Overexpression Of OsiICK4 Remarkably Influences The Rice Seed Development And Plant Growth

Cell division and its circulation is the primary power for plant (including other organisms) growth and development. Normal cell cycle, an active course that started from end of last division to the end of next division, is controlled by highly conserved molecular machinery. Inhibitors of cyclin-dependent kinases (ICKs) are a central unit of this molecular machinery. In rice, there are six putative ICK genes predicted genome sequences. In addition to Oyrza;ICKl (cloned from japonica) and OsiICK6 (cloned from indica), the function of the other ICK genes have been not verified yet. In the present study, OsiICK4 was chosen to carry out molecular characterization and overexpression analysis due to having the shortest encoding product among all rice ICKs. And then painstaking observations were made to see if this overexpression had any influence on plant growth and development. The promoter used was cloned from rice 33 kDa secretory protein promoter (33SP) by our laboratory. The method used for introduction of the overexpression construct into recipient genome was Agrobacterium-mediated gene transformation. The major results and conclusions attained were described as follow:The OsiICK4 cloned by use of RT-PCR has full length for 261bp, which encode 85 amino acids. MEME analysis revealed that the C-terminal of OsiICK4 contains two conserved regions, the typical CDK and CYC binding domains. Further analysis indicated that OsiICK4 also contained a putative nuclear localization signal. But it is different from other rice ICKs that OsiICK4 has no PEST domain as well as CDK phosphorylation site.The data from sub-cellular localization confirmed that the OsiICK4 is a nuclear protein, which could interact with OsiCDKA and OsiCYCD, but not with OsiCDKB as verified by yeast two hybridization.Genetic transformation has produced 23 independent transformants harboring OsiICK4 overexpression construct. The semi-quantitative RT-PCR was applied to analyze T2 homozygous lines that were derived from four of these independent transformants. The results indicated that their expression levels of OsiICK4 were all remarkably higher than those of control. Further phenotype analysis demonstrated that those T2 homozygous lines under examination had the plant height 20% shorter than control on average. The reason identified for that was due to remarkable length increasing of transgenic stem cell but large decreasing in cell numbers based on data from microscope dissection.Overexpression of OsiICK4 also caused leaf abnormally roiling toward reverse direction. In the same way, the reason identified for that based on data from the microscope dissection was due to decreasing of leaf's motion cell numbers but large increasing in cell size under overexpression of OsiICK4. Those results thus demonstrated that overexpression of OsiICK4 could effectively impress cell division and consequently led to decreasing of cell numbers but compensated in increasing of cell size.Overexpression of OsiICK4 could even more lead to decreasing of pollen fertility and abnormal development of seed, so that the end production of rice largely cut down. The data obtained from the pollen fertility detection and DAPI staining of nuclear revealed that the OsiICK4-overexpression lines had many pollens stained faintly or not stained and stopped developing at mononuclear stage. Therefore, the plants were highly sterile and even if able to set some seeds but their endosperm couldn't fill well and part of their embryos died eventually. Those results hence proved that the OsiICK4 gene plays an important role during formation of pollens and development of embryo and endosperm.In summary, our experimental results have already demonstrated a critical role for OsiICK4 in the processes of vegetative growth and reproduction development in rice. These processes include stem- and leaf-cell division-expansion, pollen development and seed formation. It is known that, however, the endosperm constitutes the largest part of the rice seed. Artificially regulating the expression of OsiICK4 gene during seed development and enforcing it to promote endosperm development could be a delightful strategy for increasing grain yield.