Functional Analysis Of SST Family In Sexual Reproduction Of Arabidopsis Thaliana

Sexual plant reproduction is the key process of plant development linking generations. The investigation on the molecular mechanism of sexual reproduction have great impact on understanding fruit and seed formation, including their size, amount, special orientation and relvevent agriculturally evaluated charactors. Based on our previeous work, we found a cDNA expressing of unique characteristy in the process of sexual reproduction. Its homologes in Arabidopsis thaliana are requied for the formation of fruit, which is an indispensable component of plant sexual reproduction. The gene is involved in the regulation of fruit shape and size, as well as the direction of its growth. Therefore, a detailed investigation was carried out and the main results are as follows:1. We did blast analysis using the cDNA sequence on NCBI web site, and revealed several homologs in different angiosperms, such as Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oryza sativa, Zea mays and Sorghum bicolor. Intriguingly, there was no homolog found with a similar domain organization outside angiosperms. There were four comparatively conserved domains scattered in the whole amino acid sequence. So far, no research data could be found about these conserved domains. The result of phylogenetic analysis suggested that this new plant gene family could possibly be derived from independent DNA duplication events.2. Constructs containing DNA fragment upstream of the AtSST coding regions were conected to a β-glucuronidase (GUS) reporter gene and several independent transgenic lines were analyzed. In the AtSST1Pro::GUS transgenic plants, GUS activity was observed in stipules, mature pollens, replum, embryo sacs, embryo and endosperm. To examine AtSST2expression, the signal was only detected at the proximal branch of the flowers or the fruits, while the AtSST1Pro::GUS and AtSST3Pro::GUS lines showed GUS signals at the same tissues. In addition, there was specific signal detected at the hypocotyls in the AtSST2Pro::GUS lines. AtSST3Pro::GUS lines showed signals in almostly all plant organs.3. sst mutants showed normal phenotype as wide type in metabolism and development of plant, while overexpression of AtSSTs, under the control of35s promoter, caused dwarfing plant, curly leaves, short silique, modified style, small inflorescence branching angle and special disorganization of oulves.4. Further investigation revealed that AtSSTs may involve in the elongation of cells. Then, we checked the distribution of auxin in the over-expression plants, which had abnormal phenotypes. It was found that auxn distribution was reduced at replum and this was proposed to be the reason for short siliques.5. The artificial micro RNA transformative lines of AtSST, simultaneously downregulating the AtSSTs, showed thinner and longer style than the wild type, and the epidermic cells of style were longer too. We speculated that the three AtSSTs have a redundant and essential role in style specialization.6. Although the mutants and artificial microRNA mutants of NGA had the similar phenotype comparing with the amiR-AtSST lines, we proposed that the two gene group regulated the style development in a same regulatory pathway. The relative expression level of AtSSTs dramtically reduced in the35s::mirNGA line, and all the three promoters of AtSSTs contained a DNA binding site of NGA transcription factors. These results suggested that SST is probably directly regulated by NGA transcription factor.7. In addition, we found the linkage arrangement across multipe genomes between SSTs and a group of NAC transcription factors. AtSSTl and AtNAC1_L, both can regulate the expression of each other, a classical mechanism exiting generally in low species. Thus, we offer a new clue for evolution in angiosperm. In summary, we found a new plant gene family, which could possibly be derived from independent DNA duplication events. SST is required for style specialization and directly regulated by NGA transcription factor in Arabidopsis thaliana. Over-expression of AtSST caused several abnormal phenotypes such as short stature, short fruits and compact inflorescence architecture, probably via regulating the growth and expansion of cells. In addition, our study also indicated there is a bidirectional promoter from the gene family, which regulates two neighbor genes through a classical mechanism commonly existing in lower species.