水稻OsGRF6的克隆及调控初生根发育功能的初步分析

OsGRF6隶属于GRF家族，参与调控植物的生长发育和调控植物对非生物胁迫的耐受性。为了研究OsGRF6调控水稻初生根发育的分子机制，该研究从水稻品种‘ZH11’中克隆得到转录因子基因OsGRF6，并对其进行了进化树构建，启动子分析，并对OsGRF6-OE和osgrf6进行了基因型分析和qRT-PCR鉴定及水稻表型观察，并通过酵母单杂交检测了OsGRF6的结合基序，进一步通过ChIP-seq和RNA-seq数据分析了OsGRF6下游的候选靶基因。结果显示：（1）OsGRF6含有一个QLQ和一个WRC保守的功能结构域，与拟南芥AtGRF1和AtGRF2以及水稻OsGRF7亲缘关系较近。OsGRF6启动子区域含有多个非生物胁迫和激素响应顺式作用元件。（2）OsGRF6在水稻种子、幼穗和根中表达量较高，而在叶中表达量较低。亚细胞定位显示，OsGRF6定位于细胞核，并且OsGRF6具有转录激活活性。（3）与野生型相比，过表达OsGRF6的转基因材料表现出初生根长度较长，而突变体材料则表现出初生根变短的表型。（4）OsGRF6能与CGGCA基序结合。（5）结合ChIP-seqs和RNA-seqs分析发现，OsGRF6靶基因中包含多个参与调控水稻根发育的基因，如OsARF7，OsARF4等。     

The OsmiR396c‐OsGRF4‐OsGIF1 regulatory module determines grain size and yield in rice

Grain weight is the most important component of rice yield and is mainly determined by grain size, which is generally controlled by quantitative trait loci (QTLs). Although numerous QTLs that regulate grain weight have been identified, the genetic network that controls grain size remains unclear. Herein, we report the cloning and functional analysis of a dominant QTL, grain length and width 2 (GLW2), which positively regulates grain weight by simultaneously increasing grain length and width. The GLW2 locus encodes OsGRF4 (growth‐regulating factor 4) and is regulated by the microRNA miR396c in vivo. The mutation in OsGRF4 perturbs the OsmiR396 target regulation of OsGRF4, generating a larger grain size and enhanced grain yield. We also demonstrate that OsGIF1 (GRF‐interacting factors 1) directly interacts with OsGRF4, and increasing its expression improves grain size. Our results suggest that the miR396c‐OsGRF4‐OsGIF1 regulatory module plays an important role in grain size determination and holds implications for rice yield improvement.     

OsGRF4 controls grain shape,panicle length and seed shattering in rice

Traits such as grain shape, panicle length and seed shattering, play important roles in grain yield and harvest. In this study, the cloning and functional analysis of PANICLE TRAITS 2 (PT2), a novel gene from the Indica rice Chuandali (CDL), is reported. PT2 is synonymous with Growth‐Regulating Factor 4 (OsGRF4), which encodes a growth‐regulating factor that positively regulates grain shape and panicle length and negatively regulates seed shattering. Higher expression of OsGRF4 is correlated with larger grain, longer panicle and lower seed shattering. A unique OsGRF4 mutation, which occurs at the OsmiRNA396 target site of OsGRF4, seems to be associated with high levels of OsGRF4 expression, and results in phenotypic difference. Further research showed that OsGRF4 regulated two cytokinin dehydrogenase precursor genes (CKX5 and CKX1) resulting in increased cytokinin levels, which might affect the panicle traits. High storage capacity and moderate seed shattering of OsGRF4 may be useful in high‐yield breeding and mechanized harvesting of rice. Our findings provide additional insight into the molecular basis of panicle growth.     

Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice

A major catabolic pathway for gibberellin (GA) is initiated by 2beta-hydroxylation, a reaction catalyzed by GA 2-oxidase. We have isolated and characterized a cDNA, designated Oryza sativa GA 2-oxidase 1 (OsGA2ox1) from rice (Oryza sativa L. cv Nipponbare) that encodes a GA 2-oxidase. The encoded protein, produced by heterologous expression in Escherichia coli, converted GA(1), GA(4), GA(9), GA(20), and GA(44) to the corresponding 2beta-hydroxylated products GA(8), GA(34), GA(51), GA(29), and GA(98), respectively. Ectopic expression of the OsGA2ox1 cDNA in transgenic rice inhibited stem elongation and the development of reproductive organs. These transgenic plants were deficient in endogenous GA(1). These results indicate that OsGA2ox1 encodes a GA 2-oxidase, which is functional not only in vitro but also in vivo. OsGA2ox1 was expressed in shoot apex and roots but not in leaves and stems. In situ hybridization analysis revealed that OsGA2ox1 mRNA was localized in a ring at the basal region of leaf primordia and young leaves. This ring-shaped expression around the shoot apex was drastically decreased after the phase transition from vegetative to reproductive growth. It was absent in the floral meristem, but it was still present in the lateral meristem that remained in the vegetative phase. These observations suggest that OsGA2ox1 controls the level of bioactive GAs in the shoot apical meristem; therefore, reduction in its expression may contribute to the early development of the inflorescence meristem.     

Isolation and characterization of a rice homebox gene, OSH15

In many eukaryotic organisms including plants, homeobox genes are thought to be master regulators that establish the cellular or regional identities and specify the fundamental body plan. We isolated and characterized a cDNA designated OSH15 (Oryza sativa homeobox 15) that encodes a KNOTTED-type homeodomain protein. Transgenic tobacco plants overexpressing the OSH15 cDNA showed a dramatically altered morphological phenotype caused by disturbance of specific aspects of tobacco development, thereby indicating the involvement of OSH15 in plant development. We analyzed the in situ mRNA localization of OSH15 through the whole plant life cycle, comparing the expression pattern with that of another rice homeobox gene, OSH1. In early embryogenesis, both genes were expressed as the same pattern at a region where the shoot apical meristem would develop later. In late embryogenesis, the expression pattern of the two genes became different. Whereas the expression of OSH1 continued within the shoot apical meristem, OSH15 expression within the shoot apical meristem ceased but became observable in a ring shaped pattern at the boundaries of some embryonic organs. This pattern of expression was similar to that observed around vegetative or reproductive shoots, or the floral meristem in mature plants. RNA in situ localization data suggest that OSH15 may play roles in the shoot organization during early embryogenesis and thereafter, OSH15 may be involved in morphogenetic events around the shoot apical meristem.     

OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408

microRNAs (miRNAs) are promising targets for crop improvement of complex agricultural traits. Coordinated activity between/among different miRNAs may fine-tune specific developmental processes in diverse organisms. Grain size is a main factor determining rice (Oryza sativa L.) crop yield, but the network of miRNAs influencing this trait remains uncharacterized. Here we show that sequestering OsmiR396 through target mimicry (MIM396) can substantially increase grain size in several japonica and indica rice subspecies and in plants with excessive tillers and a high panicle density. Thus, OsmiR396 has a major role related to the regulation of rice grain size. The grain shape of Growth Regulating Factor8 (OsGRF8)-overexpressing transgenic plants was most similar to that of MIM396 plants, suggesting OsGRF8 is a major mediator of OsmiR396 in grain size regulation. A miRNA microarray analysis revealed changes to the expression of many miRNAs, including OsmiR408, in the MIM396 plants. Analyses of gene expression patterns and functions indicated OsmiR408 is an embryo-specific miRNA that positively regulates grain size. Silencing OsmiR408 expression (miR408KO) using CRISPR technology resulted in small grains. Moreover, we revealed the direct regulatory effects of OsGRF8 on OsMIR408 expression. A genetic analysis further showed that the large-grain phenotype of MIM396 plants could be complemented by miR408KO. Also, several hormone signaling pathways might be involved in the OsmiR396/GRF-meditated grain size regulation. Our findings suggest that genetic regulatory networks comprising various miRNAs, such as OsmiR396 and OsmiR408, may be crucial for controlling rice grain size. Furthermore, the OsmiR396/GRF module may be important for breeding new high-yielding rice varieties.

The OsmiR396/Growth Regulating Factor module plays a pivotal role in rice grain size regulation and genetically regulates OsmiR408, which acts as an embryo-specific grain size regulator.