Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight

Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci （QTLs）. We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.

Identification and fine mapping of two blast resistance genes in rice cultivar 93-11

Rice blast, caused by Magnaporthe oryzae, is a major disease of rice almost worldwide. The Chinese indica cultivar 93-11 is resistant to numerous isolates of the blast fungus in China, and can be used as broad-spectrum resistance resource, particularly in japonica rice breeding programs. In this study, we identified and mapped two blast resistance genes, Pi60(t) and Pi61(t), in cv. 93-11 using F2 and F3 populations derived from a cross between the susceptible cv. Lijiangxintuanheigu(LTH) and resistant cv. 93-11 and inoculated with M. oryzae isolates from different geographic origins. Pi60(t) was delimited to a 274 kb region on the short arm of chromosome 11, flanked by InDel markers K1-4 and E12 and cosegregated with InDel markers B1 and Y10. Pi61(t) was mapped to a 200 kb region on the short arm(near the centromere) of chromosome 12, flanked by InDel markers M2 and S29 and cosegregating with InDel marker M9. In the 274 kb region of Pi60(t), 93-11 contains six NBS-LRR genes including the two Pia/ PiCO39 alleles(BGIOSGA034263 and BGIOSGA035032) which are quite close to the two Pia/ PiCO39 alleles(SasRGA4 and SasRGA5) in Sasanishiki and CO39, with only nine amino acids differing in the protein sequences of BGIOSGA035032 and SasRGA5. In the 200 kb region of Pi61(t), 93-11 contains four NBS-LRR genes, all of which show high identities in protein sequence with their corresponding NBS-LRR alleles in susceptible cv. Nipponbare. Comparison of the response spectra and physical positions between the target genes and other R genes in the same chromosome regions indicated that Pi60(t) could be Pia/PiCO39 or its allele, whereas Pi61(t) appears to be different from Pita, Pita-2, Pi19(t), Pi39(t) and Pi42(t) in the same R gene cluster. DNA markers tightly linked to Pi60(t) and Pi61(t) will enable marker-assisted breeding and map-based cloning.

Mitochondrion-targeted PENTATRICOPEPTIDE REPEAT5 is required for cis-splicing of nad4 intron 3 and endosperm development in rice

Endosperm as the storage organ of starch and protein in cereal crops largely determines grain yield and quality.Despite the fact that several pentatricopeptide repeat(PPR)proteins required for endosperm development have been identified in rice,the molecular mechanisms of many P-type PPR proteins in endosperm development remains unclear.Here,we isolated a rice floury endosperm mutant ppr5 that developed small starch grains and an abnormal aleurone layer,accompanied by decreased starch,protein,and amylose contents.Map-based cloning combined with a complementation test demonstrated that PPR5 encodes a P-type PPR protein that is localized to the mitochondria.The mutation in PPR5 caused reduced splicing efficiency of mitochondrial NADH dehydrogenase 4(nad4)gene intron 3 and reduced complex I assembly and activity.Loss of PPR5 function greatly upregulated expression of alternative oxidases(AOXs),reduced ATP production,and affected mitochondrial morphology.We demonstrate that PPR5,as a P-type PPR protein,is required for mitochondrial function and endosperm development by controlling the cis-splicing of mitochondrial nad4 intron 3.

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.

ESCRT-Ⅲ component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice

The endosomal sorting complex required for transport(ESCRT)is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes.Although ESCRT components affect a variety of plant growth and development processes,their impact on leaf development is rarely reported.Here,we found that OsSNF7.2,an ESCRT-Ⅲ component,controls leaf rolling in rice(Oryza sativa).The Ossnf7.2 mutant rolled leaf 17(rl17)has adaxially rolled leaves due to the decreased number and size of the bulliform cells.OsSNF7.2is expressed ubiquitously in all tissues,and its protein is localized in the endosomal compartments.OsSNF7.2 homologs,including OsSNF7,OsSNF7.3,and OsSNF7.4,can physically interact with OsSNF7.2,but their single mutation did not result in leaf rolling.Other ESCRT complex subunits,namely OsVPS20,OsVPS24,and OsBRO1,also interact with OsSNF7.2.Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation.Both Osyuc8and rl17 Osyuc8 showed rolled leaves,indicating that OsYUC8 and OsSNF7.2 function in the same pathway,conferring leaf development.This study reveals a new biological function for the ESCRT-Ⅲcomponents,and provides new insights into the molecular mechanisms underlying leaf rolling.

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.

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.

GW5-Like, a homolog of GW5, negatively regulates grain width, weight and salt resistance in rice

Grain size is an important determinant of yield potential in crops. We previously demonstrated that natural mutations in the regulatory sequences of qSW5/GW5 confer grain width diversity in rice. However, the biological function of a GW5 homolog, named GW5-Like(GW5 L), remains unknown. In this study, we report on GW5 L knockout mutants in Kitaake, a japonica cultivar(cv.)considered to have a weak gw5 variant allele that confers shorter and wider grains. GW5 L is evenly expressed in various tissues, and its protein product is localized to the plasma membrane. Biochemical assays verified that GW5 L functions in a similar fashion to GW5. It positively regulates brassinosteroid(BR) signaling through repression of the phosphorylation activity of GSK2. Genetic data show that GW5 L overexpression in either Kitaake or a GW5 knockout line, Kasaorf3(indica cv. Kasalath background), causes more slender, longer grains relative to the wild-type. We also show that GW5 L could confer salt stress resistance through an association with calmodulin protein OsCa M1-1. These findings identify GW5 L as a negative regulator of both grain size and salt stress tolerance, and provide a potential target for breeders to improve grain yield and salt stress resistance in rice.

Rice FLOURY ENDOSPERM 18 encodes a pentatricopeptide repeat protein required for 5′ processing of mitochondrial nad5 messenger RNA and endosperm development

Pentatricopeptide repeat(PPR) proteins, composing one of the largest protein families in plants,are involved in RNA binding and regulation of organelle RNA metabolism at the posttranscriptional level. Although several PPR proteins have been implicated in endosperm development in rice(Oryza sativa), the molecular functions of many PPRs remain obscure. Here, we identified a rice endosperm mutant named floury endosperm 18(flo18) with pleiotropic defects in both reproductive and vegetative development.Map-based cloning and complementation tests showed that FLO18 encodes a mitochondriontargeted P-type PPR protein with 15 PPR motifs.Mitochondrial function was disrupted in the flo18 mutant, as evidenced by decreased assembly of Complex I in the mitochondrial electron transport chain and altered mitochondrial morphology. Loss of FLO18 function resulted in defective 5′-end processing of mitochondrial nad5 transcripts encoding subunit 5 of nicotinamide adenine dinucleotide hydrogenase. These results suggested that FLO18 is involved in 5′-end processing of nad5 messenger RNA and plays an important role in mitochondrial function and endosperm development.

DHD4,a CONSTANS-like family transcription factor,delays heading date by affecting the formation of the FAC complex in rice

Heading date(or flowering time)is one of the most important agronomic traits in rice,influencing its regional adaptability and crop yield.Many major-effect genes for rice heading date have been identified,but in practice they are difficult to be used for rice molecular breeding because of their dramatic effects on heading date.Genes with minor effects on heading date,which are more desirable for fine-tuning flowering time without significant yield penalty,were seldom reported.In this study,we identified a new minor-effect heading date repressor,Delayed Heading Date 4(DHD4).The dhd4 mutant shows a slightly earlier flowering phenotype without a notable yield penalty compared with wild-type plants under natural long-day conditions.DHD4 encodes a CONSTANS-like transcription factor localized in the nucleus.Molecular,biochemical,and genetic assays show that DHD4 can compete with 14-3-3 to interact with OsFD1,thus affecting the formation of the Hd3a-14-3-3-OsFD1 triprotein FAC complex,resulting in reduced expression of OsMADS14 and OsMADS15,and ultimately delaying flowering.Taken together,these results shed new light on the regulation of flowering time in rice and provide a promising target for fine-tuning flowering time to improve the regional adaptability of rice.