In eukaryotes, histone acetylation is a major modification on histone N‐terminal tails that is tightly connected to transcriptional activation. HDA6 is a histone deacetylase involved in the transcriptional regulation of genes and transposable elements (TEs) in Arabidopsis thaliana. HDA6 has been shown to participate in several complexes in plants, including a conserved SIN3 complex. Here, we uncover a novel protein complex containing HDA6, several Harbinger transposon‐derived proteins (HHP1, SANT1, SANT2, SANT3, and SANT4), and MBD domain‐containing proteins (MBD1, MBD2, and MBD4). We show that mutations of all four SANT genes in the sant‐null mutant cause increased expression of the flowering repressors FLC, MAF4, and MAF5, resulting in a late flowering phenotype. Transcriptome deep sequencing reveals that while the SANT proteins and HDA6 regulate the expression of largely overlapping sets of genes, TE silencing is unaffected in sant‐null mutants. Our global histone H3 acetylation profiling shows that SANT proteins and HDA6 modulate gene expression through deacetylation. Collectively, our findings suggest that Harbinger transposon‐derived SANT domain‐containing proteins are required for histone deacetylation and flowering time control in plants.

In plants, 5mC DNA methylation is an important and conserved epistatic mark involving genomic stability, gene transcriptional regulation, developmental regulation, abiotic stress response, metabolite synthesis, etc. However, the roles of 5mC DNA methylation modification (5mC methylation) in tea plant growth and development (in pre-harvest processing) and flavor substance synthesis in pre- and post-harvest processing are unknown. We therefore conducted a comprehensive methylation analysis of four key pre-harvest tissues (root, leaf, flower, and fruit) and two processed leaves during oolong tea post-harvest processing. We found that differential 5mC methylation among four key tissues is closely related to tissue functional differentiation and that genes expressed tissue-specifically, responsible for tissue-specific functions, maintain relatively low 5mC methylation levels relative to non-tissue-specifically expressed genes. Importantly, hypomethylation modifications of CsAlaDC and TS/GS genes in roots provided the molecular basis for the dominant synthesis of theanine in roots. In addition, integration of 5mC DNA methylationomics, metabolomics, and transcriptomics of post-harvest leaves revealed that content changes in flavor metabolites during oolong tea processing were closely associated with transcription level changes in corresponding metabolite synthesis genes, and changes in transcript levels of these important synthesis genes were strictly regulated by 5mC methylation. We further report that some key genes during processing are regulated by 5mC methylation, which can effectively explain the content changes of important aroma metabolites, including α-farnesene, nerolidol, lipids, and taste substances such as catechins. Our results not only highlight the key roles of 5mC methylation in important flavor substance synthesis in pre- and post-harvest processing, but also provide epimutation-related gene targets for future improvement of tea quality or breeding of whole-tissue high-theanine varieties.

MicroRNAs (miRNAs) target specific mRNA molecules based on sequence complementarity for their degradation or repression of translation, thereby regulating various developmental and physiological processes in eukaryotic organisms. Expressing the target mimicry (MIM) and short tandem target mimicry (STTM) can block endogenous activity of mature miRNAs and eliminate the inhibition of their target genes, resulting in phenotypic changes due to higher expression of the target genes. Here, we report a strategy to achieve derepression of interested miRNA-target genes through CRISPR/Cas9-based generation of in-frame mutants within the miRNA-complementary sequence of the target gene. We show that two rice genes, OsGRF4 (GROWTH REGULATING FACTOR 4) and OsGRF8 carrying in-frame mutants with disruption of the miR396 recognition sites, escape from miR396-mediated post-transcriptional silencing, resulting in enlarged grain size and increase in brown planthopper (BPH) resistance, in their respective transgenic rice lines. These results demonstrate that CRISPR/Cas9-mediated disruption of miRNA target sites can be effectively employed to precisely derepress particular target genes of functional importance for trait improvement in plants.

Continuously increasing global temperatures present great challenges to food security. Grain size, one of the critical components determining grain yield in rice (Oryza sativa L.), is a prime target for genetic breeding. Thus, there is an immediate need for genetic improvement in rice to maintain grain yield under heat stress. However, quantitative trait loci (QTLs) endowing heat stress tolerance and grain size in rice are extremely rare. Here, we identified a novel negative regulator with pleiotropic effects, Thermo‐Tolerance and grain Length 1 (TTL1), from the super pan‐genomic and transcriptomic data. Loss‐of‐function mutations in TTL1 enhanced heat tolerance, and caused an increase in grain size by coordinating cell expansion and proliferation. TTL1 was shown to function as a transcriptional regulator and localized to the nucleus and cell membrane. Furthermore, haplotype analysis showed that hap^L and hap^S of TTL1 were obviously correlated with variations of thermotolerance and grain size in a core collection of cultivars. Genome evolution analysis of available rice germplasms suggested that TTL1 was selected during domestication of the indica and japonica rice subspecies, but still had much breeding potential for increasing grain length and thermotolerance. These findings provide insights into TTL1 as a novel potential target for the development of high‐yield and thermotolerant rice varieties.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the two-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multi-omics analysis of small RNA, degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt small RNA (sRNA) species in rice anthers and are sensitive to low temperature and markedly down-regulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5 and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss-of-function of 21-nt phasiRNAs can result in up-regulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in two-line hybrid rice breeding in the future.