Cloning And Functional Analysis Of A Key Gene SLG Involved In Regulation Of Grain Size And Leaf Angle In Rice

Rice (Oryza sativa L.) feeds more than half of the world's population as one of the world's most important cereal crops. Given the rapid increase in world population and decrease in cultivated land area,improving rice production remains a great challenge for rice breeding programs. Grain size is one of the important agronomic traits that determine rice yield. Moreover, it also influences appearance, processing,cooking and eating quality of rice. Leaf angle, as an important agronomic trait, influences the plant architecture and grain yields. A more erect leaf increases photosynthetic efficiency and nitrogen storage for grain filling, and facilitates dense planting. Up to date, although many genes that influence rice grain size and leaf angle have been reported and we have a certain degree of recognition of their genetic and molecular mechanism, the precise molecular mechanisms that control these two traits have not been fully elucidated.BRs are a group of steroidal phytohormones that regulate diverse plant growth and developmental process. It has been reported that BRs play aimportant roles in regulating rice grain size, leaf angle and yield potential. BRs homeostasis is vital for normal growth and development of plants. Despite the rapid progress in elucidating the BR biosynthesis, metabolism and signalling pathway in recent decades, the mechanisms that modulate BRs homeostasis remain poorly understood.In this study, we isolated and characterized a rice semi-dominant mutant slender grain Dominant(slg-D) showing phenotypes of slender grain and increased lamina joint inclination from a collection of activation-tagging T-DNA insertion rice lines. Cloning and functional analysis of the slg-D mutant reveal the genetic and molecular mechanism that causes the phenotype of slg-D. We also studied the slg-D in physiology and cell morphology. These results promote our knowledge of the mechanisms that regulate grain size and leaf angle in rice, and provid theoretical basis for rice breeding. The main results are as follows:1. The slg-D mutant had slender grains and larger leaf angles phenotypes compared with the wild-type (WT). Cell morphology observation via light microscopy and scanning electron microscopy(SEM) indicates that the alteration in cell size, especially the cell length change, is responsible for the mutant phenotypes of slg-D. The result of genetic analyses indicates that the slg-D mutation behaves in a semi-dominant manner controlled by a single locus.2. We found that transcript levels of LOC_Os08g44840 and LOC_Os08g44830 were obviously increased in the slg-D mutant plants because of the T-DNA insertion in slg-D. Our results prove that the phenotype of the slg-D mutant is caused by overexpression of LOC_Os08g44840. Thus, we designated LOC_Os08g44840 as SLENDER GRAIN (SLG). SLG encodes a putative BAHD family of acyltransferases protein consisting of 445 amino acids. Phylogenetic analysis reveals that SLG-like proteins can largely be classified into two groups: dicot and monocot. SLG is a member of monocot group. However, to date, none of the genes in this group have been functionally characterized in other plant species.3. Quantitative RT-PCR revealed that expression of SLG is strong in young panicles, relatively high in lamina joints, low in shoot apices, culms and leaves, and very little in roots and leaf sheaths. Analysis of GUS staining activity in transgenic lines showed the same expression pattern of SLG as quantitative real-time RT-PCR. It was also showed that the GUS signals were mainly restricted to the vasculature.RNA in situ hybridization identified that the mRNA of SLG was mainly localized in the spikelet meristem primordia, lemma primordia, palea primordia, floral meristem primordia and vasculature regions. The predominant expression of SLG in young panicles and lamina joints implies its role in controlling grain shape and leaf angle. The result of subcellular localization of SLG reveals that the GFP signals is found in both the cytoplasm and the nucleus.4. BR response assay revealed that the sensitivity to BL treatment was similar in slg-D and WT,suggesting that SLG do not affect BR signaling. We found that slg-D was restored to WT by brassinazole(BRZ) treatment, an inhibitor of BR biosynthesis. Overexpression of SLG in d11-2 (deficient in BR synthesis) and d61-1 (deficient in BR signaling) did not change the existing phenotypes. The slg-D plants had elevated BR contents and accordingly, expressions of several BR biosynthesis and signaling genes had the expected transcription level change as a response to the elevated BR levels. These results show that SLG positively regulates endogenous BR levels and is a new regulator of BR homeostasis in rice5. The SLG RNAi plants displayed a more compact architecture, reduced plant height, smaller leaf angle, shorter and rounder grain (Fig. 7). These phenotypes are similar to those of BR-deficient mutants,such as d61 and d11, further highlighting a role of SLG in regulating BR homeostasis. This observation also suggests that an optimized expression level of SLG may help create a compact and semi-dwarf ideal plant type.6. Yeast two-hybrid, pull-down and BiFC assay confirmed that SLG could interact with itself to form homomers and the 30-amino-acid in the N-terminal region of SLG (SLG?C4) was responsible for the self-interaction of SLG.We found that overexpression of a truncated SLG CDS SLG?C1 in both WT and slg-D mutant conferred smaller leaf angles, shorter and rounder grains, and dwarf phenotypes similar to that of SLG RNAi plants. These results provide genetic evidence that SLG indeed functions as homomers in vivo.