| Cotyledons of soybean are the main nutrient storage area, which determine the whole development process of plant. Recently, the use of molecular biology and genetics allowed us to unravel partial molecular mechanisms of cotyledon development, which involved many gene hierarchies, and co-ordinated spatially and temporally regulated programmes of cell divisions, gene expression, and hormone function. But even so, there are still many blind spots in cotyledon development. In this research, we selected curled-cotyledon (ceo) mutant as experimental material. We studied the physiology, morphology and molecular mechanism of the cco mutant, and screened two related genes of ceo mutant Glyma17G138200 and Glyma03G02300 for further function analysis with genetic engineering methods.Firstly, we analyzed the phenotype of cco mutant. A majority of agronomic traits of cco mutant were same as wild-type (WT). The growth period of cco mutant was significantly prolonged compared to WT. In addition, plant height, hundred grain weight, hypocotyl length and germination rate were significantly reduced. Aming to detect embyrogensis defects of cco mutant, we traced the phenotype back to the globular stage of embryogenesis. Compared with WT, anatomical analysis revealed that the cco cotyledons at the torpedo stage became more slender and grew outward. The entire embryos of cco mutant resembled the "tail of swallow". In addition, cco seeds displayed reduced germination rate and gibberellic acid (GA3) level, whereas the abscisic acid (ABA) and auxin (IAA) levels were increased.To characterize the transcriptional regulatory network of cco, the pods of 7 DAF (Days After Fertilization) were selected for high-throughput mRNA sequencing (RNA-Seq) on Illumina HiSeq2000. Using False Discovery Rate (FDR)<0.001 and|log2Ratio|≥1,1,093 genes were differentially expressed between WT and the cco mutant. Many differentially expressed genes (DEGs) (20.46%) were mapped to the hormone biosynthesis and signal transduction pathways, including auxin, cytokinins (CKs), gibberellic acids (GAs), abscisic acid (ABA), ethylene and brassinosteroids (BRs). Transcriptome analysis indicated that auxin and ABA biosynthesis and signal transduction in cco were more active than in WT. However, an early step in GA biosynthesis was blocked, and the rate of conversion of inactive GAs to bioactive GAs was limited for the cco mutant. These results were consistent with our HPLC analysis. Multiple phytohormone biosynthesis and signalling pathways are reprogrammed in the cco mutant, which probably resulted in pleiotropic defects of cco mutant. Using RT-PCR, we screened two genes related cco mutant Glymal 7G138200 (YABBY) and Glyma03G02300 (LBD) for further study.In this research, we identified 17 YABBY genes in soybean genome by bioinformatics. Phylogenetic reconstruction classed YABBY genes family into five subfamilies:FIL, YAB2, YAB5, INO and CRC. Glyma17G138200 belonged to FIL subfamily, named GmFILa. GmFILa expressed highly in leaves and developing seeds. Detailed expression analysis was performed using mRNA in situ hybridization. The expression of GmFILa is tightly restricted to the abaxial cell layers of leaves and sepals. Sequence of GmFILa ORF was 648bp, encoding 215 amino acids, which had a two conserved domains, a C2C2 zinc finger-like domain towards the N terminus and a YABBY domain in the C terminus. Subcellular localization displayed that GmFILa protein located on cell nucleus. In order to further understand the function of GmFILa, transgenic plants were generated with the ectopic expression of GmFILa in Arabidopsis driven by a CaMV 35S promoter. The GmFILa transgenic plants showed small, narrow, curled leaves, and arrested shoot apical meristem. Ectopic expression of GmFILa could also result in partial abaxialization of adaxial leaf surfaces. The free IAA content of leaves of transgenic plants was reduced compared to WT. Subsequent transcriptomic analysis of WT and transgenic plants revealed DEGs were mainly related to hormones and stress response. In general, GmFILa is tightly restricted to the abaxial cell layers of lateral organs, and plays important roles in initiation and adaxial-abaxial surfaces outgrowth of leaves. Expression level of GmFILa in early development of seeds of cco mutant was inhibited, the content of free IAA in which was reduced. We speculated that the different expression level of GmFILa contributed to the change of IAA content in cco.Glyma03G02300 encoded LBD transcription factor, named GmLBD1, which showed significantly decreased expression level in vegatative organs of cco mutant compared to WT, especially in root. We identified 86 LBD genes in soybean genome by bioinformatics. GmLBDl was clustered into a branch with AtLBD18, AtLBD19 and AtLBD31, indicating their factional similarity. GmLBD1 located on chromosone 3. The sequence of GmLBD1 ORF was 756 bp, encoding 251 amino acids, which contained two exons and one intron. Subcellular localization displayed that GmLBDl protein located on cell membrane and nucleus. To further investigate function of GmLBDl, we constructed plant expression vector pBI101-GmLBDl. And it was transformed into Arabidopsis using Agrobacterium-mediated method. Overexpression of GmLBDl in Arabidopsis increased numbers of lateral roots and free IAA level. GmLBDl could be auxin-inducible, and overexpression of GmLBD1 in Arabidopsis increased auxin content. In this study, we found that cco mutant displayed reduced root system, in which the expression of GmLBD1 was remarkable depressed. Therefore, we speculated the down-regulation of GmLBDl, causing a decline in auxin content of root, which leaded to reduced root system of cco mutant. GmLBDl may promote lateral root development by auxin. |
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