Structural basis for enzymatic photocatalysis in chlorophyll biosynthesis.

The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth1-3. POR, which is one of three known light-dependent enzymes4,5, catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways.

A widespread coral-infecting apicomplexan with chlorophyll biosynthesis genes.

Apicomplexa is a group of obligate intracellular parasites that includes the causative agents of human diseases such as malaria and toxoplasmosis. Apicomplexans evolved from free-living phototrophic ancestors, but how this transition to parasitism occurred remains unknown. One potential clue lies in coral reefs, of which environmental DNA surveys have uncovered several lineages of uncharacterized basally branching apicomplexans1,2. Reef-building corals have a well-studied symbiotic relationship with photosynthetic Symbiodiniaceae dinoflagellates (for example, Symbiodinium3), but the identification of other key microbial symbionts of corals has proven to be challenging4,5. Here we use community surveys, genomics and microscopy analyses to identify an apicomplexan lineage-which we informally name 'corallicolids'-that was found at a high prevalence (over 80% of samples, 70% of genera) across all major groups of corals. Corallicolids were the second most abundant coral-associated microeukaryotes after the Symbiodiniaceae, and are therefore core members of the coral microbiome. In situ fluorescence and electron microscopy confirmed that corallicolids live intracellularly within the tissues of the coral gastric cavity, and that they possess apicomplexan ultrastructural features. We sequenced the genome of the corallicolid plastid, which lacked all genes for photosystem proteins; this indicates that corallicolids probably contain a non-photosynthetic plastid (an apicoplast6). However, the corallicolid plastid differs from all other known apicoplasts because it retains the four ancestral genes that are involved in chlorophyll biosynthesis. Corallicolids thus share characteristics with both their parasitic and their free-living relatives, which suggests that they are evolutionary intermediates and implies the existence of a unique biochemistry during the transition from phototrophy to parasitism.

Barley Viridis-k links an evolutionarily conserved C-type ferredoxin to chlorophyll biosynthesis.

Ferredoxins are single-electron carrier proteins involved in various cellular reactions. In chloroplasts, the most abundant ferredoxin accepts electrons from photosystem I and shuttles electrons via ferredoxin NADP+ oxidoreductase to generate NADPH or directly to ferredoxin dependent enzymes. In addition, plants contain other isoforms of ferredoxins. Two of these, named FdC1 and FdC2 in Arabidopsis thaliana, have C-terminal extensions and functions that are poorly understood. Here we identified disruption of the orthologous FdC2 gene in barley (Hordeum vulgare L.) mutants at the Viridis-k locus; these mutants are deficient in the aerobic cyclase reaction of chlorophyll biosynthesis. The magnesium-protoporphyrin IX monomethyl ester cyclase is one of the least characterized enzymes of the chlorophyll biosynthetic pathway and its electron donor has long been sought. Agroinfiltrations showed that the viridis-k phenotype could be complemented in vivo by Viridis-k but not by canonical ferredoxin. VirK could drive the cyclase reaction in vitro and analysis of cyclase mutants showed that in vivo accumulation of VirK is dependent on cyclase enzyme levels. The chlorophyll deficient phenotype of viridis-k mutants suggests that VirK plays an essential role in chlorophyll biosynthesis that cannot be replaced by other ferredoxins, thus assigning a specific function to this isoform of C-type ferredoxins.

Arabidopsis cryptochrome 1 controls photomorphogenesis through regulation of H2A.Z deposition.

Light is a key environmental cue that fundamentally regulates plant growth and development, which is mediated by the multiple photoreceptors including the blue light (BL) photoreceptor cryptochrome 1 (CRY1). The signaling mechanism of Arabidopsis thaliana CRY1 involves direct interactions with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)/SUPPRESSOR OF PHYA-105 1 and stabilization of COP1 substrate ELONGATED HYPOCOTYL 5 (HY5). H2A.Z is an evolutionarily conserved histone variant, which plays a critical role in transcriptional regulation through its deposition in chromatin catalyzed by SWR1 complex. Here we show that CRY1 physically interacts with SWC6 and ARP6, the SWR1 complex core subunits that are essential for mediating H2A.Z deposition, in a BL-dependent manner, and that BL-activated CRY1 enhances the interaction of SWC6 with ARP6. Moreover, HY5 physically interacts with SWC6 and ARP6 to direct the recruitment of SWR1 complex to HY5 target loci. Based on previous studies and our findings, we propose that CRY1 promotes H2A.Z deposition to regulate HY5 target gene expression and photomorphogenesis in BL through the enhancement of both SWR1 complex activity and HY5 recruitment of SWR1 complex to HY5 target loci, which is likely mediated by interactions of CRY1 with SWC6 and ARP6, and CRY1 stabilization of HY5, respectively.

Bilin-dependent regulation of chlorophyll biosynthesis by GUN4.

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, CrGUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding CrCHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXα reverses the loss of CrCHLH1 in the bilin-deficient heme oxygenase (hmox1) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4:bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment.

Functional Characterization of PmDXR, a Critical Rate-Limiting Enzyme, for Turpentine Biosynthesis in Masson Pine (Pinus massoniana Lamb.).

As one of the largest and most diverse classes of specialized metabolites in plants, terpenoids (oprenoid compounds, a type of bio-based material) are widely used in the fields of medicine and light chemical products. They are the most important secondary metabolites in coniferous species and play an important role in the defense system of conifers. Terpene synthesis can be promoted by regulating the expressions of terpene synthase genes, and the terpene biosynthesis pathway has basically been clarified in Pinus massoniana, in which there are multiple rate-limiting enzymes and the rate-limiting steps are difficult to determine, so the terpene synthase gene regulation mechanism has become a hot spot in research. Herein, we amplified a PmDXR gene (GenBank accession no. MK969119.1) of the MEP pathway (methyl-erythritol 4-phosphate) from Pinus massoniana. The DXR enzyme activity and chlorophyll a, chlorophyll b and carotenoid contents of overexpressed Arabidopsis showed positive regulation. The PmDXR gene promoter was a tissue-specific promoter and can respond to ABA, MeJA and GA stresses to drive the expression of the GUS reporter gene in N. benthamiana. The DXR enzyme was identified as a key rate-limiting enzyme in the MEP pathway and an effective target for terpene synthesis regulation in coniferous species, which can further lay the theoretical foundation for the molecularly assisted selection of high-yielding lipid germplasm of P. massoniana, as well as provide help in the pathogenesis of pine wood nematode disease.

The miR396-GRFs Module Mediates the Prevention of Photo-oxidative Damage by Brassinosteroids during Seedling De-Etiolation in Arabidopsis.

The switch from dark- to light-mediated development is critical for the survival and growth of seedlings, but the underlying regulatory mechanisms are incomplete. Here, we show that the steroids phytohormone brassinosteroids play crucial roles during this developmental transition by regulating chlorophyll biosynthesis to promote greening of etiolated seedlings upon light exposure. Etiolated seedlings of the brassinosteroids-deficient det2-1 (de-etiolated2) mutant accumulated excess protochlorophyllide, resulting in photo-oxidative damage upon exposure to light. Conversely, the gain-of-function mutant bzr1-1D (brassinazole-resistant 1-1D) suppressed the protochlorophyllide accumulation of det2-1, thereby promoting greening of etiolated seedlings. Genetic analysis indicated that phytochrome-interacting factors (PIFs) were required for BZR1-mediated seedling greening. Furthermore, we reveal that GROWTH REGULATING FACTOR 7 (GRF7) and GRF8 are induced by BZR1 and PIF4 to repress chlorophyll biosynthesis and promote seedling greening. Suppression of GRFs function by overexpressing microRNA396a caused an accumulation of protochlorophyllide in the dark and severe photobleaching upon light exposure. Additionally, BZR1, PIF4, and GRF7 interact with each other and precisely regulate the expression of chlorophyll biosynthetic genes. Our findings reveal an essential role for BRs in promoting seedling development and survival during the initial emergence of seedlings from subterranean darkness into sunlight.

Disruption of Hydrogen Gas Synthesis Enhances the Cellular Levels of NAD(P)H, Glycogen, Poly(3-hydroxybutyrate) and Photosynthetic Pigments Under Specific Nutrient Condition(s) in Cyanobacterium Synechocystis sp. PCC 6803.

In photoautotrophic Synechocystis sp. PCC 6803, NADPH is generated from photosynthesis and utilized in various metabolism, including the biosynthesis of glyceraldehyde 3-phosphate (the upstream substrate for carbon metabolism), poly(3-hydroxybutyrate) (PHB), photosynthetic pigments, and hydrogen gas (H2). Redirecting NADPH flow from one biosynthesis pathway to another has yet to be studied. Synechocystis's H2 synthesis, one of the pathways consuming NAD(P)H, was disrupted by the inactivation of hoxY and hoxH genes encoding the two catalytic subunits of hydrogenase. Such inactivation with a complete disruption of H2 synthesis led to 1.4-, 1.9-, and 2.1-fold increased cellular NAD(P)H levels when cells were cultured in normal medium (BG11), the medium without nitrate (-N), and the medium without phosphate (-P), respectively. After 49-52 d of cultivation in BG11 (when the nitrogen source in the media was depleted), the cells with disrupted H2 synthesis had 1.3-fold increased glycogen level compared to wild type of 83-85% (w/w dry weight), the highest level reported for cyanobacterial glycogen. The increased glycogen content observed by transmission electron microscopy was correlated with the increased levels of glucose 6-phosphate and glucose 1-phosphate, the two substrates in glycogen synthesis. Disrupted H2 synthesis also enhanced PHB accumulation up to 1.4-fold under -P and 1.6-fold under -N and increased levels of photosynthetic pigments (chlorophyll a, phycocyanin, and allophycocyanin) by 1.3- to 1.5-fold under BG11. Thus, disrupted H2 synthesis increased levels of NAD(P)H, which may be utilized for the biosynthesis of glycogen, PHB, and pigments. This strategy might be applicable for enhancing other biosynthetic pathways that utilize NAD(P)H.

DEEP GREEN PANICLE1 suppresses GOLDEN2-LIKE activity to reduce chlorophyll synthesis in rice glumes.

Understanding the regulation mechanisms of photosynthesis is key to improving its efficiency and, ultimately, crop yield. In this study, we report that DEEP GREEN PANICLE1 (DGP1) is involved in photosynthesis regulation in rice (Oryza sativa L.). We identified the dgp1 mutant, which has increased chlorophyll content in glumes. The mutated gene was isolated by map-based cloning. Knockout plants, generated using a gene editing approach, mimic the phenotype of dgp1. Overexpression of DGP1 leads to chlorotic leaves and glumes. DGP1 is a plant-specific protein with a conserved TIGR01589 domain. The expression of DGP1 was detected in green tissues and is induced by light. Moreover, genes involved in key steps of chlorophyll synthesis are upregulated in the glumes of dgp1. Importantly, we found that DGP1 interacts with the rice proteins GOLDEN2-LIKE1 (OsGLK1) and GOLDEN2-LIKE2 (OsGLK2), the two transcription factors involved in the regulation of photosynthesis. Transactivation assays showed that DGP1 represses the activation activity of OsGLK1 on its target genes. Our results demonstrate that DGP1 is a repressor of OsGLK activity and thus photosynthesis in rice. Manipulation of this gene and its homologs in other crops may provide new approaches for high photosynthetic efficiency breeding.

A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly.

Oxygenic photosynthesis relies on accessory factors to promote the assembly and maintenance of the photosynthetic apparatus in the thylakoid membranes. The highly conserved membrane-bound rubredoxin-like protein RubA has previously been implicated in the accumulation of both PSI and PSII, but its mode of action remains unclear. Here, we show that RubA in the cyanobacterium Synechocystis sp PCC 6803 is required for photoautotrophic growth in fluctuating light and acts early in PSII biogenesis by promoting the formation of the heterodimeric D1/D2 reaction center complex, the site of primary photochemistry. We find that RubA, like the accessory factor Ycf48, is a component of the initial D1 assembly module as well as larger PSII assembly intermediates and that the redox-responsive rubredoxin-like domain is located on the cytoplasmic surface of PSII complexes. Fusion of RubA to Ycf48 still permits normal PSII assembly, suggesting a spatiotemporal proximity of both proteins during their action. RubA is also important for the accumulation of PSI, but this is an indirect effect stemming from the downregulation of light-dependent chlorophyll biosynthesis induced by PSII deficiency. Overall, our data support the involvement of RubA in the redox control of PSII biogenesis.

Photosystem Biogenesis Is Localized to the Translation Zone in the Chloroplast of Chlamydomonas.

Intracellular processes can be localized for efficiency or regulation. For example, localized mRNA translation by chloroplastic ribosomes occurs in the biogenesis of PSII, one of the two photosystems of the photosynthetic electron transport chain in the chloroplasts of plants and algae. The biogenesis of PSI and PSII requires the synthesis and assembly of their constituent polypeptide subunits, pigments, and cofactors. Although these biosynthetic pathways are well characterized, less is known about when and where they occur in developing chloroplasts. Here, we used fluorescence microscopy in the unicellular alga Chlamydomonas reinhardtii to reveal spatiotemporal organization in photosystem biogenesis. We focused on translation by chloroplastic ribosomes and chlorophyll biosynthesis in two developmental contexts of active photosystem biogenesis: (1) growth of the mature chloroplast and (2) greening of a nonphotosynthetic chloroplast. The results reveal that a translation zone is the primary location of the biogenesis of PSI and PSII. This discretely localized region within the chloroplast contrasts with the distributions of photosystems throughout this organelle and, therefore, is likely a hub where anabolic pathways converge for photosystem biogenesis.plantcell;31/12/3057/FX1F1fx1.

ORANGE Represses Chloroplast Biogenesis in Etiolated Arabidopsis Cotyledons via Interaction with TCP14.

The conversion of etioplasts into chloroplasts in germinating cotyledons is a crucial transition for higher plants, enabling photoautotrophic growth upon illumination. Tight coordination of chlorophyll biosynthesis and photosynthetic complex assembly is critical for this process. ORANGE (OR), a DnaJ-like zinc finger domain-containing protein, was reported to trigger the biogenesis of carotenoid-accumulating plastids by promoting carotenoid biosynthesis and sequestration. Both nuclear and plastidic localizations of OR have been observed. Here, we show that Arabidopsis (Arabidopsis thaliana) OR physically interacts with the transcription factor TCP14 in the nucleus and represses its transactivation activity. Through this interaction, the nucleus-localized OR negatively regulates expression of EARLY LIGHT-INDUCIBLE PROTEINS (ELIPs), reduces chlorophyll biosynthesis, and delays development of thylakoid membranes in the plastids of germinating cotyledons. Nuclear abundance of OR decreased upon illumination. Together with an accumulation of TCP14 in the nucleus, this derepresses chloroplast biogenesis during de-etiolation. TCP14 is epistatic to OR and expression of ELIPs is directly regulated by the binding of TCP14 to Up1 elements in the ELIP promoter regions. Our results demonstrate that the interaction between OR and TCP14 in the nucleus leads to repression of chloroplast biogenesis in etiolated seedlings and provide new insights into the regulation of early chloroplast development.plantcell;31/12/2996/FX1F1fx1.

Cold acclimation can specifically inhibit chlorophyll biosynthesis in young leaves of Pakchoi.

BACKGROUND: Leaf color is an important trait in breeding of leafy vegetables. Y-05, a pakchoi (Brassica rapa ssp. chinensis) cultivar, displays yellow inner (YIN) and green outer leaves (GOU) after cold acclimation. However, the mechanism of this special phenotype remains elusive. RESULTS: We assumed that the yellow leaf phenotype of Y-05 maybe caused by low chlorophyll content. Pigments measurements and transmission electron microscopy (TEM) analysis showed that the yellow phenotype is closely related with decreased chlorophyll content and undeveloped thylakoids in chloroplast. Transcriptomes and metabolomes sequencing were next performed on YIN and GOU. The transcriptomes data showed that 4887 differentially expressed genes (DEGs) between the YIN and GOU leaves were mostly enriched in the chloroplast- and chlorophyll-related categories, indicating that the chlorophyll biosynthesis is mainly affected during cold acclimation. Together with metabolomes data, the inhibition of chlorophyll biosynthesis is contributed by blocked 5-aminolevulinic acid (ALA) synthesis in yellow inner leaves, which is further verified by complementary and inhibitory experiments of ALA. Furthermore, we found that the blocked ALA is closely associated with increased BrFLU expression, which is indirectly altered by cold acclimation. In BrFLU-silenced pakchoi Y-05, cold-acclimated leaves still showed green phenotype and higher chlorophyll content compared with control, meaning silencing of BrFLU can rescue the leaf yellowing induced by cold acclimation. CONCLUSIONS: Our findings suggested that cold acclimation can indirectly promote the expression of BrFLU in inner leaves of Y-05 to block ALA synthesis, resulting in decreased chlorophyll content and leaf yellowing. This study revealed the underlying mechanisms of leaves color change in cold-acclimated Y-05.

Effect of chlorophyll biosynthesis-related genes on the leaf color in Hosta (Hosta plantaginea Aschers) and tobacco (Nicotiana tabacum L.).

BACKGROUND: 'Regal Splendour' (Hosta variety) is famous for its multi-color leaves, which are useful resources for exploring chloroplast development and color changes. The expressions of chlorophyll biosynthesis-related genes (HrHEMA, HrPOR and HrCAO) in Hosta have been demonstrated to be associated with leaf color. Herein, we isolated, sequenced, and analyzed HrHEMA, HrPOR and HrCAO genes. Subcellular localization was also performed to determine the location of the corresponding enzymes. After plasmid construction, virus-induced gene silencing (VIGS) was carried out to reduce the expressions of those genes. In addition, HrHEMA-, HrPOR- and HrCAO-overexpressing tobacco plants were made to verify the genes function. Changes of transgenic tobacco were recorded under 2000 lx, 6000 lx and 10,000 lx light intensity. Additionally, the contents of enzyme 5-aminolevulinic acid (5-ALA), porphobilinogen (PBG), chlorophyll a and b (Chla and Chlb), carotenoid (Cxc), superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), proline (Pro) and catalase (CAT) under different light intensities were evaluated. RESULTS: The silencing of HrHEMA, HrPOR and HrCAO genes can induce leaf yellowing and chloroplast structure changes in Hosta. Specifically, leaves of Hosta with HrCAO silencing were the most affected, while those with HrPOR silencing were the least affected. Moreover, all three genes in tobacco were highly expressed, whereas no expression was detected in wild-type (WT). However, the sensitivities of the three genes to different light intensities were different. The highest expression level of HrHEMA and HrPOR was detected under 10,000 lx of illumination, while HrCAO showed the highest expression level under 6000 lx. Lastly, the 5-ALA, Chla, Cxc, SOD, POD, MDA, Pro and CAT contents in different transgenic tobaccos changed significantly under different light intensities. CONCLUSION: The overexpression of these three genes in tobacco enhanced photosynthesis by accumulating chlorophyll content, but the influential level varied under different light intensities. Furthermore, HrHEMA-, HrPOR- and HrCAO- overexpressing in tobacco can enhance the antioxidant capacity of plants to cope with stress under higher light intensity. However, under lower light intensity, the antioxidant capacity was declined in HrHEMA-, HrPOR- and HrCAO- overexpressing tobaccos.

Enhanced SA and Ca2+ signaling results in PCD-mediated spontaneous leaf necrosis in wheat mutant wsl.

Leaf is the major photosynthesis organ and the key source of wheat (Triticum aestivum L.) grain. Spotted leaf (spl) mutant is a kind of leaf lesion mimic mutants (LMMs) in plants, which is an ideal material for studying the mechanisms of leaf development. In this study, we report the leaf abnormal development molecular mechanism of a spl mutant named white stripe leaf (wsl) derived from wheat cultivar Guomai 301 (WT). Histochemical observation indicated that the leaf mesophyll cells of the wsl were destroyed in the necrosis regions. To explore the molecular regulatory network of the leaf development in mutant wsl, we employed transcriptome analysis, histochemistry, quantitative real-time PCR (qRT-PCR), and observations of the key metabolites and photosynthesis parameters. Compared to WT, the expressions of the chlorophyll synthesis and photosynthesis-related homeotic genes were repressed; many genes in the WRKY transcription factor (TF) families were highly expressed; the salicylic acid (SA) and Ca2+ signal transductions were enhanced in wsl. Both the chlorophyll contents and the photosynthesis rate were lower in wsl. The contents of SA and reactive oxygen species (ROS) were significantly higher, and the leaf rust resistance was enhanced in wsl. Based on the experimental data, a primary molecular regulatory model for leaf development in wsl was established. The results indicated that the SA accumulation and enhanced Ca2+ signaling led to programmed cell death (PCD), and ultimately resulted in spontaneous leaf necrosis of wsl. These results laid a solid foundation for further research on the molecular mechanism of leaf development in wheat.

Identification and fine genetic mapping of the golden pod gene (pv-ye) from the snap bean (Phaseolus vulgaris L.).

KEY MESSAGE: Using bulked segregant analysis combined with next-generation sequencing, we delimited the pv-ye gene responsible for the golden pod trait of snap bean cultivar A18-1. Sequence analysis identified Phvul.002G006200 as the candidate gene. The pod is the main edible part of snap beans (Phaseolus vulgaris L.). The commercial use of the pods is mainly affected by their color. Consumers seem to prefer golden pods. The aim of the present study was to identify the gene responsible for the golden pod trait in the snap bean. 'A18-1' (a golden bean cultivar) and 'Renaya' (a green bean cultivar) were chosen as the experimental materials. Genetic analysis indicated that a single recessive gene, pv-ye, controls the golden pod trait. A candidate region of 4.24 Mb was mapped to chromosome Pv 02 using bulked-segregant analysis coupled with whole-genome sequencing. In this region, linkage analysis in an F2 population localized the pv-ye gene to an interval of 182.9 kb between the simple sequence repeat markers SSR77 and SSR93. This region comprised 16 genes (12 annotated genes from the P. vulgaris database and 4 functionally unknown genes). Combined with transcriptome sequencing results, we identified Phvul.002G006200 as the potential candidate gene for pv-ye. Sequencing of Phvul.002G006200 identified a single-nucleotide polymorphism (SNP) in pv-ye. A pair of primers covering the SNP were designed, and the fragment was sequenced to screen 1086 F2 plants with the 'A18-1' phenotype. Our findings showed that among the 1086 mapped individuals, the SNP cosegregated with the 'A18-1' phenotype. The findings presented here could form the basis to reveal the molecular mechanism of the golden pod trait in the snap bean.

Distribution and functional analysis of the two types of 8-vinyl reductase involved in chlorophyll biosynthesis in marine cyanobacteria.

In the chlorophyll biosynthesis pathway, the 8-vinyl group of the chlorophyll precursor is reduced to an ethyl group by 8-vinyl reductase. Two isozymes of 8-vinyl reductase have been described in oxygenic photosynthetic organisms: one encoded by BciA and another by BciB. Only BciB contains an [Fe-S] cluster and most cyanobacteria harbor this form; whereas a few contain BciA. Given this disparity in distribution, cyanobacterial BciA has remained largely overlooked, which has limited understanding of chlorophyll biosynthesis in these microorganisms. Here, we reveal that cyanobacterial BciA encodes a functional 8-vinyl reductase, as evidenced by measuring the in vitro activity of recombinant Synechococcus and Acaryochloris BciA. Genomic comparison revealed that BciB had been replaced by BciA during evolution of the marine cyanobacterium Synechococcus, and coincided with replacement of Fe-superoxide dismutase (SOD) with Ni-SOD. These findings imply that the acquisition of BciA confers an adaptive advantage to cyanobacteria living in low-iron oceanic environments.

Transcriptomic and physiological analysis of OsCAO1 knockout lines using the CRISPR/Cas9 system in rice.

KEY MESSAGE: The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T0 plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type 'Dongjin' plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout of OsCAO1 influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests that OsCAO1 also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.

Phytoplasma-induced Changes in the Acetylome and Succinylome of Paulownia tomentosa Provide Evidence for Involvement of Acetylated Proteins in Witches' Broom Disease.

Lysine acetylation and succinylation are post-translational modifications of proteins that have been shown to play roles in plants response to pathogen infection. Phytoplasma infection can directly alter multiple metabolic processes in the deciduous plant Paulownia and lead to Paulownia witches' broom (PaWB) disease, the major cause of Paulownia mortality worldwide. However, the extent and function of lysine aceylation and succinylation during phytoplasma infection have yet to be explored. Here, we investigated the changes in the proteome, acetylome, and succinylome of phytoplasma-infected Paulownia tomentosa seedlings using quantitative mass spectrometry. In total, we identified 8963 proteins, 2893 acetylated proteins (5558 acetylation sites), and 1271 succinylated proteins (1970 succinylation sites), with 425 (533 sites) simultaneously acetylated and succinylated. Comparative analysis revealed that 276 proteins, 546 acetylated proteins (741 acetylation sites) and 5 succinylated proteins (5 succinylation sites) were regulated in response to phytoplasma infection, suggesting that acetylation may be more important than succinylation in PaWB. Enzymatic assays showed that acetylation of specific sites in protochlorophyllide reductase and RuBisCO, key enzymes in chlorophyll and starch biosynthesis, respectively, modifies their activity in phytoplasma-infected seedlings. On the basis of these results, we propose a model to elucidate the molecular mechanism of responses to PaWB and offer a resource for functional studies on the effects of acetylation on protein function.

Hemolytic Activity in Relation to the Photosynthetic System in Chattonella marina and Chattonella ovata.

Chattonella species, C. marina and C. ovata, are harmful raphidophycean flagellates known to have hemolytic effects on many marine organisms and resulting in massive ecological damage worldwide. However, knowledge of the toxigenic mechanism of these ichthyotoxic flagellates is still limited. Light was reported to be responsible for the hemolytic activity (HA) of Chattonella species. Therefore, the response of photoprotective, photosynthetic accessory pigments, the photosystem II (PSII) electron transport chain, as well as HA were investigated in non-axenic C. marina and C. ovata cultures under variable environmental conditions (light, iron and addition of photosynthetic inhibitors). HA and hydrogen peroxide (H2O2) were quantified using erythrocytes and pHPA assay. Results confirmed that% HA of Chattonella was initiated by light, but was not always elicited during cell division. Exponential growth of C. marina and C. ovata under the light over 100 µmol m-2 s-1 or iron-sufficient conditions elicited high hemolytic activity. Inhibitors of PSII reduced the HA of C. marina, but had no effect on C. ovata. The toxicological response indicated that HA in Chattonella was not associated with the photoprotective system, i.e., xanthophyll cycle and regulation of reactive oxygen species, nor the PSII electron transport chain, but most likely occurred during energy transport through the light-harvesting antenna pigments. A positive, highly significant relationship between HA and chlorophyll (chl) biosynthesis pigments, especially chl c2 and chl a, in both species, indicated that hemolytic toxin may be generated during electron/energy transfer through the chl c2 biosynthesis pathway.