Rice TCD8 Encoding a Multi-Domain GTPase Is Crucial for Chloroplast Development of Early Leaf Stage at Low Temperatures.

The multi-domain GTPase (MnmE) is conservative from bacteria to human and participates in tRNA modified synthesis. However, our understanding of how the MnmE is involved in plant chloroplast development is scarce, let alone in rice. A novel rice mutant, thermo-sensitive chlorophyll-deficient mutant 8 (tcd8) was identified in this study, which apparently presented an albino phenotype at 20 °C but a normal green over 24 °C, coincided with chloroplast development and chlorophyll content. Map-based cloning and complementary test revealed the TCD8 encoded a multi-domain GTPase localized in chloroplasts. In addition, the disturbance of TCD8 suppressed the transcripts of certain chloroplast-related genes at low temperature, although the genes were recoverable to nearly normal levels at high temperature (32 °C), indicating that TCD8 governs chloroplast development at low temperature. The multi-domain GTPase gene in rice is first reported in this study, which endorses the importance in exploring chloroplast development in rice.

Rice TSV2 encoding threonyl-tRNA synthetase is needed for early chloroplast development and seedling growth under cold stress.

Threonyl-tRNA synthetase (ThrRS), one of the aminoacyl-tRNA synthetases (AARSs), plays a crucial role in protein synthesis. However, the AARS functions on rice chloroplast development and growth were not fully appraised. In this study, a thermo-sensitive virescent mutant tsv2, which showed albino phenotype and lethal after the 4-leaf stage at 20°C but recovered to normal when the temperatures rose, was identified and characterized. Map-based cloning and complementation tests showed that TSV2 encoded a chloroplast-located ThrRS protein in rice. The Lys-to-Arg mutation in the anticodon-binding domain hampered chloroplast development under cold stress, while the loss of function of the ThrRS core domain in TSV2 fatally led to seedling death regardless of growing temperatures. In addition, TSV2 had a specific expression in early leaves. Its disruption obviously resulted in the downregulation of certain genes associated with chlorophyll biosynthesis, photosynthesis, and chloroplast development at cold conditions. Our observations revealed that rice nuclear-encoded TSV2 plays an important role in chloroplast development at the early leaf stage under cold stress.

Chloroplast development at low temperature requires the pseudouridine synthase gene TCD3 in rice.

Low temperature affects a broad spectrum of cellular components in plants, such as chloroplasts, as well as plant metabolism. On the other hand, pseudouridine (Ψ) synthases are required for the most abundant post-transcriptional modification of RNA in Escherichia coli. However, the role of rice Ψ synthases in regulating chloroplast development at low temperature remains elusive. In this study, we identified the rice thermo-sensitive chlorophyll-deficient (tcd3) mutant, which displays an albino phenotype before the 4-leaf stage and ultimately dies when grown at 20 °C, but can grow normally at 32 °C. Genetic analysis showed that the mutant trait is controlled by a single recessive nuclear gene (tcd3). Map-based cloning, complementation and knockout tests revealed that TCD3 encodes a chloroplast-localized Ψ synthase. TCD3 is a cold-induced gene that is mainly expressed in leaves. The disruption of TCD3 severely affected the transcript levels of various chloroplast-associated genes, as well as ribosomal genes involved in chloroplast rRNA assembly at low temperature (20 °C), whereas the transcript levels of these genes were normal at high temperature (32 °C). These results provide a first glimpse into the importance of rice Ψ synthase gene in chloroplast development at low temperatures.

Rice TCM1 Encoding a Component of the TAC Complex is Required for Chloroplast Development under Cold Stress.

Transcriptionally active chromosome (TAC) is a component of protein-DNA complexes with RNA polymerase activity, expressed in the plastid. However, the function of rice TAC proteins is still poorly understood. In this paper, we first report the identification of a new rice ( L.) mutant () in the gene encoding TAC. The mutant displayed an albino phenotype and malformed chloroplasts before the three-leaf stage when grown at low temperatures (20°C) and a normal phenotype at higher temperatures (>28°C). Map-based cloning revealed that encodes a novel chloroplast-targeted TAC protein in rice. In addition, the transcript levels of all examined plastid-encoded polymerase (PEP)-dependent genes were clearly downregulated in mutants at low temperatures, although partially recovering levels were obtained at high temperatures, comparable to wild-type plants. Furthermore, the transcripts were ubiquitously expressed in all examined tissues, with high expression levels in green tissues. The data suggest that the rice nuclear-encoded TAC protein TCM1 is essential for proper chloroplast development and maintaining PEP activity under cold stress.

Rice TSV3 Encoding Obg-Like GTPase Protein Is Essential for Chloroplast Development During the Early Leaf Stage Under Cold Stress.

The Spo0B-associated GTP-binding (Obg) proteins are essential for the viability of nearly all bacteria. However, the detailed roles of Obg proteins in higher plants have not yet been elucidated. In this study, we identified a novel rice (Oryza sativa L.) thermo-sensitive virescent mutant (tsv3) that displayed an albino phenotype at 20° before the three-leaf stage while being a normal green at 32° or even at 20° after the four-leaf stage. The mutant phenotype was consistent with altered chlorophyll content and chloroplast structure in leaves. Map-based cloning and complementation experiments showed that TSV3 encoded a small GTP-binding protein. Subcellular localization studies revealed that TSV3 was localized to the chloroplasts. Expression of TSV3 was high in leaves and weak or undetectable in other tissues, suggesting a tissue-specific expression of TSV3 In the tsv3 mutant, expression levels of genes associated with the biogenesis of the chloroplast ribosome 50S subunit were severely decreased at the three-leaf stage under cold stress (20°), but could be recovered to normal levels at a higher temperature (32°). These observations suggest that the rice nuclear-encoded TSV3 plays important roles in chloroplast development at the early leaf stage under cold stress.

The rice TCD11 encoding plastid ribosomal protein S6 is essential for chloroplast development at low temperature.

Plastid ribosome proteins (PRPs) are important components for chloroplast biogenesis and early chloroplast development. Although it has been known that chloroplast ribosomes are similar to bacterial ones, the precise molecular function of ribosomal proteins remains to be elucidated in rice. Here, we identified a novel rice mutant, designated tcd11 (thermo-sensitive chlorophyll-deficient mutant 11), characterized by the albino phenotype until it died at 20°C, while displaying normal phenotype at 32°C. The alteration of leaf color in tcd11 mutants was aligned with chlorophyll (Chl) content and chloroplast development. The map-based cloning and molecular complementation showed that TCD11 encodes the ribosomal small subunit protein S6 in chloroplasts (RPS6). TCD11 was abundantly expressed in leaves, suggesting its different expressions in tissues. In addition, the disruption of TCD11 greatly reduced the transcript levels of certain chloroplasts-associated genes and prevented the assembly of ribosome in chloroplasts at low temperature (20°C), whereas they recovered to nearly normal levels at high temperature (32°C). Thus, our data indicate that TCD11 plays an important role in chloroplast development at low temperature. Upon our knowledge, the observations from this study provide a first glimpse into the importance of RPS6 function in rice chloroplast development.

The Rice Pentatricopeptide Repeat Gene TCD10 is Needed for Chloroplast Development under Cold Stress.

BACKGROUND: Chloroplast plays a vital role in plant development and growth. The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants. In addition, cold stress affects a broad spectrum of cellular components, e.g. chloroplast, and metabolism in plants. However, the regulatory mechanism for rice PPR genes on chloroplast development still remains elusive under cold stress. RESULT: In this paper, we characterized a new rice PPR gene mutant tcd10 (thermo-sensitive chlorophyll-deficient mutant 10) that exhibits the albino phenotype, malformed chloroplast and could not survive after the 5-leaf stage when grown at 20 °C, but does the normal phenotype at 32 °C. Map-based cloning, followed by RNA interference and CRISPR/Cas9 genome editing techniques, revealed that TCD10 encoding a novel PPR protein, mainly localized to the chloroplasts, with 27 PPR motifs, is responsible for the mutant phenotype. In addition, TCD10 is specific expression in tissues. The disruption of TCD10 resulted in an evidently reduced expression of chloroplast-associated genes under cold stress (20 °C), whereas they did recovered to normal levels at high temperature (32 °C). These results showed an important role of TCD10 for chloroplast development under cold stress. CONCLUSIONS: The TCD10 encodes a novel rice PPR protein, mainly located in chloroplasts, which is important for chloroplast development, growth and the maintenance of photosynthetic electron transport and its disorder would lead to an aberrant chloroplast and abnormal expressions in these genes for chloroplast development and photosynthesis in rice under cold stress.

Temperature-sensitive albino gene TCD5, encoding a monooxygenase, affects chloroplast development at low temperatures.

Chloroplasts are essential for photosynthesis and play critical roles in plant development. In this study, we characterized the temperature-sensitive chlorophyll-deficient rice mutant tcd5, which develops albino leaves at low temperatures (20 °C) and normal green leaves at high temperatures (32 °C). The development of chloroplasts and etioplasts is impaired in tcd5 plants at 20 °C, and the temperature-sensitive period for the albino phenotype is the P4 stage of leaf development. The development of thylakoid membranes is arrested at the mid-P4 stage in tcd5 plants at 20 °C. We performed positional cloning of TCD5 and then complementation and knock-down experiments, and the results showed that the transcript LOC_Os05g34040.1 from the LOC_Os05g34040 gene corresponded to the tcd5 phenotype. TCD5 encodes a conserved plastid-targeted monooxygenase family protein which has not been previously reported associated with a temperature-sensitive albino phenotype in plants. TCD5 is abundantly expressed in young leaves and immature spikes, and low temperatures increased this expression. The transcription of some genes involved in plastid transcription/translation and photosynthesis varied in the tcd5 mutant. Although the phenotype and temperature dependence of the TCD5 orthologous mutant phenotype were different in rice and Arabidopsis, OsTCD5 could rescue the phenotype of the Arabidopsis mutant, suggesting that TCD5 function is conserved between monocots and dicots.

The Rice TCM5 Gene Encoding a Novel Deg Protease Protein is Essential for Chloroplast Development under High Temperatures.

BACKGROUND: High temperature affects a broad spectrum of cellular components and metabolism in plants. The Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms. Deg proteases are required for the survival of Escherichia coli at high temperatures. However, it is still unclear whether rice Deg proteases are required for chloroplast development under high temperatures. RESULTS: In this study, we reported the first rice deg mutant tcm5 (thermo-sensitive chlorophyll-deficient mutant 5) that has an albino phenotype, defective chloroplasts and could not survive after the 4-5 leaf seedling stage when grown at high temperature (32 °C). However, when grown at low temperatures (20 °C), tcm5 has a normal phenotype. Map-based cloning showed that TCM5 encoding a chloroplast-targeted Deg protease protein. The TCM5 transcripts were highly expressed in all green tissues and undetectable in other tissues, showing the tissue-specific expression. In tcm5 mutants grown at high temperatures, the transcript levels of certain genes associated with chloroplast development especially PSII-associated genes were severely affected, but recovered to normal levels at low temperatures. These results showed important role of TCM5 for chloroplast development under high temperatures. CONCLUSIONS: The TCM5 encodes chloroplast-targeted Deg protease protein which is important for chloroplast development and the maintenance of PSII function and its disruption would lead to a defective chloroplast and affected expression levels of genes associated with chloroplast development and photosynthesis at early rice seedling stage under high temperatures.

The rice ALS3 encoding a novel pentatricopeptide repeat protein is required for chloroplast development and seedling growth.

BACKGROUND: Pentatricopeptide repeat (PPR) proteins play essential roles in modulating the expression of organelle genes and have expanded greatly in higher plants. However, molecular mechanisms of most rice PPR genes remain unclear. RESULTS: In this study, a new rice PPR mutant, asl3 (albino seedling lethality3) exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered photosynthetic-pigment and chloroplast development. Map-based cloning showed that ASL3 encodes a novel rice PPR protein with 10 tandem PPR motifs, which localizes to the chloroplast. ASL3 showed tissue-specific expression, as it was highly expressed in the chlorenchyma, but expressed at much lower levels in roots and panicles. RNAi of ASL3 confirmed that ASL3 plays an essential role in the early development and chloroplast development in rice. Moreover, expression analysis revealed that the asl3 mutation severely affected the transcriptional levels of important genes associated with plastid translation machinery and photosynthesis, which may impair photosynthesis and finally led to the seedling death in asl3 mutant. These results evidenced the important role of ASL3 in the early development of rice, especially chloroplast development. CONCLUSIONS: The ASL3 gene encoded a novel chloroplast-targeted PPR protein with 10 tandem PPR motifs in rice. Disruption of the ASL3 would lead to a defective chloroplast and seedling lethality, and affected expression levels of genes associated with chloroplast development and photosynthesis at early leaf stage of rice.

The rice OsDG2 encoding a glycine-rich protein is involved in the regulation of chloroplast development during early seedling stage.

KEY MESSAGE: OsDG2 gene encoded a novel chloroplast-targeted GRP in rice. Disruption of the OsDG2 would lead to delayed greening phenotype and affected expression levels of genes associated with chloroplast development at early leaf stage of rice. Glycine-rich proteins (GRPs) participate in various biological processes in plants. However, the evidence of GRPs involved in chloroplast development in plants is quite limited. In this study, we identified a rice GRP gene mutant named osdg2 (O ryza s ativa d elayed g reening 2), which exhibits delayed greening phenotype characterized as bright yellow leaves before the three-leaf stage and thereafter turns to normal green. Further study showed that the mutant phenotype was consistent with changes in chlorophyll content and chloroplast development. The rice OsDG2 gene, encoding a novel GRP protein, was located on chromosome 2 through map-based cloning method and confirmed by molecular complementation tests. Subcellular localization results showed that OsDG2 was targeted in chloroplasts. In addition, the OsDG2 transcripts were highly expressed in leaves and undetectable in other tissues, showing the tissue-specific expression. In osdg2 mutant, the expression levels of most genes associated with chloroplast development were severely decreased in the 3rd leaves, but almost recovered to wild-type level in the 4th leaves. Our findings indicated that the nuclear-encoded OsDG2 plays important roles in chloroplast development at early leaf stage of rice.

Importance of the rice TCD9 encoding α subunit of chaperonin protein 60 (Cpn60α) for the chloroplast development during the early leaf stage.

The chloroplast development governs plant growth and metabolism. This study characterized a novel rice thermo-sensitive chloroplast development 9 (tcd9) mutant, which exhibited the albino phenotype before the 3-leaf stage grown below 24 °C whereas displayed normal green at over 28 °C or even at 20 °C after 5-leaf stage. The obvious decrease in Chl levels, abnormal chloroplasts with few thylakoid lamella and abnormal thylakoids were observed for the albino mutant seedlings at low temperature, but the mutant was apparently normal green at high temperature, suggesting the thermo-sensitivity of albino phenotype. Genetic analysis showed that the albino phenotype was controlled by a single recessive nuclear gene (tcd9). The map-based cloning and molecular complementation tests revealed that the mutation of TCD9 encoding α subunit of Cpn60 protein (Cpn60α), localized in chloroplasts, was responsible for albino phenotype. In addition, TCD9 exhibited the high expression in all tested tissues, especially in young leaves. The transcriptional analysis indicated that all expression levels of the studied genes related to chloroplast development in tcd9 mutant were seriously affected in the albino seedlings at 20 °C, whereas some of them recovered into normal levels in green-seedlings at 32 °C. Our observations suggest that the nuclear-encoded Cpn60α protein TCD9 plays a crucial role in chloroplast development at early leaf stage of rice.

The rice OsV4 encoding a novel pentatricopeptide repeat protein is required for chloroplast development during the early leaf stage under cold stress.

Pentatricopeptide repeat (PPR) proteins, characterized by tandem arrays of a 35 amino acid motif, have been suggested to play central and broad roles in modulating the expression of organelle genes in plants. However, the molecular mechanisms of most rice PPR genes remains unclear. In this paper, we isolated and characterized a temperature-conditional virescent mutant, OsV4, in rice (Oryza sativa cultivar Jiahua1 (WT, japonica rice variety)). The mutant displays albino phenotype and abnormal chloroplasts at the three leaf stage, which gradually turns green after the four leaf stage at a low temperature (20 °C). But the mutant always develops green leaves and well-developed chloroplasts at a high temperature (32 °C). Genetic and molecular analyses uncovered that OsV4 encodes a novel chloroplast-targeted PPR protein including four PPR motifs. Further investigations show that the mutant phenotype is associated with changes in chlorophyll content and chloroplast development. The OsV4 transcripts only accumulate to high levels in young leaves, indicating that its expression is tissue-specific. In addition, transcript levels of some ribosomal components and plastid-encoded polymerase-dependent genes are dramatically reduced in the albino mutants grown at 20 °C. These findings suggest that OsV4 plays an important role during early chloroplast development under cold stress in rice.

Disruption of the rice plastid ribosomal protein s20 leads to chloroplast developmental defects and seedling lethality.

Plastid ribosomal proteins (PRPs) are essential for ribosome biogenesis, plastid protein biosynthesis, chloroplast differentiation, and early chloroplast development. This study identifies the first rice PRP mutant, asl1 (albino seedling lethality1), which exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered chlorophyll (Chl) content and chloroplast development. Map-based cloning revealed that ASL1 encodes PRP S20 (PRPS20), which localizes to the chloroplast. ASL1 showed tissue-specific expression, as it was highly expressed in plumule and young seedlings but expressed at much lower levels in other tissues. In addition, ASL1 expression was regulated by light. The transcript levels of nuclear genes for Chl biosynthesis and chloroplast development were strongly affected in asl1 mutants; transcripts of some plastid genes for photosynthesis were undetectable. Our findings indicate that nuclear-encoded PRPS20 plays an important role in chloroplast development in rice.

[Characteristics and molecular mapping of a novel chlorophyll-deficient yellow-leaf mutant in rice].

A yellow-leaf mutant (yl11) with chlorophyll-deficient in rice (Oryza sativa L.) was selected from the progeny of a japonica rice variety "Jiahua 1" treated with 60Co γ-radiation. In comparison with the wild-type parent, "Jiahua 1", the mutant had yellow-leaves at whole growth stages and displayed significantly decreased in chlorophyll content and net photosynthetic rate. Underdeveloped chloroplast and alterations of the major agronomic traits, such as plant-heights, were also observed in the mutant. Genetic analysis confirmed that the yellow-leaf mutant trait was controlled by a single recessive nuclear gene (yl11). Using SSR and In/Del molecular markers and 920 F2 and F3 plants from the cross of yl11 with the indica variety Peiai 64S, the yl11 was mapped between the molecular markers MM2199 and InDel21039 with a physical distance of 110 kb on the long arm of chromosome 11, in which no known functional genes for chlorophyll synthesis or chloroplast development in rice has been found. These findings will provide a foundation for the cloning and functional analysis of this gene in the future.

Molecular genetic analysis of QTLs for ferulic acid content in dried straw of rice (Oryza sativa L.).

Phenolic acids are secondary metabolic organic compounds produced by plants and often are mentioned as allelochemicals. This study was conducted to determine the genetic basis controlling the ferulic acid content of rice straw in a recombinant inbred (RI) population derived from a cross between a japonica variety, Asominori, with a higher content of ferulic acid, and an indica variety, IR24, with a lower content, using 289 RFLP markers. Continuous distributions and transgressive segregations of ferulic acid content were observed in the RI population, which showed that ferulic acid content in rice straw was quantitatively inherited. Single marker analysis and composite interval mapping identified three quantitative trait loci (QTLs) for ferulic acid content with LOD values of 2.03 (chromosome 3), 3.16 (chromosome 6), and 3.06 (chromosome 7); all three had increased additive effects (13.5, 18.3, and 18.1 microg g(-1)) from the Asominori parent and accounted for 5.5, 16.9, and 12.8% of total phenotypic variation, respectively. This is the first report on the identification of QTLs associated with ferulic acid and their chromosomal localization on the molecular map of rice. The tightly linked molecular markers that flank the QTLs might be useful in breeding and selection of varieties with higher phenolic acid content.