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Determination of Accurate Extinction Coefficients and Simultaneous Equations for Assaying Chlorophyll a and b Extracted with Four Different Solvents: Verification of the Concentration of Chlorophyll Standards by Atomic Absorption Spectroscopy
Abstract
The extinction coefficients for chlorophylls a and b in diethylether (Smith, J.H.C. and Benitez, A. (1955) in Modern Methods of Plant Analysis (Paech, K. and Tracey, M.V., eds.), Vol. 4, pp. 143–196, Springer-Verlag, Berlin), used in this paper as primary standards, were verified, to within an error of less than 1%, by magnesium determination using atomic absorbance spectrophotometry. We also report the determination of accurate extinction coefficients for chlorophylls a and b in N,N′-dimethylformamide, methanol or buffered 80% aqueous acetone. Highly purified chlorophylls were used and methods were employed which not only minimize errors due to evaporation of the volatile solvents employed in their estimation but also eliminate variable micro-contamination by chlorophyll degradation products, a potential source of inconsistency between the extinction coefficients obtained in each of these three solvents. Using these new coefficients, expressed as both millimolar and specific coefficients, we have derived new simultaneous equations to obtain chlorophyll concentrations as nmol/ml and μg/ml, respectively. These equations were applied to data obtained with leaf discs from spinach and Flindersia brayleyana extracted with the three specified solvents and to a concentrated solution (in N,N′ -dimethylformamide) of a chlorophyll a + b mixture added to the threesolvent systems. The validity of these equations is proven by the consistency of the chlorophyll determinations and of the chlorophyll a/b ratios. New simultaneous equations, compatible with the equations derived for the threesolvents, are presented for the assay of chlorophylls a and b converted to their cyclic hydroxylactone derivatives by extraction with alkaline pyridine reagent (2.1 M pyridine in 0.35 M NaOH). Most chlorophyll analyses in higher plants, including the chlorophyll content and chlorophyll a/b ratios of plant thylakoids and chlorophyll-protein complexes, have been obtained in 80% aqueous acetone with the much used simultaneous equations of Arnon (Arnon, D.I. (1949) Plant Physiol. 24, 1–15). For this reason we include conversion factors whichcorrect these earlier data and make it compatible with data calculated with the simultaneous equations presented in this paper. The importance of these corrections to the formulation of meaningful models of the photosynthetic apparatus is demonstrated. Our results also indicate that grinding leaf discs with N,N′-dimethylformamide is a more reliable method for extracting all chlorophylls than shaking with this solvent for 24 h.
... Enzymatic mechanisms occurring in the cells of microorganisms can lead to the elimination of toxic chemical substances from the petroleum industry [31][32][33][34], such as naphthenic acids and surfactants [32,35]. The toxic effects of certain chemicals generate enzymatic changes in algae cells, making it necessary to study certain enzymes influenced by chemical stress, including peroxidases (Px), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) [36][37][38][39]. Considering this principle, in our previous tests, we studied the removal of metabolites after the hydrolysis of the substance CBM by using the algal suspension, and the results indicated that the suspension of Chlorella sp. ...
... Enzymatic mechanisms occurring in the cells of m ination of toxic chemical substances from the petroleu thenic acids and surfactants [32,35]. The toxic effects o matic changes in algae cells, making it necessary to stu chemical stress, including peroxidases (Px), superoxid and glutathione reductase (GR) [36][37][38][39]. Considering t we studied the removal of metabolites after the hydrol the algal suspension, and the results indicated that th sumed the entire amount of CBM from the samples [19 ...
... The concentration of chlorophyll "a" and chlorophyll "b" were calculated using the following Equations no. (7) and (8) [31,32,36,37]. ...
Chemical compounds, such as the CS gas employed in military operations, have a number of characteristics that impact the ecosystem by upsetting its natural balance. In this work, the toxicity limit and microorganism’s reaction to the oxidative stress induced by O-chlorobenzylidenemalonitrile, a chemical found in CS gas, were assessed in relation to the green algae Chlorella pyrenoidosa. A number of parameters, including the cell growth curve, the percent inhibition in yield, the dry cell weight, the percentage viability and productivity of algal biomass flocculation activity, and the change in oxygen production, were analyzed in order to comprehend the toxicological mechanisms of O-chlorobenzylidenemalonitrile on algal culture. Using fluorescence and Fourier transform infrared spectroscopy (FTIR), the content of chlorophyll pigments was determined. The values obtained for pH during the adaptation period of the C. pyrenoidosa culture were between 6.0 and 6.8, O2 had values between 6.5 and 7.0 mg/L, and the conductivity was 165–210 µS/cm. For the 20 µg/mL O-chlorobenzylidenemalonitrile concentration, the cell viability percentage was over 97.4%, and for the 150 µg/mL O-chlorobenzylidenemalonitrile concentration was 74%. The ECb50 value for C. pyrenoidosa was determined from the slope of the calibration curve; it was estimated by extrapolation to the value of 298.24 µg/mL. With the help of this study, basic information on the toxicity of O-chlorobenzylidenemalonitrile to aquatic creatures will be available, which will serve as a foundation for evaluating the possible effects on aquatic ecosystems. The management of the decontamination of the impacted areas could take the results into consideration.
... Concentration values are then derived using well-established equations. Previous investigations have explored a range of methods to evaluate chlorophyll content in higher plant leaves, encompassing non-destructive chlorophyll meters [12], fluorometry [13], photo-acoustic spectroscopy [14], chromatographic approaches [15], and spectrophotometry [4,[16][17][18][19]. Numerous studies have demonstrated that chlorophylls, which are lipid-soluble compounds present in plant tissues, can be extracted using water-miscible organic solvents. ...
... Numerous studies have demonstrated that chlorophylls, which are lipid-soluble compounds present in plant tissues, can be extracted using water-miscible organic solvents. These solvents include acetone, pyridine, methanol, ethanol, diethyl ether, DMF, and DMSO, all of which have the ability to absorb water [4,16,[20][21][22][23][24][25][26][27][28]. ...
... These findings are in accordance with previous research, which has consistently demonstrated that DMSO and 96% ethanol are highly efficient solvents for chlorophyll extraction [12,25,32]. Previous investigations have explored several methods for evaluating chlorophyll content in higher plant leaves, and several studies have substantiated that chlorophylls, which are lipid-soluble compounds found in plant tissues, can be effectively extracted using watermiscible organic solvents [4,16,[20][21][22][23][24][25][26][27][28]. However, consideration of various factors including, cost effectiveness, time, safety and environmental regulations in the laboratory or research setting is also crucial when choosing an appropriate solvent. ...
: This study focuses on optimizing chlorophyll extraction techniques and strategically placing leaf disks to enhance chlorophyll concentration in sesame (Sesamum indicum L.) leaves. Thirty sesame genotypes, categorized into light green (LG), middle green (MG), and deep green (DG) pigment groups based on leaf coloration, were selected from a larger pool of field-grown accessions. The investigation involved determining optimal SPAD value index measurements, quantifying pigment concentrations, exploring extraction solvents, and selecting suitable leaf disk positions. Significant variations in chlorophyll content were observed across genotypes, greenness categories, and leaf disk positions. The categorization of genotypes into DG, MG, and LG groups revealed a correlation between leaf appearance and chlorophyll content. The study highlighted a consistent relationship between carotenoids and chlorophyll, indicating their role in adaptation to warm environments. Examination of leaf disk positions revealed a significant chlorophyll gradient along the leaf blade, emphasizing the need for standardized protocols. Chlorophyll extraction experiments identified DMSO and 96% ethanol, particularly the later incubated for 10 minutes at 85°C, as effective choices. This recommendation considers factors like cost-effectiveness, time efficiency, safety, and environmental regulations, ensuring consistent and simplified extraction processes. For higher chlorophyll extraction, focusing on leaf tips and the 75% position along the sesame leaf blade is suggested, consistently yielding higher chlorophyll content. Our findings therefore, underscores insightful knowledge on chlorophyll gradients and encouraging for standardized protocols, that enabling researchers to refine experimental designs for precise and comparable chlorophyll measurements. Recommended solvent choices ensure reliable outcomes in plant physiology, ecology, and environmental studies.
... After incubation, the sample was centrifuged at 14,000 Xg for 7 minutes at 4 ºC. The supernatant was read at 470, 665 and 720 nm to compute the chlorophyll a content (Porra et al., 1989). ...
... The method observed to give the highest percent quality extraction was the 80% buffered acetone extraction. A study extracted chlorophyll from rice leaves through several methods, and the 80% acetone extraction gave a 24% higher yield (Porra et al., 1989;Pramanik and Bera, 2013). ...
... The difference in chlorophyll a content of all treatments is not statistically significant after 20 days of incubation maybe because the plant is still in its early stage and is not fully matured to analyze its chlorophyll a content. Based on a previous study, the chlorophyll a content of rice seedlings decreases slightly after 15 days, indicating another step or maturation in its growing stage (Porra et al., 1989;Pramanik and Bera, 2013). ...
Nostoc is a blue-green cyanobacteria that produce their food through photosynthesis and nitrogen fixation. These organisms undergo nitrogen fixation and provide a potential nitrogen source for growth and development. Since rice is known as one of the world's staple foods, especially in Asia, this study aims to determine the utilization of Nostoc piscinale as a potential biofertilizer for planting rice crops. N. piscinale was inoculated into three subcultures and incubated for 87 to 170 days, and then analyzed for nitrogen-fixing activity and rice plant development. Growth of cyanobacteria showed a significant increase in chlorophyll a starting from day 30 up to day 170 while nitrogen-fixing activity remained constant from day 4. On the other hand, the growth and development of rice treated with cyanobacteria showed correlated trends with commercial fertilizer (CSF) in terms of root and shoot (growth and fresh weight) and chlorophyll a content with no statistical differences (p-value ≥ 0.05). Nitrogen tests indicate the utilization of ammonia produced by N. piscinale and the change in soil pH. After harvesting the samples at 20 days and measuring the soil pH, the cyanobacterial samples were seen to lower the soil pH before planting, which is significantly different from the untreated and CSF-treated samples. The utilization of nitrogen for the growth and development of Oryza sativa subsp. indica proved that N. piscinale would be a positive alternative source of nitrogen due to the results obtained from the soil nitrogen composition and soil pH.
... The absorbance of each extract solution was determined at 664 nm, 647 nm, and 480 nm using a spectrophotometer (PV1; Shanghai Mapada Instruments Co., Ltd..; Shanghai, China). The total chlorophyll (total chl), chlorophyll a (chl a), chlorophyll b (chl b), and carotenoids in each kale sample were calculated and compared to their fresh weight (FW) according to Porra et al. [16] and Wellburn [17]. In addition, the chl a-to-chl b ratio was calculated. ...
... However, as the UV-A intensity increased to 15 W/m 2 , the efficiency of photosystem II declined (Figure 4b). The increase in Y(II) indicated that kale was able to absorb the UV-A for utilization in photosystem II, as evidenced by the increase in photochemical quenching (qP), as shown on Figure 4c, where qP is the fraction of absorbed light energy that is used for photochemistry in photosynthesis [16]. Photoreceptors that respond to UV-A might play a role in the signaling pathways that enhance PSII and photoprotective activities. ...
UV-A (315–400 nm) is a component of solar radiation impacting plant physiology. Consequently, the impact was investigated of UV-A supplementation on various aspects of kale cultivation in a plant factory with artificial lighting. Three UV-A intensity treatments (5, 10, or 15 W/m2) were applied to kale plants for 5 days before harvesting. The results revealed that 5 or 10 W/m2 UV-A supplementation provided significant advantages for kale growth. The fresh weight of the kale shoots increased by over 35–50% after UV-A exposure. In particular, the 10 W/m2 UV-A exposure significantly improved the photosynthetic rate, water use efficiency and quantum efficiency of photosystem II (Y(II)). In addition, the treatment with UV-A positively impacted changes in leaf spectral indices, resulting in decreases in the simple ratio index (SR), the nor-malized phaeophytinization index (NPQI), and the normalized difference red edge index (NDRE). Furthermore, the phytonutrients in kale (vitamin C, soluble protein, and total phenolics) were enhanced using the 10 W/m2 UV-A treatment. One notable outcome was the consistent reduction in nitrate contents across all UV-A treatment intensities. Therefore, supplementing kale cultiva-tion with UV-A radiation was a valuable strategy for enhancing kale yield and quality in plant factory production.
... The chlorophyll a/b ratio remained constant at 3.75 as total concentration was altered. The maximum pigment concentration of the filters approximated concentrations that are found commonly in well-nourished leaves (Porra, Thompson, and Kriedemann, 1989). ...
... The disks were placed immediately into 8 mL of 100% methanol, and pigments were allowed to extract in the dark at 30ЊC for 24 h. Absorbances of the clear extract at 652.0, 665.2, and 750 nm were recorded and concentrations of chlorophylls a, b, and a ϩ b were computed after Porra, Thompson, and Kriedemann (1989). Chlorophyll concentration of the extract and the total disk surface area of 1.84 cm 2 were used to compute leaf chlorophyll concentrations per unit projected area. ...
A number of studies have linked responses in leaf spectral reflectance, transmittance, or absorptance to physiological stress. A variety of stressors including dehydration, flooding, freezing, ozone, herbicides, competition, disease, insects, and deficiencies in ectomycorrhizal development and N fertilization have been imposed on species ranging from grasses to conifers and deciduous trees. In all cases, the maximum difference in reflectance within the 400-850 nm wavelength range between control and stressed states occurred as a reflectance increase at wavelengths near 700 nm. In studies that included transmittance and absorptance as well as reflectance, maximum differences occurred as increases and decreases, respectively, near 700 nm. This common optical response to stress could be simulated closely by varying the chlorophyll concentration of model leaves (fiberglass filter pads) and by the natural variability in leaf chlorophyll concentrations in senescent leaves of five species. The optical response to stress near 700 nm, as well as corresponding changes in reflectance that occur in the green-yellow spectrum, can be explained by the general tendency of stress to reduce leaf chlorophyll concentration.
... Chlorophyll was extracted from C. urium and C. reinhardtii cells and analyzed according to Porra et al. (1989). The absorption at 750 nm (OD 750 ) was used as a reference. ...
... The absorption at 750 nm (OD 750 ) was used as a reference. Chla and Chlb were then photometrically quantified using the equation of Porra et al. (1989). ...
Autophagy is a central degradative pathway highly conserved among eukaryotes, including microalgae, which remains unexplored in extremophilic organisms. In this study, we described and characterized autophagy in the newly identified extremophilic green microalga Chlamydomonas urium, which was isolated from an acidic environment.
The nuclear genome of C. urium was sequenced, assembled and annotated in order to identify autophagy‐related genes. Transmission electron microscopy, immunoblotting, metabolomic and photosynthetic analyses were performed to investigate autophagy in this extremophilic microalga.
The analysis of the C. urium genome revealed the conservation of core autophagy‐related genes. We investigated the role of autophagy in C. urium by blocking autophagic flux with the vacuolar ATPase inhibitor concanamycin A. Our results indicated that inhibition of autophagic flux in this microalga resulted in a pronounced accumulation of triacylglycerols and lipid droplets (LDs). Metabolomic and photosynthetic analyses indicated that C. urium cells with impaired vacuolar function maintained an active metabolism. Such effects were not observed in the neutrophilic microalga Chlamydomonas reinhardtii.
Inhibition of autophagic flux in C. urium uncovered an active recycling of LDs through lipophagy, a selective autophagy pathway for lipid turnover. This study provided the metabolic basis by which extremophilic algae are able to catabolize lipids in the vacuole.
... After centrifugation for 10 min at 12,100 × g, absorbance was read at 646.6 and 663.6 nm for chlorophyll and at 480 nm for carotenoids. The concentrations of chlorophyll a and b (mg/g DW) and total carotenoids (%) were calculated according to Ref. 30 . The total phenol content of the methanolic extract was determined according to Ref. 31 . ...
In light of the multitude of olive trees cultivated and the lack of the genetic diversity of available genotypes to select varieties and lines that are characterized by high diversity and better performance under the corresponding conditions, A comparison analysis of the genotyping and morphological characteristics of eight olive cultivars growing in Saudi Arabia’s Al-Jouf region was conducted and analyzed. Morpho-anatomical and chemical characteristics along with both inter-simple-sequence repeats (ISSRs) and start-codon-targeted (SCoT) markers were used to evaluate the genetic diversity among eight olive varieties in Al-Jouf, Saudi Arabia. Analyses of 27 morphological, chemical, and anatomical characteristics concluded the existence of genetic differences among the studied varieties. Moreover, six ISSR and eight SCoT primer combinations produced a total of 48 loci, of which 18 (10 ISSR and 8 SCoT) were polymorphic. The average polymorphism information content (PIC values of 0.48 and 0.44, respectively) and marker index (MI of 0.79 and 0.48, respectively) detected for ISSR and SCoT markers revealed the prevalence of high genetic diversity among the studied olive varieties. Based on chemical and anatomical characteristics and the selected molecular markers, the eight olive cultivars were grouped into two distinct clusters. Clusters in the adjacent joint dendrogram produced using ISSR, SCoT and combined data were similar, and grouped all individuals into two groups. However, the dendrogram generated on the basis of SCoT separated individuals into subgroups containing at least two varieties. The findings showed that both methods were effective in assessing diversity, and that SCoT markers can be used as a reliable and informative method for assessing genetic diversity and relationships among olive varieties and can serve as a complementary tool to provide a more complete understanding of the genetic diversity available in Olea europaea populations in Saudi Arabia.
... 100mg of leaves were extracted overnight at 4°C using 10mL of 80% (v/v) acetone and the total chlorophyll levels were assessed following the previously described method (Porra et al., 1989). 100mg of leaves were extracted overnight at 4°C using 10mL of methanol-HCl (99:1) and the total anthocyanin levels were determined following the previously described procedure (Rabino and Mancinelli, 1986). ...
Aluminum (Al), prevalent in the crust of the Earth, jeopardizes plant health in acidic soils, hindering root growth and overall development. In this study, we first analysed the Al- and pH- tolerance of the Penicillium olsonii TLL1 strain (POT1; NRRL:68252) and investigated the potential for enhancing plant resilience under Al-rich acidic soil conditions. Our research illustrates the extraordinary tolerance of POT1 to both high Al concentrations and acidic conditions, showcasing its potential to alleviate Al-induced stress in plants. Metabolite analysis revealed that POT1 detoxifies Al through organic acid-dependent chelation mechanisms, significantly reducing Al stress in Arabidopsis and Pak Choi plants. Consequently, plant growth conditions improved, and the Al content in plant tissues decreased. Transcriptome analysis indicated that POT1 treatment downregulates genes associated with Al and oxidative stress such as MATE, ALS3, NIP1–2 and several peroxidases, highlighting its effectiveness in lessening Al-induced damage. Comparative assessments highlight the superior performance of POT1 compared to other Al-tolerant Penicillium species, attributed to its ability to thrive in diverse pH levels and effectively detoxify Al. These findings position POT1 as a promising agent for enhancing crop resilience in Al-compromised acidic soils, offering new avenues for promoting plant health and bolstering food security through increased crop yield and safety.
... Chlorophyll analysis was performed according to the method developed by Porra et al. (1989). Accordingly, chlorophyll a was determined at 646.6 nm and chlorophyll b was determined at 663.6 nm. ...
Microalgae are among the important microorganisms for a sustainable world as a source of renewable energy. In this study, three new microalgae were isolated from different regions of Türkiye and identified by molecular techniques. First isolate was Chlorella sorokiniana Shihira and Krauss, 1965 which was isolated from Dim River, second was Pseudochloris wilhelmii Somogyi et al., 2013 from Tokat and the third was Tetradesmus obliquus (Turpin) Wynne and Hallan, 2016 from Tunca River. The maximum biomass of C. sorokiniana was 1.02 g/L, 1.86 g/L for P. wilhelmii and 0.80 g/L for T. obliquus. The chlorophyll (a+b) concentrations were 0.146, 0.278 and 0.181 µg/mL for C. sorokiniana, P. wilhelmii and T. obliquus, respectively. The biotechnological utilization capacities of new isolates were revealed with the support of literature.
... In order to harness the potential of this trait in genotypic selection especially under stress conditions, the chlorophyll estimation should be reliable and high throughput especially in situations when the germplasm set to be evaluated is large sample and scale of experiment is big [7]. The estimation of leaf chlorophyll content is based on the destructive solvent extraction of chlorophyll from leaves followed by spectrophotometric determination using absorbances [8,9]. Equipments such as spectrophotometer, a fluorometer, or a high-performance liquid chromatography (HPLC) are often used to measure light absorptions at a various range of wavelength [10,11], which are then used to determine leaf chlorophyll. ...
... The absorbance (A) of extracted chlorophyll was measured at 663 and 645 nm, respectively (Spectramax mini, Molecular Devices, USA). Total chlorophyll contents were calculated using the following equation: Chl a + Chl b=8.04 * A663 + 20.29 * A645, and the results were expressed as micrograms per gram fresh weight (Porra et al., 1989). ...
Static magnetic field (SMF) plays important roles in various biological processes of many organisms including plants, though the molecular mechanism remains largely unclear. Here in this study, we evaluated different magnetic setups to test their effects on growth and development on Arabidopsis (Arabidopsis thaliana), and discovered that plant growth was significantly enhanced by inhomogeneous SMF generated by a regular triangular prism magnet perpendicular to the direction of gravity. Comparative transcriptomic analysis revealed that auxin synthesis and signal transduction genes were upregulated by SMF exposure. SMF also facilitated plants to maintain the iron homeostasis. The expression of iron metabolism-related genes was downregulated by SMF, however, the iron content in plant tissues remains relatively unchanged. Furthermore, SMF exposure also helped the plants to reduce ROS level and synergistically maintain the oxidant balance by enhanced activity of antioxidant enzymes and accumulation of nicotinamide. Taken together, our data suggested that SMF is involved in regulating the growth and development of Arabidopsis thaliana through maintaining iron homeostasis and balancing oxidative stress, which could be beneficial for plant survival and growth. The work presented here would extend our understanding of the mechanism and the regulatory network of how magnetic field affects the plant growth, which would provide insights into the development of novel plant synthetic biology technologies to engineer stress-resistant and high-yielding crops.
... Absorbance at 647 nm and 664 nm was measured using a spectrophotometer. Chlorophyll content was calculated according to the formula by Porra et al. (1989), and then normalized to the weight of the beans. ...
Key message
Hyperspectral features enable accurate classification of soybean seeds using linear discriminant analysis and GWAS for novel seed trait genes.
Abstract
Evaluating crop seed traits such as size, shape, and color is crucial for assessing seed quality and improving agricultural productivity. The introduction of the SUnSet toolbox, which employs hyperspectral sensor-derived image analysis, addresses this necessity. In a validation test involving 420 seed accessions from the Korean Soybean Core Collections, the pixel purity index algorithm identified seed- specific hyperspectral endmembers to facilitate segmentation. Various metrics extracted from ventral and lateral side images facilitated the categorization of seeds into three size groups and four shape groups. Additionally, quantitative RGB triplets representing seven seed coat colors, averaged reflectance spectra, and pigment indices were acquired. Machine learning models, trained on a dataset comprising 420 accession seeds and 199 predictors encompassing seed size, shape, and reflectance spectra, achieved accuracy rates of 95.8% for linear discriminant analysis model. Furthermore, a genome-wide association study utilizing hyperspectral features uncovered associations between seed traits and genes governing seed pigmentation and shapes. This comprehensive approach underscores the effectiveness of SUnSet in advancing precision agriculture through meticulous seed trait analysis.
... These findings are in accordance with previous research, which has consistently demonstrated that DMSO and 96% ethanol are highly efficient solvents for chlorophyll extraction [12,24,31]. Previous investigations have explored several methods for evaluating chlorophyll content in higher plant leaves, and several studies have substantiated that chlorophylls, which are lipid-soluble compounds found in plant tissues, can be effectively extracted using water-miscible organic solvents [4,16,[19][20][21][22][23][24][25][26][27]. ...
This study focuses on optimizing chlorophyll extraction techniques, in which leaf discs are cut from places on the leaf blade to enhance chlorophyll concentration in sesame (Sesamum indicum L.) leaves. Thirty sesame genotypes, categorized into light green (LG), middle green (MG), and deep green (DG) pigment groups based on leaf coloration, were selected from a larger pool of field-grown accessions. The investigation involved determining optimal Soil Plant Analysis Development (SPAD) value index measurements, quantifying pigment concentrations, exploring extraction solvents, and selecting suitable leaf disk positions. Significant variations in chlorophyll content were observed across genotypes, greenness categories, and leaf disk positions. The categorization of genotypes into DG, MG, and LG groups revealed a correlation between leaf appearance and chlorophyll content. The study highlighted a consistent relationship between carotenoids and chlorophyll, indicating their role in adaptation to warm environments. An examination of leaf disk positions revealed a significant chlorophyll gradient along the leaf blade, emphasizing the need for standardized protocols. Chlorophyll extraction experiments identified DMSO and 96% ethanol, particularly in those incubated for 10 min at 85 °C, as effective choices. This recommendation considers factors like cost-effectiveness, time efficiency, safety, and environmental regulations, ensuring consistent and simplified extraction processes. For higher chlorophyll extraction, focusing on leaf tips and the 75% localization along the sesame leaf blade is suggested, as this consistently yields increased chlorophyll content. Furthermore, our examination revealed significant anatomical variations in the internal structure of the mesophyll tissue leaves between deep green and light green sesame plants, primarily linked to chloroplast density and pigment-producing structures. Our findings, therefore, provide insightful knowledge of chlorophyll gradients and encourage the use of standardized protocols that enable researchers to refine their experimental designs for precise and comparable chlorophyll measurements. The recommended solvent choices ensure reliable outcomes in plant physiology, ecology, and environmental studies.
... Chlorophyll quanti cation was performed using a batch of 30-40 cotyledons, as described by Porra et al. (1989). ...
Nucleotides are the building blocks of living organisms and their biosynthesis must be tightly regulated. Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in GTP synthesis that is essential for biological activities, such as RNA synthesis. In animals, the suppression of IMPDH function causes ribosomal stress (also known as nucleolar stress), a disorder in ribosome biogenesis that results in cell proliferation defects and apoptosis. Despite its importance, plant IMPDH has not been analyzed in detail. Therefore, we analyzed the phenotypes of mutants of the two IMPDH genes in Arabidopsis thaliana and investigated their relationship with ribosomal stress. Double mutants of IMPDH1 and IMPDH2 were lethal, and only the impdh2 mutants showed growth defects and transient chlorophyll deficiency. These results suggested that IMPDH1 and IMPDH2 are redundant and essential, whereas IMPDH2 has a crucial role. In addition, the impdh2 mutants showed a reduction in nucleolus size and resistance to several translation inhibitors, which is a known response to ribosomal stress. Furthermore, the IMPDH1 / impdh1 impdh2 mutants showed more severe growth defects and phenotypes such as reduced plastid rRNA levels and abnormal processing patterns than the impdh2 mutants. Finally, multiple mutations of impdh with as2 , which has abnormal leaf polarity, caused the development of needle-like leaves because of the enhancement of the as2 phenotype, which is a typical effect observed in mutants of genes involved in ribosome biogenesis. These results indicated that IMPDH is closely related to ribosome biogenesis, and that mutations in the genes lead to not only known responses to ribosomal stress, but also plant-specific responses.
... Leaf samples (30 mg) were subjected to methanol extraction according to [57]. The photosynthetic pigments were determined as described by [58], using a microplate spectrophotometer (EPOCH, BioTek Instruments, Inc., Winooski, VT, USA) and measuring absorbances at 653 (chlorophyll a), 666 (chlorophyll b), and 470 nm (carotenoids) on a spectrophotometer (Genesys 10UV, Thermo Spectronic, Madison, WI, USA). ...
Boron (B) is a micronutrient crucial for the growth, development, productivity, and quality of crops. However, in areas characterized by acid soil (pHwater < 5.0) and high rainfall, soil B concentration tends to decrease, leading to insufficient supply to crops. This study was aimed at determining the optimal rate of B fertilization to enhance Vaccinium corymbosum L. performance in acid conditions. One-year-old cultivars with contrasting Al resistance (Al-sensitive Star and Al-resistant Cargo) were used. Plants were conditioned in plastic pots containing 18 L of half-ionic-strength Hoagland solution (pH 4.5) for 2 weeks. Thereafter, the following B treatments were applied foliarly: control, without B application (distilled water), 200, 400, and 800 mg L−1 of B as Solubor® for up to 72 h. Photosynthetic performance, root and shoot B levels, antioxidants, and oxidative stress were evaluated. Root and shoot B concentrations increased with the increasing B application, being higher in leaves than in roots of both cultivars. Net photosynthesis decreased at 800 mg L−1 B supply and effective quantum yield of PSII at 72 h in all B treatments. Lipid peroxidation increased in both cultivars at 800 mg L−1 B treatment. Antioxidant activity increased in all B treatments in both cultivars; while, at 400 and 800 mg L−1 B, total phenols increased in leaves of cultivar Star and decreased in cultivar Cargo. In conclusion, optimal B foliar application for highbush blueberry appears to be around 400 mg L−1 B. The appropriate B foliar application could help mitigate potential stress-induced problems in highbush blueberry cultivation. However, the optimal foliar B application should be confirmed in field experiments to help the farmers manage B nutrition.
... Chlorophyll measurement was done using methanol extraction, as previously described (Porra et al., 1989). Cells were grown in TAP under low light conditions, to avoid any possible secondary effects on the photosynthetic machinery in the mutants. ...
Translation of psbA , the chloroplast gene that encodes the D1 subunit of Photosystem II (PSII), is important for both PSII biogenesis and repair. The translation of the psbA transcript in the chloroplast is under the control of nuclear gene products. Using a Chlamydomonas forward genetic screen and whole genome sequencing, we found a mutant defective in PSII activity and mapped the causative gene to be the homolog of Arabidopsis High Fluorescence ( HCF244 ) gene, or CrHCF244 . We then demonstrated that CrHCF244 is required for psbA translation in the alga, consistent with the function of HCF244 in Arabidopsis . The Arabidopsis HCF244 gene also complemented the algal mutant. These results experimentally support the functional conservation of the homologs in green algae and land plants. However, these studies also revealed differences in psbA translation in Chlamydomonas and Arabidopsis . Loss of HCF244 in Arabidopsis results in a large decrease in chlorophyll. In contrast, there is no significant loss of chlorophyll in Chlamydomonas when CrHCF244 is knocked out. This observation supports the uncoupling of D1 translation and chlorophyll association in algae as reported recently in an ohp2 mutant, which is defective in chlorophyll delivery. Intriguingly, the CrHCF244 mutant also exhibited a relatively high rate of suppressor mutants, pointing to the presence of alternative pathway(s) for D1 translation control. The characterization of both the conserved aspects and the differences in psbA translation control between algae and plants will help elucidate how this process is regulated.
Highlight
We identified CrHCF244 as a translation factor of psbA in Chlamydomonas .
Characterization of this protein and genetic examinations of other previously identified psbA translation factors in Chlamydomonas reveal similarities and differences in psbA translation between Chlamydomonas and Arabidopsis .
... The powder of the rice seedlings (100 mg) was extracted with 1 mL of 0.1 mM NH4OH (containing 80% acetone). Chlorophyll concentrations were determined at wavelengths of 647, 644, and 750 nm using a spectrophotometer (MicroDigital Nabi, GyungGi, Republic of Korea) according to Porra et al. [64]. As for measuring the malondialdehyde (MDA) levels, the powder (50 mg) was extracted with 1.5 mL of reaction buffer containing 0.5% thiobarbituric acid and 20% trichloroacetic acid. ...
The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.
... The lycopene content of extracts was expressed as mg 100 g −1 FW. The β-carotene content was performed following the method described by Porra et al. [35], with minor modifications. Fresh samples (0.1 g) were extracted in 4 mL of 80% (v/v) acetone solution and agitated in the dark at 4 • C for 3 days. ...
Tomatoes are globally renowned for their nutritional value and culinary versatility. However, environmental stresses, particularly salinity, present significant challenges to tomato production, impacting both yield and fruit quality. In light of these challenges, this study investigates the effect of salinity on yield and fruit quality of a local cultivar tomato named ‘Pisanello’ in a closed soilless rockwool cultivation system. Total yield, fruit size, and number were investigated in both control (10 mM of NaCl) and salinity-treated plants (salinity 1 (S1)~30 mM of NaCl and salinity 2 (S2)~60 mM of NaCl), alongside various physicochemical parameters in fully ripened tomato fruits. The results indicated a decrease in crop production with rising sodium chloride concentration in the nutrient solution (25% and 41% for S1 and S2 treatment, respectively). Conversely, salinity-treated fruits exhibited an increase in total phenolic content of +21.9% in S1 and +36.7% in S2 and in antioxidant capacity (+33.5% and +34.7%, for the S1 and S2 treatments, respectively). Salinity treatments registered in general higher quality parameters such as titratable acidity (+8.9 for S1 and +16.5% for S2), total soluble solids (+18.5% for S1 and +43.0% for S2) and fruit firmness (+30.7% for S1 and +60.3% for S2) in comparison with control tomato fruits. Sensory profile analysis further validated the preference for fresh consumption of tomato fruits grown with saline water. These findings suggests that salinity stress can enhance the nutritional quality and taste of the Pisanello tomato. Further investigation could explore the optimal NaCl concentration to balance tomato production and nutritional quality.
... Absorbance measurements were performed at three wavelengths: 750 nm (turbidity), 665 nm (chlorophyll a), and 652 nm (chlorophyll b) using a V-1200 spectrophotometer (VWR International China Co., Ltd., Shanghai, China). Values were used to determine the chlorophyll a concentration (μg/mL) using Equation (1) [45]: ...
... sol. (ml) × toluene used (ml)} / {115.13 μg mole -1 g sample} Estimation of chlorophyll content: The youngest fully extended leaf sample was used to estimate the Chlorophyll contents following Porra et al. (1989). The equation for measuring the chlorophyll a, b, and total chlorophyll is: Here, A is the absorbance at specific wavelengths. ...
An experiment was conducted to investigate the impact of exogenous Ca in alleviating salinity stress of mungbean. Two mungbean genotypes, BD 6895 and BD 6905, were used with four different Ca levels (5, 10, 15 and 20 mM) under 10 dSm-1 salinity conditions in a hydroponic culture. The application of exogenous Ca positively influenced on overall plant growth and development. Although both genotypes exhibited similar plant heights, they gave the peak (56.65 and 48.6 cm for BD 6895 and BD 6905, respectively) at 10 mM Ca. Higher levels of exogenous Ca were associated with increased chlorophyll content. Maximum Chlorophyll (1.6 mg g-1) was observed at 15 mM Ca treatment. Furthermore, exogenous Ca application reduced Malondialdehyde levels (at 15 mM Ca: 1.14 and 1.69 μmole/g for BD 6895 and BD 6905, respectively). Both the genotypes exhibited a proline content pattern, demonstrating proline content upsurge with increased Ca application. Microscopic analysis revealed larger vascular areas with exogenous Ca (BD 6895: 402 µm, BD 6905: 398.3 µm) compared to smaller areas under salinity stress (BD 6895: 258.7 µm, BD 6905: 248.4 µm). Salinity stress induced changes in upper epidermis thickness, leaf tissue compactness, chloroplast breakdown and chlorosis in mungbean plants. However, exogenous Ca application counteracted these detrimental effects, enabling mungbean plants to thrive in saline conditions. In conclusion, the study highlights the positive influence of exogenous Ca (10-15 mM) in promoting mungbean growth and managing salt stress. Ann. Bangladesh Agric. (2023) 27 (2): 91-104
... Absorbance values at 710 nm were subtracted from these measurements. From these data, the pigment contents were calculated using equations from Porra et al. [101] for chlorophyll and from Wellburn [102] for Crt. The pigment content was normalized by the leaf area. ...
Many contaminated soils contain several toxic elements (TEs) in elevated contents, and plant–TE interactions can differ from single TE contamination. Therefore, this study investigated the impact of combined contamination (As, Cd, Pb, Zn) on the physiological and metabolic processes of lettuce. After 45 days of exposure, TE excess in soil resulted in the inhibition of root and leaf biomass by 40 and 48%, respectively. Oxidative stress by TE accumulation was indicated by markers—malondialdehyde and 5-methylcytosine—and visible symptoms of toxicity (leaf chlorosis, root browning) and morpho-anatomical changes, which were related to the change in water regime (water potential decrease). An analysis of free amino acids (AAs) indicated that TEs disturbed N and C metabolism, especially in leaves, increasing the total content of free AAs and their families. Stress-induced senescence by TEs suggested changes in gas exchange parameters (increase in transpiration rate, stomatal conductance, and intercellular CO2 concentration), photosynthetic pigments (decrease in chlorophylls and carotenoids), a decrease in water use efficiency, and the maximum quantum yield of photosystem II. These results confirmed that the toxicity of combined contamination significantly affected the processes of lettuce by damaging the antioxidant system and expressing higher leaf sensitivity to TE multicontamination.
... The assessment of photosynthetic pigment content in old leaves exposed to various Mn concentrations (5,35,100,165, and 230 µM) was conducted following the methodology outlined by Porra et al. [69]. For each sample, 0.2 g of fresh leaf material was placed into a glass test tube. ...
Manganese (Mn) is a heavy metal that can cause excessive Mn poisoning in plants, disrupting microstructural homeostasis and impairing growth and development. However, the specific response mechanisms of leaves to Mn poisoning have not been fully elucidated. This study revealed that Mn poisoning of soybean plants resulted in yellowing of old leaves. Physiological assessments of these old leaves revealed significant increases in the antioxidant enzymes activities (peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)) and elevated levels of malondialdehyde (MDA), proline, indoleacetic acid (IAA), and salicylic acid (SA), under 100 μM Mn toxicity. Conversely, the levels of abscisic acid (ABA), gibberellin 3 (GA3), and jasmonic acid (JA) significantly decreased. The Mn content in the affected leaves significantly increased, while the levels of Ca, Na, K, and Cu decreased. Transcriptome analysis revealed 2258 differentially expressed genes in the Mn-stressed leaves, 744 of which were upregulated and 1514 were downregulated; these genes included genes associated with ion transporters, hormone synthesis, and various enzymes. Quantitative RT-PCR (qRT-PCR) verification of fifteen genes confirmed altered gene expression in the Mn-stressed leaves. These findings suggest a complex gene regulatory mechanism under Mn toxicity and stress, providing a foundation for further exploration of Mn tolerance-related gene regulatory mechanisms in soybean leaves. Using the methods described above, this study will investigate the molecular mechanism of old soybean leaves’ response to Mn poisoning, identify key genes that play regulatory roles in Mn toxicity stress, and lay the groundwork for cultivating high-quality soybean varieties with Mn toxicity tolerance traits.
... Fresh leaves (0.08 g) were soaked in 2 mL 80% acetone for 24 h. The absorbance of the leaf extracts was measured at 663 and 645 nm using an ultraviolet-visible spectrophotometer (V-750; JASCO Co., Ltd., Japan), according to the method described by Porra et al. (1989). ...
Compared with conventional crop cultivation in greenhouses or fields, plant factories with artificial light (PFAL) have advantages in the highly efficient use of space, energy, and resources available for cultivation. However, few studies on environmental controls for improving the space use efficacy (SUE) of PFAL in the production of edamame, a vegetable soybean, have been reported. Therefore, developing an environmental control method for high productivity with minimal space and energy requirements is of high priority. The aims of this study were to (1) identify the optimal photosynthetic photon flux density (PPFD) and light quality to enhance the SUE of edamame at the vegetative growth stage, and (2) examine the effects of PPFD, light quality, and their interaction on edamame plant growth at the vegetative stage. SUE is defined as the crop biomass produced per unit cubic volume of cultivation during the growth period. We examined three PPFD treatments (300, 500, and 700 μmol m⁻² s⁻¹) with three color temperature LED lamps (3,000, 5,000, and 6,500 K), for a total of nine treatments. The results demonstrated that, under the same light quality treatment, higher PPFDs resulted in larger fresh and dry weights of all organs, higher stem length, and lower specific leaf area. Under the same PPFD treatment, a high ratio of blue (400–499 nm) to red (600–699 nm) photon flux density increased the plant height but decreased the projected leaf area. The values of SUE at 700 μmol m⁻² s⁻¹ increased by 213, 163, and 92% with 3,000, 5,000, and 6,500 K, respectively compared with those at 300 μmol m⁻² s⁻¹. The values of SUE at 700 μmol m⁻² s⁻¹ increased by 34 and 23% in 5,000 and 6,500 K treatments, respectively compared with that in the 3,000 K treatment. In conclusion, a combination of 700 μmol m⁻² s⁻¹ PPFD and 5,000 K color temperature is the suitable condition to increase the SUE of edamame at the vegetative growth stage in a PFAL.
... The supernatant was collected, and absorbances were taken for chlorophyll a (chl-a) and chlorophyll b (chl-b) at 663 nm and 645 nm with a multi-detection microplate reader, CYTATION3, BioTek USA. Total chlorophyll content was measured by adding chla and chl-b [63]. For transmission electron microscopy, 1-2 mm leaf pieces in fixative solution (2.5% glutaraldehyde in 0.1 M of phosphate buffer) were taken, and after dehydration, the samples were stained and imaged with a ThermoScientific Tecnai F20 microscope, USA [64]. ...
Salinity stress is a type of abiotic stress which negatively affects the signaling pathways and cellular compartments of plants. Melatonin (MT) has been found to be a bioactive compound that can mitigate these adverse effects, which makes it necessary to understand the function of MT and its role in salt stress. During this study, plants were treated exogenously with 100 µM of MT for 7 days and subjected to 200 mM of salt stress, and samples were collected after 1 and 7 days for different indicators and transcriptome analysis. The results showed that salt reduced chlorophyll contents and damaged the chloroplast structure, which was confirmed by the downregulation of key genes involved in the photosynthesis pathway after transcriptome analysis and qRT-PCR confirmation. Meanwhile, MT increased the chlorophyll contents, reduced the electrolyte leakage, and protected the chloroplast structure during salt stress by upregulating several photosynthesis pathway genes. MT also decreased the H2O2 level and increased the ascorbic acid contents and APX activity by upregulating genes involved in the ascorbic acid pathway during salt stress, as confirmed by the transcriptome and qRT-PCR analyses. Transcriptome profiling also showed that 321 and 441 DEGs were expressed after 1 and 7 days of treatment, respectively. The KEGG enrichment analysis showed that 76 DEGs were involved in the photosynthesis pathway, while 35 DEGs were involved in the ascorbic acid metabolism pathway, respectively. These results suggest that the ex-ogenous application of MT in plants provides important insight into understanding MT-induced stress-responsive mechanisms and protecting Brassica campestris against salt stress by regulating the photosynthesis and ascorbic acid pathway genes.
... Comparando os dados do padrão com os dos solventes utilizados nota-se que o extrato aquoso começa a absorver a clorofila em um comprimento de onda 235 nm e alcança o seu maior pico de absorção em 350 nm, isto pode estar associado à ISSN 2178-6925 7 absorção parcial que a água realiza pois abstrai parcialmente os pigmentos que tem afinidade com o meio aquoso e tendem a ser arrastadas com maior facilidade Mussi et al. (2003); Porra., (1989). ...
As plantas medicinais são produtoras de diversos metabólitos e substâncias, dentre estas pode-se citar a clorofila que confere a coloração verde a folhas das plantas. Dentre os gêneros botânicos e famílias botânicas utilizadas em experimentos científicos destacam-se a família Piperaceae e o gênero Piper. O gênero Piper e a espécie Piper marginatum Jacq. que possui em suas folhas um alto teor de clorofila o que confere a coloração verde nas mesmas e muitas das vezes este teor não é quantificado e dentre as técnicas utilizadas para se analisar o teor de concentração de clorofila destaca-se a técnica de Espectrofotometria no UV-Vis. O objetivo deste trabalho foi determinar a concentração de clorofila de diferentes extratos presente em Piper marginatum Jacq coletadas em Itacoatiara por meio da técnica de espectrofotometria no UV-Vis. As folhas de Piper marginatum Jacq. foram coletadas no Campus I da UFAM em Itacoatiara e foram levadas para o laboratório de Química onde foram limpas e submetidas ao processo de extração separadamente nos solventes (água e etanol) os extratos foram filtrados separadamente e armazenados em frasco âmbar e mantidos em temperatura adequada. Os extratos foram submetidos a diferentes tipos de testes: Teor de clorofila em UV-Vis, Refratividade, Condutividade elétrica e pH. As análises foram submetidas ao teste estatístico usando o software SISVAR para os parâmetros testados e as absorbâncias foram analisadas pelo software SciDavis. Os resultados obtidos avaliaram que o melhor solvente para extração de clorofila foi o etanol, todavia ambos extratos se apresentaram estáveis durante os ensaios. Estes resultados são um indicativo que a espécie possui estabilidade em ambos solventes todavia devem ser realizados testes complementares sobre a espécie.
... The acetone extract was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used for the analysis. The analysis was performed by measuring absorbance at 750.0, 636.6, 646.6, and 470.0 nm using a UV-Vis-NIR spectrophotometer (UV-2700, Shimadzu Corporation, Kyoto, Japan), and the concentrations of chlorophyll and carotenoids were determined using equations derived from the previous studies (Lichtenthaler, 1987;Porra et al., 1989). A reflectometer (RQ Flex plus, Merck Darmstadt, Germany) quantified the ascorbic acid in the leaves of plants in each treatment (Yamori et al., 2022). ...
While it is commonly understood that air temperature can greatly affect the process of photosynthesis and the growth of higher plants, the impact of root zone temperature (RZT) on plant growth, metabolism, essential elements, as well as key metabolites like chlorophyll and carotenoids, remains an area that necessitates extensive research. Therefore, this study aimed to investigate the impact of raising the RZT on the growth, metabolites, elements, and proteins of red leaf lettuce. Lettuce was hydroponically grown in a plant factory with artificial light at four different air temperatures (17, 22, 27, and 30°C) and two treatments with different RZTs. The RZT was raised 3°C above the air temperature in one group, while it was not in the other group. Increasing the RZT 3°C above the air temperature improved plant growth and metabolites, including carotenoids, ascorbic acids, and chlorophyll, in all four air temperature treatments. Moreover, raising the RZT increased Mg, K, Fe, Cu, Se, Rb, amino acids, and total soluble proteins in the leaf tissue at all four air temperatures. These results showed that raising the RZT by 3°C improved plant productivity and the metabolites of the hydroponic lettuce by enhancing nutrient uptake and activating the metabolism in the roots at all four air temperatures. Overall, this research demonstrates that plant growth and metabolites can be improved simultaneously with an increased RZT relative to air temperature. This study serves as a foundation for future research on optimizing RZT in relation to air temperature. Further recommended studies include investigating the differential effects of multiple RZT variations relative to air temperature for increased optimization, examining the effects of RZT during nighttime versus daytime, and exploring the impact of stem heating. This research has the potential to make a valuable contribution to the ongoing growth and progress of the plant factory industry and fundamental advancements in root zone physiology. Overall, this research demonstrates that plant growth and metabolites can be improved simultaneously with an increased RZT relative to air temperature. This study serves as a foundation for future research on optimizing RZT in relation to air temperature. Further recommended studies include investigating the differential effects of multiple RZT variations relative to air temperature for increased optimization, examining the effects of RZT during nighttime versus daytime, and exploring the impact of stem heating. This research has the potential to make a valuable contribution to the ongoing growth and progress of the plant factory industry and fundamental advancements in root zone physiology.
... Pigment and growth analysis: The content of photosynthetic pigments extracted in 80% acetone (Chl a and b, carotenoids in mg/g fresh leaves) was determined spectrophotometrically (Thermo scientific) following standard protocol [28]. ...
The current study was aimed to explore the effects of two priming treatments, Nano-Priming (NP) and Magneto-Priming (MP) on improving the drought tolerance potential in wheat. Study revealed increase in germination efficiency in both the priming treatments compared to control Unprimed (UP) seeds. NP seeds exhibited a significant improvement in parameters such as germination percentage, seedling length, and seedling vigor when compared to both MP and UP seeds. The impact of Drought Stress (DS) on photosynthetic efficiency measured using Chl a fluorescence parameters, revealed a drastic reduction in the number of active Reaction Centers (RC) per chlorophyll molecule, performance of water splitting complex at donor side of PSII (Fv/Fo) and plant Performance Index (PI) in UP and MP plants wheat plants. However, NP+DS plants seemed to have improved the efficiency of primary photochemistry of PSII by showing significantly lesser reduction in these parameters under DS, ultimately resulting in better growth. Furthermore, the investigation of the oxidative status of plants revealed a reduction in the activity of antioxidant enzymes in NP + DS plants, potentially linked to reduced levels of reactive oxygen species production when compared to UP + DS plants. These findings suggest NP as an effective yet simple way for augmenting the drought tolerance potential in wheat.
Nitrogen (N) deposition levels and the frequency of lead (Pb) contamination events are increasing globally. In an effort to improve our understanding of plant responses to these stressors, we investigated moss responses to single and combined Pb and N stress. Three mosses from different habitats (Syntrichia caninervis, Bryum argenteum and Plagiomnium acutum) were studied and simulated Pb/N single and complex stresses were applied to them indoors. The chlorophyll (Chl) content, osmotic adjustment substances content, and antioxidant enzyme activities were measured at 7, 14, 21, and 28 days. The results revealed that the tolerance of the three bryophyte species to Pb or N stress was in the order of P. acutum > B. argenteum > S. caninervis, which was closely related to the conditions of their respective natural habitats. S. caninervis and B. argenteum were stress tolerant for 7 days and P. acutum for 14 days. The bryophytes were tolerant to Pb or N stress after the contents of osmoregulatory substances and antioxidant enzyme activities increased; however, as toxicity accumulated over time, all three species suffered irreversible damage, as indicated by an abrupt decrease in the Chl content and osmoregulatory substances, as well as a sudden drop in antioxidant enzyme activities. Under the combined effects of Pb-N stress, the Chl content, osmoregulatory substance contents, and antioxidant enzyme activities were significantly higher in the N-loving P. acutum (N produced significant benefits) than in P. acutum exposed to Pb stress alone. This phenomenon is likely because Pb and N have antagonistic effects on the growth of P. acutum; thus, their recombination generates a counter-balancing effect. In the N-sensitive species, S. caninervis and B. argenteum (N caused obvious toxicity), the indicators were slightly better than under N tress alone (indicated by the reduction of membrane lipid peroxidation and increased osmoregulatory substance contents and enzyme activities), suggesting that there is a certain antagonistic effect exerted by the simultaneous addition of Pb and N. Therefore, the detrimental effects of a single abiotic stress (Pb or N) on bryophytes may be diminished under the combined conditions of N deposition and presence of the heavy metal, Pb.
Four cyanobacterial strains isolated from arsenic (As) contaminated area in Minas Gerais, Brazil namely: Pseudanabaena spp. CCM-UFV063 and CCM-UFV065, Tolypothrix sp CCM-UFV067, and Desmonostoc sp. CCM-UFV070 were grown on BG-11 and BG-110 culture media respectively supplemented with di-sodium hydrogen arsenate heptahydrate (As⁵⁺) at concentrations of 0, 1, 10, 100, 1000 and 5000 mg L⁻¹. The growth was measured by means of optical density daily for seven (7) days. Contents of chlorophyll a, carbohydrates, protein, and amino acids were also evaluated. Fluorescence Spectroscopy and ICP-OES were used to quantify As in cells. The cellular growth, based on OD750nm, was significantly lower (P < 0.05) in 5000 mg L⁻¹ As. Chlorophyll a, carbohydrates and protein contents were significantly (P < 0.05) lowest in 5000 mg L⁻¹ As. Amino Acid content of studied cyanobacteria varied and were higher in higher As concentrations except for Pseudanabaena sp. CCM-UFV065. Similarly, As cells accumulation was dependent on concentration and showed the order of 5000 mg L⁻¹ > 1000 mg L⁻¹ > 100 mg L⁻¹ > 10 mg L⁻¹ > 1 mg L⁻¹ All studied cyanobacteria showed ability to absorb As(V) especially Pseudanabaena sp. CCM-UFV065 and could all be potential isolates for bioremediation of As-contaminated waters.
Increasing the amount of cellular space allocated to plastids will lead to increases in the quality and yield of crop plants. However, mechanisms that allocate cellular space to plastids remain poorly understood. To test whether the tomato (Solanum lycopersicum L.) REDUCED CHLOROPLAST COVERAGE (SlREC) gene products serve as central components of the mechanism that allocates cellular space to plastids and contribute to the quality of tomato fruit, we knocked out the four-member SlREC gene family. We found that slrec mutants accumulated lower levels of chlorophyll in leaves and fruit, accumulated lower levels of carotenoids in flowers and fruits, allocated less cellular space to plastids in leaf mesophyll and fruit pericarp cells, and developed abnormal plastids in flowers and fruits. Fruit produced by slrec mutants initiated ripening later than wild type and produced abnormal levels of ethylene and ABA. Metabolome and transcriptome analyses of slrec mutant fruit indicated that the SlREC gene products markedly influence plastid-related gene expression, primary and specialized metabolism, and the response to biotic stress. Our findings and previous work with distinct species indicate that REC proteins help allocate cellular space to plastids in diverse species and cell types and, thus, play a central role in allocating cellular space to plastids. Moreover, the SlREC proteins are required for the high-level accumulation of chlorophyll and carotenoids in diverse organs, including fruit, promote the development of plastids, and influence fruit ripening by acting both upstream and downstream of ABA biosynthesis in a complex network.
Plants exposed to incidences of excessive temperatures activate heat-stress responses to cope with the physiological challenge and stimulate long-term acclimation1,2. The mechanism that senses cellular temperature for inducing thermotolerance is still unclear³. Here we show that TWA1 is a temperature-sensing transcriptional co-regulator that is needed for basal and acquired thermotolerance in Arabidopsis thaliana. At elevated temperatures, TWA1 changes its conformation and allows physical interaction with JASMONATE-ASSOCIATED MYC-LIKE (JAM) transcription factors and TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins for repressor complex assembly. TWA1 is a predicted intrinsically disordered protein that has a key thermosensory role functioning through an amino-terminal highly variable region. At elevated temperatures, TWA1 accumulates in nuclear subdomains, and physical interactions with JAM2 and TPL appear to be restricted to these nuclear subdomains. The transcriptional upregulation of the heat shock transcription factor A2 (HSFA2) and heat shock proteins depended on TWA1, and TWA1 orthologues provided different temperature thresholds, consistent with the sensor function in early signalling of heat stress. The identification of the plant thermosensors offers a molecular tool for adjusting thermal acclimation responses of crops by breeding and biotechnology, and a sensitive temperature switch for thermogenetics.
Photosynthetic organisms use sunlight as energy source but rely on respiration during the night and in non-photosynthetic tissues. Respiration is also active in photosynthetically active cells, where its role is still unclear due to a lack of viable mutants.
Plants lacking cytochrome c oxidase (complex IV) activity are generally lethal but were here isolated exploiting the possibility of generating knockout lines through vegetative propagation in the moss Physcomitrium patens .
The mutants showed severely impaired growth, with an altered composition of the respiratory apparatus and increased electron transfer through the alternative oxidase. The light phase of photosynthesis remained largely unaffected while the efficiency of carbon fixation was moderately reduced. Transcriptomic and metabolomic analyses showed that the disruption of the cytochrome pathway had broad consequences for carbon and nitrogen metabolism. A major alteration in nitrogen assimilation was observed with a general reduction in amino acid abundance. A partial rescue of the growth could be obtained by growing the plants with an external supply of amino acids but not with sugars, demonstrating that respiration in plant photosynthetic cells plays an essential role at the interface between carbon and nitrogen metabolism and a key role in providing carbon skeletons for amino acid biosynthesis.
Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria, and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.
Agriculture faces new, increasingly complicated challenges in the face of a constantly changing environment that causes different types of stress in crops, one of them being salinity. Numerous investigations have been carried out to understand the effect of salt stress on the physiology of plants, as well as different ways of dealing with it and creating tolerance and/or resistance so that the productivity of crops is not affected. Among these alternatives we find biostimulants. The biostimulant CBX-103 is composed of carboxylic acids, succinic acids, oligogalacturonides, among others, which are obtained through a controlled enzymatic fermentation of the plant extract of Saccharum officinarum L. In the present study it is demonstrated that foliar applications of the biostimulant CBX- 103 in Arabidopsis thaliana Col 0 plants grown under salt stress increase leaf proline content, significantly reducing the harmful effects of stress, which is demonstrated at a biometric level and regulating growth states. A pathway in the energy regulation of plants is proposed as a mode of action due to the increase in the accumulation of proline obtained and the effect of its components.
A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (¹O2*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of ¹O2* in photodamage and cell signaling, few studies directly correlate ¹O2* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of ¹O2* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of ¹O2* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of ¹O2* in chlorophyll-containing photosynthetic membranes. We find that the apparent ¹O2* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of ¹O2* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of ¹O2* during high light. Upon saturation of NPQ, the concentration of ¹O2* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).
It has been thoroughly documented, by using ³¹P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300–500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our ³¹P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.
Arabidopsis thaliana possesses two different ion‐export mechanisms in the plastid inner envelope membrane. Due to a genome duplication, the transport proteins are encoded by partly redundant loci: K ⁺ ‐efflux antiporter1 (KEA1) and KEA2 and mechanosensitive channel of small conductance‐like2 (MSL2) and MSL3. Thus far, a functional link between these two mechanisms has not been established. Here, we show that kea1msl2 loss‐of‐function mutants exhibit phenotypes such as slow growth, reduced photosynthesis and changes in chloroplast morphology, several of which are distinct from either single mutants and do not resemble kea1kea2 or msl2msl3 double mutants. Our data suggest that KEA1 and MSL2 function in concert to maintain plastid ion homeostasis and osmoregulation. Their interplay is critical for proper chloroplast development, organelle function, and plant performance.
The biological role of lipids goes far beyond the formation of a structural membrane bilayer platform for membrane proteins and controlling fluxes across the membranes. For example, in photosynthetic thylakoid membranes, lipids occupy well-defined binding niches within protein complexes and determine the structural organization of membrane proteins and their function by controlling generic physicochemical membrane properties. In this chapter, two-dimensional thin-layer chromatography (2D TLC) and gas chromatography (GC) techniques are presented for quantitative analysis of lipid classes and fatty acids in thylakoid membranes. In addition, lipid extraction methods from isolated thylakoid membranes and leaves are described together with a procedure for the derivatization of fatty acids to fatty acid methyl esters (FAME) that is required for GC analysis.
Photosynthetic activity is established during chloroplast biogenesis, but whether photosynthesis itself is required for chloroplast biogenesis has not been clarified. In this study we use 680nm red light to overexcite Photosystem II and disrupt photosynthesis in two conditional mutants (var2 and abc1k1) which reversibly arrests chloroplast biogenesis. During their biogenesis, chloroplasts import most proteins associated with photosynthesis. Some of these must be transported further across the thylakoid membrane into the thylakoid lumen by the evolutionarily conserved SEC (Secretory) and TAT (Twin Arginine Translocation) pathways, energized by ATP and the trans-thylakoid proton gradient, respectively. Most luminal thylakoid proteins are synthesized in the cytoplasm with bi-partite, cleavable targeting sequences (first for the chloroplast envelope, second for the thylakoid membrane). Two-stage cleavage of these peptides is a critical step of chloroplast biogenesis. Linked to the photosynthetic defect in var2 and abc1k1 under red light, six essential proteins at the thylakoid lumen side of Photosystems I and II were incompletely cleaved and intermediate forms accumulated to high levels in the mutants. The results show that the processing of a specific module of Photosystem-associated proteins and concomitantly progression of chloroplast biogenesis depend on active photosynthesis at the earliest stages of seedling development.
Publisher Summary This chapter presents detailed information on chlorophylls and carotenoids to give practical directions toward their quantitative isolation and determination in extracts from leaves, chloroplasts, thylakoid particles, and pigment proteins. The chapter focuses on the spectral characteristics and absorption coefficients of chlorophylls, pheophytins, and carotenoids, which are the basis for establishing equations to quantitatively determine them. Therefore, the specific absorption coefficients of the pigments are re-evaluated. This is achieved by using a two-beam spectrophotometer of the new generation, which allows programmed automatic recording and printing out of the proper wavelengths and absorbancy values. Several procedures have been developed for the separation of the photosynthetic pigments, including column (CC), paper (PC), and thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). All chloroplast carotenoids exhibit a typical absorption spectrum that is characterized by three absorption maxima (violaxanthin, neoxanthin) or two maxima with one shoulder (lutein and β-carotene) in the blue spectral region.
Abstract— Highly purified Chi a and b were prepared from spinach leaves in a short time by a combined use of the column chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B. The former chromatography eliminated carotenoids, phaeophytin and chlorophyllide, and the latter chromatography efficiently separated Chi a and b.
We found inconsistencies in the commonly used data for chlorophyll analysis in 80% acetone. Recently developed extinction coefficients for chlorophyll b in N,N-dimethylformamide (DMF) based on values from 80% acetone are low as a result of these inconsistencies. We determined extinction coefficients of chlorophyll a (Chl a) and chlorophyll b (Chl b) in DMF for wavelengths of 618 to 665 nanometers. The simultaneous equations necessary for quantifying Chl a, Chl b, or total Chl in DMF in the absence of other chlorophyllous pigments are: Chl a = 12.70A(664.5) - 2.79A(647); Chl b = 20.70 A(647) - 4.62A(664.5); total Chl = 17.90A(647) + 8.08A(664.5), where A = absorbance in 1.00 centimeter cuvettes and Chl = milligrams per liter.N,N-Dimethylformamide is a very convenient solvent for Chl extraction since it is effective on intact plant parts and Chl is quite stable in DMF. There was no difference in the amount of Chl extracted when plant tissue was stored for 1 or 3 days at three temperatures, with or without solvent added.
New equations are presented for spectrophotometric determination of chlorophylls, based on revised extinction coefficients of chlorophylls a, b, c1 and c2. These equations may be used for determining chlorophylls a and b in higher plants and green algae, chlorophylls a and c1 + c2 in brown algae, diatoms and chrysomonads, chlorophylls a and c2 in dinoflagellates and cryptomonads, and chlorophylls a, b, and c1 + c2 in natural phytoplankton.
1. A chlorophyll containing protein fraction ("structure protein"), insoluble in aqueous media at pH 10, has been isolated from unicellular green algae.
The allomerization of chlorophyll has been investigated by reversed-phase HPLC. Four major product peaks (two sets of doublets) are observed in the HPLC chromatogram. These are shown to be pairs of stereoisomers of two major components. Similar results have been obtained from the allomerization of fully deuterated chlorophyll a. Attempts to characterize the reaction products and to delineate the reaction mechanism were initially studied by the oxidation of chlorophyll in the absence of extraneous nucleophiles. The structure of the reaction products of chlorophyll allomerization were conclusively identified as 10-hydroxychlorophyll and the 10-methoxylactone by co-chromatography, NMR, and 252Cf plasma-desorption mass spectroscopy. Exploratory studies on the allomerization products of bacteriochlorophyll have also been carried out.
The spectral absorption properties of fully deuteriated chlorophylls a
and b, ordinary chlorophylls a and b, and methyl chlorophyllides a and b,
isolated in a state of high purity, were compared. Substitution of phytyl by
methyl did not affect the wavelengths of the absorption maxima or the ratio of
the absorption at the principal maxima. Deuteriation reduced the absorption in
proportion to the molecular weight of the deuteriated pigments. Deuteriation
also shifted the absorption maxima to slightly shorter wavelengths, an effect
attributable primarily to the 6 deuterium atoms attached to C atoms in the
conjugated system. (auth)
The ¹³ C‐NMR spectrum of chlorophyll a formed when excised etiolated maize leaves were greened in the presence of [1‐ ¹³ C]glutamate showed that the four methine bridge carbons (C‐5, C‐10, C‐15 and C‐20) and four pyrrole α‐carbons (C‐4, C‐9, C‐14 and C‐16) were considerably enriched relative to the natural‐abundance spectrum thus providing unequivocal proof of the operation of the C 5 pathway of 5‐aminolaevulinate formation during chlorophyll biosynthesis in maize. [The carbon numbering system used is that recommended by IUB, see Pure Appl. Chem. 51 , 2251–2304 (1979).]
The ¹³ C‐NMR spectrum of chlorophyll a formed during greening in the presence of [2‐ ¹³ C]glycine showed that little or no enrichment of the above‐mentioned methine bridge or pyrrole α‐carbons had occurred thus indicating that the Shemin pathway makes little or no contribution to chlorophyll formation in greening maize. There was, however, considerable enrichment of the methoxyl carbon of the methoxycarbonyl group attached to isocyclic ring carbon C‐13 ² . This shows that the C‐2 carbon of glycine is used to label the C 1 pool and subsequently the methyl ester carbon of magnesium‐protoporphyrin IX‐monomethyl ester which is an intermediate in chlorophyll biosynthesis.
The ¹³ C‐NMR spectrum of chlorophyll a obtained when greening occurs in the presence of [5‐ ¹³ C]glutamate showed considerable enrichment of the two carboxyl carbons of chlorophyll a (C‐13 ³ and C‐17 ³ ). This spectrum also showed that enrichment, equivalent to 15% of that occurring at the carboxyl carbons, occurred at the four methine bridge carbons and four relevant pyrrole α‐carbons. This is consistent with the conversion of [5‐ ¹³ C]glutamate to [1‐ ¹³ C]2‐oxoglutarate (or glutamate) via [5‐ ¹³ C]2‐oxoglutarate and recycling in the tricarboxylic acid cycle: the soformed 1‐ ¹³ C‐labelled substrate would then be incorporated into chlorophyll by the C 5 pathway. This assessment of the amount of [5‐ ¹³ C]glutamate incorporated into chlorophyll after recycling as 2‐oxoglutarate in the tricarboxylic acid cycle is used not only to confirm the view that the C 5 pathway is the major, possibly sole, route for chlorophyll biosynthesis in higher plants but is also used to negate previous assertions of the involvement of the Shemin pathway based on comparison of the incorporation of 2‐oxoglutarate ¹⁴ C‐labelled at C‐1 or C‐5.
In these studies we also found that chlorophyll a purified by silica‐gel thin‐layer chromatography contained allomerization products and was unsuitable for ¹³ C‐NMR spectroscopy.
Die am Botanischen Institut in Heidelberg seither gebruchliche Methode fr quantitative Chlorophyllbestimmungen wird berprft. Hierzu ist die Aufnahme der spezifischen Extinktionsspektren der Komponenten a und b in therischer Lsung erforderlich. Die fr die Berechnung der spez. Extinktionskoeffizienten bentigte. Kenntnis der Lsungskonzentration kann auf zweierlei Weise erlangt werden:a)
mit Hilfe von Magnesium-Mikrobestimmungen chromatographisch gereinigter Chlorophylle nach colorimetrischen und komplexometrischen Verfahren. Dabei liegen die Pigmente an keiner Stelle des Arbeitsganges in festem Zustand vor, die Aufarbeitung erfolgt unter denselben Bedingungen wie bei Serienbestimmungen.
b)
durch Einwaage von kuflichen Chlorophyllkristallisaten.
Der Vergleich der mittels a) und b) gefundenen Kurven ergibt Unterschiede, deren Ursache diskutiert wird. Fr die quantitative Bestimmung der Chlorophylle ist die nach a) ermittelte spez. Extinktionskurve vorzuzichen. Sie stimmt mit der vonSmith undBenitez (1955) verffentlichten berein.Die quantitativen Angaben vonSeybold u. Mitarb. (1937–1956) bedrfen einer Korrektur deren Ausma jedoch nichts Grundstzliches an den gewonnenen Erkenntnissen ndert.
The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-¹⁴C]glycine or 2-[1-¹⁴C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spheroides and Zea mays (maize), respectively, as examples of each pathway.
Orange, chlorophyll-deficient cells of Chlorella fusca were obtained by prolonged exposure (6 wk) to light and CO2 (1.5% in air) in a nitrogen-sparse medium: growth ceased after 6 days, chlorophyll formation after 3 days, and then chlorophyll degradation followed with a drop in chlorophyll a:b ratio. When the 6-wk-old cells were exposed to light in a nitrogen-rich medium and sparged with CO2 (1.5% in air) rapid chlorophyll synthesis ensued with preferential synthesis of chlorophyll a. Regreening under these conditions was complete in approximately 24–30 hr and during this period no cell division occurred. We were unable to demonstrate 5-aminolaevulinate synthase (EC 2.3.1.37) in cell-free extracts of regreening Chlorella but demonstrated aminolaevulinate formation by whole regreening cells incubated in the presence of laevulinate, an inhibitor of aminolaevulinate dehydratase (EC 4.2.1.24). Chlorophyll synthesis was almost completely inhibited by 100 mm laevulinate, and a stoichiometric relationship exists between aminolaevulinate formation and the chlorophyll deficit caused by the presence of laevulinate: thus, the use of the inhibitor provides a true indication of the ability of the cells to form aminolaevulinate.
A new method is deseribed for the extraction and determination of chlorophylls a and b. The method is suitable for use with both normal and regreening nitrogen-deficient Chlorella fusca. The assay involves extraction of chlorophylls by an alkaline pyridine reagent which converts the isocyclic ring of the pigment to a cyclic hydroxylactone. Millimolar extinetion coefficients for the hydroxylactone derivatives of both chlorophylls a and b have been determined at 419 and 454 nm. Using these coefficients, equations have been derived for the calculation of chlorophyll a and b concentrations. The new chlorophyll assay has been compared with other assays which involve the extraction of the pigments with 80% acetone or methanol. The new procedure extracts chlorophylls from rormally grown C. fusca more readily than methanol; the chlorophylls of normal Chlorella cannot be extracted with 80% acetone. The new assay is especially useful in the study of chlorophyll synthesis in regreening nitrogen-deficient C. fusca since the chlorophylls present in these deficient cells cannot be completely extracted with acetone, methanol, methanol-dimethylsulphoxide mixtures, or KOH-methanol.
1.1. Analysis by radial paper chromatography indicates that the principal pigments of the leaves of cocklebur, wheat, oats, barley, violet and Elodea are chlorophylls a and b, lutein with or without zeaxanthin, violaxanthin, neoxanthin and carotene. Additional xanthophylls such as neozeaxanthin, lutein epoxide and eloxanthin were not detectable. These results were confirmed by column chromatography using a variety of adsorbents such as powdered sugar, cellulose, starch, ZnCO3 and activated magnesia. The separability of the pigments and the chromatographic sequence varied with the adsorbent as well as with the solvent. Inseparability of neoxanthin and violaxanthin mixtures in columns of ZnCO3 with benzene as the wash liquid may account for some reports that leaves contain lutein epoxide but not neoxanthin and violaxanthin.2.2. The chromatographic behavior of the epoxy carotenoid pigments varies with the adsorbent. With magnesia as the absorbent, the epoxy carotenoids were less sorbed than the parent pigments, consequently the epoxy groups are less sorbed than the double bonds that they replace. Conversely, with sugar as the adsorbent, the epoxy carotenoids are more sorbed than the parent pigments, hence, on sugar, the epoxy groups are more sorbed than the double bonds that they replace. When treated with acids, the epoxides were not reconverted to the parent carotenoid pigments.3.3. The postulation that O2 evolution in photosynthetic organisms results from deoxygenation of xanthophyll epoxides is not supported by chemical experience and by the that fact epoxides do not occur in some autotrophic organisms. Accummulating evidence suggests a non-chemical role for the chloroplast pigments in photoxynthesis.
Green substances produced by enzymatic oxidation of chlorophylls a and b are shown by chemical analysis, chromatography, nmr, and infrared spectroscopy to be 10-hydroxychlorophylls. These compounds are also shown to be major products of the allomerization reaction of the chlorophylls in CH3OH. Similar allomerization of the fully deuterated chlorophylls, with H substituted at C-10 by normal exchange, provides the corresponding fully deuterated oxidation products with the C-10 H replaced by OH, as shown by nmr. These results provide critical evidence for the molecular structure of the oxidized chlorophylls and strong support for the structure of the methoxy lactones that are also formed in the allomerization reactions.
The phase test intermediate of methyl chlorophyllide a is generated in organic solvents by alkoxyl and hydroxyl ions. The intermediate is oxidized by oxygen and such oxidants as iodine, p-benzoquinone, and potassium permanganate. Different products are obtained depending on the solvent, the base used to generate the intermediate, and the oxidant. Three fractions are obtained from methanol containing oxygen and traces of magnesium methoxide. These are (1) Mg-purpurin 7-trimethyl ester, (2) Mg-purpurin 7-lactone-methyl ether-dimethyl ester, and (3) Mg-10-oxy-methyl pheophorbide a. The first and third fractions each make up 10–15% of the total oxidized pigment. Mg-"unstable" chlorin diester is obtained by oxidation with potassium permanganate of the phase test intermediate generated in pyridine by aqueous potassium hydroxide. Mg-10-methoxy-methyl pheophorbide a is obtained by oxidation with iodine of the phase test intermediate generated in methanol by magnesium methoxide (ca. 7%, w/v). It is concluded that allomerization of chlorophyll in alcoholic solution is due to oxidation by oxygen of traces of phase test intermediate.The visible and infrared absorption spectra of the allomerized derivatives are presented and discussed.
Photosynthetic pigments from etiolated cucumber (Cucumis sativus var. Beit Alpha improved, Hazera Co., Gedera) cotyledons were extracted by direct immersion of the intact cotyledons into the solvent N,N-dimethylformamide (DMF). The solvent is especially efficient when pigment concentration is low; time and tools are saved and the loss of pigment that usually occurs in more complicated extraction procedures is prevented. The specific absorption coefficient of chlorophyll a in DMF was also determined.
- Mackinney