Full-length RPB1 Modulates In Vitro Shoot Regeneration In Arabidopsis

Totipotency of plant cells is an important theoretical basis of plant tissue culture,and now there is a quite established tissue culture system.But in vitro shoot regeneration of Arabidopsis is a complex biological process,and its mechanism has long been explored.The regeneration in Arabidopsis includes two steps,first,the explants from differentiated plant tissues such as roots or hypocotyls are induced to generate callus,then the shoots regenerate upon the callus.The phytohormone auxin and cytokinin play important parts.The genes which are related to auxin and cytokinin signals involved in the regeneration have been studied widely.As we reported before,the Arabidopsis full length RNA Polymerase II's largest subunit RPB1 is necessary for keeping normal cell cycling and maintaining the stem cell niches.Therefore,to explore the effect of full-length RPB1 in Arabidopsis shoot regeneration process in vitro,we made a series of studies about shoots regeneration process of RPB1 mutants.We obtained the following preliminary results:GUS activity was detected in all the stages of shoot regeneration.After the induction of 3,6,9 days on CIM,GUS activity was detected in the whole calli,while after induced 3,6,9 days on SIM,GUS activity was largely restricted to inner cell layers.Staining was observed in the young leaf and the growing edge of the leaf in regenerated shoot.WT generated more calli than card1-1,card1-2,card1-c6,so we concluded that mutations in RPB1 result in defects in callus formation on CIM.In RPB1 mutants the auxin gradient formation is subdued,and the lower fold change of ARF7,ARF19 and their downstream genes LBDs(LBD16,LBD17,LBD18,LBD29)contributed to the callus growth retardation.The pSCR::GFP signals were arranged disorderly in card1-1 with blurred cell layers in the callus.In callus of card1-1,the pSHR::SHR-GFP signal was frequently detected in the epidermis.These results indicated that in callus formation the identity of cell layers may also be affected in card1-1.Shoot regeneration is inhibited in RPB1 mutants and the full length of RPB1 performs a critical role in shoot regeneration.Cytokinin response is repressed in card1-1 during shoot regeneration from root explants.In RPB1 mutants the difference in expression fold change of genes related to shoot emergence leads to the lower frequency of shoot regeneration.The number of root regenerated on callus is the least in WT,all RPB1 mutants generated more root.Here,we show that the mutants of card1 s have significant defects in the regeneration progress for both the induction of callus and the formation of shoot.All these results further proved the importance of intact RPB1 from a distinctive perspective.