plants, they play indispensable roles in photosynthesis, furnish colors to flowers and fruit and serve as precursor molecules for the synthesis of apocarotenoids, including aroma and scent, phytohormones and other signaling molecules. Dietary carotenoids are vital to human health as a source of provitamin A and antioxidants. Hence, the enormous interest in carotenoids of crop plants. Over the past three decades, the carotenoid biosynthesis pathway has been mainly deciphered due to the characterization of natural and induced mutations that impair this process. Over the year, numerous mutations have been studied in dozens of plant species. Their phenotypes have significantly expanded our understanding of the biochemical and molecular processes underlying carotenoid accumulation in crops. Several of them were employed in the breeding of crops with higher nutritional value. This compendium of all known random and targeted mutants available in the carotenoid metabolic pathway in plants provides a valuable resource for future research on carotenoid biosynthesis in plant species.
Multiple chloroplast-to-nucleus signaling pathways contribute to the regulation of chloroplast biogenesis during plant greening. Here, we provide evidence for the direct implication of the atypical kinase ABC1K1. ABC1K1 is required for sufficient plastoquinone (PQ) allocation to the photosynthetic electron transport chain. Unexpectedly, mutation of abc1k1 suppresses greening and results in pale cotyledons under red light. This phenotype was not observed in other photosynthetic mutants and points to a specific signaling defect. Under red light, abc1k1 accumulated EXECUTER1 (EX1), a trigger of singlet oxygen (1O2) signaling. Consistent with the role of the FTSH metalloprotease in chloroplast biogenesis and EX1 degradation, the ftsh2 mutant var2, mimicked the greening defect of abc1k1 and accumulated EX1 under red light. We propose that this novel ABC1K1-dependent signal is required for chloroplast biogenesis to progress in challenging light conditions.
pH homeostasis in the chloroplast is crucial for the control of photosynthesis and other metabolic processes in plants. Recently, nuclear-encoded Day-Length-dependent Delayed Greening1 (DLDG1) and Fluctuating-Light Acclimation Protein1 (FLAP1) that are required for the light-inducible optimization of plastidial pH in Arabidopsis thaliana were identified. DLDG1 and FLAP1 homologs are specifically conserved in oxygenic phototrophs, and a DLDG1 homolog, Ycf10, is encoded in the chloroplast genome in plant cells. However, the function of Ycf10 and its physiological significance are unknown. To address this, we constructed ycf10 tobacco Nicotiana tabacum mutants and characterized their phenotypes. The ycf10 tobacco mutants grown under continuous-light conditions showed a pale-green phenotype only in developing leaves, and it was suppressed in short-day conditions. The ycf10 mutants also induced excessive non-photochemical quenching (NPQ) compared with those in the wild-type at the induction stage of photosynthesis. These phenotypes resemble those of Arabidopsis dldg1 mutants, suggesting that they have similar functions. However, there are distinct differences between the two mutant phenotypes: The highly induced NPQ in tobacco ycf10 and the Arabidopsis dldg1 mutants are diminished and enhanced, respectively, with increasing duration of the fluctuating actinic-light illumination. Ycf10 and DLDG1 were previously shown to localize in chloroplast envelope-membranes, suggesting that Ycf10 and DLDG1 differentially control H+ exchange across these membranes in a light-dependent manner to control photosynthesis.
Main conclusion
OsVDE, a lipocalin-like protein in chloroplasts, negatively regulated the ABA biosynthesis and stomatal closure under salt stress in rice seedlings.
Abstract
Violaxanthin de-epoxidase (VDE) is a key enzyme of xanthophyll cycle. It plays a critical role in abscisic acid (ABA) biosynthesis, growth and stress responses in plants. Although functions of several VDE genes have been characterized, it is largely unknown whether OsVDE regulates the ABA biosynthesis and salt stress tolerance in rice. In this study, we generated the OsVDE overexpressing and CRISPR-Cas9-mediated gene-editing transgenic lines, and identified that the gene-editing mutant lines showed the dwarfism, shorter panicle and lower seed-setting rate than the wild type whereas the overexpression lines did not exhibit the difference from the wild type. In addition, the gene-editing transgenic lines were hypersensitive to exogenous ABA during germination. Under salt stress, the gene-editing transgenic seedlings had a higher ABA level, higher stomatal closure percentage and higher survival rate than the wild type. The qRT-PCR analysis confirmed that OsVDE negatively regulated the OsNECD2/4/5 expressions, ABA biosynthesis and salt stress tolerance in rice seedlings. These results provide new evidence that VDE plays an essential role in ABA biosynthesis and salt stress tolerance in plants.
Green plants use the xanthophyll cycle to regulate the flow of energy to chlorophylla within photosynthetic proteins. Under conditions of low light intensity violaxanthin, a carotenoid possessing nine conjugated double bonds, functions as an antenna pigment by transferring energy from its lowest excited singlet state to that of chlorophylla within light-harvesting proteins. When the light intensity increases, violaxanthin is biochemically transformed into zeaxanthin, a carotenoid that possesses eleven conjugated double bonds. The results presented here show that extension of the [Symbol: see text] conjugation of the polyene lowers the energy of the lowest excited singlet state of the carotenoid below that of chlorophylla. As a consequence zeaxanthin can act as a trap for the excess excitation energy on chlorophylla pigments within the protein, thus regulating the flow of energy within photosynthetic light-harvesting proteins.
The role of electron transport to O2 in mitigating against photoinactivation of Photosystem (PS) II was investigated in leaves of pea (Pisum sativum L.) grown in moderate light (250 μmol m(-2) s(-1)). During short-term illumination, the electron flux at PS II and non-radiative dissipation of absorbed quanta, calculated from chlorophyll fluorescence quenching, increased with increasing O2 concentration at each light regime tested. The photoinactivation of PS II in pea leaves was monitored by the oxygen yield per repetitive flash as a function of photon exposure (mol photons m(-2)). The number of functional PS II complexes decreased nonlinearly with increasing photon exposure, with greater photoinactivation of PS II at a lower O2 concentration. The results suggest that electron transport to O2, via the twin processes of oxygenase photorespiration and the Mehler reaction, mitigates against the photoinactivation of PS II in vivo, through both utilization of photons in electron transport and increased nonradiative dissipation of excitation. Photoprotection via electron transport to O2 in vivo is a useful addition to the large extent of photoprotection mediated by carbon-assimilatory electron transport in 1.1% CO2 alone.
Photosystem II (PS II) chlorophyll (Chl) a fluorescence lifetimes were measured in thylakoids and leaves of barley wild-type and chlorina f104 and f2 mutants to determine the effects of the PS II Chl a+b antenna size on the deexcitation of absorbed light energy. These barley chlorina mutants have drastically reduced levels of PS II light-harvesting Chls and pigment-proteins when compared to wild-type plants. However, the mutant and wild-type PS II Chl a fluorescence lifetimes and intensity parameters were remarkably similar and thus independent of the PS II light-harvesting antenna size for both maximal (at minimum Chl fluorescence level, Fo) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, Fm). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (ΔpH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a ΔpH, the xanthophyll cycle-dependent processes increased the fractional intensity of a Chl a fluorescence lifetime distribution centered around 0.4-0.5 ns, at the expense of a 1.6 ns lifetime distribution (see Gilmore et al. (1995) Proc Natl Acad Sci USA 92: 2273-2277). When the zeaxanthin and antheraxanthin concentrations were measured relative to the number of PS II reaction center units, the ratios of fluorescence quenching to [xanthophyll] were similar between the wild-type and chlorina f104. However, the chlorina f104, compared to the wild-type, required around 2.5 times higher concentrations of these xanthophylls relative to Chl a+b to obtain the same levels of xanthophyll cycle-dependent fluorescence quenching. We thus suggest that, at a constant ΔpH, the fraction of the short lifetime distribution is determined by the concentration and thus binding frequency of the xanthophylls in the PS II inner antenna. The ΔpH also affected both the widths and centers of the lifetime distributions independent of the xanthophyll cycle. We suggest that the combined effects of the xanthophyll cycle and ΔpH cause major conformational changes in the pigment-protein complexes of the PS II inner or core antennae that switch a normal PS II unit to an increased rate constant of heat dissipation. We discuss a model of the PS II photochemical apparatus where PS II photochemistry and xanthophyll cycle-dependent energy dissipation are independent of the Peripheral antenna size.
The factors controlling the relationship between the formation of the transthylakoid pH gradient, as measured by quenching of 9-aminoacridine fluorescence, and the induction of non-radiative de-excitation, measured by the non-photochemical quenching of chlorophyll fluorescence, have been measured in isolated spinach thylakoids. It was found that the following are potentiators of chlorophyll fluorescence quenching: high concentrations of Mg2+; pre-illumination to induce zeaxanthin synthesis; dibucaine. These treatments tended to be antagonistic to the action of antimycin, an inhibitor of quenching, in a way consistent with the LHCII model for fluorescence quenching. The reagent dibucaine allowed quenching to occur in the absence of a pH gradient as measured by the quenching of 9-aminoacridine fluorescence and markedly increased the rate of its formation. In all of these experiments quenching was correlated with the formation of an absorbance change with a peak near 530 nm. This absorbance band was shifted from 522 nm to 530 nm in the presence of zeaxanthin.
Synthese des mecanismes de photoprotection chez les vegetaux, en reponse a un eclairement energetique; implication des carotenoides, en particulier la zeaxanthine, a la dissipation d'energie, en relation avec le cycle des xanthophylles
The light-dependent generation of active oxygen species is termed photooxidative stress. This can occur in two ways: (1) the donation of energy or electrons directly to oxygen as a result of photosynthetic activity; (2) exposure of tissues to ultraviolet irradiation. The light-dependent destruction of catalase compounds the problem. Although generally detrimental to metabolism, superoxide and hydrogen peroxide may serve useful functions if rigorously controlled and compartmentalised. During photosynthesis the formation of active oxygen species is minimised by a number of complex and refined regulatory mechanisms. When produced, active oxygen species are eliminated rapidly by efficient antioxidative systems. The chloroplast is able to use the production and destruction of hydrogen peroxide to regulate the thermal dissipation of excess excitation energy. This is an intrinsic feature of the regulation of photosynthetic electron transport. Photoinhibition and photooxidation only usually occur when plants are exposed to stress. Active oxygen species are part of the alarm-signalling processes in plants. These serve to modify metabolism and gene expression so that the plant can respond to adverse environmental conditions, invading organisms and ultraviolet irradiation. The capacity of the antioxidative defense system is often increased at such times but if the response is not sufficient, radical production will exceed scavenging and ultimately lead to the disruption of metabolism. Oxidative damage arises in high light principally when the latter is in synergy with additional stress factors such as chilling temperatures or pollution. Environmental stress can modify the photooxidative processes in various ways ranging from direct involvement in light-induced free radical formation to the inhibition of metabolism that renders previously optimal light levels excessive. It is in just such situations that the capacity for the production of active oxygen species can exceed that for scavenging by the antioxidative defense systems. The advent of plant transformation, however, may have placed within our grasp the possibility of engineering greater stress tolerance in plants by enhancement of the antioxidative defence system.
The mechanism of rapidly-relaxing non-photochemical quenching in two plant species,Chenopodium album L. andDigitalis purpurea L., that differ considerably in their capacity for such quenching has been investigated (Johnson G.N. et al. 1993, Plant Cell Environ.16, 673–679). Illumination of leaves of both species in the presence of 2% O2 balance N2 led to the formation of zeaxanthin. When thylakoids were isolated from leaves of each species that had been so treated it was found that inD. purpurea non-photochemical quenching was activated relative to the control; a higher level of quenching was found for a given trans-thylakoid pH gradient. No such activation of non-photochemical quenching was observed inC. album. Similar conclusions were drawn when comparing quenching in intact leaves. It is concluded that light activation of quenching is a process that cannot readily be induced inC. album. Measurement of the sensitivity of non-photochemical quenching in leaves ofC. album andD. purpurea to dithiothreitol (DTT; a reagent that inhibits formation of zeaxanthin) showed differences between the two species. In both cases, feeding leaves with DTT inhibited the light-induced formation of zeaxanthin. InC. album this was accompanied by complete inhibition of reversible non-photochemical quenching, whereas inD. purpurea this inhibition was only partial. Data are discussed in relation to studies on the mechanism of quenching and the role of zeaxanthin in this process.
Chlorsulfuron-resistant mutants of Arabidopsis thaliana were isolated by screening for growth of seedlings in the presence of the herbicide. Both whole plants and derived tissue cultures were resistant to concentrations of the herbicide approximately 300-fold higher than that required to prevent growth of the wild-type. The resistance is due to a single dominant nuclear mutation at a locus designated csr which has been genetically mapped to chromosome-3. Acetohydroxy acid synthase activity in extracts from chlorsulfuron-resistant plants was much less-susceptible to inhibition by chlorsulfuron and a structurally related inhibitor than the activity in wild-type extracts. This suggests that the csr locus is the structural gene for acetohydroxy acid synthase.
Photosystem II (PS II) efficiency, nonphotochemical fluorescence quenching, and xanthophyll cycle composition were determined in situ in the natural environment at midday in (i) a range of differently angled sun leaves ofEuonymus kiautschovicus Loesener and (ii) in sun leaves of a wide range of different plant species, including trees, shrubs, and herbs. Very different degrees of light stress were experienced by these leaves (i) in response to different levels of incident photon flux densities at similar photosynthetic capacities amongEuonymus leaves and (ii) as a result of very different photosynthetic capacities among species at similar incident photon flux densities (that were equivalent to full sunlight). ForEuonymus as well as the interspecific comparison all data fell on one single, close relationship for changes in intrinsic PSII efficiency, nonphotochemical fluorescence quenching, or the levels of zeaxanthin + antheraxanthin in leaves, respectively, as a function of the actual level of light stress. Thus, the same conversion state of the xanthophyll cycle and the same level of energy dissipation were observed for a given degree of light stress independent of species or conditions causing the light stress. Since all increases in thermal energy dissipation were associated with increases in the levels of zeaxanthin + antheraxanthin in these leaves, there was thus no indication of any form of xanthophyll cycle-independent energy dissipation in any of the twenty-four species or varieties of plants examined in their natural environment. It is also concluded that transient diurnal changes in intrinsic PSII efficiency in nature are caused by changes in the efficiency with which excitation energy is delivered from the antennae to PSII centers, and are thus likely to be purely photoprotective. Consequently, the possibility of quantifying the allocation of absorbed light into PSII photochemistry versus energy dissipation in the antennae from changes in intrinsic PSII efficiency is explored.
Attached intact leaves of Schefflera arboricola grown at three different photon flux densities (PFDs) were subjected to 24-h exposures to a high PFD and subsequent recovery
at a low PFD. While sun leaves showed virtually no sustained effects on photosystem II (PSII), shade-grown leaves exhibited
pronounced photoinhibition of PSII that required several days at low PFD to recover. Upon transfer to high PFD, levels of
nonphotochemical quenching in PSII as well as levels of zeaxanthin were initially low in shade leaves but continued to increase
gradually during the 24-h exposure. The xanthophyll cycle pool size rose gradually during and also subsequent to the photoinhibitory
treatment in shade leaves. Upon return to low PFD, a marked and extremely long-lasting retention of zeaxanthin and antheraxanthin
was observed in shade but not sun leaves. During recovery, changes in the conversion state of the xanthophyll cycle therefore
closely mirrored the slow increases in PSII efficiency. This novel report of a close association between zeaxanthin retention
and lasting PSII depressions in these shade leaves clearly suggests a role for zeaxanthin in photoinhibition of shade leaves.
In addition, there was a decrease in β-carotene levels, some decrease in chlorophyll, but no change in lutein and neoxanthin
(all per leaf area) in the shade leaves during and subsequent to the photoinhibitory treatment. These data may be consistent
with a degradation of a portion of core complexes but not of peripheral light-harvesting complexes. A possible conversion
of β-carotene to form additional zeaxanthin is discussed.
Thirty microsatellite loci were assigned to the Arabidopsis linkage map. Several microsatellite sequences in Arabidopsis DNA were found by searching the EMBL and GenBank databases, and a number of these were subsequently found to detect polymorphisms between different Arabidopsis strains by the polymerase chain reaction (PCR). After the presence of microsatellites in Arabidopsis and their utility for genetic mapping had been demonstrated, systematic screening for (CA)n and (GA)n sequences was carried out on marker-selected plasmid libraries and a small-insert genomic library. Positive clones were sequenced, PCR primers flanking the repeats were synthesized, and PCR was carried out on different strains to look for useful polymorphisms. Surprisingly, of 18 (CA)n repeats (n > 13), only one was polymorphic. In contrast, 25 of 30 (GA)n repeats, 2 of 3 (AT)n repeats, and 2 of 4 (A)n repeats were polymorphic. The majority of the (CA)n repeats were complex, with adjacent short di-, tri-, or tetranucleotide repeats, whereas most of the (GA)n, (TA)n, and (A)n repeats were simple. The (CA)n repeats were also refractory to PCR analysis, requiring extensive optimization of PCR conditions, whereas the other repeat classes were mostly amplified with a single set of standard conditions. When polymorphisms were detected, the microsatellites were mapped using a set of recombinant inbred lines originating from a cross between the strains Columbia and Landsberg erecta.
Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses. An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon. The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype. The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction. ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous. ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain. ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity. This is the first report of the isolation of a gene of the ABA biosynthetic pathway. The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location.
Lutein, a dihydroxy beta, epsilon-carotenoid, is the predominant carotenoid in photosynthetic plant tissue and plays a critical role in light-harvesting complex assembly and function. To further understand lutein synthesis and function, we isolated four lutein-deficient mutants of Arabidopsis that define two loci, lut1 and lut2 (for lutein deficient). These loci are required for lutein biosynthesis but not for the biosynthesis of beta, beta-carotenoids. The lut1 mutations are recessive, accumulate high levels of zeinoxanthin, which is the immediate precursor of lutein, and define lut1 as a disruption in epsilon ring hydroxylation. The lut2 mutations are semidominant, and their biochemical phenotype is consistent with a disruption of epsilon ring cyclization. The lut2 locus cosegregates with the recently isolated epsilon cyclase gene, thus, providing additional evidence that the lut2 alleles are mutations in the epsilon cyclase gene. It appears likely that the epsilon cyclase is a key step in regulating lutein levels and the ratio of lutein to beta,beta-carotenoids. Surprisingly, despite the absence of lutein, neither the lut1 nor lut2 mutation causes a visible deleterious phenotype or altered chlorophyll content, but both mutants have significantly higher levels of beta, beta-carotenoids. In particular, there is a stable increase in the xanthophyll cycle pigments (violaxanthin, antheraxanthin, and zeaxanthin) in both lut1 and lut2 mutants as well as an increase in zeinoxanthin in lut1 and beta-carotene in lut2. The accumulation of specific carotenoids is discussed as it pertains to the regulation of carotenoid biosynthesis and incorporation into the photosynthetic apparatus. Presumably, particular beta, beta-carotenoids are able to compensate functionally and structurally for lutein in the photosystems of Arabidopsis.
The thermal dissipation of absorbed light energy by the light-harvesting apparatus of higher plants is important in protecting the photosynthetic machinery from the effects of excess illumination. A major mechanism for such photoprotection, known as trans-thylakoid delta pH-dependent chlorophyll fluorescence quenching (qE), is induced by acidification of the lumen, is correlated with the interconversion of xanthophyll pigments, and is manifested as quenching of chloropyll fluorescence. The mechanistic basis for qE remains unknown. The reagent N, N'-dicyclohexylcarbodiimide (DCCD) specifically inhibits qE and covalently binds to two minor light-harvesting pigment-protein complexes (LHCII), LHCIIa and LHCIIc. It is shown that DCCD treatment of isolated LHCIIc complexes reverses acid-induced chlorophyll fluorescence quenching in an in vitro system. Fingerprinting of [14C]DCCD-labeled LHCIIc demonstrates that there are two DCCD-sensitive amino acid residues on this complex, and these are shown to be glutamate residues, each of which is located near the lumen. In view of the effects of DCCD on the pattern of proton release from photosystem II during photosynthesis, we propose a model for the mechanism of the induction of qE--that these residues from part of a proton pathway, the lumen pH being sensed via its effects on the rate of proton release. One possibility is that the resulting changes in the protonation state of these carboxyl side chains may modulate the structural and energetic organization of LHCII.
Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the alpha- or beta-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (alpha-carotene branch), zeaxanthin, and antheraxanthin (beta-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), alpha-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.
The three mutant alleles of the ABA locus of Arabidopsis thaliana result in plants that are deficient in the plant growth regulator abscisic acid (ABA). We have used 18O2 to label ABA in water-stressed leaves of mutant and wild-type Arabidopsis. Analysis by selected ion monitoring and tandem mass spectrometry of [18O]ABA and its catabolites, phaseic acid and ABA-glucose ester (beta-D-glucopyranosyl abscisate), indicates that the aba genotypes are impaired in ABA biosynthesis and have a small ABA precursor pool of compounds that contain oxygens on the ring, presumably oxygenated carotenoids (xanthophylls). Quantitation of the carotenoids from mutant and wild-type leaves establishes that the aba alleles cause a deficiency of the epoxy-carotenoids violaxanthin and neoxanthin and an accumulation of their biosynthetic precursor, zeaxanthin. These results provide evidence that ABA is synthesized by oxidative cleavage of epoxy-carotenoids (the "indirect pathway"). Furthermore the carotenoid mutant we describe undergoes normal greening. Thus the aba alleles provide an opportunity to study the physiological roles of epoxy-carotenoids in photosynthesis in a higher plant.
:CO2 assimilation, transpiration and modulated chlorophyll fluorescence of leaves of Chenopodium bonus-henricus (L.) were measured in the laboratory and, at a high altitude location, in the field. Direct calibration of chlorophyll fluorescence parameters against carbon assimilation in the presence of 1 or 0.5% oxygen (plus CO2) proved necessary to calculate electron transport under photorespiratory conditions in individual experiments. Even when stomata were open in the field, total electron transport was two to three times higher in sunlight than indicated by net carbon gain. It decreased when stomata were blocked by submerging leaves under water or by forcing them to close in air by cutting the petiole. Even under these conditions, electron transport behind closed stomata approached 10 nmol electrons m−2 leaf area s−1 at temperatures between 25 and 30 °C. No photoinactivation of photosystem II was indicated by fluorescence analysis after a day's exposure to full sunlight. Only when leaves were submerged in ice was appreciable photoinactivation noticeable after 4 h exposure to sunlight. Even then almost full recovery occurred overnight. Electron transport behind blocked stomata was much decreased when leaves were darkened for 70 min (in order to deactivate light-regulated enzymes of the Calvin cycle) before exposure to full sunlight. Brief exposure of leaves to HCN (to inhibit photoassimilation and photorespiration) also decreased electron transport drastically compared to electron transport in unpoisoned leaves with blocked stomata. Non-photochemical fluorescence quenching and reduction of QA, the primary electron acceptor of photosystem II was increased by HCN-poisoning. Very similar observations were made when glyceraldehyde was used instead of HCN to inhibit photosynthesis and photorespiration. In HCN-poisoned leaves, residual electron transport increased linearly with temperature and showed early light saturation revealing characteristics of the Mehler reaction. During short exposure of these leaves to photon flux densities equivalent to 25% of sunlight, no or only little photoinactivation of photosystem II was observed. However, prolonged exposure to sunlight caused inactivation even though non-photochemical quenching of chlorophyll fluorescence was extensive. Simultaneously, oxidation of cellular ascorbate and glutathione increased. Inactivation of photosystem II was reversible in dim light and in the dark only after short times of exposure to sunlight. Glyceraldehyde was very similar to HCN in increasing the sensitivity of photosystem II in leaves to sunlight. We conclude from the observations that the electron transport permitted by the interplay of photoassimilatory and photorespiratory electron transport is essential to prevent the photoinactivation of photosynthetic electron transport. The Mehler and Asada reactions, which give rise to strong nonphotochemical fluorescence quenching, are insufficient to protect the chloroplast electron transport chain against photoinactivation.
Sustained decreases in intrinsic photosystem II efficiency (i.e. Fv/Fm) in response to high light and chilling temperatures were examined in eight species, and were found to be accompanied by the retention of zeaxanthin (Z) and antheraxanthin (A) overnight. The quantitative relationship between changes in Fv/Fm and the A + Z level during these sustained changes on cold days was similar to that obtained for rapidly reversible changes on warm days. Furthermore, upon removal of leaves from the field, recovery from 'photoinhibition' (the reversal of the depression of Fv/Fm) matched the timecourse of the epoxidation of Z and A to violaxanthin (V). These findings suggest that the 'photoinhibition' occuring in these species might be due to the sustained engagement of these de-epoxidised components of the xanthophyll cycle in photoprotective energy dissipation. When examined over the course of several days during the winter, the predawn conversion state of the xanthophyll cycle responded to the daily changes in minimum air (and leaf) temperature, such that the xanthophyll cycle was largely de-epoxidised prior to sunrise on cold nights and was present predominantly as V after nights when the nocturnal temperatures were above freezing. In addition, in some of the species examined, there was a large acclimation of the xanthophyll cycle pool size to the level of excessive light, with a much larger pool present in the leaves examined during the winter and that pool being de-epoxidised to Z and A to a much greater degree at midday than from similar leaves examined during the summer. The xanthophyll cycle, and the photoprotective energy dissipation process associated with it, would thus appear to provide plants the flexibility required to deal with the excessive levels of light absorbed by chlorophyll under a wide range of climatic conditions, and can quite possibly account for the 'photoinhibition' observed during winter stress.
Zeaxanthin has been correlated with high-energy non-photochemical fluorescence quenching but whether antheraxanthin, the intermediate in the pathway from violaxanthin to zeaxanthin, also relates to quenching is unknown. The relationships of zeaxanthin, antheraxanthin and ΔpH to fluorescence quenching were examined in chloroplasts ofPisum sativum L. cv. Oregon andLactuca sativa L. cv. Romaine. Data matrices as five levels of violaxanthin de-epoxidation against five levels of light-induced lumen-proton concentrations were obtained for both species. The matrices included high levels of antheraxanthin as well as lumen-proton concentrations induced by subsaturating to saturation light levels. Analyses of the matrices by simple linear and multiple regression showed that quenching is predicted by models where the major independent variable is the product of lumen acidity and de-epoxidized xanthophylls, the latter as the sum of zeaxanthin and antheraxanthin. The interactions of lumen acidity and xanthophyll concentration are shown in three-dimensional plots of the best-fit multiple regression models. Antheraxanthin apparently contributes to quenching as effectively as zeaxanthin and explains quenching previously not accounted for by zeaxanthin. Hence, we propose that all high-energy dependent quenching is xanthophyll dependent. Quenching requires a threshold lumen pH that varies with xanthophyll composition. After the threshold, quenching is linear with lumen acidity or xanthophyll composition.
This paper discusses biochemical and regulatory aspects of the violaxanthin cycle as well as its possible role in photoprotection. The violaxanthin cycle responds to environmental conditions in the short-term and long-term by adjusting rates of pigment conversions and pool sizes of cycle pigments, respectively. Experimental evidence indicating a relationship between zeaxanthin formation and non-photochemical energy dissipation is reviewed. Zeaxanthin-associated energy dissipation appears to be dependent on transthylakoid ΔpH. The involvement of light-harvesting complex II in this quenching process is indicated by several studies. The current hypotheses on the underlying mechanism of zeaxanthin-dependent quenching are alterations of membrane properties, including conformational changes of the light-harvesting complex II, and singlet-singlet energy transfer from chlorophyll to zeaxanthin.
By selecting for germinating seeds in the progeny of mutagen-treated non-germinating gibberellin responsive dwarf mutants of the ga-1 locus in Arabidopsis thaliana, germinating lines (revertants) could be isolated. About half of the revertants were homozygous recessive for a gene (aba), which probably regulates the presence of abscisic acid (ABA). Arguments for the function of this gene were obtained from lines homozygous recessive for this locus only, obtained by selection from the F2 progeny of revertant X wild-type crosses. These lines are characterized by a reduced seed dormancy, symptoms of withering, increased transpiration and a lowered ABA content in developing and ripe seeds and leaves.
Superoxide dismutases (SODs) are metal-containing enzymes that catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined where it plays a major role in the defense against toxic-reduced oxygen species, which are generated as byproducts of many biological oxidations. The generation of oxygen radicals can be further exacerbated during environmental adversity and consequently SOD has been proposed to be important for plant stress tolerance. In plants, three forms of the enzyme exist, as classified by their active site metal ion: copper/zinc, manganese, and iron forms. The distribution of these enzymes has been studied both at the subcellular level and at the phylogenic level. It is only in plants that all three different types of SOD coexist. Their occurrence in the different subcellular compartments of plant cells allows a study of their molecular evolution and the possibility of understanding why three functionally equivalent but structurally different types of SOD have been maintained. Several cDNA sequences that encode the different SODs have recently become available, and the use of molecular techniques have greatly increased our knowledge about this enzyme system and about oxidative stress in plants in general, such that now is an appropriate time to review our current knowledge.
Higher plants must dissipate absorbed light energy that exceeds the photosynthetic capacity to avoid molecular damage to the pigments and proteins that comprise the photosynthetic apparatus. Described in this minireview is a current view of the biochemical, biophysical and bioenergetic aspects of the primary photoprotective mechanism responsive for dissipating excess excitation energy as heat from photosystem II (PSII). The photoprotective heat dissipation is measured as nonphotochemical quenching (NPQ) of the PSII chlorophyll a (Chl a) fluorescence. The NPQ mechanism is controlled by the trans-thylakoid membrane PH gradient (ApH) and the special xanthophyll cycle pigments. In the NPQ mechanism, the de-epoxidized endgroup moieties and the trans-thylakoid membrane orientations of antheraxanthin (A) and zeaxanthin (Z) strongly affect their interactions with protonated chlorophyll binding proteins (CPs) of the PSII inner antenna. The CP protonation sites and steps are influenced by proton domains sequestered within the proteo-lipid cone of the thylakoid membrane. Xanthophyll cycle enrichment around the CPs may explain why changes in the peripheral PSII antenna size do not necessarily affect either the concentration of the xanthophyll cycle pigments on a per PSII unit basis or the NPQ mechanism. Recent time-resolved PSII Chl a fluorescence studies suggest the NPQ mechanism switches PSII units to an increased rate constant of heat dissipation in a series of steps that include xanthophyll de-epoxidation, CP-protonation and binding of the xanthophylls to the protonated CPs; the concerted process can be described with a simple two-step, pH-activation model. The xanthophyll cycle-dependent NPQ mechanism is profoundly influenced by temperatures suboptimal for photosynthesis via their effects on the trans-thylakoid membrane energy coupling system. Further, low temperature effects can be grouped into either short term (minutes to hours) or long term (days to seasonal) series of changes in the content and composition of the PSII pigment-proteins. This minireview concludes by briefly highlighting primary areas of future research interest regarding the NPQ mechanism.
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The use of solar energy in photosynthesis depends on the ability to safely dissipate excess energy. The key dissipation process employed by plants in their natural environment is mediated by a particular group of carotenoids. Multiple levels of control allow adjustments in energy dissipation activity in response to changing levels of light stress in the natural environment. Recent advances in the understanding of the photophysics, biochemical regulation and ecophysiology of this essential photoprotective process are reviewed.
PLANTS absorb light for photosynthesis but as light can itself be dangerous to plants, they need to protect themselves against its damaging effects. Here we show that photorespiration can act as such a defence mechanism. We constructed transgenic tobacco plants enriched or reduced in plastidic glutamine synthetase (GS2), a key enzyme in photorespiration. Those transgenic plants having twice the normal amount of GS2 had an improved capacity for photorespiration and an increased tolerance to high-intensity light, whereas those with a reduced amount of GS2 had a diminished capacity for photorespiration and were photoinhib-ited more severely by high-intensity light compared with control plants. We conclude that photorespiration protects C3 plants from photoinhibition.
We have investigated the binding to proteins of the photosynthetic apparatus of the carboxy-modifying agent dicyclohexylcarbodiimide, (cHxN)2C; this inhibits the protective dissipation of excess absorbed light energy (qE) by the light-harvesting apparatus of photosystem II (LHCII), suggesting that carboxyl amino-acid side chains within hydrophobic protein domains may be involved in qE. (cHxN)214C was used to label thylakoids and photosystem II particles, so as to identify proteins which may be involved in the detection of lumen pH during qE induction. Of six thylakoid proteins labelled with (cHxN)2C under conditions where qE is efficiently induced, three are associated with photosystem I, and none with the bulk LHCII. PSII-associated label is bound to three minor components of LHCII, identified as LHCIIa (two species) and LHCIIc, as shown by protein sequencing of tryptic fragments of purified complexes. pH titration of qE formation and protein labelling in coupled thylakoids showed that both qE and labelling of LHCIIa increased at pH 7–8.
Conifers of the boreal zone encounter considerable combined stress of low temperature and high light during winter, when photosynthetic consumption of excitation energy is blocked. In the evergreen Pinus sylvestris L. these stresses coincided with major seasonal changes in photosystem II (PSII) organisation and pigment composition. The earliest changes occurred in September, before any freezing stress, with initial losses of chlorophyll, the D1-protein of the PSII reaction centre and of PSII light-harvesting-complex (LHC II) proteins. In October there was a transient increase in F0, resulting from detachment of the light-harvesting antennae as reaction centres lost D1. The D1-protein content eventually decreased to 90%, reaching a minimum by December, but PSII photochemical efficiency [variable fluorescence (Fv)/maximum fluorescence (Fm)] did not reach the winter minimum until mid-February. The carotenoid composition varied seasonally with a twofold increase in lutein and the carotenoids of the xanthophyll cycle during winter, while the epoxidation state of the xanthophylls decreased from 0.9 to 0.1 from October to January. The loss of chlorophyll was complete by October and during winter much of the remaining chlorophyll was reorganised in aggregates of specific polypeptide composition, which apparently efficiently quench excitation energy through non-radiative dissipation. The timing of the autumn and winter changes indicated that xanthophyll de-epoxidation correlates with winter quenching of chlorophyll fluorescence while the drop in photochemical efficiency relates more to loss of D1-protein. In April and May recovery of the photochemistry of PSII, protein synthesis, pigment rearrangements and zeaxanthin epoxidation occurred concomitantly. Indoor recovery of photosynthesis in winter-stressed branches under favourable conditions was completed within 3 d, with rapid increases in F0, the epoxidation state of the xanthophylls and in light-harvesting polypeptides, followed by recovery of D1-protein content and Fv/Fm, all without net increase in chlorophyll. The fall and winter reorganisation allow Pinus sylvestris to maintain a large stock of chlorophyll in a quenched, photoprotected state, allowing rapid recovery of photosynthesis in spring.
The role of the xanthophyll cycle in regulating the energy flow to the PS II reaction centers and therefore in photoprotection was studied by measurements of light-induced absorbance changes, Chl fluorescence, and photosynthetic O2 evolution in sun and shade leaves of Hedera canariensis. The light-induced absorbance change at 510 nm (A510) was used for continuous monitoring of zeaxanthin formation by de-epoxidation of violaxanthin. Non-radiative energy dissipation (NRD) was estimated from non-photochemical fluorescence quenching (NPQ).High capacity for zeaxanthin formation in sun leaves was accompanied by large NRD in the pigment bed at high PFDs as indicated by a very strong NPQ both when all PS II centers are closed (F'm) and when all centers are open (F'o). Such Fo quenching, although present, was less pronounced in shade leaves which have a much smaller xanthophyll cycle pool.Dithiothreitol (DTT) provided through the cut petiole completely blocked zeaxanthin formation. DTT had no detectable effect on photosynthetic O2 evolution or the photochemical yield of PS II in the short term but fully inhibited the quenching of Fo and 75% of the quenching of Fm, indicating that NRD in the antenna was largely blocked. This inhibition of quenching was accompanied by an increased closure of the PS II reaction centers.In the presence of DTT a photoinhibitory treatment at a PFD of 200 mol m-2 s-1, followed by a 45 min recovery period at a low PFD, caused a 35% decrease in the photon yield of O2 evolution, compared to a decrease of less than 5% in the absence of DTT. The Fv/Fm ratio, measured in darkness showed a much greater decrease in the presence than in the absence of DTT. In the presence of DTT Fo rose by 15–20% whereas no change was detected in control leaves.The results support the conclusion that the xanthophyll cycle has a central role in regulating the energy flow to the PS II reaction centers and also provide direct evidence that zeaxanthin protects against photoinhibitory injury to the photosynthetic system.
The generation of non-photochemical fluorescence quenching under photoinhibitory illumination and its relaxation under subsequent low light illumination in leaves from intermittent-light-grown pea (Pisum sativum L.) plants (IML-plants) has been investigated. In parallel, we studied (i) the activity of the xanthophyll cycle with emphasis on zeaxanthin formation and reconversion to violaxanthin and (ii) the degradation rate of D1 protein. In comparison to control plants grown in continuous light, IML-plants were much more susceptible to photoinhibition as determined from the increase of slowly (halftimes > 20 min) relaxing quenching (qI) of variable chlorophyll fluorescence. The relaxation (recovery) kinetics of qI (under weak light) in both types of plant depended on the photon flux density, temperature and duration of pre-illumination. The recovery time generally increased with an increasing degree of qI. In IML-plants, relaxation of qI was kinetically closely related to the epoxidation of zeaxanthin. At high degrees of photosystem II inhibition the kinetics resembled those of D1 degradation. The results are discussed in terms of the mechanisms of photosystem II inactivation in vivo.
Light-induced chloroplast movements were found to cause changes in chlorophyll fluorescence emission, closely matching those in leaf absorptance, both in terms of the kinetics and the maximum extent of the changes observed in different species. The results demonstrate that chloroplast movements can have a significant effect on the efficiency of light utilization in photosynthesis. They further show that chloroplast movements need to be taken into account in measurements of fluorescence quenching and especially in measurements of light-induced optical changes used to monitor zeaxanthin formation and pH associated light scattering in leaves. Means of minimizing and of adjusting for the influences of chloroplast movements in such measurements are discussed.
The kinetics and temperature dependencies of development and relaxation of light-induced absorbance changes caused by deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (Z; peak at 506 nm) and by light scattering (S; peak around 540 nm) as well as of nonphotochemical quenching of chlorophyll fluorescence (NPQ) were followed in cotton leaves. Measurements were made in the absence and the presence of dithiothreitol (DTT), an inhibitor of violaxanthin deepoxidase. The amount of NPQ was calculated from the Stern-Volmer equation. A procedure was developed to correct gross AS (Sg) for absorbance changes around 540 nm that are due to a spectral overlap with Z. This protocol isolated the component which is caused by light-scattering changes alone (Sn). In control leaves, the kinetics and temperature dependence of the initial rate of rise in Sn that takes place upon illumination, closely matched that of Z. Application of DTT to leaves, containing little zeaxanthin or antheraxanthin, strongly inhibited both Sn and NPQ, but DTT had no inhibitory effect in leaves in which these xanthophylls had already been preformed, showing that the effect of DTT on An and NPQ results solely from the inhibition of violaxanthin deepoxidation. The rates and maximum extents of Sn and NPQ therefore depend on the amount of zeaxanthin (and/or antheraxanthin) present in the leaf. In contrast to the situation during induction, relaxation of Z upon darkening was much slower than the relaxation of Sn and NPQ. The relaxation of Sn and NPQ showed quantitatively similar kinetics and temperature dependencies (Q10=2.4). These results are consistent with the following hypotheses: The increase in lumen-proton concentration resulting from thylakoid membrane energization causes deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The presence of these xanthophylls is not sufficient to cause Sn or NPQ but, together with an increased lumen-proton concentration, these xanthophylls cause a conformational change, reflected by Sn. The conformational change facilititates nonradiative energy dissipation, thereby causing NPQ. Membrane energization is prerequisite to conformational changes in the thylakoid membrane and resultant nonradiative energy dissipation but the capacity for such changes in intact leaves is quite limited unless zeaxanthin (and/or antheraxanthin) is present in the membrane. The sustained Sn and NPQ levels that remain after darkening may be attributable to a sustained high lumen-proton concentration.
The quantitative relationship between energy-dependent quenching of chlorophyll fluorescence (qE) and trans-thylakoid pH difference (ΔpH, estimated with 9-aminoacridine) was compared in chloroplasts from leaves preilluminated at low oxygen in the absence of CO2 with chloroplasts from leaves darkened under the same conditions. The extent of both phenomena was varied by changes in actinic light intensity. Chloroplasts from preilluminated leaves contained high levels of zeaxanthin (up to 15% of total carotenoid) and were capable of forming qE at lower ΔpH values than chloroplasts from dark-adapted leaves, which lacked zeaxanthin. Infusion of dithiothreitol into leaves prior to preillumination prevented the light-induced formation of zeaxanthin; chloroplasts prepared from these leaves showed the same relationship between qE and ΔpH observed in chloroplasts fom dark-adapted leaves. The rate of appearance and disappearance of the change in relationship between qE and ΔpH upon leaf preillumination and darkening was closely matched by the kinetics of synthesis and degradation of zeaxanthin. The quantitative relationship between qE and quenching of the dark-level of fluorescence (F0) was very similar in chloroplasts from preilluminated or dark-adapted leaves, containing disparate amounts of zeaxanthin. In both sets of chloroplasts, qE was inhibited by antimycin A. These latter observations suggest that the formation of qE involves mechanistically similar features in chloroplasts containing or lacking zeaxanthin. Overall, the data are discussed in terms of action of zeaxanthin as a ‘quenching amplifier’, functioning physiologically to allow qE formation in the absence of high ΔpH potentially prohibitive to high rates of CO2 fixation.
In photosynthesis of higher plants, photosystem II drives electron transfer from the water-oxidizing manganese centre at the lumenal side to bound plastoquinone at the stromal side of the thylakoid membrane. Proton release into the lumen and proton uptake from the stroma, i.e. net proton pumping, follows as consequence of vectoral electron transport. The proton pumping activity can be short circuited by covalent modification with N,N'-dicyclohexylcarbodiimide (cHxN)2C of certain proteins in the 20-28-kDa range. After modification, protons from water oxidation are no longer released into the thylakoid lumen, but instead transferred through the photosystem complex to protonate the photoreduced bound quinone at the other side of the membrane [Jahns, P., Polle, A. & Junge, W. (1988) EMBO J. 7, 589-594]. Here we identify the pertinent (cHxN)2C-binding proteins by amino acid sequence analysis and localize (cHxN)2C-binding sites within their primary structure. The proteins that are associated with the proton short circuit are light-harvesting chlorophyll-a/b-binding proteins. Our results imply that in addition to acting as antennae they may serve another function: the funneling into the thylakoid lumen of protons, which are liberated in the water-oxidizing Mn centre.
Carotenoids are essential for the survival of photosynthetic organisms. They function as light-harvesting molecules and provide photoprotection. In this review, the molecular features which determine the efficiencies of the various photophysical and photochemical processes of carotenoids are discussed. The behavior of carotenoids in photosynthetic bacterial reaction centers and light-harvesting complexes is correlated with data from experiments carried out on carotenoids and model systems in vitro. The status of the carotenoid structural determinations in vivo is reviewed.
The effects of dithiothreitol on absorbance changes at 505 and 515 nm in isolated lettuce chloroplasts were investigated. Dithiothreitol inhibited the ascorbate-dependent 505-nm change that is due to the de-epoxidation of violaxanthin to zeaxanthin. Dithiothreitol was effective for both light-induced de-epoxidation at pH 7 and dark de-epoxidation at pH 5. Titration of de-epoxidase activity with dithiothreitol resulted in complete inhibition at about 5 μmoles dithiothreitol per mg chlorophyll. Removal of dithiothreitol restored de-epoxidase activity. These results are consistent with the view that dithiothreitol inhibits violaxanthin de-epoxidation and the corresponding 505-nm change by reducing a disulfide that is required for de-epoxidase activity.Dithiothreitol was effective in resolving absorbance changes due to violaxanthin de-epoxidation and other changes that were superimposed under some conditions. At 515 nm and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), phenazine methosulfate, and ascorbate, dithiothreitol inhibited the large, slow and irreversible change which was due to de-epoxidation but not the fast and reversible so-called 515-nm change. At 505 nm and under similar conditions, dithiothreitol revealed the presence of a slow reversible change in addition to the one from de-epoxidation. Results with dithiothreitol showed that the absorbance change at 505 nm in the presence of DCMU, 2,6-dichlorophenolindophenol and ascorbate was due entirely to de-epoxidation. Similarly, absorbance changes at 515 nm also appeared to be mainly from de-epoxidation but with the presence of a small transient change due to some other components. It is suggested that dithiothreitol may be useful in resolving complex light-induced absorbance changes in other photosynthetic systems as well as in enabling new studies on reversible absorbance changes in the 500-nm region.
We discuss energy-dependent fluorescence lowering (qE-quenching), and suggest a model to explain the experimental data currently available. The main elements of the model are: (a) the qE-quenching reflects a mechanism associated with a component of the light-harvesting antenna rather than the reaction center of photosystem (PS) II--we suggest that it occurs through formation of an efficient quencher in one of the minor chlorophyll protein (CP) complexes; (b) the minor CPs have glutamate residues instead of glutamines at positions shown in light-harvesting complex II (LHCII) to be ligands to chlorophylls near the lumenal interface. We suggest that the quenching reflects a change in ligation of chlorophyll on protonation of these glutamate residues leading to formation of an exciton coupled dimer with a neighboring pigment, in which additional energy levels allow vibrational relaxation of the excited singlet. The model accounts for the dependence on low lumenal pH, the ligand residue changes between LHCII and the minor CPs, the preferential distribution of components of the xanthophyll cycle in the minor CPs, the inhibition of qE-quenching by DCCD, and the specific binding of DCCD to the minor CPs.
Even though light is the source of energy for photosynthesis, it can also be harmful to plants. Light-induced damage is targetted mainly to Photosystem II and leads to inactivation of electron transport and subsequent oxidative damage of the reaction centre, in particular to the D1 protein. Inactivation and protein damage can be induced by two different mechanisms, either from the acceptor side or from donor side of P680. The damaged D1 protein is triggered for degradation and digested by at least one serine-type proteinase that is tightly associated with the Photosystem II complex itself. The damaged Photosystem II complex dissociates from the light-harvesting antenna and migrates from appressed to non-appressed thylakoid regions where a new D1 protein is co-translationally inserted into the partially disassembled Photosystem II complex. D1 protein phosphorylation probably allows for coordinated biodegradation and biosynthesis of the D1 protein. After religation of cofactors and assembly of subunits, the repaired Photosystem II complex can again be found in the appressed membrane regions. Various protective mechanisms and an efficient repair cycle of Photosystem II allow plants to survive light stress.
The embryo of the flowering plant Arabidopsis develops by a regular pattern of cell divisions and cell shape changes. Mutations in the KNOLLE (KN) gene affect the rate and plane of cell divisions as well as cell morphology, resulting in mutant seedlings with a disturbed radial organization of tissue layers. At the cellular level, mutant embryos are characterized by incomplete cross walls and enlarged cells with polyploid nuclei. The KN gene was isolated by positional cloning. The predicted KN protein has similarity to syntaxins, a protein family involved in vesicular trafficking. During embryogenesis, KN transcripts are detected in patches of single cells or small cell groups. Our results suggest a function for KN in cytokinesis.
The function of the long-chain, highly unsaturated carotenoids of higher plants in photoprotection is becoming increasingly well understood, while at the same time their function in other processes, such as light collection, needs to be reexamined. Recent progress in this area has been fueled by more accurate determinations of the photophysical properties of these molecules, as well as extensive characterization of the physiology and ecology of a particular group of carotenoids, those of the xanthophyll cycle, that play a key role in the photoprotection of photosynthesis under environmental stress. The deepoxidized xanthophylls zeaxanthin and antheraxanthin, together with a low pH within the photosynthetic membrane, facilitate the harmless dissipation of excess excitation energy directly within the light-collecting chlorophyll antennae. Evidence for this function as well as current contrasting hypotheses concerning its molecular mechanism are reviewed. In addition, the acclimation of the xanthophyll cycle content and composition of leaves to contrasting environments with different demands for photoprotection is summarized.
Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.
The abscisic-acid-deficient aba-1 mutant of Arabidopsis thaliana is unable to epoxidize zeaxanthin. As a consequence, it contains large amounts of this carotenoid and lacks epoxy-xanthophylls. HPLC analysis of pigment contents in leaves, isolated thylakoids and preparations of the major light-harvesting complex of photosystem II (PSII) (LHC-II) indicated that zeaxanthin replaced neoxanthin, violaxanthin and antheraxanthin in the light-harvesting system of PSII in aba-1. Non-denaturing electrophoretic fractionation of solubilized thylakoids showed that the xanthophyll imbalance in aba-1 was associated with a pronounced decrease in trimeric LHC-II in favour of monomeric complexes, with a substantial increase in free pigments (mainly zeaxanthin and chlorophyll b), suggesting a decreased stability of LHC-II. The reduced thermostability of PSII in aba-1 was also deduced from in vivo chlorophyll fluorescence measurements. Wild-type and aba-1 leaves could not be distinguished on the basis of their photosynthetic performance: no significant difference was observed between the two types of leaves for light-limited and light-saturated photosynthetic oxygen evolution, PSII photochemistry and PSII to PSI electron flow. When dark-adapted leaves (grown in white light of 80 mumol m-2s-1) were suddenly exposed to red light of 150 mumol m-2s-1, there was a strong nonphotochemical quenching of chlorophyll fluorescence, the amplitude of which was virtually identical (at steady state) in aba-1 and wild-type leaves, despite the fact that the xanthophyll cycle pigment pool was completely in the form of zeaxanthin in aba-1 and almost exclusively in the form of violaxanthin in the wild type. A high concentration of zeaxanthin in aba-1 thylakoids did not, in itself, provide any particular protection against the photoinhibition of PSII. Taken together, the presented results indicate the following: (1) zeaxanthin can replace epoxy-xanthophylls in LHC-II without significantly affecting the photochemical efficiency of PSII; (2) zeaxanthin does not play any specific role in direct (thermal) energy dissipation in PSII; (3) the photoprotective action of the xanthophyll cycle (rapid photoconversion of violaxanthin to zeaxanthin) is not based on the mere substitution of violaxanthin by zeaxanthin in the chlorophyll antennae.
This article focuses on light-harvesting complexes (LHCs) in oxygen evolving photosynthetic organisms. These organisms include cyanobacteria, red algae, plants, green algae, brown algae, diatoms, chrysophytes, and dinoflagellates. We highlight the diversity of pigment-protein complexes that fuel the conversion of radiant energy to chemical bond energy in land plants and the diverse groups of the algae, detail the ways in which environmental parameters (i.e. light quantity and quality, nutrients) modulate the synthesis of these complexes, and discuss the evolutionary relationships among the LHC structural polypeptides.
Carotenoids have a key role in photosynthesis in photosynthetic systems, transferring excitation energy to chlorophyll (Chl) during light harvesting. These pigments also protect the photosynthetic apparatus from photodamage by quenching the Chl triplet state and singlet oxygen. In addition, in higher plants and some algae, a number of xanthophylls also have the ability to deactivate excited Chl under conditions of excess excitation via the operation of the xanthophyll cycle (violaxanthin<-->antheraxanthin<-->zeaxanthin or diadinoxanthin<-->diatoxanthin). The formation of zexanthin (or diatoxanthin) can be clearly correlated with the non-photochemical quenching of Chl fluorescence, and is now recognized as a major photoprotective process in higher plants and a number of algal genera. The interconversion of these xanthophylls in response to a changing light environment alters the extent of their carbon-carbon double bond conjugation, which, in turn, affects the excited state energies and lifetimes of the carotenoids and may also alter their structure/conformation and hydrophobicity. The possible roles of these photophysical and physicochemical changes in the mechanism(s) of xanthophyll-mediated energy dissipation via quenching of Chl fluorescence are discussed.
Energy-dependent quenching of chlorophyll fluorescence (qE) reflects the action of a powerful mechanism of protection from photoinhibition in which the low pH in the chloroplast lumen induces dissipation of excess excitation energy. Dicyclohexylcarbodiimide (DCCD), a protein-modifying agent, is a powerful inhibitor of qE and has been shown to react with acidic residues, in a hydrophobic environment, involved in proton translocation. The CP29 subunit of photosystem II has been proposed to be the site of qE quenching and shown to bind DCCD. We have hypothesised, on the basis of the CP29 protein sequence and of the structure of light-harvesting complex II protein, that glutamic acid 166 is the DCCD binding site. In this study, we have produced recombinant proteins either with wild-type sequence or carrying a mutation on the 166 position. We show that the mutant protein does not bind DCCD. This identifies E166 as the site whose protonation may lead to a conformational change triggering qE.