Clock component OsPRR59 delays heading date by repressing transcription of Ehd3 in rice

Heading date(or flowering time),an important agronomic trait in crop species,is closely associated with regional adaptation and yield.Members of the Pseudo-Response Regulator(PRR)family play key roles in regulating flowering.However,their role and molecular mechanism controlling heading date in rice is not very clear.Here,we identified rice OsPRR protein,OsPRR59,which delayed heading under longday conditions.OsPRR59 positively regulates yield by affecting plant height,secondary branches number per panicle,grain number per panicle,seed setting rate,and grain weight per plant.OsPRR59 is expressed in most tissues and its protein is localized to the nucleus.We also found that OsPRR59 directly binds to the promoter of Ehd3 to inhibit its expression.Compared with the WT,osprr59 ehd3 showed a significantly delayed heading phenotype,as did the ehd3 mutant.This was opposite to the phenotype of the osprr59 mutant,confirming that Ehd3 acted downstream of OsPRR59 in regulating rice flowering.Our results identified a direct regulator of Ehd3,and revealed a novel molecular mechanism of clock component OsPRR proteins in regulating heading date and provide a new genetic resource for fine-tuning heading date in rice.

OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice

Although both protein arginine methylation(PRMT)and jasmonate(JA)signaling are crucial for regulating plant development,the relationship between these processes in the control of spikelet development remains unclear.In this study,we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures.Interestingly,we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7.We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs,thereby promoting the ubiquitination of OsJAZ1 by the SCF^(OsCOI1a/OsCOI1b) complex and degradation via the 26S proteasome.This process ultimately releases OsMYC2,a core transcriptional regulator in the JA signaling pathway,to activate or repress JA-responsive genes,thereby maintaining normal plant(spikelet)development.However,in the osprmt6a-1 mutant,reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs.As a result,OsJAZ1 proteins become more stable,repressing JA responses,thus causing the formation of abnormal spikelet structures.Moreover,we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner,thereby establishing a negative feedback loop to balance JA signaling.We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures.Collectively,our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

A Novel Chloroplast-Localized Pentatricopeptide Repeat Protein Involved in Splicing Affects Chloroplast Development and Abiotic Stress Response in Rice

Pentatricopeptide repeat （PPR） proteins comprise a large family in higher plants and modulate organellar gene expression by participating in various aspects of organellar RNA metabolism. In rice, the family contains 477 members, and the majority of their functions remain unclear. In this study, we isolated and characterized a rice mutant, white stripe leaf （wsl）, which displays chlorotic striations early in development. Map-based cloning revealed that WSL encodes a newly identified rice PPR protein which targets the chloroplasts. In wsl mutants, PEP-dependent plastid gene expression was significantly down-regulated, and plastid rRNAs and translation products accumulate to very low levels. Consistently with the observations, wsl shows a strong defect in the splicing of chloroplast transcript rpl2, resulting in aberrant transcript accumulation and its product reduction in the mutant. The wsl shows enhanced sensitivity to ABA, salinity, and sugar, and it accumulates more H2O2 than wild-type. These results suggest the reduced translation efficiency may affect the response of the mutant to abiotic stress.

Identification of a Stable Quantitative Trait Locus for Percentage Grains with White Chalkiness in Rice (Oryza sativa)

High chalkiness is a major problem in many rice-producing areas of the world, especially in hybrid rice （Oryza sativa L.） in China. We previously showed a major quantitative trait locus for the percentage of grains with white chalkiness （QTLqPGWC-8） in the interval G1149-R727 on chromosome 8 using a chromosome segment substitution line （CSSL）. Here, we selected the line-CSSL50 harboring the QTLqPGWC-8 allele from the CSSLs derived from a cross between Asominori （as a recurrent parent） and IR24 （as a donor parent）, which had higher percentage chalkiness, markedly different from that of Asominori. There were also significant differences in starch granules, appearance of amylose content （AAC） and milling qualities between Asominori and CSSL50, but not in grain size or thousand grain weight （TGW）. The BC4F2 and BC4F3 populations from a cross between CSSL50 and Asominori were used for fine mapping of qPGWC-8. We narrowed down the location of this QTL to a 142 kb region between Indel markers 8G-7 and 8G-9. QTLqPGWC-8 accounted for 50.9% of the difference in PGWC between the parents. The markers tightly linked to qPGWC-8 should facilitate cloning of the gene underlying this QTL and will be of value for marker-assisted selection in breeding rice varieties with better grain quality.

Construction of a sustainable 3-hydroxybutyrate-producing probiotic Escherichia coli for treatment of colitis

Colitis is a common disease of the colon that is very difficult to treat.Probiotic bacteria could be an effective treatment.The probiotic Escherichia coli Nissle 1917(EcN)was engineered to synthesize the ketone body(R)-3-hydroxybutyrate(3HB)for sustainable production in the gut lumen of mice suffering from colitis.Components of heterologous 3HB synthesis routes were constructed,expressed,optimized,and inserted into the EcN genome,combined with deletions in competitive branch pathways.The genome-engineered EcN produced the highest 3HB level of 0.6 g/L under microaerobic conditions.The live therapeutic was found to colonize the mouse gastrointestinal tract over 14 days,elevating gut 3HB and short-chain-length fatty acid(SCFA)levels 8.7-and 3.1-fold compared to those of wild-type EcN,respectively.The sustainable presence of 3HB in mouse guts promoted the growth of probiotic bacteria,especially Akkermansia spp.,to over 31%from the initial 2%of all the microbiome.As a result,the engineered EcN termed EcNL4 ameliorated colitis induced via dextran sulfate sodium(DSS)in mice.Compared to wild-type EcN or oral administration of 3HB,oral EcNL4 uptake demonstrated better effects on mouse weights,colon lengths,occult blood levels,gut tissue myeloperoxidase activity and proinflammatory cytokine concentrations.Thus,a promising live bacterium was developed to improve colonic microenvironments and further treat colitis.This proof-of-concept design can be employed to treat other diseases of the colon.

Transcriptional activation and phosphorylation of OsCNGC9 confer enhanced chilling tolerance in rice

Low temperature is a major environmental factor that limits plant growth and productivity.Although transient elevation of cytoplasmic calcium has long been recognized as a critical signal for plant cold tolerance,the calcium channels responsible for this process have remained largely elusive.Here we report that OsCNGC9,a cyclic nucleotide-gated channel,positively regulates chilling tolerance by mediating cytoplasmic calcium elevation in rice(Oryza sativa).We showed that the loss-of-function mutant of OsCNGC9 is defective in cold-induced calcium influx and more sensitive to prolonged cold treatment,whereas OsCNGC9 overexpression confers enhanced cold tolerance.Mechanistically,we demonstrated that in response to chilling stress,OsSAPK8,a homolog of Arabidopsis thaliana OST1,phosphorylates and activates OsCNGC9 to trigger Ca2+influx.Moreover,we found that the transcription of OsCNGC9 is activated by a rice dehydration-responsive element-binding transcription factor,OsDREB1A.Taken together,our results suggest that OsCNGC9 enhances chilling tolerance in rice through regulating cold-induced calcium influx and cytoplasmic calcium elevation.

3-Hydroxybutyrate ameliorates insulin resistance by inhibiting PPARγSer273 phosphorylation in type 2 diabetic mice

3-Hydroxybutyrate(3HB)is a small ketone body molecule produced endogenously by the body in the liver.Previous studies have shown that 3HB can reduce blood glucose level in type 2 diabetic(T2D)patients.However,there is no systematic study and clear mechanism to evaluate and explain the hypoglycemic effect of 3HB.Here we demonstrate that 3HB reduces fasting blood glucose level,improves glucose tolerance,and ameliorates insulin resistance in type 2 diabetic mice through hydroxycarboxylic acid receptor 2(HCAR2).Mechanistically,3HB increases intracellular calcium ion(Ca^(2+))levels by activating HCAR2,thereby stimulating adenylate cyclase(AC)to increase cyclic adenosine monophosphate(cAMP)concentration,and then activating protein kinase A(PKA).Activated PKA inhibits Raf1 proto-oncogene serine/threonine-protein kinase(Raf1)activity,resulting in a decrease in extracellular signal-regulated kinases 1/2(ERK1/2)activity and ultimately inhibiting peroxisome proliferator-activated receptorγ(PPARγ)Ser273 phosphorylation in adipocytes.Inhibition of PPARγSer273 phosphorylation by 3HB altered the expression of PPARγregulated genes and reduced insulin resistance.Collectively,3HB ameliorates insulin resistance in type 2 diabetic mice through a pathway of HCAR2/Ca^(2+)/cAMP/PKA/Raf1/ERK1/2/PPARγ.