Regulation Of Chloroplast Development Research Articles

Bulked Segregant Analysis by Sequencing-Based Genetic Mapping of the Green Spotted Fruit Rind Regulating Locus in Wild Melon XNM020 Reveals Four Possible Candidate Genes

Fruit rind patterns are vital commercial quality traits in melon, in which the spotted or striped fruit rinds contribute to the commercial value of melon and can directly affect the choice of consumers. Although the spotted or non-spotted fruit rind pattern was studied in several cultivated melon accessions, the inheritance and regulating locus of this trait in wild melons are still unknown. Therefore, in this study, the inheritance and regulating loci of the green spotted fruit rind in a wild melon accession XNM020 were explored with F2 segregating populations derived from crossing between XNM020 and a cultivated melon XNM125. Segregating ratios of phenotypic data indicated that the green spotted fruit rind in XNM020 has a monogenic dominant inheritance. BSA-Seq showed that two potential genomic regions on chromosomes 4 (from 0.00 to 2.97 Mb) and 5 (from 0.00 to 2.34 Mb) regulate the formation of the green spotted fruit rind in wild melon XNM020. According to the annotations of polymorphic SNPs (single-nucleotide polymorphisms) and small InDels (insertions and deletions) in target genomic regions and the predicted gene functions, four genes MELO3C003316, MELO3C003375, MELO3C003388, and MELO3C014660 regulating chloroplast development or chlorophyll biosynthesis may be the best candidate genes. The results of this study enriched the inheritances of spotted fruit rinds in melon and also provided target genomic regions for marker-assisted selection breeding of melon focusing on fruit rinds.

Genome-Wide Analysis of the GLK Gene Family and Its Expression at Different Leaf Ages in the Citrus Cultivar Kanpei.

The GLK gene family plays a crucial role in the regulation of chloroplast development and participates in chlorophyll synthesis. However, the precise mechanism by which GLK contributes to citrus's chlorophyll synthesis remains elusive. The GLK gene family causes variations in the photosynthetic capacity and chlorophyll synthesis of different citrus varieties. In this study, we identified tissue-specific members and the key CcGLKs involved in chlorophyll synthesis. A total of thirty CcGLK transcription factors (TFs) were discovered in the citrus genome, distributed across all nine chromosomes. The low occurrence of gene tandem duplication events and intronic variability suggests that intronic variation may be the primary mode of evolution for CcGLK TFs. Tissue-specific expression patterns were observed for various GLK family members; for instance, CcGLK12 and CcGLK15 were specifically expressed in the skin, while CcGLK30 was specific to the ovary, and CcGLK10, CcGLK6, CcGLK21, CcGLK2, CcGLK18, CcGLK9, CcGLK28, and CcGLK8 were specifically expressed in the leaves. CcGLK4, CcGLK5, CcGLK11, CcGLK23, CcGLKl7, CcGLK26, and CcGLK20 may participate in the regulation of the ALA, prochlorophylate, protoporphyrin IX, Mg-protoporphyrin IX, Chl b, T-Chl, MG-ProtoIX ME, and POR contents in citrus.

YGL3 Encoding an IPP and DMAPP Synthase Interacts with OsPIL11 to Regulate Chloroplast Development in Rice

As the source of isoprenoid precursors, the plastidial methylerythritol phosphate (MEP) pathway plays an essential role in plant development. Here, we report a novel rice (Oryza sativa L.) mutant ygl3 (yellow-green leaf3) that exhibits yellow-green leaves and lower photosynthetic efficiency compared to the wild type due to abnormal chloroplast ultrastructure and reduced chlorophyll content. Map-based cloning showed that YGL3, one of the major genes involved in the MEP pathway, encodes 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, which is localized in the thylakoid membrane. A single base substitution in ygl3 plants resulted in lower 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity and lower contents of isopentenyl diphosphate (IPP) compared to the wild type. The transcript levels of genes involved in the syntheses of chlorophyll and thylakoid membrane proteins were significantly reduced in the ygl3 mutant compared to the wild type. The phytochrome interacting factor-like gene OsPIL11 regulated chlorophyll synthesis during the de-etiolation process by directly binding to the promoter of YGL3 to activate its expression. The findings provides a theoretical basis for understanding the molecular mechanisms by which the MEP pathway regulate chloroplast development in rice.

BPG4 regulates chloroplast development and homeostasis by suppressing GLK transcription factors and involving light and brassinosteroid signaling

Chloroplast development adapts to the environment for performing suitable photosynthesis. Brassinosteroids (BRs), plant steroid hormones, have crucial effects on not only plant growth but also chloroplast development. However, the detailed molecular mechanisms of BR signaling in chloroplast development remain unclear. Here, we identify a regulator of chloroplast development, BPG4, involved in light and BR signaling. BPG4 interacts with GOLDEN2-LIKE (GLK) transcription factors that promote the expression of photosynthesis-associated nuclear genes (PhANGs), and suppresses their activities, thereby causing a decrease in the amounts of chlorophylls and the size of light-harvesting complexes. BPG4 expression is induced by BR deficiency and light, and is regulated by the circadian rhythm. BPG4 deficiency causes increased reactive oxygen species (ROS) generation and damage to photosynthetic activity under excessive high-light conditions. Our findings suggest that BPG4 acts as a chloroplast homeostasis factor by fine-tuning the expression of PhANGs, optimizing chloroplast development, and avoiding ROS generation.

WLP3 Encodes the Ribosomal Protein L18 and Regulates Chloroplast Development in Rice

Plastid ribosomal proteins play a crucial role in the growth and development of plants, mainly in the gene expression and translation of key genes in chloroplasts. While some information is known about the regulatory processes of plastid ribosomal proteins in various plant species, there is limited knowledge about the underlying mechanisms in rice. In this study, ethyl methanesulfonate (EMS) mutagenesis was used to generate a new mutant called wlp3 (white leaf and panicle3), characterized by white or albino leaves and panicles, which exhibited this phenotype from the second leaf stage until tillering. Furthermore, after a certain period, the newly emerging leaves developed the same phenotype as the rice variety ZH11, while the albino leaves of wlp3 showed an incomplete chloroplast structure and significantly low chlorophyll content. A transition mutation (T to C) at position 380 was identified in the coding region of the LOC_Os03g61260 gene, resulting in the substitution of isoleucine by threonine during translation. WLP3 encodes the ribosomal L18 subunit, which is localized in the chloroplast. Complementation experiments confirmed that LOC_Os03g61260 was responsible for the albino phenotype in rice. WLP3 has high expression in the coleoptile, leaves at the three-leaf stage, and panicles at the heading stage. Compared to the wild-type (WT), wlp3 exhibited reduced chlorophyll synthesis and significantly decreased expression levels of genes associated with plastid development. Yeast two-hybrid (Y2H) analysis revealed that WLP3 interacts with other ribosomal subunits, to influence chloroplast development. These results contribute to a better understanding of the underlying molecular mechanisms of chloroplast development and plastid gene translation.

Plastid terminal oxidase is required for chloroplast biogenesis in barley.

Chloroplast biogenesis is critical for crop biomass and economic yield. However, chloroplast development is a very complicated process coordinated by cross-communication between the nucleus and plastids, and the underlying mechanisms have not been fully revealed. To explore the regulatory machinery for chloroplast biogenesis, we conducted map-based cloning of the Grandpa 1 (Gpa1) gene regulating chloroplast development in barley. The spontaneous mutation gpa1.a caused a variegation phenotype of the leaf, dwarfed growth, reduced grain yield, and increased tiller number. Genetic mapping anchored the Gpa1 gene onto 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. One gene (HORVU.MOREX.r3.2HG0213170) in the delimited region encodes a putative plastid terminal oxidase (PTOX) in thylakoid membranes, which is homologous to IMMUTANS (IM) of Arabidopsis. The IM gene is required for chloroplast biogenesis and maintenance of functional thylakoids in Arabidopsis. Using CRISPR technology and gene transformation, we functionally validated that the PTOX-encoding gene, HORVU.MOREX.r3.2HG0213170, is the causal gene of Gpa1. Gene expression and chemical analysis revealed that the carotenoid biosynthesis pathway is suppressed by the gpa1 mutation, rendering mutants vulnerable to photobleaching. Our results showed that the overtillering associated with the gpa1 mutation was caused by the lower accumulation of carotenoid-derived strigolactones (SLs) in the mutant. The cloning of Gpa1 not only improves our understanding of the molecular mechanisms underlying chloroplast biosynthesis but also indicates that the PTOX activity is conserved between monocots and dicots for the establishment of the photosynthesis factory.

Physiological, Cytological, and Transcriptomic Analysis of Magnesium Protoporphyrin IX Methyltransferase Mutant Reveal Complex Genetic Regulatory Network Linking Chlorophyll Synthesis and Chloroplast Development in Rice.

Functional defects in key genes for chlorophyll synthesis usually cause abnormal chloroplast development, but the genetic regulatory network for these key genes in regulating chloroplast development is still unclear. Magnesium protoporphyrin IX methyltransferase (ChlM) is a key rate-limiting enzyme in the process of chlorophyll synthesis. Physiological analysis showed that the chlorophyll and carotenoid contents were significantly decreased in the chlm mutant. Transmission electron microscopy demonstrated that the chloroplasts of the chlm mutant were not well developed, with poor, loose, and indistinct thylakoid membranes. Hormone content analysis found that jasmonic acid, salicylic acid, and auxin accumulated in the mutant. A comparative transcriptome profiling identified 1534 differentially expressed genes (DEGs) between chlm and the wild type, including 876 up-regulated genes and 658 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these DEGs were highly involved in chlorophyll metabolism, chloroplast development, and photosynthesis. Protein-protein interaction network analysis found that protein translation played an essential role in the ChlM gene-regulated process. Specifically, 62 and 6 DEGs were annotated to regulate chlorophyll and carotenoid metabolism, respectively; 278 DEGs were predicted to be involved in regulating chloroplast development; 59 DEGs were found to regulate hormone regulatory pathways; 192 DEGs were annotated to regulate signal pathways; and 49 DEGs were putatively identified as transcription factors. Dozens of these genes have been well studied and reported to play essential roles in chlorophyll accumulation or chloroplast development, providing direct evidence for the reliability of the role of the identified DEGs. These findings suggest that chlorophyll synthesis and chloroplast development are actively regulated by the ChlM gene. And it is suggested that hormones, signal pathways, and transcription regulation were all involved in these regulation processes. The accuracy of transcriptome data was validated by quantitative real-time PCR (qRT-PCR) analysis. This study reveals a complex genetic regulatory network of the ChlM gene regulating chlorophyll synthesis and chloroplast development. The ChlM gene's role in retrograde signaling was discussed. Jasmonic acid, salicylic acid, or their derivatives in a certain unknown state were proposed as retrograde signaling molecules in one of the signaling pathways from the chloroplast to nucleus.

The G2-Like gene family in Populus trichocarpa: identification, evolution and expression profiles

The Golden2-like (GLK) transcription factors are plant-specific transcription factors (TFs) that perform extensive and significant roles in regulating chloroplast development. Here, genome-wide identification, classification, conserved motifs, cis-elements, chromosomal locations, evolution and expression patterns of the PtGLK genes in the woody model plant Populus trichocarpa were analyzed in detail. In total, 55 putative PtGLKs (PtGLK1-PtGLK55) were identified and divided into 11 distinct subfamilies according to the gene structure, motif composition and phylogenetic analysis. Synteny analysis showed that 22 orthologous pairs and highly conservation between regions of GLK genes across P. trichocarpa and Arabidopsis were identified. Furthermore, analysis of the duplication events and divergence times provided insight into the evolutionary patterns of GLK genes. The previously published transcriptome data indicated that PtGLK genes exhibited distinct expression patterns in various tissues and different stages. Additionally, several PtGLKs were significantly upregulated under the responses of cold stress, osmotic stress, and methyl jasmonate (MeJA) and gibberellic acid (GA) treatments, implying that they might take part in abiotic stress and phytohormone responses. Overall, our results provide comprehensive information on the PtGLK gene family and elucidate the potential functional characterization of PtGLK genes in P. trichocarpa.

A Golden2-like transcription factor, BnGLK1a, improves chloroplast development, photosynthesis, and seed weight in rapeseed

Enhancing photosynthetic efficiency is a major goal for improving crop yields under agricultural field conditions and is associated with chloroplast biosynthesis and development. In this study, we demonstrate that Golden2-like 1a (BnGLK1a) plays an important role in regulating chloroplast development and photosynthetic efficiency. Overexpressing BnGLK1a resulted in significant increases in chlorophyll content, the number of thylakoid membrane layers and photosynthetic efficiency in Brassica napus, while knocking down BnGLK1a transcript levels through RNA interference (RNAi) had the opposite effects. A yeast two-hybrid screen revealed that BnGLK1a interacts with the abscisic acid receptor PYRABACTIN RESISTANCE 1-LIKE 1–2 (BnPYL1–2) and CONSTITUTIVE PHOTOMORPHOGENIC 9 SIGNALOSOME 5A subunit (BnCSN5A), which play essential roles in regulating chloroplast development and photosynthesis. Consistent with this, BnGLK1a-RNAi lines of B. napus display hypersensitivity to the abscisic acid (ABA) response. Importantly, overexpression of BnGLK1a resulted in a 10% increase in thousand-seed weight, whereas seeds from BnGLK1a-RNAi lines were 16% lighter than wild type. We propose that BnGLK1a could be a potential target in breeding for improving rapeseed productivity. Our results not only provide insights into the mechanisms of BnGLK1a function, but also offer a potential approach for improving the productivity of Brassica species.

Identification of candidate genes that regulate the trade-off between seedling cold tolerance and fruit quality in melon (Cucumis melo L.).

Trade-offs between survival and growth are widely observed in plants. Melon is an annual, trailing herb that produces economically valuable fruits that are traditionally cultivated in early spring in China. Melon seedlings are sensitive to low temperatures, and thus usually suffer from cold stress during the early growth period. However, little is known about the mechanism behind the trade-offs between seedling cold tolerance and fruit quality in melon. In this study, a total of 31 primary metabolites were detected from the mature fruits of eight melon lines that differ with respect to seedling cold tolerance; these included 12 amino acids, 10 organic acids, and 9 soluble sugars. Our results showed that concentrations of most of the primary metabolites in the cold-resistant melons were generally lower than in the cold-sensitive melons; the greatest difference in metabolite levels was observed between the cold-resistant line H581 and the moderately cold-resistant line HH09. The metabolite and transcriptome data for these two lines were then subjected to weighted correlation network analysis, resulting in the identification of five key candidate genes underlying the balancing between seedling cold tolerance and fruit quality. Among these genes, CmEAF7 might play multiple roles in regulating chloroplast development, photosynthesis, and the ABA pathway. Furthermore, multi-method functional analysis showed that CmEAF7 can certainly improve both seedling cold tolerance and fruit quality in melon. Our study identified an agriculturally important gene, CmEAF7, and provides a new insight into breeding methods to develop melon cultivars with seedling cold tolerance and high fruit quality.

Transcriptomics and Metabolomics Analysis Provides Insight into Leaf Color and Photosynthesis Variation of the Yellow-Green Leaf Mutant of Hami Melon (Cucumis melo L.).

Leaf color mutants are ideal materials for studying the regulatory mechanism of chloroplast development and photosynthesis. We isolated a cucumis melo spontaneous mutant (MT), which showed yellow-green leaf phenotype in the whole growing period and could be inherited stably. We compared its leaves with the wild type (WT) in terms of cytology, physiology, transcriptome and metabolism. The results showed that the thylakoid grana lamellae of MT were loosely arranged and fewer in number than WT. Physiological experiments also showed that MT had less chlorophyll content and more accumulation of reactive oxygen species (ROS) than WT. Furthermore, the activity of several key enzymes in C4 photosynthetic carbon assimilation pathway was more enhanced in MT than WT. Transcriptomic and metabolomic analyses showed that differential expression genes and differentially accumulated metabolites in MT were mainly co-enriched in the pathways related to photosystem-antenna proteins, central carbon metabolism, glutathione metabolism, phenylpropanoid biosynthesis and flavonoid metabolism. We also analyzed several key proteins in photosynthesis and chloroplast transport by Western blot. In summary, the results may provide a new insight into the understanding of how plants respond to the impaired photosynthesis by regulating chloroplast development and photosynthetic carbon assimilation pathways.

A natural variation in the promoter of GRA117 affects carbon assimilation in rice.

GRA117 is crucial in the process of carbon assimilation in rice as it regulates the development of chloroplasts, which in turn facilitates the Calvin-Benson cycle. Carbon assimilation is a critical process for plant growth, and despite numerous relevant studies, there are still unknown constraints. In this study, we isolated a rice mutant, gra117, which exhibited seedling albinism, delayed chloroplast development, decreased chlorophyll content, reduced yield, and seedling stress susceptibility, as compared to WT. Our further investigations revealed that gra117 had a significantly lower net photosynthetic carbon assimilation rate, as well as reduced levels of Rubisco enzyme activity, RUBP, PGA, carbohydrate, protein content, and dry matter accumulation. These findings provide evidence for decreased carbon assimilation in gra117. By mapping cloning, we discovered a 665bp insertion in the GRA117 promoter region that decreases GRA117 transcriptional activity and causes the gra117 phenotype. GRA117 encodes PfkB-type fructokinase-like 2, which is subcellularly localized in chloroplasts and is widely expressed in various rice tissues, particularly at high levels in leaf tissues. GRA117 transcription is regulated by the core region 1029bp before the start codon. Our quantitative RT-PCR and Western blot assays showed that GRA117 promotes the expression and translation of photosynthetic genes. RNA-Seq analysis revealed that GRA117 plays a significant role in photosynthetic carbon fixation, carbon metabolism, and chloroplast ribosome-related pathways. Our study supports that GRA117 promotes the Calvin-Benson cycle by regulating chloroplast development, ultimately leading to enhanced carbon assimilation in rice.

OsDXR interacts with OsMORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice

Plant chlorophyll biosynthesis and chloroplast development are two complex processes regulated by exogenous and endogenous factors. In this study, we identified OsDXR , a gene expressing a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice. OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process. OsDXR was highly expressed in rice leaves, and subcellular localization indicated that OsDXR was a chloroplast protein. Many genes involved in chlorophyll biosynthesis and chloroplast development were differentially expressed in the OsDXR knock-out lines compared to the wild type. Moreover, we found that the RNA editing efficiency of ndhA -1019 and rpl2 -1 were significantly reduced in the OsDXR knock-out lines. Furthermore, OsDXR interacted with the RNA editing factor OsMORF1 in a yeast two-hybrid screen and bimolecular fluorescence complementation assay. We demonstrate that disruption of the plastidial 2-C-methyl-derythritol-4-phosphate pathway results in defects in chloroplast development and RNA editing of chloroplast genes.

Genomic Survey and Expression Analysis of GLKs in Watermelon (Citrullus lanatus)

Golden2-like (GLK) genes positively regulate chloroplast development, increase crop yields, and improve fruit quality. However, there has been no comprehensive identification and characterization of GLKs in watermelon. In this study, a total of 48 ClGLKs were identified in the watermelon genome. Based on phylogenetic analysis, they were divided into five groups. ClGLKs within the same group showed a similar gene structure and conserved motif compositions. Promoter analysis indicated that cis-elements responsive to light were the most abundant, though cis-elements associated with hormones, stress, and developmental regulation were also identified in ClGLKs promoters. Expression analysis indicated significant responses of some ClGLKs to drought and CGMMV stress, suggesting that these genes may participate in responses to biotic and abiotic stresses. Phenotypic analyses revealed enhanced chloroplast development and increased thylakoid density and chlorophyll content in the pericarp of a “dark green” watermelon cultivar. ClGLK8 was identified as the homolog of GLK1-2, the genes that promote chloroplast development and chlorophyll biosynthesis in fruits, and showed significantly increased expression in accordance with chloroplast development and chlorophyll accumulation. Our results provide detailed knowledge of the ClGLKs, which will enhance efforts to further improve the fruit quality of watermelon.

Arabidopsis Sec14 proteins (SFH5 and SFH7) mediate interorganelle transport of phosphatidic acid and regulate chloroplast development

Lipids establish the specialized thylakoid membrane of chloroplast in eukaryotic photosynthetic organisms, while the molecular basis of lipid transfer from other organelles to chloroplast remains further elucidation. Here we revealed the structural basis of Arabidopsis Sec14 homology proteins AtSFH5 and AtSFH7 in transferring phosphatidic acid (PA) from endoplasmic reticulum (ER) to chloroplast, and whose function in regulating the lipid composition of chloroplast and thylakoid development. AtSFH5 and AtSFH7 localize at both ER and chloroplast, whose deficiency resulted in an abnormal chloroplast structure and a decreased thickness of stacked thylakoid membranes. We demonstrated that AtSFH5, but not yeast and human Sec14 proteins, could specifically recognize and transfer PA invitro. Crystal structures of the AtSFH5-Sec14 domain in complex with L-α-phosphatidic acid (L-α-PA) and 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) revealed that two PA ligands nestled in the central cavity with different configurations, elucidating the specific binding mode of PA to AtSFH5, different from the reported phosphatidylethanolamine (PE)/phosphatidylcholine (PC)/phosphatidylinositol (PI) binding modes. Quantitative lipidomic analysis of chloroplast lipids showed that PA and monogalactosyldiacylglycerol (MGDG), particularly the C18 fatty acids at sn-2 position in MGDG were significantly decreased, indicating a disrupted ER-to-plastid (chloroplast) lipid transfer, under deficiency of AtSFH5 and AtSFH7. Our studies identified the role and elucidated the structural basis of plant SFH proteins in transferring PA between organelles, and suggested a model for ER-chloroplast interorganelle phospholipid transport from inherent ER to chloroplast derived from endosymbiosis of a cyanobacteriumproviding a mechanism involved in the adaptive evolution of cellular plastids.

Genome-Wide Analysis of the GLK Gene Family and the Expression under Different Growth Stages and Dark Stress in Sweet Orange (Citrus sinensis)

The GLK gene family is of great significance in regulating chloroplast development and participating in chlorophyll synthesis. However, the mechanism of GLK involvement in Citrus chlorophyll synthesis remains unclear. In this study, bioinformatics methods were used to analyze the gene structure, protein evolution, chromosome distribution, promoter elements and expression profile of GLK gene family in Citrus. Overall, 27 CsGLK TFs were identified from Citrus genome and divided into three subgroups according to the conserved domains. All members were distributed on nine chromosomes. The tandem replication events (ka/ks < 1) indicated that CsGLK TFs underwent a purification selection evolutionary process. The intron variation might be a vital configuration for the evolution of CsGLK genes. The expression pattern of CsGLKs showed that family members had higher expression levels in different tissues and at different growth stages and could actively respond to dark stress. CsGLK TFs of the same group had similar structures, but their expression patterns were quite different, indicating that they may have different functions and not be redundant. Correlation analysis showed that CsGLK2, CsGLK9, CsGLK10, CsGLK11, CsGLK20 and CsGLK24 were significantly positive correlations with Chl a and Chl b contents. In addition, CsGLK2, CsGLK5, CsGLK10, CsGLK11, CsGLK12, CsGLK15, CsGLK20 and CsGLK24 were significantly positive related to Mg-Proto IX, Proto IX and Pchl.

Integrating physiology, genetics, and transcriptome to decipher a new thermo-sensitive and light-sensitive virescent leaf gene mutant in cucumber.

Chloroplasts are the material basis of photosynthesis, and temperature and light severely affect chloroplast development and thus influence photosynthetic efficiency. This study identified a spontaneous virescent leaf mutant, SC311Y, whose cotyledons and true leaves were yellow and gradually turned green. However, temperature and light affected the process of turning green. In addition, this mutant (except at the seedling stage) had ruffled leaves with white stripes, sterile males, and poorly fertile female flowers. Genetic characteristics analysis revealed that the recessive gene controlled the virescent leaf. Two F2 populations mapped v-3 to the interval of 33.54–35.66 Mb on chromosome 3. In this interval, BSA-Seq, RNA-Seq, and cDNA sequence analyses revealed only one nonsynonymous mutation in the Csa3G042730 gene, which encoded the RNA exosome supercomplex subunit resurrection1 (RST1). Csa3G042730 was predicted to be the candidate gene controlling the virescent leaf, and the candidate gene may regulate chloroplast development by regulating plastid division2 (PDV2). A transcriptome analysis showed that different factors caused the reduced chlorophyll and carotenoid content in the mutants. To our knowledge, this study is the first report of map-based cloning related to virescent leaf, male-sterile, and chloroplast RNA regulation in cucumber. The results could accelerate the study of the RNA exosome supercomplex for the dynamic regulation of chloroplast RNA.

OsPPR9 encodes a DYW-type PPR protein that affects editing efficiency of multiple RNA editing sites and is essential for chloroplast development

Photosynthesis occurs mainly in chloroplasts, whose development is regulated by proteins encoded by nuclear genes. Among them, pentapeptide repeat (PPR) proteins participate in organelle RNA editing. Although there are more than 450 members of the PPR protein family in rice, only a few affect RNA editing in rice chloroplasts. Gene editing technology has created new rice germplasm and mutants, which could be used for rice breeding and gene function study. This study evaluated the functions of OsPPR9 in chloroplast RNA editing in rice. The osppr9 mutants were obtained by CRISPR/Cas9, which showed yellowing leaves and a lethal phenotype, with suppressed expression of genes associated with chloroplast development and accumulation of photosynthetic-related proteins. In addition, loss of OsPPR9 protein function reduces the editing efficiency of rps8-C182, rpoC2-C4106, rps14-C80, and ndhB-C611 RNA editing sites, which affects chloroplast growth and development in rice. Our data showed that OsPPR9 is highly expressed in rice leaves and encodes a DYW-PPR protein localized in chloroplasts. Besides, the OsPPR9 protein was shown to interact with OsMORF2 and OsMORF9. Together, our findings provide insights into the role of the PPR protein in regulating chloroplast development in rice.

STRIPE3, encoding a human dNTPase SAMHD1 homolog, regulates chloroplast development in rice

Chloroplast is an important organelle for photosynthesis and numerous essential metabolic processes, thus ensuring plant fitness or survival. Although many genes involved in chloroplast development have been identified, mechanisms underlying such development are not fully understood. Here, we isolated and characterized the stripe3 (st3) mutant which exhibited white-striped leaves with reduced chlorophyll content and abnormal chloroplast development during the seedling stage, but gradually produced nearly normal green leaves as it developed. Map-based cloning and transgenic tests demonstrated that a splicing mutation in ST3, encoding a human deoxynucleoside triphosphate triphosphohydrolase (dNTPase) SAMHD1 homolog, was responsible for st3 phenotypes. ST3 is highly expressed in the third leaf at three-leaf stage and expressed constitutively in root, stem, leaf, sheath, and panicle, and the encoded protein, OsSAMHD1, is localized to the cytoplasm. The st3 mutant showed more severe albino leaf phenotype under exogenous 1-mM dATP/dA, dCTP/dC, and dGTP/dG treatments compared with the control conditions, indicating that ST3 is involved in dNTP metabolism. This study reveals a gene associated with dNTP catabolism, and propose a model in which chloroplast development in rice is regulated by the dNTP pool, providing a potential application of these results to hybrid rice breeding.

WAL3 encoding a PLS-type PPR protein regulates chloroplast development in rice

Chloroplast development is a complex process that is critical for the growth and development of plants. Pentapeptide repeat (PPR) proteins contain large members but only few of them have been characterized in rice. In this study, we identified a new PLS-type protein, WAL3 (Whole Albino Leaf on Chromosome 3), playing important roles in plant chloroplast development. Knockout of WAL3 gene in Nipponbare variety caused abnormal chloroplast development and showed an albino lethal phenotype. Expression analysis showed that WAL3 gene was constitutively expressed with the highest expression in leaves. The WAL3 protein localized in chloroplasts and affected the splicing of multiple group II introns. Transcriptome sequencing showed that WAL3 involved in multiple metabolic pathways including the chlorophyll synthesis and photosynthetic related metabolic pathways. The decreased abundance of photosynthesis-related proteins in wal3 mutants indicated WAL3 influence photosynthesis. In summary, our study revealed that WAL3 is essential for chloroplast development in rice.