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MAIN CONCLUSION: Ectopic expression of OsWOX9A induces narrow adaxially rolled rice leaves with larger bulliform cells and fewer large veins, probably through regulating the expression of auxin-related and expansin genes. The WUSCHEL-related homeobox (WOX) family plays a pivotal role in plant development by regulating genes involved in various aspects of growth and differentiation. OsWOX9A (DWT1) has been linked to tiller growth, uniform plant growth, and flower meristem activity. However, its impact on leaf growth and development in rice has not been studied. In this study, we investigated the biological role of OsWOX9A in rice growth and development using transgenic plants. Overexpression of OsWOX9A conferred narrow adaxially rolled rice leaves and altered plant architecture. These plants exhibited larger bulliform cells and fewer larger veins compared to wild-type plants. OsWOX9A overexpression also reduced plant height, tiller number, and seed-setting rate. Comparative transcriptome analysis revealed several differentially expressed auxin-related and expansin genes in OsWOX9A overexpressing plants, consistent with their roles in leaf and plant development. These results indicate that the ectopic expression of OsWOX9A may have multiple effects on the development and growth of rice, providing a more comprehensive picture of how the WOX9 subfamily contributes to leaf development and plant architecture.

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Plant architecture and organ size are considered as important traits in crop breeding and germplasm improvement. Although several factors affecting plant architecture and organ size have been identified in rice, the genetic and regulatory mechanisms remain to be elucidated. Here, we identified and characterized the small plant and organ 1 (spo1) mutant in rice (Oryza sativa), which exhibits narrow and rolled leaf, reductions in plant height, root length, and grain width, and other morphological defects. Map-based cloning revealed that SPO1 is allelic with OsCSLD4, a gene encoding the cellulose synthase-like protein D4, and is highly expressed in the roots at the seedling and tillering stages. Microscopic observation revealed the spo1 mutant had reduced number and width in leaf veins, smaller size of leaf bulliform cells, reduced cell length and cell area in the culm, and decreased width of epidermal cells in the outer glume of the grain. These results indicate the role of SPO1 in modulating cell division and cell expansion, which modulates plant architecture and organ size. It is showed that the contents of endogenous hormones including auxin, abscisic acid, gibberellin, and zeatin tested in the spo1 mutant were significantly altered, compared to the wild type. Furthermore, the transcriptome analysis revealed that the differentially expressed genes (DEGs) are significantly enriched in the pathways associated with plant hormone signal transduction, cell cycle progression, and cell wall formation. These results indicated that the loss of SPO1/OsCSLD4 function disrupted cell wall cellulose synthase and hormones homeostasis and signaling, thus leading to smaller plant and organ size in spo1. Taken together, we suggest the functional role of SPO1/OsCSLD4 in the control of rice plant and organ size by modulating cell division and expansion, likely through the effects of multiple hormonal pathways on cell wall formation.

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Protoporphyrinogen IX (Protogen IX) oxidase (PPO) catalyzes the oxidation of Protogen IX to Proto IX. PPO is also the target site for diphenyl ether-type herbicides. In plants, there are two PPO encoding genes, PPO1 and PPO2. To date, no PPO gene or mutant has been characterized in monocotyledonous plants. In this study, we isolated a spotted and rolled leaf (sprl1) mutant in rice (Oryza sativa). The spotted leaf phenotype was sensitive to high light intensity and low temperature, but the rolled leaf phenotype was insensitive. We confirmed that the sprl1 phenotypes were caused by a single nucleotide substitution in the OsPPO1 (LOC_Os01g18320) gene. This gene is constitutively expressed, and its encoded product is localized to the chloroplast. The sprl1 mutant accumulated excess Proto(gen) IX and reactive oxygen species (ROS), resulting in necrotic lesions. The expressions of 26 genes associated with tetrapyrrole biosynthesis, photosynthesis, ROS accumulation, and rolled leaf were significantly altered in sprl1, demonstrating that these expression changes were coincident with the mutant phenotypes. Importantly, OsPPO1-overexpression transgenic plants were resistant to the herbicides oxyfluorfen and acifluorfen under field conditions, while having no distinct influence on plant growth and grain yield. These finding indicate that the OsPPO1 gene has the potential to engineer herbicide resistance in rice.

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Tropical ectotherms are particularly vulnerable to global warming because their physiologies are assumed to be adapted to narrow temperature ranges. This study explores three mechanisms potentially constraining thermal adaptation to global warming in tropical insects: (a) Trade-offs in genotypic performance at different temperatures (the jack-of-all-trades hypothesis), (b) positive genetic covariance in performance, with some genotypes performing better than others at viable temperatures (the 'winner' and 'loser' genotypes hypothesis), or (c) limited genetic variation as the potential result of relaxed selection and the loss of genes associated with responses to extreme temperatures (the gene decay hypothesis). We estimated changes in growth and survival rates at multiple temperatures for three tropical rain forest insect herbivores (Cephaloleia rolled-leaf beetles, Chrysomelidae). We reared 2,746 individuals in a full sibling experimental design, at temperatures known to be experienced by this genus of beetles in nature (i.e. 10-35°C). Significant genetic covariance was positive for 16 traits, supporting the 'winner' and 'loser' genotypes hypothesis. Only two traits displayed negative cross-temperature performance correlations. We detected a substantial contribution of genetic variance in traits associated with size and mass (0%-44%), but low heritability in plastic traits such as development time (0%-6%) or survival (0%-4%). Lowland insect populations will most likely decline if current temperatures increase between 2 and 5°C. It is concerning that local adaption is already lagging behind current temperatures. The consequences of maintaining the current global warming trajectory would be devastating for tropical insects. However, if humans can limit or slow warming, many tropical ectotherms might persist in their current locations and potentially adapt to warmer temperatures.

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BACKGROUND: Abscisic acid (ABA) acts as a signaling hormone in plants against abiotic stress, but its function in energy homeostasis under heat stress is unclear. RESULTS: Two rice genotypes, Nipponbare (wild-type, WT) with flat leaves and its mutant high temperature susceptibility (hts) plant with semi-rolled leaves, were subjected to heat stress. We found significantly higher tissue temperature, respiration rate, and ABA and H2O2 contents in leaves as well as a lower transpiration rate and stomatal conductance in hts than WT plants. Additionally, increased expression of HSP71.1 and HSP24.1 as well as greater increases in carbohydrate content, ATP, NAD (H), and dry matter weight, were detected in WT than hts plants under heat stress. More importantly, exogenous ABA significantly decreased heat tolerance of hts plants, but clearly enhanced heat resistance of WT plants. The increases in carbohydrates, ATP, NAD (H), and heat shock proteins in WT plants were enhanced by ABA under heat stress, whereas these increases were reduced in hts plants. CONCLUSION: It was concluded that ABA is a negative regulator of heat tolerance in hts plants with semi-rolled leaves by modulating energy homeostasis.

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The leaf blade is the main photosynthetic organ and its morphology is related to light energy capture and conversion efficiency. We isolated a novel rice Dynamic Narrow-Rolled Leaf 1 (dnrl1) mutant showing reduced width of leaf blades, rolled leaves and lower chlorophyll content. The narrow-rolled leaf phenotype resulted from the reduced number of small longitudinal veins per leaf, smaller size and irregular arrangement of bulliform cells compared with the wild-type. DNRL1 was mapped to chromosome 7 and encoded a putative 3-deoxy-7-phosphoheptulonate synthase (DAHPS) which catalyzes the conversion of phosphoenolpyruvate and D-erythrose 4-phosphate to DAHP and phosphate. Sequence analysis revealed that a single base substitution (T-A) was detected in dnrl1, leading to a single amino acid change (L376H) in the coding protein. The mutation led to a lower expression level of DNRL1 as well as the lower activity of DAHPS in the mutant compared with the wild type. Genetic complementation and over-expression of DNRL1 could rescue the narrow-rolled phenotype. DNRL1 was constitutively expressed in all tested organs and exhibited different expression patterns from other narrow-rolled leaf genes. DNRL1-GFP located to chloroplasts. The lower level of chlorophyll in dnrl1 was associated with the downregulation of the genes responsible for chlorophyll biosynthesis and photosynthesis. Furthermore, dnrl1 showed significantly reduced levels of aromatic amino acids including Trp, Phe and Tyr. We conclude that OsDAHPS, encoded by DNRL1, plays a critical role in leaf morphogenesis by mediating the biosynthesis of amino acids in rice.

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Rice yield is closely related to plant leaf shape and chlorophyll content. In this study, we isolated and identified a narrow and rolled leaf mutant, temporarily named nrl3 with darker green leaves. Histological analysis showed that nrl3 has a reduced number of vascular bundles and undergoes abnormal abaxial sclerenchymatous cell differentiation. The NRL3 mutant phenotype was controlled by a single recessive gene, fine-mapped to a 221 kb interval between Indel3 and RM2322 on Chr3. There are 42 ORF in this interval. Sequencing identified an SNP mutant leading to a premature stop in ORF 18, the candidate gene. Bioinformation analysis indicated that NRL3 encodes a novel protein with unknown function. NRL3 is localized in cytoplasm, membrane and nucleus. Expression analysis of nrl3 showed that genes involved in chlorophyll synthesis were significantly up-regulated while those involved in chlorophyll degradation and programmed cell death (PCD) were significantly down-regulated. The expression levels of photosynthesis genes were also affected. Y2H and BIFC assays indicated that NRL3 interacts directly with NAL9/VYL to regulate leaf morphology in rice. Thus, NRL3 plays an important role in leaf morphogenesis and chlorophyll accumulation, and can be used as a new gene resource for constructing improved rice.

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We previously established the genetic locus of the rolled-leaf mutant, γ-rl, to chromosome 3. In this study, we performed a comparative genomic hybridization (CGH) analysis to identify the genes responsible for the γ-rl mutant phenotype. This was combined with RNA transcriptome sequencing (RNA-seq) to analyze differences in the mRNA expression in seeds 12 h after germination. Using the reference genome of the "indica type" rice from GenBank, we created a chip with 386,000 high density DNA probes designed to target chromosome 3. The genomic DNA from γ-rl and Qinghuazhan (the wild-type) was used for hybridization against the chip to compare signal differences. We uncovered 49 regions with significant differences in hybridization signals including deletions and insertions. RNA-seq analysis between γ-rl and QHZ identified 1060 differentially expressed genes, which potentially regulate numerous biological activities. Moreover, we identified 72 annotated genes in the 49 regions discovered in CGH. Among these, 44 genes showed differential expression in RNA-seq. qRT-PCR validation of the candidate genes confirmed that seven of the 44 genes showed a significant change in their expression levels. Among these, four genes [OsI_10125 (LOC_Os03g06654), OsI_14045 (LOC_Os03g62490), OsI_14279 (LOC_Os03g62620) and OsI_14326 (LOC_Os03g63250)] were down regulated and three genes [(OsI_10794 (LOC_Os03g14950), OsI_11412 (LOC_Os03g21250) and OsI_14152 (LOC_Os03g61360)] were up regulated with a fold change ≥2.0 and a P value ≤ 0.01. Finally, we constructed transgenic plants to study the in vivo functions of these genes. RNAi knock down of LOC_Os03g62620 resulted in rolled-leaf, lower seed-setting and decreased seed growth phenotypes. Transgenic plants with LOC_Os03g14950 over-expression showed dwarf plants with a shortened leaf phenotype. Our results, LOC_Os03g62620 and LOC_Os03g14950 as the essential genes responsible for creating the γ-rl mutant phenotypes suggested that these genes may play crucial roles in regulating rice leaf development and seed growth.

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Grain yield in rice (Oryza sativa L.) is closely related to leaf and flower development. Coordinative regulation of leaf, pollen, and seed development in rice as a critical biological and agricultural question should be addressed. Here we identified two allelic rice mutants with narrow and semi-rolled leaves, named narrow and rolled leaf 2-1 (nrl2-1) and nrl2-2. Map-based molecular cloning revealed that NRL2 encodes a novel protein with unknown biochemical function. The mutation of NRL2 caused pleiotropic effects, including a reduction in the number of longitudinal veins, defective abaxial sclerenchymatous cell differentiation, abnormal tapetum degeneration and microspore development, and the formation of more slender seeds compared with the wild type (WT). The NRL2 protein interacted with Rolling-leaf (RL14), causing the leaves of the nrl2 mutants to have a higher cellulose content and lower lignin content than the WT, which may have been related to sclerenchymatous cell differentiation and tapetum degeneration. Thus, this gene is an essential developmental regulator controlling fundamental cellular and developmental processes, serving as a potential breeding target for high-yielding rice cultivars.

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BACKGROUND: The Class III homeodomain Leu zipper (HD-Zip III) gene family plays important roles in plant growth and development. Here, we analyze the function of OsHox32, an HD-Zip III family member, and show that it exhibits pleiotropic effects on regulating plant type architecture and leaf development in rice. RESULTS: Transgenic lines overexpressing OsHox32 (OsHox32-OV) produce narrow leaves that roll towards the adaxial side. Histological analysis revealed a decreased number of bulliform cells in OsHox32-OV lines. In addition, the angle between the leaf and culm was reduced, resulting in an erect plant phenotype. The height of the plants was reduced, resulting in a semi-dwarf phenotype. In addition, the chlorophyll level was reduced, resulting in a decrease in the photosynthetic rate, but water use efficiency was significantly improved, presumably due to the rolled leaf phenotype. OsHox32 exhibited constitutive expression in different organs, with higher mRNA levels in the stem, leaf sheath, shoot apical meristems and young roots, suggesting a role in plant-type and leaf development. Moreover, OsHox32 mRNA levels were higher in light and lower in the dark under both long-day and short-day conditions, indicating that OsHox32 may be associated with light regulation. Photosynthesis-associated and chlorophyll biosynthesis-associated genes were down-regulated to result in the reduction of photosynthetic capacity in OsHox32-OV lines. mRNA level of six rice YABBY genes is up-regulated or down-regulated by OsHox32, suggesting that OsHox32 may regulate the architecture of plant type and leaf development by controlling the expression of YABBY genes in rice. In addition, OsHox32 mRNA level was induced by the phytohormones, indicating that OsHox32 may be involved in phytohormones regulatory pathways. CONCLUSIONS: OsHox32, an HD-Zip III family member, plays pleiotropic effects on plant type architecture and leaf development in rice.

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Leaf morphology, particularly in crop, is one of the most important agronomic traits because it influences the yield through the manipulation of photosynthetic capacity and transpiration. To understand the regulatory mechanism of leaf morphogenesis, an Oryza sativa dominant mutant, rolled and erect leaf 1 (rel1) has been characterized. This mutant has a predominant rolled leaf, increased leaf angle, and reduced plant height phenotype that results in a reduction in grain yield. Electron microscope observations indicated that the leaf incurvations of rel1 dominant mutants result from the alteration of the size and number of bulliform cells. Molecular cloning revealed that the rel1 dominant mutant phenotype is caused by the activation of the REL1 gene, which encodes a novel unknown protein, despite its high degree of conservation among monocot plants. Moreover, the downregulation of the REL1 gene in the rel1 dominant mutant restored the phenotype of this dominant mutant. Alternatively, overexpression of REL1 in wild-type plants induced a phenotype similar to that of the dominant rel1 mutant, indicating that REL1 plays a positive role in leaf rolling and bending. Consistent with the observed rel1 phenotype, the REL1 gene was predominantly expressed in the meristem of various tissues during plant growth and development. Nevertheless, the responsiveness of both rel1 dominant mutants and REL1-overexpressing plants to exogenous brassinosteroid (BR) was reduced. Moreover, transcript levels of BR response genes in the rel1 dominant mutants and REL1-overexpressing lines were significantly altered. Additionally, seven REL1-interacting proteins were also identified from a yeast two-hybrid screen. Taken together, these findings suggest that REL1 regulates leaf morphology, particularly in leaf rolling and bending, through the coordination of BR signalling transduction.

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