Functional Analysis Of Cyclin-dependent Kinase Inhibitors KRP1 And KRP2 In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is a staple food for over half of the world population and more than 3.5 billion people depend on rice for their daily calories.However,Asia accounts for 90% of global rice consumption.It is also the fastest-growing staple food in Africa,as well as in Latin America.Cyclin-dependent kinases and a number of other proteins control the progression of the plant cell cycle and play a major role in the regulation of the eukaryotic cell cycle.The activities of CDKs can be inhibited by some low molecular proteins known as KRP(kip-related protein).Oryza;KRP1 has been reported to cause a reduction in cell production during leaf development and significant reduction in seed filling.It also plays an important role in endoreduplication and the exit from the mitotic cell cycle during rice grain formation.In order to gain more insight into the role of Oryza;KRP1,rice plant overexpressing Oryza;KRP1 and Knock out double mutant krp1/krp2 and single mutant krp2 were developed using CRISPR/Cas9 technology.The effect of different phytohormones on the relative transcriptional level of Oryza;KRP1 and Oryza;KRP2 were examined.The effect of knock out of Oryza;KRP1 and Oryza;KRP2 gene in the plant growth,seed production,seed germination were also explored.Finally,the possible interaction between the Oryza;KRP1 and some selected CDKs were examined.The subcellular localization result showed that Oryza;KRP1 is localized in the nucleus.The KRP1 mRNA abundances were extremely low in callus,panicle,stem,3DAP and 9DAP but predominantly expressed in 6DAP and root followed by leaf and sheath.Upon ABA treatment,the KRP1 and KRP2 transcription level increased.KRP1 and KRP2 are mainly expressed in developing seeds,roots,and leaves.Overexpression of krp1 and knock-out of krp1/krp2 affects seedling growth the mutant seeds growth rate was slower than the wild type.The seeds of the overexpressed and knock-out plants showed a reduction in seed weight.The transgenic plants overexpressing krp1 are significantly shorter plants.Using yeast two-hybrid system we discovered that KRP1 is able to interact with CDKC;2 and CDKE;1 directly but whereas CDKA;2 or CDKF;3 was unable to interact with KRP1.KRP1 and KRP2 are linked to inhibition of mitosis and therefore to cell division.Elucidating the cellular and molecular events contributing to normal endosperm development might provide a clear understanding of the components of seed production and the possibility to improve grain yield.