| With the population increasing and economy developing,the consumption of nitrogen(N)and phosphate(P)fertilizer constantly increase as the rigid demand for grain yield in China,However,high yield mainly depending on a large amount of fertilizer has brought serious environmental problems.In 2015,Ministry of Agriculture put forward the action plan of"Fertilizer Reduction and Efficiency Improvement".The key to achieve both high yield and high resource efficiency is to modulate crop population structure in order to achieve nutrient supply matching with demand of high-yield crop population.Tillering is one of the important agronomic characters of rice and other gramineae crops.Controlling tiller development is an important mean to shape high quality crop population and to improve crop yield.Supplying N and P affect rice tillering process and quality of crop population,and thus illustrating mechanism of N and P regulating rice tiller bud growth is vital to achieve high grain yield and resource utilization efficiency.Rice tiller growth is not only influenced by environmental factors such as fluctuating N and P supplies,but also by internal hormone level.Cytokinins,as currently recognized to positively regulate shoot branching or tillering,might be involved rice tiller growth regulated by N and P supplies,but the mechanism remains obscure.Recently,strigolactones,have received more attention due to its inhibiting shoot branching or tillering.The D53 protein,which is located in the nucleus,is the key gene of the strigolactones signaling pathway.Our previous studies showed that,with N and P concentration decreasing,the synthesis and secretion of strigolactones were significantly increased and D53 protein was degraded in rice,suggesting that strigolactones may be involved in rice plant growth and development regulated by N and P supplies.However,mechanism by which strigolactones are involved in the N-and P-modulated rice tillering still is not clear,nor is the crosstalk between cytokinins and strigolactones.All above question mentioned will be discussed in this paper.In this paper,we first reported a reporting system that can detect cytokinins signaling in rice.Then effects of N-and P-deficiency on the formation and elongation of rice tillering buds were analyzed.Afterwards,crosstalk between cytokinins and strigolactones during rice tillering regulated by N-and P-deficiency were studied.The results are listed as follows:1 A reporting system(TCSn)has been reported to detect cytokinin signaling in Arabidopsis thaliana,but it is not clear whether this system can be used in rice.Bioinformatic analysis showed that genes belonging to cytokinin signaling pathway have high homology in Arabidopsis thaliana and rice,containing conservative functional domains.The results of RT-qPCR analysis showed that the expression of seven type-A OsRRs genes were up-regulated by exogenous cytokinin in both root and shoot of rice.The synthetic promoter TCSn(tandem element A(A/G)GAT(C/T)TT)fused with GUS reporter gene was transferred into the wild type rice to obtain positive transgenic materials.Results showed that TCSn::GUS was actively expressed in the meristem of germinated seeds and rice seedlings.Among exogenous application of hormones,only cytokinin induced TCSn::GUS expression in transgenic seedlings.More importantly,TCSn::GUS expression didn’t weaken after three generations of self-interbreeding,indicating that the reporting system could be inherited stably.The above results suggested that TCSn::GUS is a stable and sensitive reporting system that can detect cytokinin signal output in rice,providing a powerful tool for further study on the biological function of cytokinins.2 We investigated rice tiller bud formation and outgrowth in response to N and P supplies.The tiller number of rice reduced significantly with N and P concentration decreasing.The reason for reduction of tiller number was that the elongation of tiller buds was inhibited rather than bud formation.Do cytokinins,as positive regulator for rice tiller,participate in N-and P-modulated rice bud elongation?Compared with control treatment,low N or P condition(LN and LP)significantly decreased cytokinins concentration in stem bases and in xylem sap of wild-type plants.The results of RT-qPCR showed that relative expression of cytokinin synthetic genes(OsIPTs)was significantly decreased in the root of wild-type plants,and similar decreasing tendency was observed in expression of most type-A OsRRs genes in the stem base.Correspondingly,expression level of TCSn::GUS in roots and in stem bases also significantly decreased in response to LN and LP.Application of 6-BA to LN-and LP-treated seedlings significantly increased tiller number to levels similar to those of the control treatment;TCSn::GUS expression was also similar in stem base between these treatments after 6-BA application.Compared with wild-type plants,Osckx2,a gain-of-function mutant of cytokinins oxidase,has higher expression level of FAD region with cytokinins oxidation function and less tiller number.These results suggest that cytokinins are involved in LN-and LP-regulated tiller bud growth in rice.3 Compared with control treatment,LN-and LP-condition reduced elongation of tiller budin wild-type plants but not in strigolactones d mutants.Application of rac-GR24 to control-treated seedlings significantly decreased tiller number to levels similar to those of the LN and LP treatments;conversely,application of strigolactones synthetic inhibitor to LN-or LP-treated seedlings significantly increased tiller number to levels similar to those of the control treatments.These suggested that strigolactones are involved in LN-and LP-regulated rice tiller bud outgrowth.4 Cytokinin-and strigolactone-related mutants were used to further illustrate the crosstalk between cytokinins and strigolactones in LN-and LP-regulated rice tiller bud outgrowth.d mutants were more sensitive to exogenous 6-BA application than wild-type plants while similar cytokinin contents was observed in d mutants and wild-type plants.At the same time,the TCSn::GUS activity and the expression of OsRR2,OsRR5 and OsRR7 under control,LN and LP treatments were significantly higher in d mutants than in wild-type plants.Moreover,application of rac-GR24 significantly reduced the TCSn::G.US activity and tiller number in wild type rice and strigolactones synthesis mutant d10,but not in signal mutant d3.RT-qPCR results showed that exogenous rac-GR24 could inhibit expression of most type-A family genes.Conversely,the TCSn::GUS activity in the stem bases of wild type was significantly enhanced by the application of exogenous strigolactones synthesis inhibitor.These results showed that the content of cytokinins in rice were not affected in d mutants,however,the expression of the type-A family genes was significantly inhibited by strigolactones.5 As mentioned before,cytokinin signaling pathways are conserved in Arabidopsis and rice.There are a large number of type-A genes and higher homology each other which are few reported in rice,suggested that they have strong functional redundancy,so it is difficult to obtain the multiple combination of mutants what we wanted in a short time to further research the mechanism of cytokinin’s involvement in N-and P-modulated rice tillering.Compared with rice,there are abundant of type-A mutants related to cytokinin signaling pathway in Arabidopsis thaliana,and the branching phenotype of Arabidopsis thaliana is easy to observe,so we chose Arabidopsis thaliana plants to continue to verify our hypothesis.We obtained arr3/4/5/6/7/15 mutant,and observed the response of the branch of mutant and wild type to LN-and LP-treatment.Compared with normal nutrition,branch numbers,plant height and shoot biomass of wild-type Arabidopsis thaliana significantly reduced and also is the TCSn::GUS activity which is reflecting cytokinins function under the condition of N-and P-deficiency,which was similar to rice phenotype.The application of exogenous strigolactone rac-GR24 could also significantly reduce the branch number of wild type Arabidopsis,but the branch number of arr3/4/5/6/7/15 had no significant change under the treatment of LN,LP and rac-GR24.We used an improved and efficient CRISPR/Cas9 system to create strogolactones synthesis mutant max1 and arr3/4/5/6/7/15/max1 without Cas9 which could be inherited steadily.In addition,compared with the sextuple mutant arr3/4/5/6/7/15,the branch numbers of arr3/4/5/6/7/15/max1 were decreased,and the difference reached a significant level.These results revealed that strigolactones repress rice tillering at least partially through cytokinin signaling.In conclusion,firstly we created a biological tool can detect cytokinin signal output in rice,and then use this tool to explore the relationship between strigolactones and cytokinins in regulation of rice tillers,finally proved the strigolactones participate in the regulation of the growth and development of rice tillering together with cytokinins under LN-and LP-condition. |
