Studies On The Molecular Mechanism Of Growth And Development Of Tumor-like Stems In Tuber Mustard(Brassica Juncea)

Tuber mustard(Brassica juncea Coss. var. tumida Tsen et Lee) is an important variant of mustard in the Cruciferae family and its swollen stems are used as excellent raw materials for processing and pickling. In the present study, tuber mustard was used as materials, the roles of cell division and endoreduplication and its molecular regulation were studied in the swelling stems. Additionally, the inherience pattern of RBR loci from its putative parents and their roles in stem development were also studied. The effect of photoperiod and temperature on the stem growth and development were also investaged to make clare the mechanism of stem swelling. Finally, the comparative proteomics of stems before swelling and after swelling were studied to obtain the key proteins involving in stem swelling. On this basic, the DNA methyltranferase genes were cloned and the possible roles was discussed. The main results were as follows:(1)A dynamic change of cell ploidy in different regions of the swollen stems were observed, with a decrease in4C proportion in P4-1and a sharp increase in8C cells that became the dominant cell type (86.33%at most) in the inner pith cells. cDNAs of14cell cycle genes and four cell expansion genes were cloned and their spatial transcripts analysed in order to understand their roles in stem development. The expression of most cell cycle genes peaked in regions of the outer pith (P2or P3), some genes regulating S/G2and G2/M significantly decrease in P5and P6, while G1/S regulators showed a relative high expression level in the inner pith (P5) where cells were undergoing endoreduplication. Coincidentally, BjXTH1and BjXTH2were exclusively expressed in the endoreduplicated cells. In conclusion, we think that cells of outer pith regions (P2and P3) mainly divide for cell proliferation, while cells of the inner pith expand through endoreduplication. Endoreduplication could trigger expression of BjXTH1and BjXTH2and thus function in cell expansion of the pith tissue. (2) In tuber mustard, the corresponding putative parental RBR loci were maintained, but underwent a different evolutionary pattern, which might imply a function divergence. Four RBR genes from both A-and B-subgenomes were expressed in tissues tested, suggesting absence of uniparental silence of RBR genes. Expression of BjRBRl and BjRBR2peaked in stage III and IV when the stems grow vigorously, while BjRBR3and BjRBR4showed constitutive expression. However, the spacial expression of BjRBRl-4showed almost the similar pattern, that is, they were down-regulated in mitosis cells (P2and P3) while up-regulated in endoredulicated cells. The results suggested the RBRs gene functioned both in cell division and endoredplication in a different way.(3) The effect of photoperiod and temperature on stem growth and development was studied, and the results suggest that both of them controlled the stem swelling, while temperature might affect the shape of tumor. And the anatomy analysis of swelling stem and unswelling stem indicated the cell number but not the cell size is one of main factors which determine the stem swelling, and the cell division of outer pith is the main source of cell increasement during stem development. The results of transcript changes of cell cycle-related genes and cell expansion genes between swelling stems (S4W/12) and unswelling (S4W/25) stems also indicated that the vigorous cell divison in the swelling stems because most of the positive regulators of cell cycle showed higher expression than those of unswelling stems.(4) The comparative proteomics between stems before swelling and after swelling were studied and the results suggest the proteins regulating stem swelling were involved in different biological pathways. And one of them, the S-adenosylmethionine synthetase (SAMS) arose our interest. The foliar spray of methionine solution on the tuber mustard in early developmental stages suppressed the stem swelling. Additionally, DNA methyltransferase were also cloned and their expression pattern during stem development indicated that the expression levels of MET (BjMETl&BjMET2) peaked in stage IV, when stems were swelling vigorously and undergoing cell division and endoreduplication, thus imply the DNA methylation might be involved in the regulation of cell cycle progress then contributed to the stem swelling in tuber mustard.