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The Arabidopsis embryo mutant schlepperless has a defect in the chaperonin-60alpha gene
Abstract
We identified a T-DNA-generated mutation in the chaperonin-60alpha gene of Arabidopsis that produces a defect in embryo development. The mutation, termed schlepperless (slp), causes retardation of embryo development before the heart stage, even though embryo morphology remains normal. Beyond the heart stage, the slp mutation results in defective embryos with highly reduced cotyledons. slp embryos exhibit a normal apical-basal pattern and radial tissue organization, but they are morphologically retarded. Even though slp embryos are competent to transcribe two late-maturation gene markers, this competence is acquired more slowly as compared with wild-type embryos. slp embryos also exhibit a defect in plastid development-they remain white during maturation in planta and in culture. Hence, the overall developmental phenotype of the slp mutant reflects a lesion in the chloroplast that affects embryo development. The slp phenotype highlights the importance of the chaperonin-60alpha protein for chloroplast development and subsequently for the proper development of the plant embryo and seedling.
Supplementary resource (1)
Nucleotide Sequence
February 1996
... Plant embryogenesis is a process generally divided in four phases such as embryo development, maturation, desiccation and germination (Apuya et al. 2001). Embryo development and conversion into plant are processes controlled by molecular mechanisms as gene expression, where emb mutants can help in understanding the correlation between phenotypes and gene functions. ...
... The resulting seedlings showed short hypocotyl and the shoot meristem twisted (Sun et al. 1998).The schlepperless is a mutant in the chaperonin-60α gene. Embryo mutants for the chaperonin-60α gene show normal development but their growth is slow and the development is arrested at the heart stage (Apuya et al. 2001). The T-DNA insertion mutant slow embryo development1 that affects a protein targeted to mitochondria showed a slower development compared to wild-type embryos. ...
... Shevell et al. (2000) SchlepperlessRetardation of embryo development before the heart stageApuya et al. (2001) Topless-1 Transformation of the shoot pole of the embryo into a root Long et al. (2002) hoc Over production of cytokinin, bushy phenotype with supernumerary rosettes Catterou et al. (2002) Pex10 null mutant Retarded development and morphological abnormalities Sparkes (2003) Embryo ball Cotyledons are absent, the apical region produced leaf-like structures with trichomes Kristof et al. (2008) bobber1 Arrest at the globular stage of development Jurkuta et al. (2009) gnom Root meristem deficient and cotyledon primordial formation fails Wolters et al. (2011) Dicer-like1 Early embryo maturation Willmann et al. (2011) lec1 Cotyledon identity altered and ectopic trichomes formation Huang et al. (2015) Slow embryo development1 Slower development in globular stage and white appearance Ju et al. (2016) Serine decarboxylase Arrest embryo development Yunus et al. (2016) Altered Meristem Program 1 Seeds develop deficient coat ...
Somatic embryogenesis is a morphogenetic event where somatic cells have the ability to produce embryos without gamete fusion. It is used as a technique for plant mass propagation. It is a process that has six well defined steps such as induction, expression, development, maturation, germination and plant conversion. These steps are characterized by distinct physiological , morphological and molecular events. Although somatic embryogenesis has been established in several plant species, there remains many problems to be solved. The main problem in somatic embryogenesis is the large number of abnormal embryos produced which cannot germinate nor convert into normal plants. Abnormalities in somatic embryos (SE) can be generated by genetic or epigenetic changes in the DNA. These changes in the DNA can be influenced by external factors such as the use of plant growth regulators and mutagenic substances or stress factors applied to the plant tissue such as high and low temperatures, drought, salinity, and heavy metals. Abnormalities generated by genetic changes in the DNA are hardly reversible; however, abnormalities generated by epigenetic changes may be reversible and the abnormal embryos are able to produce normal plants in most cases. This review focuses on the identification of the main factors that can cause abnormal SE development in different plant species, suggest how SE abnormalities are related to somaclonal variations and identify which genes may be involved with embryo abnormalities. Zygotic embryo abnormalities in Arabidopsis thaliana mutants are listed with the aim to understand the main genetic mechanisms involved in embryo aberrations. Key message The abnormalities in somatic embryos are related to the use of 2,4-D in most of the published protocols, this sintetic auxin disrupts the endogenous auxin balance and the auxin polar transportation interfering with the embryo apical-basal polarity.
... Both Cpn60α and Cpn60β are required for proper plastid division, and the disruption of Cpn60α results in general defects in plastid function (Henderson et al., 2013). A cpn60α1 mutant showed general defects in plastid function, leading to embryonic lethality, which highlights the importance of the critical role of Cpn60α1 in chloroplast development (Apuya et al., 2001). In rice, TCD9, which encodes the Cpn60α subunit of Cpn60 protein, localized in chloroplasts, was shown to be responsible for the albino phenotype (Jiang et al., 2014). ...
... According to gene annotation and BlastP analysis, a total of nine Cpn60-like genes were identified in the SGN database (http://solgenomics.net) (Supplementary Dataset 1). Phylogenetic analysis of Cpn60 family proteins placed Solyc11g069790.1 into the same clade with AtCpn60α1, which plays a key role in regulating chloroplast and embryo development in Arabidopsis (Fig. 4B) (Apuya et al., 2001). Therefore, the candidate gene Solyc11g069790.1, ...
As an indispensable biological process of plants, photosynthesis produces organic substances for plant growth, during which photorespiration occurs to oxidize carbohydrates to a homeostasis. Although the molecular mechanism underlying photosynthesis and photorespiration has been widely explored, the crosstalk between photosynthesis and photorespiration remains largely unknown. In this study, we isolated and characterized T-DNA insertion mutant of tomato (Solanum lycopersicum) named yellow leaf (yl) with yellowish leaves, retarded growth, and chloroplast collapse that hampered both photosynthesis and photorespiration. Genetic and expression analyses demonstrated that the phenotype of yl was caused by a loss of function mutation resulting from a single copy T-DNA insertion in chaperonin 60α1 (SlCPN60α1). SlCPN60α1 showed high expression levels in leaves and located in both chloroplast and mitochondria. Silencing of SlCPN60α1using the virus-induced gene silencing and RNA interference mimic the phenotype of yl. Two-dimensional electrophoresis (2-DE) and yeast-two hybrid (Y2H) results suggest that SlCPN60α1 potentially interact with proteins which are involved in chlorophyll synthesis, photosynthetic electron transport and the Calvin cycle, and further affect photosynthesis. Moreover, SlCPN60α1 directly interacted with serine hydroxymethyltransferase (SlSHMT1) in mitochondria, thereby regulating photorespiration in tomato. This study outlines the importance of SlCPN60α1 for both photosynthesis and photorespiration and provides molecular insights towards plant genetic improvement.
... Plant embryogenesis is a process generally divided in four phases such as embryo development, maturation, desiccation and germination ( Apuya et al. 2001). Embryo development and conversion into plant are processes controlled by molecular mechanisms as gene expression, where emb mutants can help in understanding the cor- relation between phenotypes and gene functions. ...
... The resulting seedlings showed short hypocotyl and the shoot meristem twisted ( Sun et al. 1998).The schlepperless is a mutant in the chaperonin-60α gene. Embryo mutants for the chaperonin-60α gene show normal development but their growth is slow and the development is arrested at the heart stage ( Apuya et al. 2001). The T-DNA inser- tion mutant slow embryo development1 that affects a pro- tein targeted to mitochondria showed a slower development compared to wild-type embryos. ...
Somatic embryogenesis is a morphogenetic event where somatic cells have the ability to produce embryos without gamete fusion. It is used as a technique for plant mass propagation. It is a process that has six well defined steps such as induction, expression, development, maturation, germination and plant conversion. These steps are characterized by distinct physiological , morphological and molecular events. Although somatic embryogenesis has been established in several plant species, there remains many problems to be solved. The main problem in somatic embryogenesis is the large number of abnormal embryos produced which cannot germinate nor convert into normal plants. Abnormalities in somatic embryos (SE) can be generated by genetic or epigenetic changes in the DNA. These changes in the DNA can be influenced by external factors such as the use of plant growth regulators and mutagenic substances or stress factors applied to the plant tissue such as high and low temperatures, drought, salinity, and heavy metals. Abnormalities generated by genetic changes in the DNA are hardly reversible; however, abnormalities generated by epigenetic changes may be reversible and the abnormal embryos are able to produce normal plants in most cases. This review focuses on the identification of the main factors that can cause abnormal SE development in different plant species, suggest how SE abnormalities are related to somaclonal variations and identify which genes may be involved with embryo abnormalities. Zygotic embryo abnormalities in Arabidopsis thaliana mutants are listed with the aim to understand the main genetic mechanisms involved in embryo aberrations. Key message The abnormalities in somatic embryos are related to the use of 2,4-D in most of the published protocols, this sintetic auxin disrupts the endogenous auxin balance and the auxin polar transportation interfering with the embryo apical-basal polarity.
... Plant embryogenesis is a process generally divided in four phases such as embryo development, maturation, desiccation and germination (Apuya et al. 2001). Embryo development and conversion into plant are processes controlled by molecular mechanisms as gene expression, where emb mutants can help in understanding the correlation between phenotypes and gene functions. ...
... The resulting seedlings showed short hypocotyl and the shoot meristem twisted (Sun et al. 1998).The schlepperless is a mutant in the chaperonin-60α gene. Embryo mutants for the chaperonin-60α gene show normal development but their growth is slow and the development is arrested at the heart stage (Apuya et al. 2001). The T-DNA insertion mutant slow embryo development1 that affects a protein targeted to mitochondria showed a slower development compared to wild-type embryos. ...
Somatic embryogenesis is a morphogenetic event where somatic cells have the ability to produce embryos without gamete fusion. It is used as a technique for plant mass propagation. It is a process that has six well defined steps such as induction, expression, development, maturation, germination and plant conversion. These steps are characterized by distinct physiological, morphological and molecular events. Although somatic embryogenesis has been established in several plant species, there remains many problems to be solved. The main problem in somatic embryogenesis is the large number of abnormal embryos produced which cannot germinate nor convert into normal plants. Abnormalities in somatic embryos (SE) can be generated by genetic or epigenetic changes in the DNA. These changes in the DNA can be influenced by external factors such as the use of plant growth regulators and mutagenic substances or stress factors applied to the plant tissue such as high and low temperatures, drought, salinity, and heavy metals. Abnormalities generated by genetic changes in the DNA are hardly reversible; however, abnormalities generated by epigenetic changes may be reversible and the abnormal embryos are able to produce normal plants in most cases. This review focuses on the identification of the main factors that can cause abnormal SE development in different plant species, suggest how SE abnormalities are related to somaclonal variations and identify which genes may be involved with embryo abnormalities. Zygotic embryo abnormalities in Arabidopsis thaliana mutants are listed with the aim to understand the main genetic mechanisms involved in embryo aberrations.
... Among them, AtCpn60α1 and AtCpn60α2 share only about 57% identity, and AtCpn60β1/2/3 share 90%-95% identity, while AtCpn60β4 is only 60% identical to the other AtCpn60β subunits [14][15]. AtCpn60α1 was the first chaperonin gene studied in detail, and its mutant, schlepperless (slp), showed retardation of embryo development before the heart stage, and defective embryos with highly reduced cotyledons [16]. Then a T-DNA insertion mutant of AtCpn60α2, emb3007, showed the embryo development arrested at the globular stage in the SeedGenes database (http://www.seedgenes.org/), ...
... Functional divergence of CPNA2 and CPNA1 occurs during Arabidopsis embryo development Hill and Hemmingsen reported that CPNA2 is the paralog of CPNA1 [15], thus it is possible that they have redundant functions. However, a CPNA1 mutant, slp (schlepperless), showed an Functional divergence of Cpn60α subunits embryo-defective phenotype that mainly appeared at the heart stage and thereafter, which is different from the cpna2 mutants [16]. To confirm the previous study, we obtained another AtCpn60α1 mutant (cpna1, SALK_006606) (S1A Fig), and then observed embryo development in the siliques of cpna1/+. ...
Chaperonins are a class of molecular chaperones that assist in the folding and assembly of a wide range of substrates. In plants, chloroplast chaperonins are composed of two different types of subunits, Cpn60α and Cpn60β, and duplication of Cpn60α and Cpn60β genes occurs in a high proportion of plants. However, the importance of multiple Cpn60α and Cpn60β genes in plants is poorly understood. In this study, we found that loss-of-function of CPNA2 (AtCpn60α2), a gene encoding the minor Cpn60α subunit in Arabidopsis thaliana, resulted in arrested embryo development at the globular stage, whereas the other AtCpn60α gene encoding the dominant Cpn60α subunit, CPNA1 (AtCpn60α1), mainly affected embryonic cotyledon development at the torpedo stage and thereafter. Further studies demonstrated that CPNA2 can form a functional chaperonin with CPNB2 (AtCpn60β2) and CPNB3 (AtCpn60β3), while the functional partners of CPNA1 are CPNB1 (AtCpn60β1) and CPNB2. We also revealed that the functional chaperonin containing CPNA2 could assist the folding of a specific substrate, KASI (β-ketoacyl-[acyl carrier protein] synthase I), and that the KASI protein level was remarkably reduced due to loss-of-function of CPNA2. Furthermore, the reduction in the KASI protein level was shown to be the possible cause for the arrest of cpna2 embryos. Our findings indicate that the two Cpn60α subunits in Arabidopsis play different roles during embryo development through forming distinct chaperonins with specific AtCpn60β to assist the folding of particular substrates, thus providing novel insights into functional divergence of Cpn60α subunits in plants.
... Genes encoding Hsfs and Hsps have been well characterized in some model plants, such as Arabidopsis and rice [12,13]. In Arabidopsis, 21,27,18,18,7, and 4 genes have been identified as Hsf, sHsp, * Correspondence: lumz@caf.ac.cn; chenjun@caf.ac.cn 1 Hsp60, Hsp70, Hsp90, and Hsp100 family members, respectively [13][14][15][16][17][18][19]. ...
... Hsp60 proteins as chaperones participate in the folding and aggregation of many proteins that are transported to organelles, such as chloroplasts and mitochondria [25,26]. Arabidopsis hsp60 mutants showed defects in chloroplast, embryo, and seedling development and also increased cell death [27,28]. Unlike other family members that are mainly expressed when the organism is subjected to environmental assaults, Hsp70s play essential functions in facilitating refolding and proteolytic degradation of abnormal proteins under both normal and stress conditions [29,30]. ...
Heat shock proteins (Hsps) are molecular chaperones that are involved in many normal cellular processes and stress responses, and heat shock factors (Hsfs) are the transcriptional activators of Hsps. Hsfs and Hsps are widely coordinated in various biological processes. Although the roles of Hsfs and Hsps in stress responses have been well characterized in Arabidopsis, their roles in perennial woody species undergoing various environmental stresses remain unclear.
Here, a comprehensive identification and analysis of Hsf and Hsp families in poplars is presented. In Populus trichocarpa, we identified 42 paralogous pairs, 66.7% resulting from a whole genome duplication. The gene structure and motif composition are relatively conserved in each subfamily. Microarray and quantitative real-time RT-PCR analyses showed that most of the Populus Hsf and Hsp genes are differentially expressed upon exposure to various stresses. A coexpression network between Populus Hsf and Hsp genes was generated based on their expression. Coordinated relationships were validated by transient overexpression and subsequent qPCR analyses.
The comprehensive analysis indicates that different sets of PtHsps are downstream of particular PtHsfs and provides a basis for functional studies aimed at revealing the roles of these families in poplar development and stress responses.
... Interestingly, cpn60β2 or cpn60β3 single mutants showed less severe phenotypes, hinting to an overlapping function of these isoforms [19] [20]. Cpn60s also appear to play a role in protecting plants during heat stress as indicated by their up-regulation during heat shock [21] [22] [23] [24] [25]. ...
... Stroma 238 ± 16.5 Albino, small growth, severely impaired plant development (A. thaliana cpn60α2 knockout schlepperless) [10] [15] [19] [20] [27] [32] ...
Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims at giving an overview of the most current insights on these plastid chaperones, their general and conserved functions but also their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given.
Copyright © 2015. Published by Elsevier B.V.
... The seventh genomic sequence seems to be a Cpn60b subunit pseudogene. Recently, schlepperless (slp) was identified as a mutation of Cpn60a in Arabidopsis (Apuya et al. 2001). This mutant exhibits defects in plastid development and also in embryo development. ...
... This mutant exhibits defects in plastid development and also in embryo development. The phenotypes of slp plants highlight the importance of Cpn60a for chloroplast development and subsequently for the proper development of the plant embryo and seedling (Apuya et al. 2001). To learn about the function of Cpn60b as molecular chaperones in planta, transgenic tobacco plants, which express antisense Cpn60b has been studied. ...
;Lesion mimic mutants develop spontaneous cell death without pathogen attack. Some of the genes defined by these mutations may function as regulators of cell death, whereas others may perturb cellular metabolism in a way that leads to cell death. To understand the molecular mechanism of cell death in lesion mimic mutants, we isolated a lesion initiation 1 (len1) mutant by a T-DNA tagging method. The len1 mutant develops lesions on its leaves and expresses systemic acquired resistance (SAR). LEN1 was identified to encode a chloroplast chaperonin 60 (Cpn60), a homologue of bacterial GroEL. The recombinant LEN1 had molecular chaperone activity for suppressing protein aggregation in vitro. Moreover, len1 plants develop accelerated cell death to heat shock stress in comparison with wild-type plants. The chlorophyll a/b binding protein (CAB) was present in len1 plants at a lower level than in the wild-type plants. These results indicate that LEN1 functions as a molecular chaperone in chloroplasts and its deletion leads to cell death in Arabidopsis.
... Transfer of newly synthesized rbcL subunits to the holoenzyme is inhibited by the addition of anti-chaperonin antibodies, support that Cpn60 plays a critical role in Rubisco assembly (Cannon et al., 1986;Milos and Roy, 1984). Functional analysis of plastid Cpn60 genes has been conducted in Arabidopsis, where a T-DNA insertion mutant in AtCpn60α1 shows an embryonic-lethal phenotype (Apuya et al., 2001). Deletion of AtCpn60β1 leads to seedling death under short-day conditions (Ishikawa et al., 2003). ...
... This outcome demonstrated that OsCpn60α1 is essential for seedling growth. Similarly, an Arabidopsis mutant lacking AtCpn60α1 has white cotyledons and fails to develop further (Apuya et al., 2001). However, the oscpn60α1 phenotypes are milder than those of that Atcpn60α1 mutant. ...
Chaperonins are involved in protein-folding. The rice genome encodes six plastid chaperonin subunits (Cpn60) - three α and three β. Our study showed that they were differentially expressed during normal plant development. Moreover, five were induced by heat stress (42°C) but not by cold (10°C). The oscpn60α1 mutant had a pale-green phenotype at the seedling stage and development ceased after the fourth leaf appeared. Transiently expressed OsCpn60α1:GFP fusion protein was localized to the chloroplast stroma. Immuno-blot analysis indicated that the level of Rubisco large subunit (rbcL) was severely reduced in the mutant while levels were unchanged for some imported proteins, e.g., stromal heat shock protein 70 (Hsp70) and chlorophyll a/b binding protein 1 (Lhcb1). This demonstrated that OsCpn60α1 is required for the folding of rbcL and that failure of that process is seedling-lethal.
... We also found that the photosynthetic complexes in the thylakoid membranes, especially the PSII core subunits, were severely disassembled (Fig. 6), again similar to the PSII defects under Mg-deficient conditions (Laing et al., 2000;Hariadi & Shabala, 2004;Yang et al., 2012;Volgusheva et al., 2017;Huang et al., 2019;Ye et al., 2019). These severe defects in the thylakoid stacking and PSII assembly in the mgr8/mgr9 mutant hindered chloroplast development, leading to defects in embryo biogenesis (Figs 4g, S7c,d), similar to previous observations of embryo-defective mutants which were also reported to result from aberrant chloroplast development (Apuya et al., 2001;Kobayashi et al., 2007;Beeler et al., 2014;Ke et al., 2017). Future work will be directed towards understanding the functional coordination of MGR8/9 and MGT-type transporters in chloroplast Mg homeostasis. ...
Magnesium (Mg²⁺) serves as a cofactor for a number of photosynthetic enzymes in the chloroplast, and is the central atom of the Chl molecule. However, little is known about the molecular mechanism of Mg²⁺ transport across the chloroplast envelope.
Here, we report the functional characterization of two transport proteins in Arabidopsis: Magnesium Release 8 (MGR8) and MGR9, of the ACDP/CNNM family, which is evolutionarily conserved across all lineages of living organisms.
Both MGR8 and MGR9 genes were expressed ubiquitously, and their encoded proteins were localized in the inner envelope of chloroplasts. Mutations of MGR8 and MGR9 together, but neither of them alone, resulted in albino ovules and chlorotic seedlings. Further analysis revealed severe defects in thylakoid biogenesis and assembly of photosynthetic complexes in the double mutant. Both MGR8 and MGR9 functionally complemented the growth of the Salmonella typhimurium mutant strain MM281, which lacks Mg²⁺ uptake capacity. The embryonic and early seedling defects of the mgr8/mgr9 double mutant were rescued by the expression of MGR9 under the embryo‐specific ABI3 promoter. The partially rescued mutant plants were hypersensitive to Mg²⁺ deficient conditions and contained less Mg²⁺ in their chloroplasts than wild‐type plants.
Taken together, we conclude that MGR8 and MGR9 serve as Mg²⁺ transporters and are responsible for chloroplast Mg²⁺ uptake.
... HSP60 is also involved in assisting proteins that help in photosynthesis such as Rubisco [99]. Studies revealed that a mutation in Chaperonin-60α gene that codes for HSP60 protein causes a defection in chloroplasts, which ultimately results in poor seedlings and embryo development in Arabidopsis plant [100]. However, deletion of this gene results in cell death [101]. ...
The demand for cotton fibres is increasing due to growing global population while its production is facing challenges from an unpredictable rise in temperature owing to rapidly changing climatic conditions. High temperature stress is a major stumbling block relative to agricultural production around the world. Therefore, the development of thermo-stable cotton cultivars is gaining popularity. Understanding the effects of heat stress on various stages of plant growth and development and its tolerance mechanism is a prerequisite for initiating cotton breeding programs to sustain lint yield without compromising its quality under high temperature stress conditions. Thus, cotton breeders should consider all possible options, such as developing superior cultivars through traditional breeding, utilizing molecular markers and transgenic technologies, or using genome editing techniques to obtain desired features. Therefore, this review article discusses the likely effects of heat stress on cotton plants, tolerance mechanisms, and possible breeding strategies.
... To investigate the significance of CPN60A-TRXL1 interaction, we analyzed the role of CPN60A in disease resistance and thermotolerance. As Arabidopsis cpn60A homozygous mutants are embryo lethal (Apuya et al., 2001;Suzuki et al., 2009), we silenced CPN60A in N. benthamiana using VIGS ( Figure 4C). NbCPN60A-silenced plants were yellowish-green with 4-foldless chlorophyll content compared to the non-silenced control (TRV2::GFP; Figures 4A and 4B). ...
Global warming and emerging plant diseases challenge agricultural food/feed production. We identify mechanism(s) regulating both plant thermotolerance and disease resistance. Using virus-induced gene silencing (VIGS)-based genetic screening, we identify a thioredoxin-like 1 (TRXL1) gene involved in plant nonhost disease resistance and thermotolerance. TRXL1 is reduced, partly degraded via proteases and proteasome, and
alters its chloroplast localization during heat stress. TRXL1 interacts with more than 400 proteins, including chaperonin CPN60A, caseinolytic protease (CLPC1), and NADP-dependent malate dehydrogenase (NADPMDH). Chaperonin 60A (CPN60A) guards TRXL1 from degradation, whereas CLPC1 degrades TRXL1 during heat stress. TRXL1 regulates NADP-MDH activity, leading to an increase in malate level and inhibition of superoxide radical formation. We show that CPN60A and NADP-MDH positively regulate nonhost resistance, and CPN60A positively and CLPC1 negatively regulate thermotolerance. This study shows an antagonistic post-translational regulation of TRXL1 by CPN60A and CLPC1 and regulation of MDH by TRXL1, leading to plant disease resistance and thermotolerance.
... Notably, expression of CPN60α was enhanced when GhSFT or GhSP expression was altered (Fig. 4), consistent with the significant impact this chaperone has for continued plant growth and development. The Arabidopsis CPN60α loss-of-function schlepperless and temperature-sensitive CPN60α2 allele show that disruptions to embryonic photosynthesis negatively impact postgerminative growth (Apuya et al., 2001;Sela et al., 2020). Collectively, these findings suggest a conserved mechanism for the determinacy status of the shoot apex to signal future source-sink relationships in developing vegetative (i.e. ...
Patterns of indeterminate and determinate growth specify plant architecture and influence crop productivity. In cotton (Gossypium hirsutum), SINGLE FLOWER TRUSS (SFT) stimulates the transition to flowering and determinate growth while its closely related antagonist SELF-PRUNING (SP) maintains meristems in indeterminate states to favor vegetative growth. Over-expressing GhSFT while simultaneously silencing GhSP produces highly determinate cotton with reduced foliage and synchronous fruiting. These findings suggest that GhSFT, GhSP, and genes in these signaling networks hold promise for enhancing "annualized" growth patterns and improving cotton productivity and management. To identify the molecular programs underlying cotton growth habits, we used comparative co-expression networks, differential gene expression, and phenotypic analyses in cotton varieties expressing altered levels of GhSFT or GhSP. Using multiple cotton and tomato datasets, we identified diverse genetic modules highly correlated with SFT or SP orthologs which shared related gene ontologies in different crop species. Notably, altering GhSFT or GhSP levels in cotton affected the expression of genes regulating meristem fate and metabolic pathways. Further phenotypic analyses of gene products involved in photosynthesis, secondary metabolism, and cell wall biosynthesis showed that early changes in GhSFT and GhSP levels profoundly impacted later development in distal tissues. Identifying the molecular underpinnings of GhSFT and GhSP activities emphasizes their broad actions in regulating cotton architecture.
... Un premier groupe renferme GroEL et mtHSP60. On positionne également dans ce groupe une HSP60 végétale connue sous le nom de RBP (Rubisco binding protein) localisée dans les chloroplastes [262,263]. Un second groupe comprend les HSP60 présents dans le cytosol (les CCT) des cellules eucaryotes et des archès qui possèdent plusieurs homologues en fonction de l'espèce. Nous allons nous intéresser ici uniquement à la protéine HSP60 du groupe 1, et dans le deuxième groupe nous présenterons quelques exemples de CCT. ...
L’inflammation est le mécanisme de base du système immunitaire. Dans le cas de pathologie inflammatoire cette inflammation persiste et devient délétère pour l’organisme. Les causes de cette persistance peuvent être variées. L’une de ces causes est la présence de molécules induisant l’inflammation. Elles peuvent être d’origine exogène comme les PAMP (Pathogen-Associated Molecular Pattern). Ce sont des molécules issues des pathogènes (LPS, peptidoglycanes, ADN CpG) capables d’activer le système immunitaire. Ces molécules peuvent également être d’origine endogène comme les DAMP (Damage Associated Molecular Pattern). Ce sont des molécules libérées par les cellules en état de danger (HMGB1, HSP60) pour prévenir et activer le système immunitaire. La présence de récepteurs (TLR2, TLR4, RAGE) capable de reconnaitre ces PAMP et DAMP est également nécessaire pour pouvoir induire une inflammation. Mes travaux explorent les mécanismes moléculaires et cellulaires des PAMP et des DAMP, dans l’installation et le maintien de l’inflammation dans le cadre des maladies inflammatoires. Pour cela mon étude se focalise sur les mécanismes de reconnaissance et d’induction de l’inflammation par les PAMP et DAMP. Nous avons ainsi mis en évidence certains mécanismes cellulaires et moléculaires dans la réponse inflammatoire liés aux DAMP et PAMP. Nous nous sommes également intéressé aux récepteurs impliqués dans ces différents mécanismes et avons même mis en évidence un potentiel nouveau récepteur CD93. Nous émettons l’hypothèse que CD93 pourrait avoir un rôle dans les pathologies inflammatoires par cette capacité à lier les DAMP et PAMP.
... Among them, the expression level of AtCpn60α1, AtCpn60β1 and AtCpn60β2 is much higher than three others. A T-DNA insertion mutant of AtCpn60α1, termed schlepperless (slp), was the firstly identified mutant of Cpn60 in Arabidopsis [28]. The slp mutant exhibited an embryonic lethality phenotype due to a defect in plastid development which affected embryo development before the heart stage. ...
The chaperonin 60 (Cpn60) protein is of great importance to plants due to its involvement in modulating the folding of numerous chloroplast protein polypeptides. In chloroplasts, Cpn60 is differentiated into two subunit types—Cpn60α and Cpn60β and the rice genome encodes three α and three β plastid chaperonin subunits. However, the functions of Cpn60 family members in rice were poorly understood. In order to investigate the molecular mechanism of OsCpn60β1, we attempted to disrupt the OsCpn60β1 gene by CRISPR/Cas9-mediated targeted mutagenesis in this study. We succeeded in the production of homozygous OsCpn60β1 knockout rice plants. The OsCpn60β1 mutant displayed a striking albino leaf phenotype and was seedling lethal. Electron microscopy observation demonstrated that chloroplasts were severely disrupted in the OsCpn60β1 mutant. In addition, OsCpn60β1 was located in the chloroplast and OsCpn60β1 is constitutively expressed in various tissues particularly in the green tissues. The label-free qualitative proteomics showed that photosynthesis-related pathways and ribosomal pathways were significantly inhibited in OsCpn60β1 mutants. These results indicate that OsCpn60β1 is essential for chloroplast development in rice.
... When large-scale genetic screens of T-DNA insertion lines for mutant phenotypes were first organized by Ken Feldmann at DuPont (Wilmington, DE) and later at the University of Arizona (Feldmann, 1991), my laboratory screened siliques from about half of the lines and identified 180 emb mutants that were subsequently analyzed in detail (Errampalli et al., 1991;Castle et al., 1993). The remaining lines were screened and specific mutants of interest characterized further by a collaborative group overseen by Robert Goldberg at UCLA (Yadegari et al., 1994;Apuya et al., 2001). Elsewhere, Martine Devic and colleagues in France began to screen the INRA collection of T-DNA lines (Devic et al., 1996;Ortega et al., 2002) and analyze emb mutants of interest (Albert et al., 1999;Lahmy et al., 2004). ...
With the emergence of high‐throughput methods in plant biology, the importance of long‐term projects characterized by incremental advances involving multiple laboratories can sometimes be overlooked. Here, I highlight my 40‐year effort to isolate and characterize the most common class of mutants encountered in Arabidopsis (Arabidopsis thaliana): those defective in embryo development. I present an updated dataset of 510 EMBRYO‐DEFECTIVE (EMB) genes identified throughout the Arabidopsis community; include important details on 2200 emb mutants and 241 pigment‐defective embryo (pde) mutants analyzed in my laboratory; provide curated datasets with key features and publication links for each EMB gene identified; revisit past estimates of 500–1000 total EMB genes in Arabidopsis; document 83 double mutant combinations reported to disrupt embryo development; emphasize the importance of following established nomenclature guidelines and acknowledging allele history in research publications; and consider how best to extend community‐based curation and screening efforts to approach saturation for this diverse class of mutants in the future. Continued advances in identifying EMB genes and characterizing their loss‐of‐function mutant alleles are needed to understand genotype‐to‐phenotype relationships in Arabidopsis on a broad scale, and to document the contributions of large numbers of essential genes to plant growth and development.
... Chaperonin CPN60-2, a mitochondrial chaperonin HSP60 that functions in an ATP-dependent manner, acts in the import and assembly of mitochondrial proteins (Wang et al. 2004). Under abiotic stress, CPN60 promotes refolding and assembly of misfolded or unfolded proteins in the mitochondrial matrix and the proper development of chloroplasts and subsequently embryo and seedling growth (Apuya et al. 2001). ...
Proteome analysis of embryo and endosperm of Givotia moluccana, a promising angiosperm tree species was carried out using two-dimensional electrophoresis combined with matrix-assisted laser desorption/ionization time of flight to identify differentially abundant proteins and their possible functions. The study revealed the presence of 492 protein spots in the embryo and 432 in the endosperm, and the relative abundance of 44 spots varied significantly between the two. Of the 21 functionally characterized protein spots, 14.3% were involved in metabolism and energy, 28.6% in protein destination and storage, 14.3% in defense and stress and 19% in cell growth and division. The embryo-specific protein ATP-dependent zinc metalloprotease FtsH is predicted to be involved in chloroplast biogenesis and the endosperm-specific protein hydroxyacyl-ACP dehydratase in fatty acid synthesis. These results suggested that the seeds have proteins required for germination and for stress responses.
... Cpn60 proteins are considered critical in the functioning of chloroplasts and various developmental processes of plants. Atcpn60α1 mutant referred to as schlepperless (slp) show general defects in embryo development and plastid function [13]. T-DNA insertion mutant of Atcpn60α2 show the arrested embryo development phenotype at the globular stage [14]. ...
Chloroplastic Cpn60 proteins are type I chaperonins comprising of Cpn60α and Cpn60β subunits. Arabidopsis genome contains six entries in Cpn60 family, out of which two are for Cpn60α subunit and four for Cpn60β subunit. We noted that the cpn60β4 knockout mutant plants (T-DNA insertion salk_064887 line) differed from the wild type Col-0 plants in the developmental programming. cpn60β4 mutant plants showed early seed germination. Radical emergence, hypocotyl emergence and cotyledons opening were faster in cpn60β4 mutant plants than WT. Importantly, cpn60β4 mutant plants showed early-flowering phenotype. The number of flowers and siliques as well as weight of the seeds were higher in cpn60β4 mutant plants as compared to Col-0 plants. These effects were reverted to wild type like growth and developmental patterns when genomic fragment of Arabidopsis encompassing Cpn60β4 gene was complemented in the mutant background. The overexpression of Cpn60β4 gene using CaMV35 promoter in wild type background (OE-Cpn60β4) delayed the floral transition as against wild type plants. The plastid division were affected in cpn60β4 mutant plants compared to Col-0. The results of this study suggest that Cpn60β4 plays important role(s) in chloroplast development and is a key factor in plant growth, development and flowering in Arabidopsis.
... Hsp60 family proteins bind to different types of proteins after their transcription and prior to folding to avoid their aggregation (Parsell and Lindquist, 1993). Arabidopsis chlCpn60a mutated species was shown to exhibit defects in chloroplast development and subsequently in the plant development (Apuya et al., 2001). Antisense Cpn60b in transgenic tobacco plants showed drastic phenotypic alterations, including slow growth, delayed flowering, stunting, and leaf chlorosis (Zabaleta et al., 1994). ...
The perception of abiotic stress and signal transduction to switch on adaptive innate responses are crucial steps in plant persistence under adverse environmental conditions. The ability of the plants to respond against different stresses by maintaining the protein in the functional active form is of high importance and essential for the cell survival. This is possible with the functional activity of heat shock proteins (Hsps), which plays a role in maintaining the cellular homeostasis, and preserves the non-native protein in a competent state for further remodeling as well as in signal transduction. Physiological, molecular and functional studies of Hsps have started much later in plants compared to animals. Hsps mediates the stress signals either directly or indirectly, and plays an important role in maintaining the homeostasis of plant cells under adverse stress conditions. The immense research has been made in understanding the complex mechanisms of the Hsps including Hsp70/90, ATPase-coupled conformational modifications and interactions with co-chaperones. In addition this, several signaling proteins, i.e., mitogen activated protein kinases (MAP kinases), Ca2+ regulated proteins, heat shock transcription factors (Hsfs), reactive oxygen species (ROS) are seen to be interacting with Hsps. Since there are various stress perception and signaling pathways, some of which are very specific and others are crosstalk at various steps of the signaling pathways. In this study, we discuss the signal transduction of different Hsps, and their role in different signaling mechanisms.
... Mutant plants defective in chloroplast chaperones display pigmentdefective phenotypes and contain aberrant chloroplasts. For example, Arabidopsis thaliana mutants in Cpn60α display albino phenotypes with impaired growth (Apuya et al., 2001), and point mutations in the Hsp90-5 gene also generate albino seedlings while null mutants are embryo-lethal (Cao et al., 2003;Inoue et al., 2013). The knockout of the major isoform of Hps93/ClpC causes chloroplast development and protein import defects (Constan et al., 2004;Kovacheva et al., 2005Kovacheva et al., , 2007. ...
The function of proteins depends on specific partners that regulate protein folding, degradation and protein-protein interactions, such partners are the chaperones and cochaperones. In chloroplasts, proteins belonging to several families of chaperones have been identified: chaperonins (Cpn60s), Hsp90s (Hsp90-5/Hsp90C), Hsp100s (Hsp93/ClpC) and Hsp70s (cpHsc70s). Several lines of evidence have demonstrated that cpHsc70 chaperones are involved in molecular processes like protein import, protein folding and oligomer formation that impact important physiological aspects in plants such as thermotolerance and thylakoid biogenesis. Despite the vast amount of data existing around the function of cpHcp70s chaperones, very little attention has been paid to the roles of DnaJ and GrpE cochaperones in the chloroplast. In this study, we performed a phylogenetic analysis of the chloroplastic GrpE (CGE) proteins from 71 species. Based on their phylogenetic relationships and on a motif enrichment analysis, we propose a classification system for land plants’ CGEs, which include two independent groups with specific primary structure traits. Furthermore, using in vivo assays we determined that the two CGEs from A. thaliana (AtCGEs) complement the mutant phenotype displayed by a knockout E. coli strain defective in the bacterial grpE gene. Moreover, we determined in planta that the two AtCGEs are bona fide chloroplastic proteins, which form the essential homodimers needed to establish direct physical interactions with the cpHsc70-1 chaperone. Finally, we found evidence suggesting that AtCGE1 is involved in specific physiological phenomena in A. thaliana, such as the chloroplastic response to heat stress, and the correct oligomerization of the photosynthesis-related LHCII complex.
... The first group includes GroEL and mtHSP60. A vegetal HSP60 known as RBP (for Rubisco binding protein) and localized in chloroplast is part this first group (Apuya et al. 2001;Balczun et al. 2006). A second group includes the cytosolic HSP60 (CCTs) of eukaryotic cells. ...
The exposure of cells or organisms to high temperature leads to the release of alert molecules such as Heat Shock Protein: the HSP. This protein family has been initially described in Drosophila. The cellular response to a heat shock involving HSP is conserved across species, from bacteria to humans and including plants. Other stresses, such as ischemia, heavy metal poisoning, nutrient deprivation, irradiations, infections, oxidative stress and inflammation, can also induce the HSP expression. HSP form a large family of proteins which are classified according to their molecular weight: HSP100, HSP90, HSP70, HSP60, HSP40, HSP from 20 to 30 kDa and HSP10. HSP60 has different functions depending on its localization. Intracellular HSP60 can be found in the cytosol, mitochondria and the chloroplast. Therein, it has a chaperone activity by assisting the proteins folding. On the cell surface or in the extracellular medium, HSP60 acts as a danger signaling molecule. Thus, stressed or damaged cells can stimulate the immune system. Indeed, this extracellular HSP60 are involved in several inflammatory pathologies.
... Mutation in Arabidopsis plastid chaperonin protein Cpn60 is associated with defect in plastid growth and, consequently reduction in embryo and seedling development (Suzuki et al. 2009). Cpn60b antisense lines of transgenic tobacco plants showed strong phenotypic changes like delayed growth pattern, deferred flowering, chlorosis and stunting of leaves (Apuya et al. 2001). ...
Plants counter an array of stresses by generation of a group of stress-related proteins, often referred to as the chaperones. Expression of these chaperones is induced in response to almost all kinds of stress. However, there are numerous evidences showing that these chaperones are vital for survival even under normal physiological conditions. They act as key modulators in physiological stress response and acquired tolerance. Research carried out over the past several years has clearly established that these chaperones are involved in diverse cellular functions such as folding, accumulation, translocation and degradation of proteins. Thus, these evolutionary conserved proteins affect a broad array of cellular processes. Gaining knowledge about this cellular chaperone machinery is of immense significance to understand the mechanism of interdependent stress-related cross talk in plants and ultimately, for the crop improvement programs.
... These mutants germinate poorly if at all, are albino, grow extremely slowly, and display aberrant leaf phenotypes reminiscent of the cp33a mutant. A similar phenotype was also observed when mutants of the alpha subunit of chaperonin 60 were grown on MS medium (Apuya et al., 2001). Chaperonin 60 is presumed to be involved in chloroplast protein folding, and thus affects the functioning of numerous proteins and pathways. ...
Chloroplast RNA metabolism depends on a multitude of nuclear-encoded RNA binding proteins (RBPs). Most known chloroplast RBPs address specific RNA targets and RNA-processing functions. However, members of the small chloroplast ribonucleoprotein family (cpRNPs) play a global role in processing and stabilizing chloroplast RNAs. Here, we show that the cpRNP, CP33A, localizes to a distinct sub-chloroplastic domain and is essential for chloroplast development. The loss of CP33A yields albino seedlings that exhibit aberrant leaf development and can only survive in the presence of an external carbon source. Genome-wide RNA association studies demonstrate that CP33A associates with all chloroplast mRNAs. For a given transcript, quantification of CP33A-bound versus free RNAs demonstrates that CP33A associates with the majority of most mRNAs analyzed. Our results further show that CP33A is required for the accumulation of a number of tested mRNAs, and is particularly relevant for unspliced and unprocessed precursor mRNAs. Finally, CP33A fails to associate with polysomes or strongly co-precipitate with ribosomal RNA, suggesting that it defines a ribodomain that is separate from the chloroplast translation machinery. Collectively, these findings suggest that CP33A contributes to globally essential RNA processes in the chloroplasts of higher plants. This article is protected by copyright. All rights reserved.
... CoIP-MS analysis has shown that they are also part of GUN1-containing protein complexes. mutant termed schlepperless (slp), causes retardation of embryo development before the heart stage and an albino seedling phenotype, indicating that ptCpn60α1 is essential for chloroplast biogenesis (Apuya et al., 2001). Conversely, plants devoid of ptCpn60β1, also known as lesion initiation 1 (len1), have leaves with wrinkled and irregular surfaces and undergo localized, spontaneous cell death in the absence of pathogen attack, i.e., lesion formation, under short-day conditions (Ishikawa et al., 2003). ...
The GENOMES UNCOUPLED 1 (GUN1) gene has been reported to encode a chloroplast-localized pentatricopeptide-repeat protein, which acts to integrate multiple indicators of plastid developmental stage and altered plastid function, as part of chloroplast-to-nucleus retrograde communication. However, the molecular mechanisms underlying signal integration by GUN1 have remained elusive, up until the recent identification of a set of GUN1-interacting proteins, by co-immunoprecipitation and mass-spectrometric analyses, as well as protein–protein interaction assays. Here, we review the molecular functions of the different GUN1 partners and propose a major role for GUN1 as coordinator of chloroplast translation, protein import, and protein degradation. This regulatory role is implemented through proteins that, in most cases, are part of multimeric protein complexes and whose precise functions vary depending on their association states. Within this framework, GUN1 may act as a platform to promote specific functions by bringing the interacting enzymes into close proximity with their substrates, or may inhibit processes by sequestering particular pools of specific interactors. Furthermore, the interactions of GUN1 with enzymes of the tetrapyrrole biosynthesis (TPB) pathway support the involvement of tetrapyrroles as signaling molecules in retrograde communication.
... A. thaliana cpn60α2 single and cpn60β2/cpn60β3 double mutants show impaired plastid division, small growth and an albino phenotype. Interestingly, cpn60β2 or cpn60β3 single mutants showed less severe phenotypes, hinting to an overlapping function of these isoforms [19,20]. Cpn60s also appear to play a role in protecting plants during heat stress as indicated by their up-regulation during heat shock [21][22][23][24][25]. ...
Molecular chaperones play essential roles in a wide variety of cellular processes, from de-novo protein folding to protein disaggregation under stress conditions, unfolding and re-folding of misfolded proteins, protein degradation, protein transport and proteome remodeling during development. Almost all cell compartments contain chaperone activity to some extent, hence it is not surprising that a large number of chaperones also play essential roles in the plastid compartment. Here, the focus of chaperone activity is on protein targeting (protein import and assembly of complexes in target membranes) as well as protection from specific chloroplast-derived stresses. Moreover, chaperones play important roles in de-novo folding of plastid-encoded proteins, in the folding of soluble proteins after import and processing of the transit peptides, and in protein degradation. The four major groups of molecular chaperones, the chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families of chaperones, are all present in plastids but many cofactors and co-chaperones have not yet been identified. Although chaperone function is generally conserved, it seems that plastid-localized chaperones have evolved some specific functions and mechanisms. Current research on plastid-localized chaperones focuses therefore on the specificities of chaperone function in the context of their plastid environment and requirements.
... It is reported that the Cpn60α deletion is lethal to plants [57][58][59]. Here, we found that CPN60α is highly cooperative with CPN60β1 to form functional oligomers potentially by regulating the allosteric movement of CPN60 oligomers (Figs. 6 and 7). ...
BACKGROUND:
Chloroplast chaperonin, consisting of multiple subunits, mediates folding of the highly abundant protein Rubisco with the assistance of co-chaperonins. ATP hydrolysis drives the chaperonin allosteric cycle to assist substrate folding and promotes disassembly of chloroplast chaperonin. The ways in which the subunits cooperate during this cycle remain unclear.
RESULTS:
Here, we report the first crystal structure of Chlamydomonas chloroplast chaperonin homo-oligomer (CPN60β1) at 3.8 Å, which shares structural topology with typical type I chaperonins but with looser compaction, and possesses a larger central cavity, less contact sites and an enlarged ATP binding pocket compared to GroEL. The overall structure of Cpn60 resembles the GroEL allosteric intermediate state. Moreover, two amino acid (aa) residues (G153, G154) conserved among Cpn60s are involved in ATPase activity regulated by co-chaperonins. Domain swapping analysis revealed that the monomeric state of CPN60α is controlled by its equatorial domain. Furthermore, the C-terminal segment (aa 484-547) of CPN60β influenced oligomer disassembly and allosteric rearrangement driven by ATP hydrolysis. The entire equatorial domain and at least one part of the intermediate domain from CPN60α are indispensable for functional cooperation with CPN60β1, and this functional cooperation is strictly dependent on a conserved aa residue (E461) in the CPN60α subunit.
CONCLUSIONS:
The first crystal structure of Chlamydomonas chloroplast chaperonin homo-oligomer (CPN60β1) is reported. The equatorial domain maintained the monomeric state of CPN60α and the C-terminus of CPN60β affected oligomer disassembly driven by ATP. The cooperative roles of CPN60 subunits were also established.
... Ch-cpn60s from various higher plants consist of stoichiometric amounts of two divergent subunits, a and b [22], which reside in the same tetradecamer [23,24]. A ch-cpn60a mutant showed a defect in chloroplast/embryo development [25], while a ch-cpn60b developed lesions and is less thermotolerant than the wild type [26]. Recently, it was shown that both ch-cpn60a and ch-cpn60b are required for plastid division in Arabidopsis [27]. ...
... It is generally agreed that they are important in assisting plastid proteins such as Rubisco [61,85]. It has been reported that a mutated species of Arabidopsis chloroplast Cpn60a exhibits defects in chloroplast development and, subsequently, in the proper development of the plant embryo and seedling [86]. Antisense Cpn60b transgenic tobacco plants showed drastic phenotypic alterations, including slow growth, delayed flowering, stunting and leaf chlorosis [87]. ...
Heat stress has significant effect on protein metabolism, including degradation of proteins, inhibition of protein accumulation and induction of certain protein synthesis. It also poses a serious damage to the growth and development of the plant. The ability of the plants to respond to this stress by maintaining protein in their functional conformation as well as preventing the accumulation of non-native proteins are highly important for the cell survival. Heat shock proteins are involved in signaling, translation, host-defence mechanisms, carbohydrate metabolism and amino acid metabolism. In fact, these proteins are now understood to mediate signaling, translation, host-defence mechanisms, carbohydrate metabolism and amino acid metabolism by playing a significant function in controlling the genome and ultimately features that are obvious. Several reviews have reported the tolerance of plants to different abiotic stresses. The topic of enhancing protection mechanisms (including HSPs) to induce heat resistance is very interesting and research in this area has many repercussions for the understanding of heat stress response. However, this review reports Heat Shock Proteins (HSPs) and their function, research progress on the association of HSPs with plant tolerance to heat stress as well as the response of the HSPs under heat stress as an adaptive defence mechanism.
... CSLA is required for synthesis of cell wall polysaccharide mannan which serves as storage reserve during plant growth and development [55]. CPN60 was essential for development of embryo and seedling [56]. ...
Background
Sinopodophyllum hexandrum is an endangered medicinal herb, which is commonly present in elevations ranging between 2,400–4,500 m and is sensitive to temperature. Medicinal property of the species is attributed to the presence of podophyllotoxin in the rhizome tissue. The present work analyzed transcriptome of rhizome tissue of S. hexandrum exposed to 15°C and 25°C to understand the temperature mediated molecular responses including those associated with podophyllotoxin biosynthesis.
Results
Deep sequencing of transcriptome with an average coverage of 88.34X yielded 60,089 assembled transcript sequences representing 20,387 unique genes having homology to known genes. Fragments per kilobase of exon per million fragments mapped (FPKM) based expression analysis revealed genes related to growth and development were over-expressed at 15°C, whereas genes involved in stress response were over-expressed at 25°C. There was a decreasing trend of podophyllotoxin accumulation at 25°C; data was well supported by the expression of corresponding genes of the pathway. FPKM data was validated by quantitative real-time polymerase chain reaction data using a total of thirty four genes and a positive correlation between the two platforms of gene expression was obtained. Also, detailed analyses yielded cytochrome P450s, methyltransferases and glycosyltransferases which could be the potential candidate hitherto unidentified genes of podophyllotoxin biosynthesis pathway.
Conclusions
The present work revealed temperature responsive transcriptome of S. hexandrum on Illumina platform. Data suggested expression of genes for growth and development and podophyllotoxin biosynthesis at 15°C, and prevalence of those associated with stress response at 25°C.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-871) contains supplementary material, which is available to authorized users.
... At present, the great majority of the many protein phosphatases identified in this study could not be assigned a putative role. The Triticeae stigma is a non-green tissue enclosed within the floret and hence it will be interesting to investigate the role of a number Arabidopsis orthologues associated with photomorphogenesis including different subunits of the CONSTITUTIVE MORPHOGENESIS9 (COP9) SIGNALOSOME (CSN), which although involved in photomorphogenesis also regulates many different cellular processes by regulating the activity of CULLIN-RING E3 ubiquitin ligases (Stratmann and Gusmaroli, 2011), TF Z-BOX BINDING FACTOR3 (ZBF3)/CALMODULIN7 (CAM7) that integrates light signals with seedling development (Gangappa et al., 2013), and THYLAKOID FORMATION1 (THF1) whose rice orthologue NYC4 was recently shown to play a role in chlorophyll-protein complex degradation during leaf senescence (Yamatani et al., 2013), as well as SCHLEPPERLESS (SLP)/CHAPERONIN60 (CPN60) and chaperones part of the chloroplast multisubunit Clp protease such as DE-REGULATED CAO ACCUMULATION1 (DCA1)/ClpC1 and ALBINO OR PALE GREEN6 (APG6)/ClpB3, which are all involved in chloroplast development (Apuya et al., 2001;Sjögren et al., 2004;Myouga et al., 2006). ...
To our knowledge, this study represents the first high-throughput characterization of a stigma proteome in the Triticeae.
A total of 2184 triticale mature stigma proteins were identified using three different gel-based approaches combined with
mass spectrometry. The great majority of these proteins are described in a Triticeae stigma for the first time. These results
revealed many proteins likely to play important roles in stigma development and pollen–stigma interactions, as well as protection
against biotic and abiotic stresses. Quantitative comparison of the triticale stigma transcriptome and proteome showed poor
correlation, highlighting the importance of having both types of analysis. This work makes a significant contribution towards
the elucidation of the Triticeae stigma proteome and provides novel insights into its role in stigma development and function.
... 1A, the 10 DAF flax embryo is in the growth phase that marks out the beginning of maturation. This timing is confirmed by these five genes known for their involvement during this phase in embryo or in maternal tissues (Jofuku et al., 1994; Souer et al., 1996; Aida et al., 1997; Apuya et al., 2001; Maes et al., 2001; Raz et al., 2001; Takada et al., 2001; Wan et al., 2002; Vroemen et al., 2003; Dong et al., 2004; Weir et al., 2004) and for which expression is specifically found in 10 DAF flax seeds (Fig. 4). Many ESTs in the database share similarities with legumin 11S (23 ESTs), vicillin 7S (four ESTs), and albumin 2S (eight ESTs) confirming the presence of these three kinds of storage proteins in flax seed as previously shown (Marcone et al., 1994Marcone et al., , 1998). ...
... Many chloroplast-development mutants of Arabidopsis can grow well when supplied with sucrose as a carbon source (Koch, 1996;Chi et al., 2008;Yu et al., 2009); further, they may show abnormal embryo development (Uwer et al., 1998;Apuya et al., 2001;Kobayashi et al., 2007). To investigate whether SG1 is involved in chloroplast development, sg1 seedlings were grown on MS medium without or with 2% sucrose, and the sg1 embryogenesis of sg1 homozygotes was observed. ...
A new gene, SG1, was identified in a slow-greening mutant (sg1) isolated from an ethylmethanesulphonate-mutagenized population of Arabidopsis thaliana. The newly formed leaves of sg1 were initially albino, but gradually became pale green. After 3 weeks, the leaves of the mutant were as green as those of
the wild-type plants. Transmission electron microscopic observations revealed that the mutant displayed delayed proplastid
to chloroplast transition. The results of map-based cloning showed that SG1 encodes a chloroplast-localized tetratricopeptide repeat-containing protein. Quantitative real-time reverse transcription–PCR
data demonstrated the presence of SG1 gene expression in all tissues, particularly young green tissues. The sg1 mutation disrupted the expression levels of several genes associated with chloroplast development, photosynthesis, and chlorophyll
biosynthesis. The results of genetic analysis indicated that gun1 and gun4 partially restored the expression patterns of the previously detected chloroplast-associated genes, thereby ameliorating
the slow-greening phenotype of sg1. Taken together, the results suggest that the newly identified protein, SG1, is required for chloroplast development in Arabidopsis.
... In Arabidopsis, both Cpn60␣ and Cpn60 are required for proper plastid division [22]. Disruption of Cpn60␣ proteins results in abnormal development of embryos and plastids and cell death when cultured at 50 mol photons m −2 s −1 condition, which highlights the critical role of Cpn60 in maintaining plastid function [23,50,51]. However, the mechanistic basis of the chloroplast division defect caused by Cpn60 disruption remains unclear. ...
... In contrast, some albino mutants like emb1303, which lack the ability to produce pigments, remain morphologically normal and are able to germinate (Budziszewski et al. 2001;Huang et al. 2009). This phenotype is different from that of mutants with aberrant plastid development like schlepperless, which shows retarded growth (Apuya et al. 2001). The Cdf1 phenotype resembles those of the Arabidopsis emb mutants, defective in plastid protein, such as Rasp-berry3 (rsy3) and emb506, which also show developmental arrest at the globular stage (Albert et al. 1999;Despres et al. 2001). ...
Arabidopsis cell growth defect factor-1 (Cdf1 in yeast, At5g23040) was originally isolated as a cell growth suppressor of yeast from genetic screening. To investigate the in vivo role of Cdf1 in plants, a T-DNA insertion line was analyzed. A homozygous T-DNA insertion mutant (cdf1/cdf1) was embryo lethal and showed arrested embryogenesis at the globular stage. The Cdf1 protein, when fused with green fluorescent protein, was localized to the plastid in stomatal guard cells and mesophyll cells. A promoter-β-glucuronidase assay found expression of Cdf1 in the early heart stage of embryogenesis, suggesting that Cdf1 was essential for Arabidopsis embryogenesis during the transition of the embryo from the globular to heart stage.
... Hsp70 also acts as negative repressor of heat-shock factors (HSFs), the TFs regulating HSP transcription (Morimoto 1998; Kim and Schoffl 2002). Arabidopsis Hsp60 mutants show defects in chloroplast , embryo and seedling development and also increased cell death (Apuya et al. 2001; Ishikawa et al. 2003). Hsp90 play an important role in signal transduction and in assembly and maintenance of 26S proteasome (Wang et al. 2004; Imai et al. 2003). ...
Plants respond to stress conditions through early stress-response factors (ESRF), which serve the function of stress sensing and/or signal transduction. These mainly comprise qualitative and/or quantitative flux in the redox molecules, calcium ions (Ca2+), phosphatidic acid, hexose sugars and phytohormones. The role of resident proteins such as phytohormone receptors and G-proteins as first messengers under stress is well established. Yet, within the modern omics context, most of the stress response at the protein level is injudiciously attributed to substantial up- or down-regulation of expression measured at the RNA or protein level. Proteins such as kinases and transcription factors (TFs) that exhibit cascade effects are primary candidates for studies in plant stress tolerance. However, resident-protein post-translational modification (PTM), specifically in response to particular conditions such as stress, is a candidate for immediate and potent ‘quick reaction force’ (QRF) kind of effects. Stress-mediated SUMOylation of TFs and other proteins have been observed. SUMOylation can change the rate of activity, function or location of the modified protein. Early SUMOylation of resident proteins can act in the stress signal transduction or in adaptive response. Here, we consider brief background information on ESRFs to establish the crosstalk between these factors that impinge on PTMs. We then illustrate connections of protein SUMOylation to phytohormones and TFs. Finally, we present results of an in silico analysis of rice Receptor-Like Kinases, heat-shock and calcium-binding proteins to identify members of these gene families, whose basal expression under drought but potential SUMOylation presents them as QRF candidates for roles in stress signaling/response.
... For instance, stromal Hsp60 and Hsp93 (belonging to the Hsp100 family) were identified as components of the chloroplast import machinery that targets specific proteins in the chloroplasts Blobel 1996, Nielsen et al. 1997). Genetic studies also demonstrated that SLP (encodes stromal chaperonin-60α) and the stromal Hsp70 and Hsp93 proteins are essential for chloroplast development or function (Apuya et al. 2001, Constan et al. 2004, Latijnhouwers et al. 2010). cpHsc70-1 and cpHsc70-2 are the two stromal Hsp70s in Arabidopsis. ...
The Arabidopsis genome contains seven members of Hsp90. Mutations in plastid AtHsp90.5 were reported to cause defects in chloroplast development and embryogenesis. However, the exact function of plastid AtHsp90.5 has not yet been defined. In this study, albino seedlings were found among AtHsp90.5 transformed Arabidopsis, which were revealed to be AtHsp90.5 co-suppressed plants. The accumulation of photosynthetic super-complexes in the albinos was decreased, and expression of genes involved in photosynthesis was significantly down-regulated. AtHsp90.5 T-DNA insertion mutants were embryo-lethal with embryo arrested at the heart stage. Further investigation showed AtHsp90.5 expression was up-regulated in the siliques at four days post anthesis (DPA). Confocal microscopy proved AtHsp90.5 was located in the chloroplasts. Plastid development in the AtHsp90.5 mutants and co-suppressed plants was seriously impaired, and few thylakoid membranes were observed, indicating the involvement of AtHsp90.5 in chloroplast biogenesis. AtHsp90.5 was found to interact with vesicle-inducing protein in plastids 1 (VIPP1) by bimolecular fluorescence complementation system. The ratio between VIPP1 oligomers and monomers in AtHsp90.5 co-suppressed plants drastically shifted toward the oligomeric state. Our study confirmed that AtHsp90.5 is vital for chloroplast biogenesis and embryogenesis. Further evidence also suggested that AtHsp90.5 may help in the disassembly of VIPP1 for thylakoid membrane formation and/or maintenance.
... Protein degradation enzymes, such as cysteine proteases were highly up regulated in E pericarp whereas cysteine protease inhibitors were down regulated (Table 2). There was also a 2-fold increase in the levels of chaperonins596061 and protein disulfide isomerases (PDI) [62] and the aspartic proteinase oryzasin-1 [63] (Tables 1, 2). ...
... Similarly, the loss of plastidic lysophosphatidic acid acyltransferase activity also causes embryo lethality (Yu et al., 2004). In addition, mutations that affect other aspects of plastid development cause the arrest of embryogenesis at various stages (Albert et al., 1999; Apuya et al., 2001; Apuya et al., 2002; Bellaoui et al., 2003; Asano et al., 2004; Garcion et al., 2006; Huang et al., 2009) (see also http://www.seedgenes.org). Therefore, proper establishment and maintenance of the plastid activity play a vital role in embryogenesis. ...
... Cell divisions proceed apparently without any significant aberrations and the mutant embryos reach their full size. This phenotype is different from mutants with aberrant plastid development like schlepperless, which show retarded growth (Apuya et al., 2001). In contrast, albino mutants, which lack the ability to produce pigments, remain morphologically normal and are able to germinate (Budziszewski et al., 2001). ...
... These proteins show different expression profiles with Cpn60α1, Cpn60β1 and Cpn60β2 being the dominant subunits and the others being expressed at low levels (Peltier et al., 2006;Weiss et al., 2009). Inactivation of the cpn60α1 gene is lethal (Apuya et al., 2001) as is a cpn60β1β2 double mutant (Suzuki et al., 2009). It can be concluded that these gene products are involved in the generation of the housekeeping chaperonin function in the chloroplast. ...
Chaperonin 60 is the prototypic molecular chaperone, an essential protein in eukaryotes and prokaryotes, whose sequence conservation provides an excellent basis for phylogenetic analysis. Escherichia coli chaperonin 60 (GroEL), the prototype of this family of proteins, has an established oligomeric-structure-based folding mechanism and a defined population of folding partners. However, there is a growing number of examples of chaperonin 60 proteins whose crystal structures and oligomeric composition are at variance with GroEL, suggesting that additional complexities in the protein-folding function of this protein should be expected. In addition, many organisms have multiple chaperonin 60 proteins, some of which have lost their protein-folding ability. It is emerging that this highly conserved protein has evolved a bewildering variety of additional biological functions - known as moonlighting functions - both within the cell and in the extracellular milieu. Indeed, in some organisms, it is these moonlighting functions that have been left after the loss of the protein-folding activity. This highlights the major paradox in the biology of chaperonin 60. This article reviews the relationship between the folding and non-folding (moonlighting) activities of the chaperonin 60 family and discusses current knowledge on their molecular evolution focusing on protein domains involved in the non-folding chaperonin functions in an attempt to understand the emerging biology of this evolutionarily ancient protein family.
... urophylla seedlings) and for the RT3 (seedlings root) libraries (Figure 3), which represent tissues in development, suggesting that Hsp60 members are important to Eucalyptus developmental stages (see discussion below). This is compatible with the fact that mutated species of Arabidopsis chloroplasts Cpn60a exhibit defects in embryo and seedlings development (Apuya et al., 2001). The expression of Hsp60 genes in leaves colonized by the Thyrinteina grub for seven days is also worthy of note (LV3 library), and may indicate a differential expression of Hsp60 under biotic stress conditions. ...
Expressed Sequence Tags (ESTs) sequencing provides reliable and useful information concerning gene expression patterns in the genomic context. Our group used bioinformatics to identify and annotate 5'EST-contigs belonging to the molecular chaperones within the Eucalyptus Genome Sequencing Project Consortium (FORESTs) database. We found that 1,959 5'EST-contigs, or approximately 1.6% of the total 5'EST-contigs, encoded chaperones, emphasizing their biological importance. About 55% of the chaperones that we found were Hsp70 chaperones and its co-chaperones, 18% were Hsp90 chaperones, 15% were Hsp60 and its co-chaperone, 8% were Hsp100 chaperones, and 4% were Small Hsps. We also investigated the digital expression profile of the chaperone genes to gain information on gene expression levels in the different libraries and we found that molecular chaperones may have differential expression. The results discussed here give important hints about the role of chaperones in Eucalyptus cells.
Chloroplasts have important roles in photosynthesis, stress sensing and retrograde signaling. However, the relationship between chloroplast peptide chain release factor and ROS-mediated plant growth is still unclear. In the present study, we obtained a loss-of-function mutant dig8 by EMS mutation. The dig8 mutant has few lateral roots and a pale green leaf phenotype. By map-based cloning, the DIG8 gene was located on AT3G62910, with a point mutation leading to amino acid substitution in functional release factor domain. Using yeast-two-hybrid and BiFC, we confirmed DIG8 protein was characterized locating in chloroplast by co-localization with plastid marker and interacting with ribosome-related proteins. Through observing by transmission electron microscopy, quantifying ROS content and measuring the transport efficiency of plasmodesmata in dig8 mutant, we found that abnormal thylakoid stack formation and chloroplast dysfunction in the dig8 mutant caused increased ROS activity leading to callose deposition and lower PD permeability. A local sugar supplement partially alleviated the growth retardation phenotype of the mutant. These findings shed light on chloroplast peptide chain release factor-affected plant growth by ROS stress.
In nature, plants are challenged by various kinds of biotic and abiotic stresses. The different kinds of abiotic stress mainly include intense light, high temperature, drought, salinity, chilling, and heavy metals, which can occur individually or in combinations of two or more. These extreme environmental conditions lead to cellular protein denaturation, dysfunction, and aggregation. Preventing protein degradation and retaining its functional conformations are essential for cellular integrity. Heat shock proteins (HSPs), often called molecular chaperones, play an important role in sustaining the integrity of protein by preventing it from being degraded, the folding of misfolded proteins and disaggregation of aggregated misfolded proteins, as well as the degradation of non‐native and unwanted proteins. When plants are challenged by abiotic stresses, the expression of some HSP‐encoding genes is induced, which triggers a cellular network to combat these stresses. Some HSPs are transcribed constitutively in all cells or specified cells under specific developmental control. Their role in establishing cellular homeostasis under stress conditions by protecting protein degradation and retaining its function is indispensable. Based on approximate molecular weight, HSPs are classified into five major classes: (i) small heat shock proteins; (ii) HSP60; (iii) HSP70; (iv) HSP90; and (v) HSP100. Additionally, HSP40 serves as co‐chaperone, assisting the function of HSP70. HSP molecular chaperones are ubiquitously present in almost all living organisms from bacteria and animals to higher plants. The number of different classes of molecular chaperones varies significantly from organism to organism. They are located in various cellular organelles such as in the cytoplasm, nucleus, chloroplast, mitochondria, and endoplasmic reticulum. Here, the structure, function, and significance of different classes of molecular chaperones during the abiotic stress response in higher plants as well as their crosstalk with molecules of other stress response pathways have been summarized.
The astonishing increase in temperature presents an alarming threat to crop production worldwide. As evident by huge yield decline in various crops, the escalating drastic impacts of heat stress (HS) are putting global food production as well as nutritional security at high risk. HS is a major abiotic stress that influences plant morphology, physiology, reproduction, and productivity worldwide. The physiological and molecular responses to HS are dynamic research areas, and molecular techniques are being adopted for producing heat tolerant crop plants. In this article, we reviewed recent findings, impacts, adoption, and tolerance at the cellular, organellar, and whole plant level and reported several approaches that are used to improve HS tolerance in crop plants. Omics approaches unravel various mechanisms underlying thermotolerance, which is imperative to understand the processes of molecular responses toward HS. Our review about physiological and molecular mechanisms may enlighten ways to develop thermo-tolerant cultivars and to produce crop plants that are agriculturally important in adverse climatic conditions.
Chaperonins are large, essential, oligomers that facilitate protein folding in chloroplasts, mitochondria, and eubacteria. Plant chloroplast chaperonins are comprised of multiple homologous subunits that exhibit unique properties. We previously characterized homogeneous, reconstituted, chloroplast-chaperonin oligomers in vitro, each composed of one of three highly homologous beta subunits from A. thaliana. In the current work, we describe alpha-type subunits from the same species and investigate their interaction with β subtypes. Neither alpha subunit was capable of forming higher-order oligomers on its own. When combined with β subunits in the presence of Mg-ATP, only the α2 subunit was able to form stable functional hetero-oligomers, which were capable of refolding denatured protein with native chloroplast co-chaperonins. Since β oligomers were able to oligomerize in the absence of α, we sought conditions under which αβ hetero-oligomers could be produced without contamination of β homo-oligomers. We found that β2 subunits are unable to oligomerize at low temperatures and used this property to obtain homogenous preparations of functional α2β2 hetero-oligomers. The results of this study highlight the importance of reaction conditions such as temperature and concentration for the reconstitution of chloroplast chaperonin oligomers in vitro.
Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead to successful reconstitution of eukaryotic Rubisco in vitro.
The development of functional chloroplasts, which is assisted by a series of nuclear-encoded auxiliary protein factors, is essential for plant autotrophic growth and development. To understand the molecular mechanisms underlying chloroplast development, we isolated and characterized a pigment-defective mutant, pdm2, and its corresponding variegated RNA interference (RNAi) lines in Arabidopsis. Sequence analysis revealed that PDM2 encodes a pentatricopeptide repeat protein that belongs to the P subgroup. Confocal microscopic analysis and immunoblotting of the chloroplast protein fraction showed that PDM2 was located in the stroma. In RNAi plants, protein-related photosynthesis was severely compromised. Furthermore, analysis of the transcript profile of chloroplast genes revealed that plastid-encoded polymerase-dependent transcript levels were markedly reduced, while nuclearencoded polymerase-dependent transcript levels were increased, in RNAi plants. In addition, PDM2 affects plastid RNA editing efficiency in most editing sites, apparently by directly interacting with multiple organellar RNA editing factor 2 (MORF2) and MORF9. Thus, our results demonstrate that PDM2 is probably involved in the regulation of plastid gene expression required for normal chloroplast development. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development.
High-temperature stress is a major abiotic stress that affects various biological
processes of plants such as biochemical and physiological response, growth,
development, and yield. High-temperature stress has critical effects at cellular and
molecular levels also. The increased concentration of regulatory proteins such as
heat shock transcription factors (Hsfs) is a major molecular response that occurs
during heat stress. These regulatory proteins in turn regulate the expression of heat
shock protein (HSP) genes that act as critical players during stress to maintain cell
homeostasis. Besides HSPs, the other metabolic and regulatory genes, signaling
compounds, compatible osmolytes, and antioxidants too play an important role during
heat stress in plants. Apart from the protein-coding genes, recent studies have
shown that noncoding microRNAs (miRNAs) also play a key role during heat stress
by modulating the gene expression at the transcription and post-transcriptional
level. The transcriptome approaches are important to understand the molecular and
cellular changes occurring in response to heat stress. The approaches rely mostly
by adopting the traditional methods like Northern blot/RNA blot and reverse transcription
PCR (RT-PCR), where the expression of the genes can be studied in different
tissues and cells, whereas the extent of their expression can be achieved by
quantitative PCR or real time PCR. Further, the genome-wide expression profiling
tools such as microarray analysis, next-generation sequencing, and RNA sequencing
offer a great potential in this direction. This chapter primarily provides the
current understanding on the role of regulatory genes (transcription factors), HSP
genes, metabolic genes, signaling compounds, osmolytes, reactive oxygen species, and miRNAs as well as other small RNAs of plants under high temperature. In
addition, it gives a brief account of various transcriptome approaches to study the
expression profiling of genes during heat stress
Type I chaperonins are large oligomeric protein ensembles that are involved in the folding and assembly of other proteins. Chloroplast chaperonins and co-chaperonins exist in multiple copies of two distinct isoforms that can combine to form a range of labile oligomeric structures. This complex system increases the potential number of chaperonin substrates and possibilities for regulation. The incorporation of unique subunits into the oligomer can modify substrate specificity. Some subunits are upregulated in response to heat shock and some show organ-specific expression, whereas others possess additional functions that are unrelated to their role in protein folding. Accumulating evidence suggests that specific subunits have distinct roles in biogenesis of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco).
Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130–140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.
Assembly of foreign prokaryotic ribulose bisphosphate carboxylases (Rubiscos) in Escherichia coli requires both heat-shock proteins groEL and groES. GroEL is related to a chloroplast protein implicated in Rubisco assembly. Bacteria and chloroplasts therefore have a conserved mechanism that uses auxiliary proteins to assist in the assembly of Rubisco.
The products of the groES and groEL genes of Escherichia coli, constituting the groE operon, are known to be required for growth at high temperature (42 degrees C) and are members of the heat shock regulon. Using a genetic approach, we examined the requirement for these gene products for bacterial growth at low temperature (17 to 30 degrees C). To do this, we constructed various groES groEL heterodiploid derivative strains. By inactivating one of the groE operons by a polar insertion, it was shown by bacteriophage P1 transduction that at least one of the groE genes was essential for growth at low temperature. Further P1 transduction experiments with strains that were heterodiploid for only one of the groE genes demonstrated that both groE gene products were required for growth at low temperature, which suggested a fundamental role for the groE proteins in E. coli growth and physiology.
Nine different proteins were imported into isolated pea chloroplasts in vitro. For seven of these [the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), beta-subunit of ATP synthase, glutamine synthetase, the light-harvesting chlorophyll a/b binding protein, chloramphenicol acetyltransferase, and pre-beta-lactamase], a fraction was found to migrate as a stable high-molecular-weight complex during nondenaturing gel electrophoresis. This complex contained the mature forms of the imported proteins and the groEL-related chloroplast chaperonin 60 (previously known as Rubisco subunit binding protein). Thus, the stable association of imported proteins with this molecular chaperone is widespread and not necessarily restricted to Rubisco subunits or to chloroplast proteins. With two of the imported proteins (ferredoxin and superoxide dismutase), such complexes were not observed. It seems likely that, in addition to its proposed role in assembly of Rubisco, the chloroplast chaperonin 60 is involved in the assembly or folding of a wide range of proteins in chloroplasts.
Plastid chaperonin-60 protein was purified to apparent homogeneity from Brassica napus using a novel protocol. The purified protein, which migrated as a single species by nondenaturing polyacrylamide gel electrophoresis, contained four polypeptides: three variants of p60cpn60 alpha and p60cpn60 beta. Partial amino acid sequence determination demonstrated that each variant of p60cpn60 alpha is a distinct translation product. During this study, additional chaperonin-60 proteins were purified. These proteins, which were free from contaminating plastid chaperonin-60, were separated into at least two high molecular weight species that were resolved only by nondenaturing polyacrylamide gel electrophoresis. These proteins contained three 60-kD polypeptides. Two of these polypeptides were recognized by existing antisera, whereas the third was not. Partial amino acid sequence data revealed that each of these, including the immunologically distinct polypeptide, is a chaperonin-60 subunit of putative mitochondrial origin. The behavior of chaperonin-60 proteins during blue A Dyematrex chromatography suggests that this matrix may be generally useful for the identification of chaperonin-60 proteins.
A two-component transposon system based on the Ac element of maize was used as a mutagen in Arabidopsis thaliana. Transposition of a Ds element marked with a hygromycin-resistance gene was activated from four different locations in the Arabidopsis genome. The progeny of 201 plants carrying independent transposition events were screened for mutants with severe, visible phenotypes. Seven mutants were identified and four of them were analyzed genetically. Three mutations were shown to be very closely linked to a transposed copy of the element. Moreover, a mutation (alb3) causing an albino phenotype was conclusively shown to be caused by insertion of the Ds element: somatic and germinal reversion of the mutation occurred in the presence of the transposase gene but not in its absence, and in three revertants the Ds had excised from its position in the mutant line. The DNA adjacent to Ds in the mutant was isolated and it was demonstrated that revertants retained part of the 8-bp duplication caused by insertion of Ds. These experiments indicate that the Ac/Ds system can be used as an insertional mutagen in the heterologous host Arabidopsis, which will permit the isolation of genes from this species by transposon tagging.
Olive (oli) is a recessive nuclear mutation of Antirrhinum majus which reduces the level of chlorophyll pigmentation and affects the ultrastructure of chloroplasts. The oli-605 allele carries a Tam3 transposon insertion which has allowed the locus to be isolated. The oli gene encodes a large putative protein of 153 kDa which shows homology to the products of two bacterial genes necessary for tetrapyrrole-metal chelation during the synthesis of bacteriochlorophyll or cobyrinic acid. We therefore propose that the product of the oli gene is necessary for a key step of chlorophyll synthesis: the chelation of magnesium by protoporphyrin IX. Somatic reversion of the oli-605 allele produces chimeric plants which indicate that the oli gene functions cell-autonomously. Expression of oli is restricted to photosynthetic cells and repressed by light, suggesting that it may be involved in regulating the rate of chlorophyll synthesis in green tissues.
The chaperonin GroEL is able to mediate protein folding in its central cavity. GroEL-bound dihydrofolate reductase assumes its native conformation when the GroES cofactor caps one end of the GroEL cylinder, thereby discharging the unfolded polypeptide into an enclosed cage. Folded dihydrofolate reductase emerges upon ATP-dependent GroES release. Other proteins, such as rhodanese, may leave GroEL after having attained a conformation that is committed to fold. Incompletely folded polypeptide rebinds to GroEL, resulting in structural rearrangement for another folding trial in the chaperonin cavity.
Recent studies of GroE-mediated protein folding indicate that substrate proteins are productively released from a cis ternary complex in which the nonnative substrate is sequestered within the GroEL channel underneath GroES. Here, we examine whether protein folding can occur in this space. Stopped-flow fluorescence anisotropy of a pyrene-rhodanese-GroEl complex indicates that addition of GroES and ATP (but not ADP) leads to a rapid change in substrate flexibility at GroEL. Strikingly, when GroES release is blocked by the use of either a nonhydrolyzable ATP analog or a single-ring GroEL mutant, substrates complete folding while remaining associated with chaperonin. We conclude that the cis ternary complex, in the presence of ATP, is the active state intermediate in the GroE-mediated folding reaction: folding is initiated in this state and for some substrates may be completed prior to the timed release of GroES triggered by ATP hydrolysis.
The albino3 (alb3) mutant of Arabidopsis forms white or light yellow cotyledons and leaves and when germinated on soil does not survive beyond the seedling stage. The chloroplasts of the mutant are abnormal, as determined by electron microscopy, and contain reduced levels of chlorophyll. However, the chloroplasts of alb3 mutants are sufficiently differentiated to enable the expression of two nuclear genes whose transcription requires the presence of chloroplasts. The ALB3 gene was isolated by transposon tagging with the Activator/Dissociation transposable element system. ALB3 is a novel plant gene whose product shows homology to a bacterial membrane protein previously identified in five bacterial species and to a yeast protein, OXA1, and its human homolog. OXA1 is required in the mitochondria for proper assembly of the cytochrome oxidase complex. ALB3 does not have a function identical to OXA1 because mitochondrial cytochrome oxidase activity is not affected in the mutant, and immunogold labeling as well as chloroplast import experiments performed in vitro demonstrated that the ALB3 protein is present in chloroplast membranes. ALB3 might have a function related to that of OXA1 and be involved in the assembly of a chloroplast enzyme complex.
The vascular tissues of flowering plants form networks of interconnected cells throughout the plant body. The molecular mechanisms directing the routes of vascular strands and ensuring tissue continuity within the vascular system are not known, but are likely to depend on general cues directing plant cell orientation along the apical-basal axis. Mutations in the Arabidopsis gene MONOPTEROS (MP) interfere with the formation of vascular strands at all stages and also with the initiation of the body axis in the early embryo. Here we report the isolation of the MP gene by positional cloning. The predicted protein product contains functional nuclear localization sequences and a DNA binding domain highly similar to a domain shown to bind to control elements of auxin inducible promoters. During embryogenesis, as well as organ development, MP is initially expressed in broad domains that become gradually confined towards the vascular tissues. These observations suggest that the MP gene has an early function in the establishment of vascular and body patterns in embryonic and post-embryonic development.
The chaperonin GroEL is an oligomeric double ring structure that, together with the cochaperonin GroES, assists protein folding. Biochemical analyses indicate that folding occurs in a cis ternary complex in which substrate is sequestered within the GroEL central cavity underneath GroES. Recently, however, studies of GroEL "minichaperones" containing only the apical substrate binding subdomain have questioned the functional importance of substrate encapsulation within GroEL-GroES complexes. Minichaperones were reported to assist folding despite the fact that they are monomeric and therefore cannot form a central cavity. Here we compare directly the folding activity of minichaperones with that of the full GroEL-GroES system. In agreement with earlier studies, minichaperones assist folding of some proteins. However, this effect is observed only under conditions where substantial spontaneous folding is also observed and is indistinguishable from that resulting from addition of the nonchaperone protein alpha-casein. By contrast, the full GroE system efficiently promotes folding of several substrates under conditions where essentially no spontaneous folding is observed. These data argue that the full GroEL folding activity requires the intact GroEL-GroES complex, and in light of previous studies, underscore the importance of substrate encapsulation for providing a folding environment distinct from the bulk solution.
gnom is one of several genes that make substantial contributions to pattern formation along the apical-basal axis of polarity in the Arabidopsis embryo as indicated by the mutant seedling phenotype. The apical and basal end regions of the body pattern, which include the meristems of the shoot and the root, fail to form, and a minority of mutant embryos lack morphological features of apical-basal polarity. We have investigated the developmental basis of the gnom mutant phenotype, taking advantage of a large number of EMS-induced mutant alleles. The seedling phenotype has been traced back to the early embryo in which the asymmetric division of the zygote is altered, now producing two nearly equal-sized cells. The apical daughter cell then undergoes abnormal divisions, resulting in an octant embryo with about twice the normal number of cells while the uppermost derivative of the basal cell fails to become the hypophysis, which normally contributes to root development. Consistent with this early effect, gnom appears to be epistatic to monopteros in doubly mutant embryos, suggesting that, without prior gnom activity, the monopteros gene cannot promote root and hypocotyl development. On the other hand, when root formation was induced in bisected seedlings, wild-type responded whereas gnom mutants failed to produce a root but formed callus instead. These results suggest that gnom activity promotes asymmetric cell division which we believe is necessary both for apical-basal pattern formation in the early embryo and for root formation in tissue culture.
Arrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.
Embryo differentiation in Arabidopsis thaliana follows the classical Capsella variation of the Onagrad type. Fertilization occurs approximately 3 h after flowering, whereupon vacuolar organization in the zygote changes and the cell elongates rapidly to approximately three times its original length. Cytoplasmic polarization is maintained. During the first two division steps there is very little increase in total cell volume, and during subsequent divisions vacuole number increases, with a concomitant decrease in size. Plastids remain undifferentiated up to the late globular stage, after which grana begin to develop. Ribosomal concentration increases significantly after fertilization. Differences between embryo proper cells become evident by the heart stage; vacuole, plastid, and mitochondrial abundance, size, and complexity vary within the embryo. There are no plasmodesmatal connections with the endosperm or integuments. Suspensor development is complete by the early globular stage, when it consists of seven to nine highly vacuolate cells, each linked by end wall plasmodesmata. Ribosome and volume densities of plastids and mitochondria are significantly lower than in the embryo proper organelles, and dictyosomes are infrequent. Embryo sac wall projections proliferate throughout the micropylar chamber, especially adjacent to the filiform apparatus and zygote base, and ingrowths form on the basal cell proximal wall. Key words: Arabidopsis, embryogenesis, embryo differentiation, wall ingrowths.
A systematic search for mutations in the flowering plant Arabidopsis
thaliana that disrupt the spatial organization of the seedling by
altering embryogenesis is described. Mutations in nine genes affect
three different aspects of the body organization: apical-basal pattern
along the single axis of polarity, radial pattern involving the primary
tissues, and shape. The results suggest principles of pattern formation
in the plant embryo.
Genetic analysis of plant em-bryogenesis has been approached in part through the isolation and characterization of recessive embryonic mutants. The most extensive studies have dealt with maize and Arabidopsis. The high frequency of mutants defective in plant embryogenesis is consistent with the presence of many target genes with essential functions at this stage of the life cycle. Some mutants are likely to be defective in genes with general housekeeping functions. Others should facilitate the identification of genes with a more direct role in the regulation of morphogesis. Over 300 embryonic mutants of Arabidopsis isolated following chemical mutagenesis and T-DNA insertional mutagenesis are currently being analyzed. This collection includes embryonic le-thals, defectives, and pattern mutants. Developmental abnormalities include the presence of fused cotyledons, twin embryos, abnormally large suspensors, distorted epidermal layers, single cotyledons, enlarged shoot apices, pattern deletions and duplications, embryos with altered patterns of symmetry, bloated embryos with giant vacuolated cells, reduced hypocotyls that fail to produce roots, and embryos that protrude through the seed coat late in maturation. This review describes the isolation and characterization of embryonic mutants of Arabidopsis and their potential application to plant biology. © 1992 Wiley-Liss, Inc.
Chaperonins are a class of molecular chaperone, present in bacteria, mitochondria and chloroplasts, that are involved in protein folding and assembly in many organisms. Plastid and β chaperonins have been suggested to be involved specifically in the assembly of Ribulose bisphosphate carboxylase/oxygenase. However, to date there is no direct evidence to confirm the in vivo role of plastid chaperonin 60 polypeptides as molecular chaperones. This paper reports on the production, by means of antisense technology, of transgenic tobacco plants with reduced levels of chaperonin 60β (Cpn60β). Antisense cpn60β plants showed drastic phenotypic alterations including slow growth, delayed flowering, stunting and leaf chlorosis. The most extreme effect appeared to be lethality suggesting that cpn60β functions are essential for viability. Cpn60β antisense plants accumulated Rubisco to specific activities equal to or higher than that of controls and had high plastid starch contents. These observations are discussed with respect to the suggestion that chaperonin is required for the assembly of active Rubisco in vivo. In addition, metabolic alterations in the antisense transgenic plants such as reduced soluble carbohydrate content as well as higher levels of starch in chloroplasts, suggest that Cpn60β may be required for import, assembly or membrane insertion of several chloroplast membrane proteins. These results are in agreements with the proposed role of Cpn60β as a molecular chaperone.
The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3′ end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium, as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.
Many late embryogenesis abundant (Lea) protein genes in plants are regulated by abscisic acid (ABA). The RNA level of a carrot gene, DC8, increases in response to ABA in developing seeds. However, DC8 cannot be induced by ABA in adult tissues. We used chimeric genes made of various DC8 promoter fragments fused to -glucuronidase (GUS) to analyze the transcriptional regulation of DC8. DC8:GUS expression was measured in electroporated carrot protoplasts and in stably transformed carrots. The region of the DC8 promoter from –170 to –51 contained ABA-responsive sequences that required a 5 upstream region for high levels of expression in embryogenic callus protoplasts. 505 bp of the DC8 promoter conferred GUS expression in stably transformed somatic and zygotic embryos. DC8:GUS was expressed only in tissues formed in the seed. This includes cells in the embryo, the endosperm and the germinating seedlings. Gel retardation and competition experiments were performed to analyze the embryo nuclear protein-DNA binding activities in vitro. No binding activity was detected on the putative ABA-responsive region; however the 5 upstream regions located between -505 and -301 interacted with embryo nuclear factors. An additional site of DNA-protein interaction was located between positions -32 and +178. The nuclear proteins that bind these sequences were found in the embryo nuclei only, not in the nuclei from leaves or roots.
Auxotrophic mutants have played an important role in the genetic dissection of biosynthetic pathways in microorganisms. Equivalent mutants have been more difficult to identify in plants. The bio1 auxotroph of Arabidopsis thaliana was shown previously to be defective in the synthesis of the biotin precursor 7, 8-diaminopelargonic acid. A second biotin auxotroph of A. thaliana has now been identified. Arrested embryos from this bio2 mutant are defective in the final step of biotin synthesis, the conversion of dethiobiotin to biotin. This enzymatic reaction, catalyzed by the bioB product (biotin synthase) in Escherichia coli, has been studied extensively in plants and bacteria because it involves the unusual addition of sulfur to form a thiophene ring. Three lines of evidence indicate that bio2 is defective in biotin synthase production: mutant embryos are rescued by biotin but not dethiobiotin, the mutant allele maps to the same chromosomal location as the cloned biotin synthase gene, and gel-blot hybridizations and polymerase chain reaction amplifications revealed that homozygous mutant plants contain a deletion spanning the entire BIO2-coding region. Here we describe how the isolation and characterization of this null allele have provided valuable insights into biotin synthesis, auxotrophy, and gene redundancy in plants.
Expression of the gene encoding the β subunit of β-conglycinin, the 7S seed storage protein of soybean ( Glycine max [L.] Merr.), has been shown to be down-regulated by exogenously applied L-methionine in in vitro soybean cotyledon cultures, whereas expression of the gene encoding the a ′ subunit is not affected. We constructed transgenic Arabidopsis thaliana (L.) Heynh. carrying the β-glucuronidase (GUS) reporter gene under the control of the promoter regions from either the a ′ or β subunit gene. An in vitro culture system of immature siliques of A. thaliana was developed for exogenous application of L-methionine to immature seeds. Expression of the fi subunit gene promoter-GUS fusion was down-regulated by the application of L-methionine, and this response was reversible. As an alternative way to apply L-methionine, A. thaliana plants were grown on rockwool and irrigated with L-methionine-supplemented medium. GUS activity driven by the β subunit gene promoter was repressed by L-methionine, and the degree of repression depended on the concentration of L-methionine in the medium. On the other hand, the a ′ subunit gene promoter-GUS fusion did not show such a response. These results indicate that A. thaliana has a system to differentially regulate the expression of the β-conglycinin genes by L-methionine at the level of transcription. This work establishes a system to genetically analyze plants′ responses to nutritional stimuli.
Chaperonins (Cpn) are implicated in the folding and assembly of multimeric proteins in plastids and mitochondria of eukaryotes and in prokaryotes. Plastid Cpn is composed of two different polypeptides termed Cpn60 alpha and Cpn60 beta. We have isolated cDNA and genomic clones encoding Cpn60 beta from Arabidopsis thaliana. The steady-state level of the cpn60 beta mRNAs is higher in etiolated leaves and sucrose-treated plants as compared to control leaves. The A. thaliana cpn60 beta gene family consists of at least three different coding units. It was confirmed that Cpn beta-encoding genes have a high level of conservation among plants.
Mitochondria contain a nuclear-encoded heat shock protein, HSP60, which functions as a chaperonin in the post-translational assembly of multimeric proteins encoded by both nuclear and mitochondrial genes. We have isolated and sequenced full-length complementary DNAs coding for this mitochondrial chaperonin in Arabidopsis thaliana and Zea mays. Southern-blot analysis indicates the presence of a single hsp60 gene in the genome of A. thaliana. There is a high degree of homology at the predicted amino acid levels (43 to 60%) between plant HSP60s and their homologues in prokaryotes and other eukaryotes which indicates that these proteins must have similar evolutionarily conserved functions in all organisms. Northern- and western-blot analyses indicate that the expression of the hsp60 gene is developmentally regulated during seed germination. It is also heat-inducible. Developmental regulation of the (beta-subunit of F1-ATPase, an enzyme complex that is involved in the cyanide-sensitive mitochondrial electron transport system, indicates that imbibed embryos undergo rapid mitochondrial biogenesis through the early stages of germination. Based on the functional role of HSP60 in macromolecular assembly, these data collectively suggest that the presence of higher levels of HSP60 is necessary during active mitochondrial biogenesis, when the need for this protein is greatest in assisting the rapid assembly of the oligomeric protein structures.
We have isolated a cytoplasmic chaperonin based on its ability to catalyze the folding of denatured beta-actin. The cytoplasmic chaperonin is organized as a multisubunit toroid and requires Mg2+ and ATP for activity. The folding reaction proceeds via the rapid ATP-independent formation of a binary complex, followed by a slower ATP-dependent release of the native product. Electron microscopic observations reveal a striking structural change that occurs upon addition of Mg2+ and ATP. The eukaryotic cytoplasm thus contains a chaperonin that is functionally analagous to its prokaryotic, mitochondrial, and chloroplastic counterparts.
A recombinant 60 kDa Brucella abortus protein expressed in Escherichia coli was recognized in immunoblots by sera from mice experimentally infected with B. abortus and a dog experimentally infected with B. canis. Sera from humans and dogs with naturally acquired brucellosis also recognized this protein, which was designated BA60K. The gene encoding BA60K was localized within an 18 kb B. abortus genomic fragment and its direction of transcription determined by subcloning and maxicell analysis of selected restriction fragments. The nucleotide sequence of 1800 bases encompassing the predicted gene location was determined, revealing an open reading frame encoding a protein of 546 amino acids (predicted relative molecular mass of 57515). Solid phase micro-sequencing of BA60K eluted from two-dimensional polyacrylamide gels confirmed the predicted amino acid sequence. Comparison of the predicted amino acid sequence of BA60K with a protein sequence database revealed that BA60K shares 67.9% identity with the GroEL protein of E. coli, a member of the Hsp60 family of chaperonins. The immunodominant Hsp60 homologs from Legionella pneumophila, Chlamydia trachomatis and Mycobacterium tuberculosis were also found to share greater than 59% amino acid sequence identity with the BA60K protein. The identification of BA60K as a member of the Hsp60 family of chaperonins supports its role in stimulating a prominent host immune response during the course of Brucella infections. It also indicates that BA60K is an important candidate for studies aimed at identifying the antigens responsible for eliciting the protective immune response to brucellosis.
A role in folding of newly translated proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide-1 (TCP1), although its molecular targets have not yet been identified. Tubulin is a major cytosolic protein whose assembly into microtubules is critical to many cellular processes. Although numerous studies have focused on the expression of tubulin, little is known about the processes whereby newly translated tubulin subunits acquire conformations that enable them to form alpha-beta-heterodimers. We examined the biogenesis of alpha- and beta-tubulin in rabbit reticulocyte lysate, and report here that newly translated tubulin subunits entered a 900K complex in a protease-sensitive conformation. Addition of Mg-ATP, but not nonhydrolysable analogues, released the tubulin subunits as assembly-competent protein with a conformation that was relatively protease-resistant. The 900K complex purified from reticulocyte lysate contained as its major constituent a 58K protein that cross-reacted with a monoclonal antiserum against mouse TCP1. We conclude that TCP1 functions as a cytosolic chaperone in the biogenesis of tubulin.
Molecular chaperones of the chaperonin family occur in prokaryotes and in plastids and mitochondria. Prokaryotic and mitochondrial chaperonin-60 oligomers (Cpn-60) are composed of a single subunit type (p60cpn-60). In contrast, preparations of purified plastid Cpn-60 contain approximately equal quantities of two polypeptides, p60cpn-60 alpha and p60cpn-60 beta, with slightly different electrophoretic mobilities. We have isolated cDNA clones encoding plastid p60cpn-60 alpha and p60cpn-60 beta polypeptides from Brassica napus and Arabidopsis thaliana. The unexpected degree of sequence divergence observed between p60cpn-60 alpha and p60cpn-60 beta raises questions concerning the structure of the oligomer and the functions of these polypeptides. We have also found an amino acid sequence motif within all p60cpn-60 sequences which resembles the p10cpn-10 sequences.
An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
The ultrastructure and composition of the Capsella embryo from the terminal cell of the 3-celled embryo through the globular (32—64-celled) embryo was examined. Starch and lipid deposits which accumulate in the terminal cell disappear after its first division. Cell size and number of organelles per cell decrease with repeated divisions through the formation of the globular embryo. The cells contain few dictyosomes and little ER but are rich in ribosomes grouped into small polysomes. The embryo stains intensely for protein and nucleic acids. There are no apparent ultrastructural and histochemical differences in the cells of the protoderm, procambium, and ground meristem until the formation of the heart-shaped embryo. The implications of this finding to the general problem of cell and tissue differentiation during embryogenesis are discussed.
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
The chaperonin GroEL is a ribosome-sized double-ring structure that assists in folding a diverse set of polypeptides. We have examined the fate of a polypeptide during a chaperonin-mediated folding reaction. Strikingly, we find that, upon addition of ATP and the cochaperonin GroES, polypeptide is released rapidly from GroEL in a predominantly nonnative conformation that can be trapped by mutant forms of GroEL that are capable of binding but not releasing substrate. Released polypeptide undergoes kinetic partitioning: a fraction completes folding while the remainder is rebound rapidly by other GroEL molecules. Folding appears to occur in an all-or-none manner, as proteolysis and tryptophan fluorescence indicate that after rebinding, polypeptide has the same structure as in the original complex. These observations suggest that GroEL functions by carrying out multiple rounds of binding aggregation-prone or kinetically trapped intermediates, maintaining them in an unfolded state, and releasing them to attempt to fold in solution.
The EMB30 gene is involved in apical-basal pattern formation in the Arabidopsis embryo. Mutations in this locus produce mutants with a wide range of seedling phenotypes, but all of the mutants lack a root and a true hypocotyl. We have cloned the EMB30 gene, and it encodes a protein that has similarity to the yeast Sec7 protein and to two other open reading frames identified in clones from humans and C. elegans. We refer to the region of similarity among these four sequences as the Sec7 domain. The emb30-1 allele has a mutation in the Sec7 domain that alters a residue conserved in all four of these sequences, suggesting that this domain may be important for EMB30 function. Molecular data and microscopy studies of emb30 seedlings presented here indicate that EMB30 affects cell division, elongation, and adhesion and functions in seedling and adult plants as well as during embryogenic pattern formation.
A homologue of the 70-kDa heat-shock protein (Hsp70) was purified from pumpkin chloroplasts. The molecular mass of the purified protein was approximately 75 kDa and its N-terminal amino acid sequence was very similar to those of homologues of Hsp70 from bacterial cells and from the mitochondrial matrix and stroma of pea chloroplasts. The purified homologue of Hsp70 was found in the stroma of chloroplasts. To investigate the role(s) of the homologue of Hsp70 in the chloroplast stroma, we examined the possibility that the homologue of Hsp70 might interact with newly imported proteins to assist in their maturation (for example, in their folding and assembly). Ferredoxin NADP+ reductase (FNR) imported into chloroplasts in vitro could be immunoprecipitated with antisera raised against the homologue of Hsp70 from pumpkin chloroplasts and against GroEL from Escherichia coli, which is a bacterial homologue of chaperonin 60 (Cpn60), in an ATP-dependent manner, an indication that newly imported FNR interacts physically with homologues of Hsp70 and Cpn60 in chloroplasts. Time-course analysis of the import of FNR showed that imported FNR interacts transiently with the homologue of Hsp70 and that the association of FNR with the homologue of Hsp70 precedes that with the homologue of Cpn60. These results suggest that homologues of Hsp70 and Cpn60 in chloroplasts might sequentially assist in the maturation of newly imported FNR in an ATP-dependent manner.
A general role for chaperonin ring structures in mediating folding of newly translated proteins has been suggested. Here we have directly examined the role of the E. coli chaperonin GroEL in the bacterial cytoplasm by production of temperature-sensitive lethal mutations in this essential gene. After shift to nonpermissive temperature, the rate of general translation in the mutant cells was reduced, but, more specifically, a defined group of cytoplasmic proteins--including citrate synthase, ketoglutarate dehydrogenase, and polynucleotide phosphorylase--were translated but failed to reach native form. Similarly, a monomeric test protein, maltose-binding protein, devoid of its signal domain, was translated but failed to fold to its native conformation. We conclude that GroEL indeed is a machine at the distal end of the pathway of transfer of genetic information, assisting a large and specific set of newly translated cytoplasmic proteins to reach their native tertiary structures.
Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.
The KNOTTED class of plant genes encodes homeodomain proteins. These genes have been found in all plant species where they have been sought and, where examined, show expression patterns that suggest they play an important role in shoot meristem function. Until now, all mutant phenotypes associated with these genes have been due to gain-of-function mutations, making it difficult to deduce their wild-type function. Here we present evidence that the Arabidopsis SHOOT-MERISTEMLESS (STM) gene, required for shoot apical meristem formation during embryogenesis, encodes a class I KNOTTED-like protein. We also describe the expression pattern of this gene in the wild-type plant. To our knowledge, STM is the first gene shown to mark a specific pattern element in the developing plant embryo both phenotypically and molecularly.
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.
Petunia embryos carrying the no apical meristem (nam) mutation fail to develop a shoot apical meristem. Occasional shoots on nam- seedlings bear flowers that develop ten instead of five primordia in the second whorl. Double mutants with the homeotic gene green petals show that nam acts independently of organ identify in whorl 2 and now also affects primordium number in whorl 3. The nam gene was isolated by transposon tagging. The encoded protein shares a conserved N-terminal domain with several other proteins of unknown function and thus represents a novel class of proteins. Strikingly, nam mRNA accumulates in cells at the boundaries of meristems and primordia. These data indicate a role for nam in determining positions of meristems and primordia.
In order to assess the importance of morphogenesis on the induction of promoter markers for storage and Lea programmes, advantage was taken of the emb mutations producing embryos arrested at a wide range of developmental stages in Arabidopsis. These embryos are viable during their stage of developmental arrest and continue to divide further, but apparently without further differentiation into the main organs and tissues of the normal embryos. Eight independent emb mutants arrested in their development prior to the cotyledon stage were selected. These emb embryos lack the normal morphology of the wild-type embryos when the synthesis of storage and Lea proteins are normally initiated. The 2S1-uidA chimeric gene, representative of the maturation programme and the Em 1-uidA chimeric gene, representative of the desiccation programme were introduced by crosses into the emb background. In the eight emb lines, the expression of the GUS reporter gene directed by the 2S1 and Em 1 promoters was observed in the aborted seeds irrespective of their stage of developmental arrest. The time of induction of the expression of both promoters was the same in the arrested embryos as compared with the normal embryos within the same silique. Thus, the activation of these two promoters is triggered by the same signal and can occur in the absence of morphogenesis. However, in the absence of normal organ formation, the expression of the reporter gene under the control of the 2S1 and Em1 promoters was evident throughout the whole seed tissues. In normal seed development, the hormone abscisic acid (ABA) activates the promoters of the 2S1 and Em 1 genes. One of the important members of the signal transduction pathway of ABA is the ABI3 protein. It has been shown previously that this protein is a prerequisite for the induction of Em 1 by ABA in seeds. A good correlation with the expression of the ABI3 promoter and the 2S1 and Em 1 promoters was found in emb seeds tissues. This observation suggests that the promoters of the 2S1 and the Em 1 genes are expressed in the mutant seeds not at a basal level, but are probably induced by ABA, as in normal seed development.
Normal mode calculations on individual subunits and a multisubunit construct are used to analyze the structural transitions that occur during the GroEL cycle. The normal modes demonstrate that the specific displacements of the domains (hinge bending, twisting) observed in the structural studies arise from the intrinsic flexibility of the subunits. The allosteric mechanism (positive cooperativity within a ring, negative cooperativity between rings) is shown to be based on coupled tertiary structural changes, rather than the quaternary transition found in classic allosteric proteins. The results unify static structural data from x-ray crystallography and cryoelectron microscopy with functional measurements of binding and cooperativity.
Embryo formation is the first patterning process during vegetative plant growth. Using transposons as insertional mutagens in Arabidopsis, we identified the mutant edd1 that shows embryo-defective development. The insertion mutation is lethal, arresting embryo growth between the globular and heart stages of embryonic development. The mutant phenotype cosegregates with a transposed Dissociation element. Sequences flanking the transposed element were isolated and used to isolate a full-length cDNA clone representing the wild-type EDD1 gene. Complementation of the mutant through Agrobacterium-mediated gene transfer of an EDD1 wild-type copy as well as loss of the transposon concomitant with phenotypic reversion demonstrated that the transposon had caused the mutation. Based on homology to Escherichia coli, the EDD1 gene is predicted to encode a novel glycyl-tRNA synthetase (GlyRS) that has not been identified previously in higher plants. An N-terminal portion of the plant protein is able to direct a marker protein into pea chloroplasts. Thus, the gene identified by the embryo-defective insertion mutation encodes a GlyRS homolog, probably acting within the plastidic compartment.