The MYB transcription factor Seed Shattering 11 controls seed shattering by repressing lignin synthesis in African rice.

African cultivated rice (Oryza glaberrima Steud.) was domesticated from its wild progenitor species (Oryza barthii) about 3000 years ago. Seed shattering is one of the main constraints on grain production in African cultivated rice, which causes severe grain losses during harvest. By contrast, Asian cultivated rice (Oryza sativa) displays greater resistance to seed shattering, allowing higher grain production. A better understanding in regulation of seed shattering would help to improve harvesting efficiency in African cultivated rice. Here, we report the map-based cloning and characterization of OgSH11, a MYB transcription factor controlling seed shattering in O. glaberrima. OgSH11 represses the expression of lignin biosynthesis genes and lignin deposition by binding to the promoter of GH2. We successfully developed a new O. glaberrima material showing significantly reduced seed shattering by knockout of SH11 in O. glaberrima using CRISPR-Cas9 mediated approach. Identification of SH11 not only supplies a new target for seed shattering improvement in African cultivated rice, but also provides new insights into the molecular mechanism of abscission layer development.

Dual-function C2H2-type zinc-finger transcription factor GmZFP7 contributes to isoflavone accumulation in soybean.

Isoflavones are a class of secondary metabolites produced by legumes and play important roles in human health and plant stress tolerance. The C2H2 zinc-finger transcription factor (TF) functions in plant stress tolerance, but little is known about its function in isoflavone regulation in soybean (Glycine max). Here, we report a C2H2 zinc-finger TF gene, GmZFP7, which regulates isoflavone accumulation in soybean. Overexpressing GmZFP7 increased the isoflavone concentration in both transgenic hairy roots and plants. By contrast, silencing GmZFP7 expression significantly reduced isoflavone levels. Metabolomic and qRT-PCR analysis revealed that GmZFP7 can increase the flux of the phenylpropanoid pathway. Furthermore, dual-luciferase and electrophoretic mobility shift assays showed that GmZFP7 regulates isoflavone accumulation by influencing the expression of Isoflavone synthase 2 (GmIFS2) and Flavanone 3 ß-hydroxylase 1 (GmF3H1). In this study, we demonstrate that GmZFP7 contributes to isoflavone accumulation by regulating the expression of the gateway enzymes (GmIFS2 and GmF3H1) of competing phenylpropanoid pathway branches to direct the metabolic flux into isoflavone. A haplotype analysis indicated that important natural variations were present in GmZFP7 promoters, with P-Hap1 and P-Hap3 being the elite haplotypes. Our findings provide insight into how GmZFP7 regulates the phenylpropanoid pathway and enhances soybean isoflavone content.

The APETALA2-Like Transcription Factor SUPERNUMERARY BRACT Controls Rice Seed Shattering and Seed Size.

The elimination of seed shattering was a crucial event during crop domestication. Improving and fine-tuning the regulation of this process will further enhance grain yield by avoiding seed losses during crop production. In this work, we identified the loss-of-shattering mutant suppression of shattering1 (ssh1) through a screen of mutagenized wild rice (Oryza rufipogon) introgression lines with naturally high shattering. Using the MutMap approach and transformation experiments, we isolated a genetic factor for seed shattering, SSH1, which is an allele of SUPERNUMERARY BRACT (SNB), a gene encoding a plant-specific APETALA2-like transcription factor. A C-to-A point mutation in the ninth intron of SNB altered the splicing of its messenger RNA, causing the reduced shattering of the ssh1 mutant by altering the development of the abscission layer and vascular bundle at the junction between the seed and the pedicel. Our data suggest that SNB positively regulates the expression of two rice REPLUMLESS orthologs, qSH1 and SH5 In addition, the ssh1 mutant had larger seeds and a higher grain weight, resulting from its increased elongation of the glume longitudinal cells. The further identification of favorable SNB alleles will be valuable for improving rice seed shattering and grain yield using molecular breeding strategies.

HIGH-TILLERING AND DWARF 12 modulates photosynthesis and plant architecture by affecting carotenoid biosynthesis in rice.

Photosynthesis and plant architecture are important factors influencing grain yield in rice (Oryza sativa L.). Here, we identified a high-tillering and dwarf 12 (htd12) mutant and analyzed the effects of the HTD12 mutation on these important factors. HTD12 encodes a 15-cis-ζ-carotene isomerase (Z-ISO) belonging to the nitrite and nitric oxide reductase U (NnrU) protein family, as revealed by positional mapping and transformation experiments. Sequence analysis showed that a single nucleotide transition from guanine (G) to adenine (A) in the 3' acceptor site between the first intron and second exon of HTD12 alters its mRNA splicing in htd12 plants, resulting in a 49-amino acid deletion that affects carotenoid biosynthesis and photosynthesis. In addition, compared with the wild type, htd12 had significantly lower concentrations of ent-2'-epi-5-deoxystrigol (epi-5DS), a native strigolactone, in both roots and root exudates, resulting in an obvious increase in tiller number and decrease in plant height. These findings indicate that HTD12, the rice homolog of Z-ISO, regulates chloroplast development and photosynthesis by functioning in carotenoid biosynthesis, and modulates plant architecture by affecting strigolactone concentrations.

Identification of an active miniature inverted-repeat transposable element mJing in rice.

Miniature inverted-repeat transposable elements (MITEs) are structurally homogeneous non-autonomous DNA transposons with high copy numbers that play important roles in genome evolution and diversification. Here, we analyzed the rice high-tillering dwarf (htd) mutant in an advanced backcross population between cultivated and wild rice, and identified an active MITE named miniature Jing (mJing). The mJing element belongs to the PIF/Harbinger superfamily. japonica rice var. Nipponbare and indica var. 93-11 harbor 72 and 79 mJing family members, respectively, have undergone multiple rounds of amplification bursts during the evolution of Asian cultivated rice (Oryza sativa L.). A heterologous transposition experiment in Arabidopsis thaliana indicated that the autonomous element Jing is likely to have provides the transposase needed for mJing mobilization. We identified 297 mJing insertion sites and their presence/absence polymorphism among 71 rice samples through targeted high-throughput sequencing. The results showed that the copy number of mJing varies dramatically among Asian cultivated rice (O. sativa), its wild ancestor (O. rufipogon), and African cultivated rice (O. glaberrima) and that some mJing insertions are subject to directional selection. These findings suggest that the amplification and removal of mJing elements have played an important role in rice genome evolution and species diversification.

ESA1 Is Involved in Embryo Sac Abortion in Interspecific Hybrid Progeny of Rice.

The emergence of sterile individuals in the hybrid backcross progeny of wild and cultivated rice limits the use of wild rice alleles for improving cultivated rice, but the molecular mechanisms underlying this sterility remain unclear. Here, we identified the semisterile introgression line YIL42, derived from a cross between the indica rice variety Teqing (Oryza sativa) and Oryza rufipogon accession YJCWR (Yuanjiang common wild rice), which exhibits semisterility. Using positional cloning, we isolated EMBRYO SAC ABORTION 1 (ESA1), which encodes a nuclear-membrane localized protein containing an armadillo repeat domain. A mutation in ESA1 at position 1819 (T1819C) converts a stop codon into an Arg (R) codon, causing delayed termination of protein translation. Analysis of transgenic lines indicated that the difference in ESA1 protein structure between O. rufipogon-derived ESA1 and Teqing-derived esa1 affects female gamete abortion during early mitosis. Fertility investigation and expression analysis indicated that the interaction between ESA1 T1819 and unknown gene(s) of Teqing affects spikelet fertility of the hybrid backcross progeny. The ESA1 T1819 allele is present in O. rufipogon but absent in O. sativa, suggesting that variation in ESA1 may be associated with interspecific hybrid incompatibility between wild and cultivated rice. Our findings provide insight into the molecular mechanism underlying female sterility, which is useful for improving the panicle seed setting rate of rice and for developing a strategy to overcome interspecific hybrid sterility between cultivated rice and wild rice.

Variation in the regulatory region of FZP causes increases in secondary inflorescence branching and grain yield in rice domestication.

Inflorescence branching is a key agronomic trait determining rice yield. The primary branch of the ancestral wild rice (Oryza rufipogon Griff.) bears few grains, due to minimal secondary branching. By contrast, Oryza sativa cultivars have been selected to produce large panicles with more secondary branches. Here we showed that the CONTROL OF SECONDARY BRANCH 1 (COS1) gene, which is identical to FRIZZY PANICLE (FZP), plays an important role in the key transition from few secondary branches in wild rice to more secondary branches in domesticated rice cultivars. A 4-bp tandem repeat deletion approximately 2.7 kb upstream of FZP may affect the binding activities of auxin response factors to the FZP promoter, decrease the expression level of FZP and significantly enhance the number of secondary branches and grain yield in cultivated rice. Functional analyses showed that NARROW LEAF 1 (NAL1), a trypsin-like serine and cysteine protease, interacted with FZP and promoted its degradation. Consistently, downregulating FZP expression or upregulating NAL1 expression in the commercial cultivar Zhonghua 17 increased the number of secondary branches per panicle, grain number per panicle and grain yield per plant. Our findings not only provide insights into the molecular mechanism of increasing grain number and yield during rice domestication, but also offer favorable genes for improving the grain yield of rice.

The domestication of plant architecture in African rice.

Plant architecture is a key agronomical factor determining crop yield and has been a major target of cereal crop domestication. The transition of plant architecture from the prostrate tiller of typical African wild rice (Oryza barthii) to the erect tiller of African cultivated rice (Oryza glaberrima) was a key step during domestication of African rice. Here we show that PROG7 (PROSTRATE GROWTH 7), a zinc-finger transcription factor gene on chromosome 7, is required for the prostrate growth of African wild rice. Mutations in the promoter region of prog7 reduced the level of gene expression in the tiller base, leading to erect growth in African cultivated rice. Sequence comparison and haplotype analysis show that 90 varieties of cultivated rice from 11 countries carry the same mutations in the prog7 region. A strong signal in a 60-kb genomic region was detected around the prog7 gene, suggesting that the region was under strong positive selection during the domestication process. Identification of the PROG7 gene provides new insights into the molecular basis of plant architecture in crops and facilitates investigation of the history of domestication of African rice.

GAD1 Encodes a Secreted Peptide That Regulates Grain Number, Grain Length, and Awn Development in Rice Domestication.

Cultivated rice (Oryza sativa) was domesticated from wild rice (Oryza rufipogon), which typically displays fewer grains per panicle and longer grains than cultivated rice. In addition, wild rice has long awns, whereas cultivated rice has short awns or lacks them altogether. These changes represent critical events in rice domestication. Here, we identified a major gene, GRAIN NUMBER, GRAIN LENGTH AND AWN DEVELOPMENT1 (GAD1), that regulates those critical changes during rice domestication. GAD1 is located on chromosome 8 and is predicted to encode a small secretary signal peptide belonging to the EPIDERMAL PATTERNING FACTOR-LIKE family. A frame-shift insertion in gad1 destroyed the conserved cysteine residues of the peptide, resulting in a loss of function, and causing the increased number of grains per panicle, shorter grains, and awnless phenotype characteristic of cultivated rice. Our findings provide a useful paradigm for revealing functions of peptide signal molecules in plant development and helps elucidate the molecular basis of rice domestication.

LABA1, a Domestication Gene Associated with Long, Barbed Awns in Wild Rice.

Common wild rice (Oryza rufipogon), the wild relative of Asian cultivated rice (Oryza sativa), flaunts long, barbed awns, which are necessary for efficient propagation and dissemination of seeds. By contrast, O. sativa cultivars have been selected to be awnless or to harbor short, barbless awns, which facilitate seed processing and storage. The transition from long, barbed awns to short, barbless awns was a crucial event in rice domestication. Here, we show that the presence of long, barbed awns in wild rice is controlled by a major gene on chromosome 4, LONG AND BARBED AWN1 (LABA1), which encodes a cytokinin-activating enzyme. A frame-shift deletion in LABA1 of cultivated rice reduces the cytokinin concentration in awn primordia, disrupting barb formation and awn elongation. Sequencing analysis demonstrated low nucleotide diversity and a selective sweep encompassing an ∼800-kb region around the derived laba1 allele in cultivated rice. Haplotype analysis revealed that the laba1 allele originated in the japonica subspecies and moved into the indica gene pool via introgression, suggesting that humans selected for this locus in early rice domestication. Identification of LABA1 provides new insights into rice domestication and also sheds light on the molecular mechanism underlying awn development.

TOND1 confers tolerance to nitrogen deficiency in rice.

Nitrogen (N), the most important mineral nutrient for plants, is critical to agricultural production systems. N deficiency severely affects rice growth and decreases rice yields. However, excessive use of N fertilizer has caused severe pollution to agricultural and ecological environments. The necessity of breeding of crops that require lower input of N fertilizer has been recognized. Here we identified a major quantitative trait locus on chromosome 12, Tolerance Of Nitrogen Deficiency 1 (TOND1), that confers tolerance to N deficiency in the indica cultivar Teqing. Sequence verification of 75 indica and 75 japonica cultivars from 18 countries and regions demonstrated that only 27.3% of cultivars (41 indica cultivars) contain TOND1, whereas 72.7% of cultivars, including the remaining 34 indica cultivars and all 75 japonica cultivars, do not harbor the TOND1 allele. Over-expression of TOND1 increased the tolerance to N deficiency in the TOND1-deficient rice cultivars. The identification of TOND1 provides a molecular basis for breeding rice varieties with improved grain yield despite decreased input of N fertilizers.

PAY1 improves plant architecture and enhances grain yield in rice.

Plant architecture, a complex of the important agronomic traits that determine grain yield, is a primary target of artificial selection of rice domestication and improvement. Some important genes affecting plant architecture and grain yield have been isolated and characterized in recent decades; however, their underlying mechanism remains to be elucidated. Here, we report genetic identification and functional analysis of the PLANT ARCHITECTURE AND YIELD 1 (PAY1) gene in rice, which affects plant architecture and grain yield in rice. Transgenic plants over-expressing PAY1 had twice the number of grains per panicle and consequently produced nearly 38% more grain yield per plant than control plants. Mechanistically, PAY1 could improve plant architecture via affecting polar auxin transport activity and altering endogenous indole-3-acetic acid distribution. Furthermore, introgression of PAY1 into elite rice cultivars, using marker-assisted background selection, dramatically increased grain yield compared with the recipient parents. Overall, these results demonstrated that PAY1 could be a new beneficial genetic resource for shaping ideal plant architecture and breeding high-yielding rice varieties.

CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF11, controls panicle architecture and seed size in rice.

Panicle architecture and seed size are important agronomic traits that directly determine grain yield in rice (Oryza sativa L.). Although a number of key genes controlling panicle architecture and seed size have been cloned and characterized in recent years, their genetic and molecular mechanisms remain unclear. In this study, we identified a mutant that produced panicles with fascicled primary branching and reduced seeds in size. We isolated the underlying CLUSTERED PRIMARY BRANCH 1 (CPB1) gene, a new allele of DWARF11 (D11) encoding a cytochrome P450 protein involved in brassinosteroid (BR) biosynthesis pathway. Genetic transformation experiments confirmed that a His360Leu amino acid substitution residing in the highly conserved region of CPB1/D11 was responsible for the panicle architecture and seed size changes in the cpb1 mutants. Overexpression of CPB1/D11 under the background of cpb1 mutant not only rescued normal panicle architecture and plant height, but also had a larger leaf angle and seed size than the controls. Furthermore, the CPB1/D11 transgenic plants driven by panicle-specific promoters can enlarge seed size and enhance grain yield without affecting other favourable agronomic traits. These results demonstrated that the specific mutation in CPB1/D11 influenced development of panicle architecture and seed size, and manipulation of CPB1/D11 expression using the panicle-specific promoter could be used to increase seed size, leading to grain yield improvement in rice.

NARROW AND ROLLED LEAF 2 regulates leaf shape, male fertility, and seed size in rice.

Grain yield in rice (Oryza sativa L.) is closely related to leaf and flower development. Coordinative regulation of leaf, pollen, and seed development in rice as a critical biological and agricultural question should be addressed. Here we identified two allelic rice mutants with narrow and semi-rolled leaves, named narrow and rolled leaf 2-1 (nrl2-1) and nrl2-2. Map-based molecular cloning revealed that NRL2 encodes a novel protein with unknown biochemical function. The mutation of NRL2 caused pleiotropic effects, including a reduction in the number of longitudinal veins, defective abaxial sclerenchymatous cell differentiation, abnormal tapetum degeneration and microspore development, and the formation of more slender seeds compared with the wild type (WT). The NRL2 protein interacted with Rolling-leaf (RL14), causing the leaves of the nrl2 mutants to have a higher cellulose content and lower lignin content than the WT, which may have been related to sclerenchymatous cell differentiation and tapetum degeneration. Thus, this gene is an essential developmental regulator controlling fundamental cellular and developmental processes, serving as a potential breeding target for high-yielding rice cultivars.

Complexity of indica-japonica varietal differentiation in Bangladesh rice landraces revealed by microsatellite markers.

To understand the genetic diversity and indica-japonica differentiation in Bangladesh rice varieties, a total of 151 accessions of rice varieties mostly Bangladesh traditional varieties including Aus, Boro, broadcast Aman, transplant Aman and Rayada varietal groups were genotyped using 47 rice nuclear SSRs. As a result, three distinct groups were detected by cluster analysis, corresponding to indica, Aus and japonica rice. Among deepwater rice varieties analyzed some having particular morphological features that mainly corresponded to the japonica varietal group. Some small seeded and aromatic varieties from Bangladesh also corresponded to the japonica varietal group. This research for the first time establishes that the japonica varietal group is a prominent component of traditional varieties in Bangladesh, particularly in deepwater areas.

GS6, a member of the GRAS gene family, negatively regulates grain size in rice.

Grain size is an important yield-related trait in rice. Intensive artificial selection for grain size during domestication is evidenced by the larger grains of most of today's cultivars compared with their wild relatives. However, the molecular genetic control of rice grain size is still not well characterized. Here, we report the identification and cloning of Grain Size 6 (GS6), which plays an important role in reducing grain size in rice. A premature stop at the +348 position in the coding sequence (CDS) of GS6 increased grain width and weight significantly. Alignment of the CDS regions of GS6 in 90 rice materials revealed three GS6 alleles. Most japonica varieties (95%) harbor the Type I haplotype, and 62.9% of indica varieties harbor the Type II haplotype. Association analysis revealed that the Type I haplotype tends to increase the width and weight of grains more than either of the Type II or Type III haplotypes. Further investigation of genetic diversity and the evolutionary mechanisms of GS6 showed that the GS6 gene was strongly selected in japonica cultivars. In addition, a "ggc" repeat region identified in the region that encodes the GRAS domain of GS6 played an important historic role in the domestication of grain size in rice. Knowledge of the function of GS6 might aid efforts to elucidate the molecular mechanisms that control grain development and evolution in rice plants, and could facilitate the genetic improvement of rice yield.

OsMADS17 simultaneously increases grain number and grain weight in rice.

During the processes of rice domestication and improvement, a trade-off effect between grain number and grain weight was a major obstacle for increasing yield. Here, we identify a critical gene COG1, encoding the transcription factor OsMADS17, with a 65-bp deletion in the 5' untranslated region (5' UTR) presented in cultivated rice increasing grain number and grain weight simultaneously through decreasing mRNA translation efficiency. OsMADS17 controls grain yield by regulating multiple genes and that the interaction with one of them, OsAP2-39, has been characterized. Besides, the expression of OsMADS17 is regulated by OsMADS1 directly. It indicates that OsMADS1-OsMADS17-OsAP2-39 participates in the regulatory network controlling grain yield, and downregulation of OsMADS17 or OsAP2-39 expression can further improve grain yield by simultaneously increasing grain number and grain weight. Our findings provide insights into understanding the molecular basis co-regulating rice yield-related traits, and offer a strategy for breeding higher-yielding rice varieties.

TH1, a DUF640 domain-like gene controls lemma and palea development in rice.

The developmental regulation of grasses lemma and palea and their relationship to the floral organs in dicots had been variously explicated and extensively debated. Here, we characterized a triangular hull mutant th1-1 from EMS-mutagenized Oryza sativa ssp. indica cv. 93-11. The th1-1 mutant exhibited obviously triangular hull with tortuous and slender lemma/palea. Using a map-based cloning strategy, the TH1 gene was narrowed down to a 60-kb region on the long arm of chromosome 2. Sequence verification revealed that the th1-1 mutant harbored 1-bp deletion in exon 2 of LOC_Os02g56610 which resulted in a frame-shift mutation. The RNA-interference transgenic plants of LOC_Os02g56610 displayed a similar phenotype to the th1 mutant. Consequently, LOC_Os02g56610 was identified as the TH1 gene which encoded 248 amino acids and contained a DUF640 domain. RT-PCR analysis and GUS staining showed that the transcripts of TH1 mainly accumulated in young inflorescence, lemma and palea of spikelet. These results suggested that TH1 was an important gene controlling the lemma and palea development in rice.

LHD1, an allele of DTH8/Ghd8, controls late heading date in common wild rice (Oryza rufipogon).

Flowering at suitable time is very important for plants to adapt to complicated environments and produce their seeds successfully for reproduction. In rice (Oryza rufipogon Griff.) photoperiod regulation is one of the important factors for controlling heading date. Common wild rice, the ancestor of cultivated rice, exhibits a late heading date and a more sensitive photoperiodic response than cultivated rice. Here, through map-based cloning, we identified a major quantitative trait loci (QTL) LHD1 (Late Heading Date 1), an allele of DTH8/Ghd8, which controls the late heading date of wild rice and encodes a putative HAP3/NF-YB/CBF-A subunit of the CCAAT-box-binding transcription factor. Sequence analysis revealed that several variants in the coding region of LHD1 were correlated with a late heading date, and a further complementary study successfully rescued the phenotype. These results suggest that a functional site for LHD1 could be among those variants present in the coding region. We also found that LHD1 could down-regulate the expression of several floral transition activators such as Ehd1, Hd3a and RFT1 under long-day conditions, but not under short-day conditions. This indicates that LHD1 may delay flowering by repressing the expression of Ehd1, Hd3a and RFT1 under long-day conditions.

Polyamine oxidase 3 is involved in salt tolerance at the germination stage in rice.

Soil salinity inhibits seed germination and reduces seedling survival rate, resulting in significant yield reductions in crops. Here, we report the identification of a polyamine oxidase, OsPAO3, conferring salt tolerance at the germination stage in rice (Oryza sativa L.), through map-based cloning approach. OsPAO3 is up-regulated under salt stress at the germination stage and highly expressed in various organs. Overexpression of OsPAO3 increases activity of polyamine oxidases, enhancing the polyamine content in seed coleoptiles. Increased polyamine may lead to the enhance of the activity of ROS-scavenging enzymes to eliminate over-accumulated H2O2 and to reduce Na+ content in seed coleoptiles to maintain ion homeostasis and weaken Na+ damage. These changes resulted in stronger salt tolerance at the germination stage in rice. Our findings not only provide a unique gene for breeding new salt-tolerant rice cultivars but also help to elucidate the mechanism of salt tolerance in rice.