Yanjun Dong

Chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are a family of plant-specific proteins associated with RNA binding. In this study, we have conducted a detailed characterization of a novel rice CRM gene (LOC_Os04g39060) mutant, yl4, which showed yellow-green leaves at all the stages, had fewer tillers, and had a decreased plant height. Map-based cloning and CRISPR/Cas9 editing techniques all showed that YL4 encoded a CRM domain protein in rice. In addition, subcellular localization revealed that YL4 was in chloroplasts. YL4 transcripts were highly expressed in all leaves and undetectable in roots and stems, and the mutation of YL4 affected the transcription of chloroplast-development-related genes. This study indicated that YL4 is essential for chloroplast development and affects some agronomic traits.

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.

Transcriptionally active chromosome (TAC) is a component of protein-DNA complexes with RNA polymerase activity found in chloroplasts. Although TAC in Arabidopsis thaliana has been extensively investigated, how the rice (Oryza sativaL.) TAC complex functions remain largely unknown. We report the characterization of the mutant thermosensitive chlorophyll-deficient7 (tcd7) and the cloning of TCD7. tcd7 mutant seedlings displayed an albino phenotype specifically at low temperatures and before the four-leaf stage. We identified TCD7 by map-based cloning followed by transgenic rescue and genome editing tests, showing that TCD7 encodes the putative TAC component FRUCTOKINASE-LIKE 2 (OsFLN2). TCD7 transcripts were highly abundant in green tissues, and the protein localized to chloroplasts. In agreement with the albino phenotype, transcript levels of genes controlling chloroplast development and the establishment of photosynthetic capacity were severely reduced in tcd7 seedlings at low temperatures, but were expressed as in the wild type at high temperatures, implying that TCD7 regulates the PEP pathway and chloroplast development. Moreover, TCD7 interacted with the thioredoxin OsTRXz to form an OsTRXz-TCD7 regulatory module, which might regulate plastid transcription under cold stress. Our results demonstrate that the nucleus-encoded TAC protein TCD7protects chloroplast development from cold stress via a TRXz-FLN regulatory module.

Plant zinc‐finger proteins play a crucial role in biosynthesis and plant development. However, it is not known whether certain zinc‐finger proteins play a role in rice chloroplast development. In this study, a novel rice zinc‐finger protein mutant asl4 (albino seedling lethality4), which exhibits an albino lethal phenotype at the seedling stage, was used. Chlorophyll fluorescence analysis and TEM were used to investigate features of the asl4 mutant. The genetic behaviour and function of ASL4 gene were then analysed thorough map‐based cloning, transgenic complement and subcellular localization. The albino lethal phenotype was caused by a single nucleotide (G*) deletion mutation on the exon of the ASL4 (LOC_Os09g21710) gene. The ASL4 gene encoded a novel zinc‐finger protein containing two ZnF‐AN1 domains, which was localized to the nucleocytoplasm. The ASL4 transcripts were highly expressed in all leaves but relatively less in other tissues, suggesting its tissue‐specific expression. The transcript levels of associated genes for Chl biosynthesis, photosynthesis and chloroplast development were severely suppressed in asl4 mutants. In conclusion, the absence of ASL4 function caused a defect in chloroplast development and seedling lethality. This is the first published report on the importance of the ZnF‐AN1 type zinc‐finger protein gene in chloroplast development in rice.

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.

Background The chloroplast is a vital photosynthetic organelle for plant growth and development. However, the genetic factors involved in chloroplast development and its relationship with environment factors are largely unknown. Threonyl-tRNA synthetase (ThrRS), one of aminoacyl-tRNA synthetases (AARSs), plays a crucial role in protein synthesis. To date, there are few studies for AARS function on chloroplast development and plant growth, much less ThrRS in rice. Result In this paper, we characterized a thermo-sensitive virescent mutant tsv2, which showed albino phenotype and could not survive after the 4-leaf stage when grown at 20 °C, but recovered the normal phenotype when the temperature rose. Map-based cloning and complementation tests showed that TSV2 encoded a chloroplast-located ThrRS protein in rice and the Lys-to-Arg mutation in the anticodon-binding domain affected chloroplast development under cold stress. Furthermore, the loss-of-function of the core domain in TSV2 led to seedling death regardless of temperatures. In addition, TSV2 had a tissue-specific expression, and its disruption resulted in an evidently down-regulation of certain genes associated with chlorophyll biosynthesis, photosynthesis and chloroplast development at cold stress. Conclusion The TSV2 encodes a rice threonyl-tRNA synthetase, located in chloroplasts, which is essential for cold-responsive regulation for chloroplast development and plant growth and closely related to the assembly of chloroplast ribosomes and functions at the first step of chloroplast differentiation.

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.

Glycolysis is a central metabolic pathway that provides energy and products of primary metabolites. 2,3‐Biphosphoglycerate‐independent phosphoglycerate mutase (iPGAM) is a key enzyme that catalyses the reversible interconversion of 3‐phosphoglycerate (3‐PGA) to 2‐phosphoglycerate (2‐PGA) in glycolysis. Low temperature is a common abiotic stress in rice production. However, the mechanism for rice iPGAM genes is not fully understood at low temperature. In this study, the rice mutant tcm12, with chlorosis, malformed chloroplasts and impaired photosynthesis, was grown at a low temperature (tcm12 mutant. The inheritance behaviour and function of TCM12 were then analysed thorough map‐based cloning, transgenic complementation and subcellular localisation. The thermo‐sensitive chlorosis phenotype was caused by a single nucleotide mutation (T→C) on the fifth exon of TCM12 (LOC_Os12g35040) encoding iPGAM, localised to both nucleus and membranes. In addition, TCM12 was constitutively expressed, and its disruption resulted in down‐regulation of some genes associated with chlorophyll biosynthesis and photosynthesis at low temperatures (20 °C). This is the first report of the involvement of rice iPGAM gene in chlorophyll synthesis, photosynthesis and chloroplast development, providing new insights into the mechanisms underlying early growth of rice at low temperatures.

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 ( Oryza sativa L.) mutant ( tcm1 ) in the gene encoding TAC. The tcm1 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 TCM1 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 tcm1 mutants at low temperatures, although partially recovering levels were obtained at high temperatures, comparable to wild‐type plants. Furthermore, the TCM1 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.