Chilling stress is considered the major abiotic stress affecting the growth, development, and yield of rice. To understand the transcriptomic responses and methylation regulation of rice in response to chilling stress, we analyzed a cold-tolerant variety of rice (Oryza sativa L. cv. P427). The physiological properties, transcriptome, and methylation of cold-tolerant P427 seedlings under low-temperature stress (2–3 °C) were investigated. We found that P427 exhibited enhanced tolerance to low temperature, likely via increasing antioxidant enzyme activity and promoting the accumulation of abscisic acid (ABA). The Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) data showed that the number of methylation-altered genes was highest in P427 (5496) and slightly lower in Nipponbare (Nip) and 9311 (4528 and 3341, respectively), and only 2.7% (292) of methylation genes were detected as common differentially methylated genes (DMGs) related to cold tolerance in the three varieties. Transcriptome analyses revealed that 1654 genes had specifically altered expression in P427 under cold stress. These genes mainly belonged to transcription factor families, such as Myeloblastosis (MYB), APETALA2/ethylene-responsive element binding proteins (AP2-EREBP), NAM-ATAF-CUC (NAC) and WRKY. Fifty-one genes showed simultaneous methylation and expression level changes. Quantitative RT-PCR (qRT-PCR) results showed that genes involved in the ICE (inducer of CBF expression)-CBF (C-repeat binding factor)—COR (cold-regulated) pathway were highly expressed under cold stress, including the WRKY genes. The homologous gene Os03g0610900 of the open stomatal 1 (OST1) in rice was obtained by evolutionary tree analysis. Methylation in Os03g0610900 gene promoter region decreased, and the expression level of Os03g0610900 increased, suggesting that cold stress may lead to demethylation and increased gene expression of Os03g0610900. The ICE-CBF-COR pathway plays a vital role in the cold tolerance of the rice cultivar P427. Overall, this study demonstrates the differences in methylation and gene expression levels of P427 in response to low-temperature stress, providing a foundation for further investigations of the relationship between environmental stress, DNA methylation, and gene expression in rice.

Black and red rice are rich in both anthocyanin and proanthocyanin content, which belong to a large class of flavonoids derived from a group of phenolic secondary metabolites. However, the molecular pathways and mechanisms underlying the flavonoid biosynthetic pathway are far from clear. Therefore, this study was undertaken to gain insight into physiological factors that are involved in the flavonoid biosynthetic pathway in rice cultivars with red, black, and white colors. RNA sequencing of caryopsis and isobaric tags for relative and absolute quantification (iTRAQ) analyses have generated a nearly complete catalog of mRNA and expressed proteins in different colored rice cultivars. A total of 31,700 genes were identified, of which 3417, 329, and 227 genes were found specific for red, white, and black rice, respectively. A total of 13,996 unique peptides corresponding to 3916 proteins were detected in the proteomes of black, white, and red rice. Coexpression network analyses of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) among the different rice cultivars showed significant differences in photosynthesis and flavonoid biosynthesis pathways. Based on a differential enrichment analysis, 32 genes involved in the flavonoid biosynthesis pathway were detected, out of which only CHI, F3H, ANS, and FLS were detected by iTRAQ. Taken together, the results point to differences in flavonoid biosynthesis pathways among different colored rice cultivars, which may reflect differences in physiological functions. The differences in contents and types of flavonoids among the different colored rice cultivars are related to changes in base sequences of Os06G0162500, Os09G0455500, Os09G0455500, and Os10G0536400. Current findings expand and deepen our understanding of flavonoid biosynthesis and concurrently provides potential candidate genes for improving the nutritional qualities of rice.

Vacuolar invertase is involved in sugar metabolism and plays a crucial role in plant growth and development, thus regulating seed size. However, information linking vacuolar invertase and seed size in rice is limited. Here we characterized a small grain mutant sg2 (grain size on chromosome 2) that showed a reduced in grain size and 1000-grain weight compared to the wild type. Map-based cloning and genetic complementation showed that OsINV3 is responsible for the observed phenotype. Loss-of-function of OsINV3 resulted in grains of smaller size when compared to the wild type, while overexpression showed increased grain size. We also obtained a T-DNA insertion mutant of OsINV2, which is a homolog of OsINV3 and generated double knockout (KO) mutants of OsINV2 and OsINV3 using CRISPR/Cas9. Genetic data showed that OsINV2, that has no effect on grain size by itself, reduces grain length and width in the absence of OsINV3. Altered sugar content with increased sucrose and decreased hexose levels, as well as changes vacuolar invertase activities and starch constitution in INV3^KO, INV2^KO, INV3^KOINV2^KO mutants indicate that OsINV2 and OsINV3 affect sucrose metabolism in sink organs. In summary, we identified OsINV3 as a positive regulator of grain size in rice, and while OsINV2 has no function on grain size by itself. In the absence of OsINV3, it is possible to detect a role of OsINV2 in the regulation of grain size. Both OsINV3 and OsINV2 are involved in sucrose metabolism, and thus regulate grain size. Our findings increase our understanding of the role of OsINV3 and its homolog, OsINV2, in grain size development and also suggest a potential strategy to improve grain yield in rice.

Salicylic acid (SA) is a stress hormone synthesized in phenylalanine ammonia-lyase (PAL) and the branching acid pathway. SA has two interconvertible forms in plants: SAG (SA O-β-glucoside) and SA (free form). The molecular mechanism of conversion of SA to SAG had been reported previously. However, which genes regulate SAG to SA remained unknown. Here, we report a cytoplasmic β-glucosidase (β-Glu) which participates in the SA pathway and is involved in the brown hull pigmentation in rice grain. In the current study, an EMS-generated mutant brown hull 1 (bh1) displayed decreased contents of SA in hulls, a lower photosynthesis rate, and high-temperature sensitivity compared to the wild type (WT). A plaque-like phenotype (brown pigmentation) was present on the hulls of bh1, which causes a significant decrease in the seed setting rate. Genetic analysis revealed a mutation in LOC_Os01g67220, which encodes a cytoplasmic Os1βGlu4. The knock-out lines displayed the phenotype of brown pigmentation on hulls and decreased seed setting rate comparable with bh1. Overexpression and complementation lines of Os1βGlu4 restored the phenotype of hulls and normal seed setting rate comparable with WT. Subcellular localization revealed that the protein of Os1βGlu4 was localized in the cytoplasm. In contrast to WT, bh1 could not hydrolyze SAG into SA in vivo. Together, our results revealed the novel role of Os1βGlu4 in the accumulation of flavonoids in hulls by regulating the level of free SA in the cellular pool.

Panicle degeneration, sometimes known as abortion, causes heavy losses in grain yield. However, the mechanism of naturally occurring panicle abortion is still elusive. In a previous study, we characterized a mutant, apical panicle abortion1331 (apa1331), exhibiting abortion in apical spikelets starting from the 6 cm stage of panicle development. In this study, we have quantified the five phytohormones, gibberellins (GA), auxins (IAA), abscisic acid (ABA), cytokinins (CTK), and brassinosteroids (BR), in the lower, middle, and upper parts of apa1331 and compared these with those exhibited in its wild type (WT). In apa331, the lower and middle parts of the panicle showed contrasting concentrations of all studied phytohormones, but highly significant changes in IAA and ABA, compared to the upper part of the panicle. A comparative transcriptome of apa1331 and WT apical spikelets was performed to explore genes causing the physiological basis of spikelet abortion. The differential expression analysis revealed a significant downregulation and upregulation of 1587 and 978 genes, respectively. Hierarchical clustering of differentially expressed genes (DEGs) revealed the correlation of gene ontology (GO) terms associated with antioxidant activity, peroxidase activity, and oxidoreductase activity. KEGG pathway analysis using parametric gene set enrichment analysis (PGSEA) revealed the downregulation of the biological processes, including cell wall polysaccharides and fatty acids derivatives, in apa1331 compared to its WT. Based on fold change (FC) value and high variation in expression during late inflorescence, early inflorescence, and antherdevelopment, we predicted a list of novel genes, which presumably can be the potential targets of inflorescence development. Our study not only provides novel insights into the role of the physiological dynamics involved in panicle abortion, but also highlights the potential targets involved in reproductive development.

Polyploidy, or whole-genome duplication (WGD) promotes genetic diversification in plants. However, whether WGD is accompanied by epigenetic regulation especially DNA methylation remains yet elusive. Methylation of different region in genomic DNA play discrete role in gene regulation and developmental processes in plants. In our study, we used an apomictic rice line (SARII-628) that produces twin seedlings of different ploidy for methylated DNA immunoprecipitation sequencing (MeDIP-seq). We compared the level of methylation and mRNA expression in three different (CG, CHG, and CHH) sequence contexts of promoter region among haploid (1X), diploid (2X), and triploid (3X) seedling. We used MeDIP-Seq analysis of 14 genes to investigate whole genome DNA methylation and found that relative level of DNA methylation across different ploidy was in following order e.g. diploid > triploid > haploid. GO functional classification of differentially methylated genes into 9 comparisons group of promoter, intergenic and intragenic region discovered, these genes were mostly enriched for cellular component, molecular function, and biological process. By the comparison of methylome data, digital gene expression (DGE), mRNA expression profile, and Q-PCR findings LOC_ Os07g31450 and LOC_ Os01g59320 were analyzed for BS-Seq (Bisulphite sequencing). We found that (1) The level of the promoter DNA methylation is negatively correlated with gene expression within each ploidy level. (2) Among all ploidy levels, CG sequence context had highest methylation frequency, and demonstrated that the high CG methylation did reduce gene expression change suggesting that DNA methylation exert repressive function and ensure genome stability during WGD. (3) Alteration in ploidy (from diploid to haploid, or diploid to triploid) reveals supreme changes in methylation frequency of CHH sequence context. Our finding will contribute an understanding towards lower stability of CHH sequence context and educate the effect of promoter region methylation during change in ploidy state in rice.