Variation in WIDTH OF LEAF AND GRAIN contributes to grain and leaf size by controlling LARGE2 stability in rice.

Grain and flag leaf size are two important agronomic traits that influence grain yield in rice (Oryza sativa). Many QTLs and genes that regulate these traits individually have been identified, however, few QTLs and genes that simultaneously control these two traits have been identified. In this study, we conducted a genome-wide association analysis in rice and detected a major locus, WIDTH OF LEAF AND GRAIN (WLG), that associated with both grain and flag leaf width. WLG encodes a RING-domain E3 ubiquitin ligase. WLGhap.B, which possesses five SNP variations compared to WLGhap.A, encodes a protein with enhanced ubiquitination activity that confers increased rice leaf width and grain size, whereas mutation of WLG leads to narrower leaves and smaller grains. Both WLGhap.A and WLGhap.B interact with LARGE2, a HETC-type E3 ligase, however, WLGhap.B exhibits stronger interaction with LARGE2, thus higher ubiquitination activity towards LARGE2 compared with WLGhap.A. Lysine1021 is crucial for the ubiquitination of LARGE2 by WLG. Loss-of-function of LARGE2 in wlg-1 phenocopies large2-c in grain and leaf width, suggesting that WLG acts upstream of LARGE2. These findings reveal the genetic and molecular mechanism by which the WLG-LARGE2 module mediates grain and leaf size in rice, and suggest the potential of WLGhap.B in improving rice yield.

Genome-wide methylation, transcriptome and characteristic metabolites reveal the balance between diosgenin and brassinosteroids in Dioscorea zingiberensis.

Diosgenin (DG) is a bioactive metabolite isolated from Dioscorea species, renowned for its medicinal properties. Brassinosteroids (BRs) are a class of crucial plant steroidal hormones. Cholesterol and campesterol are important intermediates of DG and BR biosynthesis, respectively. DG and BRs are structurally similar components; however, the regulatory network and metabolic interplays have not been fully elucidated. In an effort to decode these complex networks, we conducted a comprehensive study integrating genome-wide methylation, transcriptome and characteristic metabolite data from Dioscorea zingiberensis. Leveraging these data, we were able to construct a comprehensive regulatory network linking DG and BRs. Mass spectrometry results enabled us to clarify the alterations in cholesterol, campesterol, diosgenin, and castasterone (one of the major active BRs). The DG content decreased by 27.72% at 6 h after brassinolide treatment, whereas the content increased by 85.34% at 6 h after brassinazole treatment. Moreover, we pinpointed DG/BR-related genes, such as CASs, CYP90s, and B3-ARFs, implicated in the metabolic pathways of DG and BRs. Moreover, CASs and CYP90s exhibit hypomethylation, which is closely related to their high transcription. These findings provide robust evidence for the homeostasis between DG and BRs. In conclusion, our research revealed the existence of a balance between DG and BRs in D. zingiberensis. Furthermore, our work not only provides new insights into the relationship between the two pathways but also offers a fresh perspective on the functions of secondary metabolites.

The genetic basis and process of inbreeding depression in an elite hybrid rice.

Inbreeding depression refers to the reduced performance arising from increased homozygosity, a phenomenon that is the reverse of heterosis and exists among plants and animals. As a natural self-pollinated crop with strong heterosis, the mechanism of inbreeding depression in rice is largely unknown. To understand the genetic basis of inbreeding depression, we constructed a successive inbreeding population from the F2 to F4 generation and observed inbreeding depression of all heterotic traits in the progeny along with the decay of heterozygosity in each generation. The expected depression effect was largely explained by 13 QTLs showing dominant effects for spikelets per panicle, 11 for primary branches, and 12 for secondary branches, and these loci constitute the main correlation between heterosis and inbreeding depression. However, the genetic basis of inbreeding depression is also distinct from that of heterosis, such that a biased transmission ratio of alleles for QTLs with either dominant or additive effects in four segregation distortion regions would result in minor effects in expected depression. Noticeably, two-locus interactions may change the extent and direction of the depression effects of the target loci, and overall interactions would promote inbreeding depression among generations. Using an F2:3 variation population, the actual performance of the loci showing expected depression was evaluated considering the heterozygosity decay in the background after inbreeding. We found inconsistent or various degrees of background depression from the F2 to F3 generation assuming different genotypes of the target locus, which may affect the actual depression effect of the locus due to epistasis. The results suggest that the genetic architecture of inbreeding depression and heterosis is closely linked but also differs in their intrinsic mechanisms, which expand our understanding of the whole-genome architecture of inbreeding depression.

Spontaneous movement of a retrotransposon generated genic dominant male sterility providing a useful tool for rice breeding.

Male sterility in plants provides valuable breeding tools in germplasm innovation and hybrid crop production. However, genetic resources for dominant genic male sterility, which hold great promise to facilitate breeding processes, are extremely rare in natural germplasm. Here we characterized the Sanming Dominant Genic Male Sterility in rice and identified the gene SDGMS using a map-based cloning approach. We found that spontaneous movement of a 1978-bp long terminal repeat (LTR) retrotransposon into the promoter region of the SDGMS gene activates its expression in anther tapetum, which causes abnormal programmed cell death of tapetal cells resulting in dominant male sterility. SDGMS encodes a ribosome inactivating protein showing N-glycosidase activity. The activation of SDGMS triggers transcription reprogramming of genes responsive to biotic stress leading to a hypersensitive response which causes sterility. The results demonstrate that an ectopic gene activation by transposon movement can give birth to a novel trait which enriches phenotypic diversity with practical utility.

Inferring Historical Introgression with Deep Learning.

Resolving phylogenetic relationships among taxa remains a challenge in the era of big data due to the presence of genetic admixture in a wide range of organisms. Rapidly developing sequencing technologies and statistical tests enable evolutionary relationships to be disentangled at a genome-wide level, yet many of these tests are computationally intensive and rely on phased genotypes, large sample sizes, restricted phylogenetic topologies, or hypothesis testing. To overcome these difficulties, we developed a deep learning-based approach, named ERICA, for inferring genome-wide evolutionary relationships and local introgressed regions from sequence data. ERICA accepts sequence alignments of both population genomic data and multiple genome assemblies, and efficiently identifies discordant genealogy patterns and exchanged regions across genomes when compared with other methods. We further tested ERICA using real population genomic data from Heliconius butterflies that have undergone adaptive radiation and frequent hybridization. Finally, we applied ERICA to characterize hybridization and introgression in wild and cultivated rice, revealing the important role of introgression in rice domestication and adaptation. Taken together, our findings demonstrate that ERICA provides an effective method for teasing apart evolutionary relationships using whole genome data, which can ultimately facilitate evolutionary studies on hybridization and introgression.

A Gγ protein regulates alkaline sensitivity in crops.

The use of alkaline salt lands for crop production is hindered by a scarcity of knowledge and breeding efforts for plant alkaline tolerance. Through genome association analysis of sorghum, a naturally high-alkaline-tolerant crop, we detected a major locus, Alkaline Tolerance 1 (AT1), specifically related to alkaline-salinity sensitivity. An at1 allele with a carboxyl-terminal truncation increased sensitivity, whereas knockout of AT1 increased tolerance to alkalinity in sorghum, millet, rice, and maize. AT1 encodes an atypical G protein γ subunit that affects the phosphorylation of aquaporins to modulate the distribution of hydrogen peroxide (H2O2). These processes appear to protect plants against oxidative stress by alkali. Designing knockouts of AT1 homologs or selecting its natural nonfunctional alleles could improve crop productivity in sodic lands.

A minimal genome design to maximally guarantee fertile inter-subspecific hybrid rice.

Hybrid rice has made considerable contributions to achieve the ambitious goal of food security for the world's population. Hybrid rice from indica/xian and japonica/geng subspecies shows much higher heterosis and is thereby an important innovation in promoting rice production in the next decade. However, such inter-subspecific hybrid rice has long suffered from serious hybrid sterility, which is a major challenge that needs to be addressed. In this study, we performed a genome design strategy to produce fertile inter-subspecific hybrid by creation of wide compatibility varieties that are able to overcome hybrid sterility. Based on combined genetic analyses in two indica-japonica crosses, we determined that four hybrid sterility loci, S5, f5, pf12 and Sc, are the major QTLs controlling inter-subspecific hybrid sterility and thus the minimal targets that can be manipulated for breeding sub-specific hybrid rice. We then cloned the pf12 locus, one of the most effective loci for hybrid male sterility, by map-based cloning, and showed that artificial disruption of pf12A gene at this locus could successfully rescue hybrid fertility. We further dissected the genetic basis of wide compatibility using three pairwise crosses from a wide-compatibility variety Dular and representative indica and japonica varieties. On this basis, we constructed and assembled different combinations of naturally compatible alleles of four loci, S5, Sc, pf12, and f5, and found that the improved lines could fully recover pollen and embryo sac fertility in test-crossed F1s, thereby completely fulfilling the demands of inter-subspecific hybrid spikelet fertility in agricultural production. This breeding scheme would facilitate redesign of future inter-subspecific hybrid rice with a higher yield potential.

A Ghd7-centered regulatory network provides a mechanistic approximation to optimal heterosis in an elite rice hybrid.

Heterosis refers to the superior performance of hybrids over their parents, which is a general phenomenon occurring in diverse organisms. Many commercial hybrids produce high yield without delayed flowering, which we refer to as optimal heterosis and is desired in hybrid breeding. Here, we attempted to illustrate the genomic basis of optimal heterosis by reinvestigating the single-locus quantitative trait loci and digenic interactions of two traits, the number of spikelets per panicle (SP) and heading date (HD), using recombinant inbred lines and 'immortalized F2 s' derived from the elite rice (Oryza sativa) hybrid Shanyou 63. Our analysis revealed a regulatory network that may provide an approximation to the genetic constitution of the optimal heterosis observed in this hybrid. In this network, Ghd7 works as the core element, and three other genes, Ghd7.1, Hd1, and Hd3a/RFT1, also have major roles. The effects of positive dominance by Ghd7 and Ghd7.1 and negative dominance by Hd1 and Hd3a/RFT1 in the hybrid background contribute the major part to the high SP without delaying HD; numerous epistatic interactions, most of which involve Ghd7, also play important roles collectively. The results expand our understanding of the genic interaction networks underlying hybrid rice breeding programs, which may be very useful in future crop genetic improvement.

Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice.

Cytoplasmic male sterility (CMS) determined by mitochondrial genes and restorer of fertility (Rf) controlled by nuclear-encoded genes provide the breeding systems of many hybrid crops for the utilization of heterosis. Although several CMS/Rf systems have been widely exploited in rice, hybrid breeding using these systems has encountered difficulties due to either fertility instability or complications of two-locus inheritance or both. In this work, we characterized a type of CMS, Fujian Abortive cytoplasmic male sterility (CMS-FA), with stable sporophytic male sterility and a nuclear restorer gene that completely restores hybrid fertility. CMS is caused by the chimeric open reading frame FA182 that specifically occurs in the mitochondrial genome of CMS-FA rice. The restorer gene OsRf19 encodes a pentatricopeptide repeat (PPR) protein targeted to mitochondria, where it mediates the cleavage of FA182 transcripts, thus restoring male fertility. Comparative sequence analysis revealed that OsRf19 originated through a recent duplication in wild rice relatives, sharing a common ancestor with OsRf1a/OsRf5, a fertility restorer gene for Boro II and Hong-Lian CMS. We developed six restorer lines by introgressing OsRf19 into parental lines of elite CMS-WA hybrids; hybrids produced from these lines showed equivalent or better agronomic performance relative to their counterparts based on the CMS-WA system. These results demonstrate that CMS-FA/OsRf19 provides a highly promising system for future hybrid rice breeding.

New Data and New Features of the FunRiceGenes (Functionally Characterized Rice Genes) Database: 2021 Update.

As a major food crop and model organism, rice has been mostly studied with the largest number of functionally characterized genes among all crops. We previously built the funRiceGenes database including ~ 2800 functionally characterized rice genes and ~ 5000 members of different gene families. Since being published, the funRiceGenes database has been accessed by more than 54,400 users with over 540,000 pageviews. The funRiceGenes database has been continuously updated with newly cloned rice genes and newly published literature, based on the progress of rice functional genomics studies. Up to Nov 2021, ~ 4100 functionally characterized rice genes and ~ 6000 members of different gene families were collected in funRiceGenes, accounting for 22.3% of the 39,045 annotated protein-coding genes in the rice genome. Here, we summarized the update of the funRiceGenes database with new data and new features in the last 5 years.

Understanding the genetic and molecular constitutions of heterosis for developing hybrid rice.

The wide adoption of hybrid rice has greatly increased rice yield in the last several decades. The utilization of heterosis facilitated by male sterility has been a common strategy for hybrid rice development. Here, we summarize our efforts in the genetic and molecular understanding of heterosis and male sterility together with the related progress from other research groups. Analyses of F1 diallel crosses show that strong heterosis widely exists in hybrids of diverse germplasms, and inter-subspecific hybrids often display higher heterosis. Using the elite hybrid Shanyou 63 as a model, an immortalized F2 population design is conducted for systematic characterization of the biological mechanism of heterosis, with identification of loci controlling heterosis of yield and yield component traits. Dominance, overdominance, and epistasis all play important roles in the genetic basis of heterosis; quantitative assessment of these components well addressed the three classical genetic hypotheses for heterosis. Environment-sensitive genic male sterility (EGMS) enables the development of two-line hybrids, and long noncoding RNAs often function as regulators of EGMS. Inter-subspecific hybrids show greatly reduced fertility; the identification and molecular characterization of hybrid sterility genes offer strategies for overcoming inter-subspecific hybrid sterility. These developments have significant implications for future hybrid rice improvement using genomic breeding.

Species-specific partial gene duplication in Arabidopsis thaliana evolved novel phenotypic effects on morphological traits under strong positive selection.

Gene duplication is increasingly recognized as an important mechanism for the origination of new genes, as revealed by comparative genomic analysis. However, how new duplicate genes contribute to phenotypic evolution remains largely unknown, especially in plants. Here, we identified the new gene EXOV, derived from a partial gene duplication of its parental gene EXOVL in Arabidopsis thaliana. EXOV is a species-specific gene that originated within the last 3.5 million years and shows strong signals of positive selection. Unexpectedly, RNA-sequencing analyses revealed that, despite its young age, EXOV has acquired many novel direct and indirect interactions in which the parental gene does not engage. This observation is consistent with the high, selection-driven substitution rate of its encoded protein, in contrast to the slowly evolving EXOVL, suggesting an important role for EXOV in phenotypic evolution. We observed significant differentiation of morphological changes for all phenotypes assessed in genome-edited and T-DNA insertional single mutants and in double T-DNA insertion mutants in EXOV and EXOVL. We discovered a substantial divergence of phenotypic effects by principal component analyses, suggesting neofunctionalization of the new gene. These results reveal a young gene that plays critical roles in biological processes that underlie morphological evolution in A. thaliana.

The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice.

G-protein signaling and ubiquitin-dependent degradation are both involved in grain development in rice, but how these pathways are coordinated in regulating this process is unknown. Here, we show that Chang Li Geng 1 (CLG1), which encodes an E3 ligase, regulates grain size by targeting the Gγ protein GS3, a negative regulator of grain length, for degradation. Overexpression of CLG1 led to increased grain length, while overexpression of mutated CLG1 with changes in three conserved amino acids decreased grain length. We found that CLG1 physically interacts with and ubiquitinats GS3which is subsequently degraded through the endosome degradation pathway, leading to increased grain size. Furthermore, we identified a critical SNP in the exon 3 of CLG1 that is significantly associated with grain size variation in a core collection of cultivated rice. This SNP results in an amino acid substitution from Arg to Ser at position 163 of CLG1 that enhances the E3 ligase activity of CLG1 and thus increases rice grain size. Both the expression level of CLG1 and the SNP CLG1163S may be useful variations for manipulating grain size in rice.

Bract suppression regulated by the miR156/529-SPLs-NL1-PLA1 module is required for the transition from vegetative to reproductive branching in rice.

Reproductive transition of grasses is characterized by switching the pattern of lateral branches, featuring the suppression of outgrowth of the subtending leaves (bracts) and rapid formation of higher-order branches in the inflorescence (panicle). However, the molecular mechanisms underlying such changes remain largely unknown. Here, we show that bract suppression is required for the reproductive branching in rice. We identified a pathway involving the intrinsic time ruler microRNA156/529, their targets SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes, NECK LEAF1 (NL1), and PLASTOCHRON1 (PLA1), which regulates the bract outgrowth and thus affects the pattern switch between vegetative and reproductive branching. Suppression of the bract results in global reprogramming of transcriptome and chromatin accessibility following the reproductive transition, while these processes are largely dysregulated in the mutants of these genes. These discoveries contribute to our understanding of the dynamic plant architecture and provide novel insights for improving crop yields.

YMR152W from Saccharomyces cerevisiae encoding a novel aldehyde reductase for detoxification of aldehydes derived from lignocellulosic biomass.

Aldehydes are the main inhibitors generated during the pretreatment of lignocellulosic biomass, which can inhibit cell growth and disturb subsequent fermentation. Saccharomyces cerevisiae has the intrinsic ability to in situ detoxify aldehydes to their less toxic or nontoxic alcohols by numerous aldehyde dehydrogenases/reductases during the lag phase. Herein, we report that an uncharacterized open reading frame YMR152W from S. cerevisiae encodes a novel aldehyde reductase with catalytic functions for reduction of at least six aldehydes, including two furan aldehydes (furfural and 5-hydroxymethylfurfural), three aliphatic aldehydes (acetaldehyde, glycolaldehyde, and 3-methylbutanal), and an aromatic aldehyde (benzaldehyde) with NADH or NADPH as the co-factor. Particularly, Ymr152wp displayed the highest specific activity (190.86 U/mg), and the best catalytic rate constant (Kcat), catalytic efficiency (Kcat/Km), and affinity (Km) when acetaldehyde was used as the substrate with NADH as the co-factor. The optimum pH of Ymr152wp is acidic (pH 5.0-6.0), but this enzyme is more stable in alkaline conditions (pH 8.0). Metal ions, chemical protective additives, salts, and substrates could stimulate or inhibit enzyme activities of Ymr152wp in varying degrees. Ymr152wp was classified into the quinone oxidoreductase (QOR) subfamily of the medium-chain dehydrogenase/reductase (MDR) family based on the results of amino acid sequence analysis and phylogenetic analysis. Although Ymr152wp was grouped into the QOR family, no quinone reductase activity was observed using typical quinones (9,10-phenanthrenequinone, 1,2-naphthoquinone, and p-benzoquinone) as the substrates. This study provides guidelines for exploring more uncharacterized aldehyde reductases in S. cerevisiae for in situ detoxification of aldehyde inhibitors derived from lignocellulosic hydrolysis.

Multiple Alleles Encoding Atypical NLRs with Unique Central Tandem Repeats in Rice Confer Resistance to Xanthomonas oryzae pv. oryzae.

Plants have developed various mechanisms for avoiding pathogen invasion, including resistance (R) genes. Most R genes encode nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs). Here, we report the isolation of three new bacterial blight R genes in rice, Xa1-2, Xa14, and Xa31(t), which were allelic to Xa1 and encoded atypical NLRs with unique central tandem repeats (CTRs). We also found that Xa31(t) was the same gene as Xa1-2. Although Xa1-2 and Xa14 conferred different resistance spectra, their performance could be attenuated by iTALEs, as has previously been reported for Xa1. XA1, XA1-2, XA14, and non-resistant RGAF differed mainly in the substructure of the leucine-rich repeat domain. They all contained unique CTRs and belonged to the CTR-NLRs, which existed only in Gramineae. We also found that interactions among these genes led to differing resistance performance. In conclusion, our results uncover a unique locus in rice consisting of at least three multiple alleles (Xa1, Xa1-2, and Xa14) that encode CTR-NLRs and confer resistance to Xanthomonas oryzae pv. oryzae (Xoo).