Summary Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF ‐Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF ‐Y heterotrimer complex NF ‐ YB 1‐ YC 12‐ bHLH 144, which is formed by the binding of NF ‐ YB 1 to NF ‐ YC 12 and then bHLH 144 in a sequential order. Knock‐out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf‐yb1 and crnf‐yc12 . RNA ‐seq analysis identified 1496 genes that were commonly regulated by NF ‐ YB 1 and NF ‐ YC 12 , including the key granule‐bound starch synthase gene Wx . NF ‐ YC 12 and bHLH 144 maintain NF ‐ YB 1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF ‐ YB 1 directly binds to the ‘G‐box’ domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF ‐ YB 1‐ YC 12‐ bHLH 144 complex, which has great potential for rice genetic improvement.

The molecular mechanisms of stress tolerance and the use of modern genetics approaches for the improvement of drought stress tolerance have been major focuses of plant molecular biologists. In the present study, we cloned the Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 (GhSnRK2) gene and investigated its functions in transgenic Arabidopsis. We further elucidated the function of this gene in transgenic cotton using virus-induced gene silencing (VIGS) techniques. We hypothesized that GhSnRK2 participates in the stress signaling pathway and elucidated its role in enhancing stress tolerance in plants via various stress-related pathways and stress-responsive genes. We determined that the subcellular localization of the GhSnRK2-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing GhSnRK2 exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that GhSnRK2 acts as a positive regulator in response to cold and drought stresses. Plants overexpressing GhSnRK2 displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of GhSnRK2 expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants. GhSnRK2 gene silencing alleviated drought tolerance in cotton plants, indicating that VIGS technique can certainly be used as an effective means to examine gene function by knocking down the expression of distinctly expressed genes. The results of this study suggested that the GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.

Vibrio alginolyticus is a food-borne marine Vibrio that causes gastroenteritis, otitis media, otitis externa, and septicemia in humans. The pathogenic mechanisms of V. alginolyticus have previously been studied in aquaculture animals; however, the underlying mechanisms in mammals remain unknown. In this study, an in vitro model of mouse peritoneal macrophages infected with V. alginolyticus was established. qPCR results revealed that V. alginolyticus induced the transcription levels of various cytokines, including IL-1β, IL-12, IL-18, TNF-α, IL-17, IL-6, IFN-γ, and IL-10, and the secretion level of IL-1β is the most significant. Inhibition assays with Ac-YVAD-CHO (a caspase-1 inhibitor) and Z-VAD-FMK (a pan-caspase inhibitor) were conducted to determine whether caspase-1 or caspase-11 is involved in V. alginolyticus-triggered IL-1β secretion. Results showed that IL-1β secretion was partly inhibited by Ac-YVAD-CHO and absolutely blocked by Z-VAD-FMK. To explore the sensed pattern recognition receptors, several NLR family members and the AIM2 receptor were detected and many receptors were upregulated especially NLRP3. Moreover, the NLRP3 protein displayed a puncta-like surrounding cell nucleus, which signified that the NLRP3 inflammasome was activated in response to V. alginolyticus infection. Inhibition assays with glyburide and CA-074 methyl ester (K+ outflow inhibitor and cathepsin B inhibitor) blocked IL-1β secretion, which demonstrated the essential role of the NLRP3 inflammasome in inflammatory response. To better understand how V. alginolyticus affects IL-1β release, the NLRP3 inflammasome was detected with doses ranging from 0.1 to 10 MOIs and time periods ranging from 3 to 12 h. Results showed that V. alginolyticus-mediated NLRP3 inflammasome activation was in a time- and dose-dependent manner and IL-1β release peaked at MOI of 1 for 12 h. Most importantly, blocking the NLRP3 inflammasome with inhibitors and the use of NLRP3-/- and caspase-1/11-/- mice could attenuate pro-inflammatory cytokine secretion, such as IL-1β, IL-6, IL-12, and TNF-α. Taken together, our study first found that the NLRP3 inflammasome plays vital roles in V. alginolyticus triggered inflammatory response in mouse peritoneal macrophages. This may provide reference information for the development of potential anti-inflammatory treatments against V. alginolyticus infection.

Notched belly grain (NBG) is a type of deformed grain shape that has been associated with inferior appearance and tastes in rice. NBG is coordinated by both environments and genetics. In this study, we report on the first map-based cloning of an NBG gene on chromosome 4, denoted NBG4, which is a novel allele of Dwarf 11 encoding a cytochrome P450 (CYP724B1) involved in brassinosteroid (BR) biosynthesis. A 10-bp deletion in the 7th exon knocked down the level of the NBG4 transcript and shifted the reading frame of the resulting protein. In addition to the dwarf and clustered panicle as previously reported in the allelic mutants, nbg4 grains also displayed retarded germination and NBG due to the physical constraint of deformed hulls caused by abnormal hull elongation. NBG4 is constitutively expressed with the highest level of expression in immature inflorescences. In all, 2294 genes were differentially expressed in nbg4 and wild-type (WT), and evidence is presented that NBG4 regulates OsPPS-2, OsPRA2, OsYUCCA1, sped1-D, and Dwarf that play critical roles in determining plant architecture, panicle development, and seed germination. This study demonstrated that NBG4 is a key node in the brassinosteroid-mediated regulation of rice grain shape.

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Aeromonas sobria, a common conditional pathogenic bacteria, is widely distributed in the environment and causes gastroenteritis in humans or septicemia in fish. Of all Aeromonas species, A. sobria is the most frequently isolated from human infections especially in immunocompromised subjects. Innate immunity is the first protection system of organism to resist non-specific pathogens invasion; however, the immune response process of hosts against A. sobria infection re\mains unexplored. The present study established an A. sobria infection model using primary mouse peritoneal macrophages (PMφs). The adherence and cytotoxicity of A. sobria on PMφs were determined by May-Grünwald Giemsa staining and LDH release measurement. Pro-inflammatory cytokine expression levels were measured using qPCR, western blotting, and ELISA methods. We also investigated the levels of ASC oligomerization and determined the roles of active caspase-1 in IL-1β secretion through inhibition assays and explored the activated pattern recognition receptors through immunofluorescence. We further elucidated the roles of activated inflammasome in regulating the host’s inflammatory response through inhibition combined with ELISA assays. Our results showed that A. sobria induced lytic cell death and LDH release, whereas it had no adhesive properties on PMφs. A. sobria triggered various proinflammatory cytokine transcription level upregulation, and IL-1β occupied the highest levels. The pro-IL-1β protein expression levels increased in a dose-dependent manner with MOI ranging from 1 to 100. This process was regulated by ASC-dependent inflammasome, which cleavage pro-IL-1β into active IL-1β p17 with activated caspase-1 p20. Meanwhile, the expression levels of NLRP3 receptor significantly increased, location analysis revealed puncta-like surrounding nuclear, and inhibition of NLRP3 inflammasome downregulated caspase-1 activation and IL-1β secretion. Blocking of NLRP3 inflammasome activation through K+ efflux and cathepsin B or caspase approaches downregulated A. sobria–induced proinflammatory cytokine production. Overall, these data indicated that A. sobria induced proinflammatory cytokine production in PMφs through activating NLRP3 inflammasome signaling pathways.

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PKA (protein lysine acetylation) is a key post-translational modification involved in the regulation of various biological processes in rice. So far, rice acetylome data is very limited due to the highly-dynamic pattern of protein expression and PKA modification. In this study, we performed a comprehensive quantitative acetylome profile on four typical rice tissues, i.e., the callus, root, leaf, and panicle, by using a mass spectrometry (MS)-based, label-free approach. The identification of 1536 acetylsites on 1454 acetylpeptides from 890 acetylproteins represented one of the largest acetylome datasets on rice. A total of 1445 peptides on 887 proteins were differentially acetylated, and are extensively involved in protein translation, chloroplast development, and photosynthesis, flowering and pollen fertility, and root meristem activity, indicating the important roles of PKA in rice tissue development and functions. The current study provides an overall view of the acetylation events in rice tissues, as well as clues to reveal the function of PKA proteins in physiologically-relevant tissues.