The combined effects of copper and zinc on Arabidopsis involve differential regulation of chlorophyll synthesis and photosystem function.

Copper (Cu) and zinc (Zn) are both oxidation-reducing metal elements that are necessary for plant growth, and their effect often depends on their concentration. However, there are few studies that have investigated how plants are stressed and affected when the two ions are present simultaneously, especially when one ion is beneficial due to a low concentration and the other is detrimental due to a high concentration. To address this question, we treated Arabidopsis plants with either high or/and low concentrations of the two ions and investigated their mutual effects and the underlying molecular mechanism, focusing on photosynthetic function. The results showed that the photosynthetic pigment content and the performance of photosynthetic systems were most affected when both metal ions were present at detrimental concentrations (60 µM Cu for Cu60 and 350 µM Zn for Zn350). These include the effective openness of the photoreaction center, the electron transport rate and efficiency of photosystem II (PSII), the NPQ-dependent energy dissipation and the activity of photosystem I (PSI). However, when the harmful concentration of one of the two metals is combined with the beneficial concentration of the other metal (Cu5+Zn350 or Zn50+Cu60), these photosynthetic indicators are compensated to different degrees but the negative effects of copper ions at high dose are more difficult to eliminate than zinc ions. These results were also confirmed by gene expression analysis, which provides a clue to understanding the interaction between heavy metal ions, reducing metal toxicity and improving the tolerance of plants to heavy metals in practice.

A missense mutation in the barley Xan-h gene encoding the Mg-chelatase subunit I leads to a viable pale green line with reduced daily transpiration rate.

KEY MESSAGE: The barley mutant xan-h.chli-1 shows phenotypic features, such as reduced leaf chlorophyll content and daily transpiration rate, typical of wild barley accessions and landraces adapted to arid climatic conditions. The pale green trait, i.e. reduced chlorophyll content, has been shown to increase the efficiency of photosynthesis and biomass accumulation when photosynthetic microorganisms and tobacco plants are cultivated at high densities. Here, we assess the effects of reducing leaf chlorophyll content in barley by altering the chlorophyll biosynthesis pathway (CBP). To this end, we have isolated and characterised the pale green barley mutant xan-h.chli-1, which carries a missense mutation in the Xan-h gene for subunit I of Mg-chelatase (HvCHLI), the first enzyme in the CBP. Intriguingly, xan-h.chli-1 is the only known viable homozygous mutant at the Xan-h locus in barley. The Arg298Lys amino-acid substitution in the ATP-binding cleft causes a slight decrease in HvCHLI protein abundance and a marked reduction in Mg-chelatase activity. Under controlled growth conditions, mutant plants display reduced accumulation of antenna and photosystem core subunits, together with reduced photosystem II yield relative to wild-type under moderate illumination, and consistently higher than wild-type levels at high light intensities. Moreover, the reduced content of leaf chlorophyll is associated with a stable reduction in daily transpiration rate, and slight decreases in total biomass accumulation and water-use efficiency, reminiscent of phenotypic features of wild barley accessions and landraces that thrive under arid climatic conditions.

Brchli1 mutation induces bright yellow leaves by disrupting magnesium chelatase I subunit function in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Magnesium chelatase (MgCh) plays a pivotal role in photosynthesis, catalyzing the insertion of magnesium into protoporphyrin IX (Proto IX), a key intermediate in chlorophyll (Chl) biosynthesis. MgCh is a heteromeric complex composed of the MgCh D subunit (CHLD), the MgCh H subunit (CHLH), and the MgCh I subunit (CHLI). The bright yellow leaves (byl) mutant was obtained through ethyl methanesulfonate (EMS) mutagenesis of the 'FT' Chinese cabbage (Brassica rapa L. ssp. pekinensis) doubled haploid line, whose Chl content, net photosynthetic rate (Pn), and non-photochemical quenching coefficient (NPQ) were decreased, and whose chloroplast development was incomplete. byl recovered to a light green phenotype under weak light conditions. Genetic analysis revealed that the bright yellow leaves phenotype of byl was caused by a single recessive nuclear gene. Using Mutmap sequencing and Kompetitive allele-specific PCR (KASP) identification, BraA01g010040.3.5C, encoding the CHLI subunit of MgCh, was identified as the candidate gene and named Brchli1. A nonsynonymous G-to-A mutation in the Brchli1 exon resulted in the substitution of aspartic acid with asparagine. Brchli1-silenced Chinese cabbage displayed bright yellow leaves with decreased Brchli1 expression. Transiently overexpressed Brchli1 in the byl mutant restored the green leaf phenotype and significantly increased relative Brchli1 expression levels. Both BrCHLI1 and its mutated variant were localized in chloroplasts. Yeast two-hybrid and luciferase complementation imaging assays demonstrated that BrCHLI1 interacted with both BrCHLD and itself. BrCHLI1 mutations did not affect its interaction with BrCHLD. Together, Brchli1 mutations impaired the function of MgCh, providing insights into the molecular mechanism of leaf coloration.

OsNF-YB7 inactivates OsGLK1 to inhibit chlorophyll biosynthesis in rice embryo.

As a master regulator of seed development, Leafy Cotyledon 1 (LEC1) promotes chlorophyll (Chl) biosynthesis in Arabidopsis, but the mechanism underlying this remains poorly understood. Here, we found that loss of function of OsNF-YB7, a LEC1 homolog of rice, leads to chlorophyllous embryo, indicating that OsNF-YB7 plays an opposite role in Chl biosynthesis in rice compared with that in Arabidopsis. OsNF-YB7 regulates the expression of a group of genes responsible for Chl biosynthesis and photosynthesis by directly binding to their promoters. In addition, OsNF-YB7 interacts with Golden 2-Like 1 (OsGLK1) to inhibit the transactivation activity of OsGLK1, a key regulator of Chl biosynthesis. Moreover, OsNF-YB7 can directly repress OsGLK1 expression by recognizing its promoter in vivo, indicating the involvement of OsNF-YB7 in multiple regulatory layers of Chl biosynthesis in rice embryo. We propose that OsNF-YB7 functions as a transcriptional repressor to regulate Chl biosynthesis in rice embryo.

Low temperature and high humidity affect dynamics of chlorophyll biosynthesis and secondary metabolites in Cucumber.

BACKGROUND: During the cold season, low temperature (LT) and high relative humidity (HRH) are significant environmental factors in greenhouses and plastic tunnels, often hindering plant growth and development. The chlorophyll (Chl) biosynthesis inhibitory mechanisms under LT and HRH stress are still widely unclear. To understand how cucumbers seedlings respond to LT and HRH stress, we investigated the impact of these stressors on Chl biosynthesis. RESULTS: Our results revealed that individual LT, HRH and combined LT + HRH stress conditions affected chlorophyll a, b, total chlorophyll and carotenoid content, reducing the levels of these pigments. The levels of Chlorophyll precursors were also markedly reduced under LT and HRH stresses, with the greatest reduction observed in cucumber seedlings exposed to LT + HRH conditions (9/5℃, 95%HRH). The activities of glutamate-1-semialdehyde transaminase (GSA-AT), ALA dehydratase (ALAD), Mg-chelatase, and protochlorophyllide oxidoreductase (POR) were increased under individual LT, HRH, conditions but decreased by combination of LT + HRH stress condition. In addition, Chl biosynthesis related genes (except PBG) were upregulated by the HRH stress but were significantly downregulated under the LT + HRH stress condition in cucumber seedlings. Furthermore, the content of phenols, flavonoids and phenolic acids (cinnamic acid and caffeic acid) were significantly surged under LT + HRH treatment over the control. Histochemical observation showed higher O2- and H2O2 content in cucumber leaves during the LT and HRH stress. CONCLUSION: The results indicate that LT + HRH stress significantly impairs chlorophyll biosynthesis in cucumber seedlings by drastically reducing pigment accumulation, altering enzyme activity and gene expression. Additionally, LT + HRH stress induces oxidative damage, which further exacerbates the decline in chlorophyll content and affects overall cucumber metabolism.

Extracellular vesicle-mediated secretion of chlorophyll biosynthetic intermediates in the cyanobacterium Leptolyngbya boryana.

Extracellular vesicles (EVs) are derived from the outer membrane (OM) in Gram-negative bacteria and have diverse physiological functions. EV-mediated secretion of monovinyl protochlorophyllide (MV-Pchlide), the chlorophyll a (Chl) biosynthetic intermediate, was previously reported in a mutant lacking dark-operative Pchlide reductase in the cyanobacterium Leptolyngbya boryana. This study showed a detailed characterization of EVs from the wild-type (WT) of L. boryana grown under photoautotrophic and dark heterotrophic conditions, focusing on the accumulation of Chl intermediates. WT L. boryana cells produce two types of EVs, low-density EVs (L-EVs) and high-density EVs (H-EVs), both under light and dark conditions. L-EVs and H-EVs showed distinct morphological features and protein compositions. L-EVs from cells grown under both light and dark conditions commonly contained carotenoids, ketomyxol glycoside, and zeaxanthin, as major pigments. Based on the protein compositions of EVs and other cellular membrane fractions, L-EVs and H-EVs are probably derived from low-density OM and high-density OM interacting with cell walls, respectively. Fluorescence detection of pigments was applied to EVs, and the two Chl intermediates, protoporphyrin IX and protoporphyrin IX monomethyl ester, were commonly detected in both L-EVs from light- and dark-grown cells, whereas L-EVs from dark-grown cells contained additional MV-Pchlide, MV-protopheophorbide, and pheophorbide. The pigment ratios of L-EVs to the total culture medium of the Chl intermediates were much higher than those of carotenoids, suggesting an active transport of the Chl intermediates from the thylakoid membrane to L-EVs. Cyanobacterial EVs may play a novel role in alleviating the accumulation of Chl intermediates in cells. (248 words).

Integrating Physiology, Cytology, and Transcriptome to Reveal the Leaf Variegation Mechanism in Phalaenopsis Chia E Yenlin Variegata Leaves.

Phalaenopsis orchids, with their unique appearance and extended flowering period, are among the most commercially valuable Orchidaceae worldwide. Particularly, the variegation in leaf color of Phalaenopsis significantly enhances the ornamental and economic value and knowledge of the molecular mechanism of leaf-color variegation in Phalaenopsis is lacking. In this study, an integrative analysis of the physiology, cytology, and transcriptome profiles was performed on Phalaenopsis Chia E Yenlin Variegata leaves between the green region (GR) and yellow region (YR) within the same leaf. The total chlorophyll and carotenoid contents in the YR exhibited a marked decrease of 72.18% and 90.21%, respectively, relative to the GR. Examination of the ultrastructure showed that the chloroplasts of the YR were fewer and smaller and exhibited indistinct stromal lamellae, ruptured thylakoids, and irregularly arranged plastoglobuli. The transcriptome sequencing between the GR and YR led to a total of 3793 differentially expressed genes, consisting of 1769 upregulated genes and 2024 downregulated genes. Among these, the chlorophyll-biosynthesis-related genes HEMA, CHLH, CRD, and CAO showed downregulation, while the chlorophyll-degradation-related gene SGR had an upregulated expression in the YR. Plant-hormone-related genes and transcription factors MYBs (37), NACs (21), ERFs (20), bHLH (13), and GLK (2), with a significant difference, were also analyzed. Furthermore, qRT-PCR experiments validated the above results. The present work establishes a genetic foundation for future studies of leaf-pigment mutations and may help to improve the economic and breeding values of Phalaenopsis.

Red-light-dependent chlorophyll synthesis kindles photosynthetic recovery of chlorotic dormant cyanobacteria using a dark-operative enzyme.

Chlorosis dormancy resulting from nitrogen starvation and its resuscitation upon available nitrogen contributes greatly to the fitness of cyanobacterial population under nitrogen-fluctuating environments. The reinstallation of the photosynthetic machinery is a key process for resuscitation from a chlorotic dormant state; however, the underlying regulatory mechanism is still elusive. Here, we reported that red light is essential for re-greening chlorotic Synechocystis sp. PCC 6803 (a non-diazotrophic cyanobacterium) after nitrogen supplement under weak light conditions. The expression of dark-operative protochlorophyllide reductase (DPOR) governed by the transcriptional factor RpaB was strikingly induced by red light in chlorotic cells, and its deficient mutant lost the capability of resuscitation from a dormant state, indicating DPOR catalyzing chlorophyll synthesis is a key step in the photosynthetic recovery of dormant cyanobacteria. Although light-dependent protochlorophyllide reductase is widely considered as a master switch in photomorphogenesis, this study unravels the primitive DPOR as a spark to activate the photosynthetic recovery of chlorotic dormant cyanobacteria. These findings provide new insight into the biological significance of DPOR in cyanobacteria and even some plants thriving in extreme environments.

Proteomic and phosphoproteomic analysis of a Haematococcus pluvialis (Chlorophyceae) mutant with a higher heterotrophic cell division rate reveals altered pathways involved in cell proliferation and nutrient partitioning.

Haematococcus pluvialis has been used to produce the ketocarotenoid antioxidant, astaxanthin. Currently, heterotrophic cultivation of H. pluvialis is limited by slow growth rates. This work aimed to address this challenge by exploring the mechanisms of acetate metabolism in Haematococcus. Chemical mutagenesis and screening identified H. pluvialis strain KREMS 23D-3 that achieved up to a 34.9% higher cell density than the wild type when grown heterotrophically on acetate. An integrative proteomics and phosphoproteomics approach was employed to quantify 4955 proteins and 5099 phosphorylation sites from 2505 phosphoproteins in the wild-type and mutant strains of H. pluvialis. Among them, 12 proteins were significantly upregulated and 22 significantly downregulated in the mutant while phosphoproteomic analysis identified 143 significantly upregulated phosphorylation sites on 106 proteins and 130 downregulated phosphorylation sites on 114 proteins. Upregulation of anaphase-promoting complex phosphoproteins and downregulation of a putative cell cycle division 20 phosphoprotein in the mutant suggests rapid mitotic progression, coinciding with higher cell division rates. Upregulated coproporphyrinogen oxidase and phosphorylated magnesium chelatase in the mutant demonstrated altered nitrogen partitioning toward chlorophyll biosynthesis. The large proportion of differentially expressed phosphoproteins suggests phosphorylation is a key regulator for protein expression and activity in Haematococcus. Taken together, this study reveals the regulation of interrelated acetate metabolic pathways in H. pluvialis and provides protein targets that may guide future strain engineering work.

Light dependent protochlorophyllide oxidoreductase: a succinct look.

Reducing protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is a major regulatory step in the chlorophyll biosynthesis pathway. This reaction is catalyzed by light-dependent protochlorophyllide oxidoreductase (LPOR) in oxygenic phototrophs, particularly angiosperms. LPOR-NADPH and Pchlide form a ternary complex to be efficiently photo-transformed to synthesize Chlide and, subsequently, chlorophyll during the transition from skotomorphogenesis to photomorphogenesis. Besides lipids, carotenoids and poly-cis xanthophylls influence the formation of the photoactive LPOR complexes and the PLBs. The crystal structure of LPOR reveals evolutionarily conserved cysteine residues implicated in the Pchlide binding and catalysis around the active site. Different isoforms of LPOR viz PORA, PORB, and PORC expressed at different stages of chloroplast development play a photoprotective role by quickly transforming the photosensitive Pchlide to Chlide. Non-photo-transformed Pchlide acts as a photosensitizer to generate singlet oxygen that causes oxidative stress and cell death. Therefore, different isoforms of LPOR have evolved and differentially expressed during plant development to protect plants from photodamage and thus play a pivotal role during photomorphogenesis. This review brings out the salient features of LPOR structure, structure-function relationships, and ultra-fast photo transformation of Pchlide to Chlide by oligomeric and polymeric forms of LPOR.

Strigolactone and nitric oxide collaborate synergistically to boost tomato seedling resilience to arsenic toxicity via modulating physiology and antioxidant system.

Arsenic (As) poses a significant environmental threat as a metalloid toxin, adversely affecting the health of both plants and animals. Strigolactones (SL) and nitric oxide (NO) are known to play crucial roles in plant physiology. Therefore, the present experiment was designed to investigate the potential cumulative role of SL (GR24-0.20 µM) and NO (100 µM) in mitigating the adverse effect of AsV (53 µM) by modulating physiological mechanisms in two genotypes of tomato (Riogrand and Super Strain 8). A sample randomized design with four replicates was used to arrange the experimental pots in the growth chamber. 45-d old both tomato cultivars under AsV toxicity exhibited reduced morphological attributes (root and shoot length, root and shoot fresh weight, and root and shoot dry weight) and physiological and biochemical characteristics [chlorophyll (Chl) a and b content, activity of δ-aminolevulinic acid dehydratase activity (an enzyme responsible for Chl biosynthesis), and carbonic anhydrase activity (an enzyme responsible for photosynthesis), and enhanced Chl degradation, overproduction of reactive oxygen species (ROS) and lipid peroxidation due to enhanced malondialdehyde (MDA) content. However, the combined application of SL and NO was more effective in enhancing the tolerance of both varieties to AsV toxicity compared to individual application. The combined application of SL and NO improved growth parameters, biosynthesis of Chls, NO and proline. However, the combined application significantly suppressed cellular damage by inhibiting MDA and overproduction of ROS in leaves and roots, as confirmed by the fluorescent microscopy study and markedly upregulated the antioxidant enzymes (catalase, peroxidase, superoxide dismutase, ascorbate dismutase and glutathione reductase) activity. This study provides clear evidence that the combined application of SL and NO supplementation significantly improves the resilience of tomato seedlings against AsV toxicity. The synergistic effect of SL and NO was confirmed by the application of cPTIO (an NO scavenger) with SL and NO. However, further molecular studies could be imperative to conclusively validate the simultaneous role of SL and NO in enhancing plant tolerance to abiotic stress.

Fluorination of a conserved tyrosine in POR offers new clues for proton transfer.

Reduction of the 17,18-double bond in the D-ring during chlorophyll biosynthesis is catalyzed by the rare, naturally occurring photoenzyme protochlorophyllide oxidoreductase (POR). A conserved tyrosine residue has been suggested to donate a proton to C18 of the substrate in the past decades. Taylor and colleagues scrutinized the model with a powerful tool that utilized a modified genetic code to introduce fluorinated tyrosine analogues into POR. The presented results show that the suggested catalytically critical tyrosine is unlikely to participate in the reaction chemistry but is required for substrate binding, and instead, a cysteine residue preceding the lid helix is proposed to have the role of proton donor.

Rice OsGATA16 is a positive regulator for chlorophyll biosynthesis and chloroplast development.

Chloroplasts are essential organelles in plants that contain chlorophylls and facilitate photosynthesis for growth and development. As photosynthetic efficiency significantly impacts crop productivity, understanding the regulatory mechanisms of chloroplast development has been crucial in increasing grain and biomass production. This study demonstrates the involvement of OsGATA16, an ortholog of Arabidopsis GATA, NITRATE INDUCIBLE, CARBON-METABOLISM INVOLVED (GNC), and GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR 1 (GNL/CGA1), in chlorophyll biosynthesis and chloroplast development in rice (Oryza sativa). The osgata16-1 knockdown mutants produced pale-green leaves, while OsGATA16-overexpressed plants (OsGATA16-OE1) generated dark-green leaves, compared to their parental japonica rice. Reverse transcription and quantitative PCR analysis revealed downregulation of genes related to chloroplast division, chlorophyll biosynthesis, and photosynthesis in the leaves of osgata16-1 and upregulation in those of OsGATA16-OE1. Additionally, in vivo binding assays showed that OsGATA16 directly binds to the promoter regions of OsHEMA, OsCHLH, OsPORA, OsPORB, and OsFtsZ, and upregulates their expression. These findings indicate that OsGATA16 serves as a positive regulator controlling chlorophyll biosynthesis and chloroplast development in rice.

Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase.

The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme-substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water-mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme-substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme.

The UV-A Receptor CRY-DASH1 Up- and Downregulates Proteins Involved in Different Plastidial Pathways.

Algae encode up to five different types of cryptochrome photoreceptors. So far, relatively little is known about the biological functions of the DASH (Drosophila, Arabidopsis, Synechocystis and Homo)-type cryptochromes. The green alga Chlamydomonas reinhardtii encodes two of them. CRY-DASH1 also called DCRY1 has its maximal absorption peak in the UV-A range. It is localized in the chloroplast and plays an important role in balancing the photosynthetic machinery. Here, we performed a comparative analysis of chloroplast proteins from wild type and a knockout mutant of CRY-DASH1 named cry-dash1mut, using label-free quantitative proteomics as well as immunoblotting. Our results show upregulation of enzymes involved in specific pathways in the mutant including key enzymes of chlorophyll and carotenoid biosynthesis consistent with increased levels of photosynthetic pigments in cry-dash1mut. There is also an increase in certain redox as well as photosystem I and II proteins, including D1. Strikingly, CRY-DASH1 is coregulated in a D1 deletion mutant, where its amount is increased. In contrast, key proteins of the central carbon metabolism, including glycolysis/gluconeogenesis, dark fermentation and the oxidative pentose phosphate pathway are downregulated in cry-dash1mut. Similarly, enzymes of histidine biosynthesis are downregulated in cry-dash1mut leading to a reduction in the amount of free histidine. Yet, transcripts encoding for several of these proteins are at a similar level in the wild type and cry-dash1mut or even opposite. We show that CRY-DASH1 can bind to RNA, taking the psbA RNA encoding D1 as target. These data suggest that CRY-DASH1 regulates plastidial metabolic pathways at the posttranscriptional level.

Integrating the multiple functions of CHLH into chloroplast-derived signaling fundamental to plant development and adaptation as well as fruit ripening.

Chlorophyll (Chl)-mediated oxygenic photosynthesis sustains life on Earth. Greening leaves play fundamental roles in plant growth and crop yield, correlating with the idea that more Chls lead to better adaptation. However, they face significant challenges from various unfavorable environments. Chl biosynthesis hinges on the first committed step, which involves inserting Mg2+ into protoporphyrin. This step is facilitated by the H subunit of magnesium chelatase (CHLH) and features a conserved mechanism from cyanobacteria to plants. For better adaptation to fluctuating land environments, especially drought, CHLH evolves multiple biological functions, including Chl biosynthesis, retrograde signaling, and abscisic acid (ABA) responses. Additionally, it integrates into various chloroplast-derived signaling pathways, encompassing both retrograde signaling and hormonal signaling. The former comprises ROS (reactive oxygen species), heme, GUN (genomes uncoupled), MEcPP (methylerythritol cyclodiphosphate), ß-CC (ß-cyclocitral), and PAP (3'-phosphoadenosine-5'-phosphate). The latter involves phytohormones like ABA, ethylene, auxin, cytokinin, gibberellin, strigolactone, brassinolide, salicylic acid, and jasmonic acid. Together, these elements create a coordinated regulatory network tailored to plant development and adaptation. An intriguing example is how drought-mediated improvement of fruit quality provides insights into chloroplast-derived signaling, aiding the shift from vegetative to reproductive growth. In this context, we explore the integration of CHLH's multifaceted roles into chloroplast-derived signaling, which lays the foundation for plant development and adaptation, as well as fruit ripening and quality. In the future, manipulating chloroplast-derived signaling may offer a promising avenue to enhance crop yield and quality through the homeostasis, function, and regulation of Chls.

Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis.

Introduction: Plant growth and greening in response to light require the synthesis of photosynthetic pigments such as chlorophylls and carotenoids, which are derived from isoprenoid precursors. In Arabidopsis, the pseudo-etiolated-in-light phenotype is caused by the overexpression of repressor of photosynthetic genes 2 (RPGE2), which regulates chlorophyll synthesis and photosynthetic genes. Methods: We investigated a homologous protein in the Russian dandelion (Taraxacum koksaghyz) to determine its influence on the rich isoprenoid network in this species, using a combination of in silico analysis, gene overexpression, transcriptomics and metabolic profiling. Results: Homology-based screening revealed a gene designated pseudo-etiolated-in-light-like (TkPEL-like), and in silico analysis identified a light-responsive G-box element in its promoter. TkPEL-like overexpression in dandelion plants and other systems reduced the levels of chlorophylls and carotenoids, but this was ameliorated by the mutation of one or both conserved cysteine residues. Comparative transcriptomics in dandelions overexpressing TkPEL-like showed that genes responsible for the synthesis of isoprenoid precursors and chlorophyll were downregulated, probably explaining the observed pale green leaf phenotype. In contrast, genes responsible for carotenoid synthesis were upregulated, possibly in response to feedback signaling. The evaluation of additional differentially expressed genes revealed interactions between pathways. Discussion: We propose that TkPEL-like negatively regulates chlorophyll- and photosynthesis-related genes in a light-dependent manner, which appears to be conserved across species. Our data will inform future studies addressing the regulation of leaf isoprenoid biosynthesis and photomorphogenesis and could be used in future breeding strategies to optimize selected plant isoprenoid profiles and generate suitable plant-based production platforms.

Transcriptome and Biochemical Analyses of a Chlorophyll-Deficient Bud Mutant of Tea Plant (Camellia sinensis).

Tea leaf-color mutants have attracted increasing attention due to their accumulation of quality-related biochemical components. However, there is limited understanding of the molecular mechanisms behind leaf-color bud mutation in tea plants. In this study, a chlorina tea shoot (HY) and a green tea shoot (LY) from the same tea plant were investigated using transcriptome and biochemical analyses. The results showed that the chlorophyll a, chlorophyll b, and total chlorophyll contents in the HY were significantly lower than the LY's, which might have been caused by the activation of several genes related to chlorophyll degradation, such as SGR and CLH. The down-regulation of the CHS, DFR, and ANS involved in flavonoid biosynthesis might result in the reduction in catechins, and the up-regulated GDHA and GS2 might bring about the accumulation of glutamate in HY. RT-qPCR assays of nine DEGs confirmed the RNA-seq results. Collectively, these findings provide insights into the molecular mechanism of the chlorophyll deficient-induced metabolic change in tea plants.

Conformational variability of cyanobacterial ChlI, the AAA+ motor of magnesium chelatase involved in chlorophyll biosynthesis.

IMPORTANCE: Photosynthesis is an essential life process that relies on chlorophyll. In photosynthetic organisms, chlorophyll synthesis involves multiple steps and depends on magnesium chelatase. This enzyme complex is responsible for inserting magnesium into the chlorophyll precursor, but the molecular mechanism of this process is not fully understood. By using cryogenic electron microscopy and conducting functional analyses, we have discovered that the motor subunit ChlI of magnesium chelatase undergoes conformational changes in the presence of ATP. Our findings offer new insights into how energy is transferred from ChlI to the other components of magnesium chelatase. This information significantly contributes to our understanding of the initial step in chlorophyll biosynthesis and lays the foundation for future studies on the entire process of chlorophyll production.

Characterization and fine mapping of a yellow leaf gene regulating chlorophyll biosynthesis and chloroplast development in cotton (Gossypium arboreum).

Chlorophyll biosynthesis and chloroplast development are essential for photosynthesis and plant growth. Gossypium arboreum, a valuable source of genetic variation for cotton improvement, remains poorly studied for the mechanisms regulating chlorophyll biosynthesis and chloroplast development. Here we created a G. arboreum etiolated leaf and stuntedness (els) mutant that displayed a distinct yellow color of leaves, bracts and stems throughout the whole growth, where chlorophyll accumulation in leaves was reduced and chloroplast development was delayed. The GaCHLH gene, which encodes the H subunit of magnesium chelatase (Mg-chelatase), was screened by MutMap and KASP analysis. Compared to GaCHLH, the gene Gachlh of the mutant had a single nucleotide transition (G to A) at 1549 bp, which causes the substitution of a glycine (G) by a serine (S) at the 517th amino acid, resulting in an abnormal secondary structure of the Gachlh protein. GaCHLH-silenced SXY1 and ZM24 plants exhibited a lower GaCHLH expression level, a lower chlorophyll content, and the yellow-leaf phenotype. Gachlh expression affected the expression of key genes in the tetrapyrrole pathway. GaCHLH and Gachlh were located in the chloroplasts and that alteration of the mutation site did not affect the final target position. The BiFC assay result indicated that Gachlh could not bind to GaCHLD properly, which prevented the assembly of Mg-chelatase and thus led to the failure of chlorophyll synthesis. In this study, the Gachlh gene of G. arboreum els was finely localized and identified for the first time, providing new insights into the chlorophyll biosynthesis pathway in cotton.