Mechanism Research Of Cytokinin Response Regulator ARR1/2 And BZIP Transcription Factor HY5 Shoot Apical Meristem Reestablishment In Arabidopsis

Shoot apical meristem(SAM)is the origin of all aerial organs.The maintenance and differentiation of stem cells are the basis of organ morphogenesis.Plant somatic cells can regenerate to a whole plants via acquiring stem cell characteristic,reestablishing stem cell niche and shoot apical meristem in vitro,which is the theoretical basis of plant cell engineering.WUS(WUSCHEL)and CLV3(CLAVATA3)are key regulators of stem cell maintenance and differentiation in SAM.WUS is also the key determinant of in vitro stem cell center reestablishment.So far,several functional genes have been found to regulate in vitro shoot regeneration,but the number of them is far from enough.Therefore,it is of great significance to carry out large-scale systematic research of genes that regulate shoot regeneration.Plant stem cell sorting and transcriptome sequencing can be used to effectively explore genes that regulate stem cell maintenance and differentiation.There are only 15-35 stem cells marked by CLV3 in the SAM of Arabidopsis thaliana,thus it is difficult to isolate these stem cells,which impedes the large-scale study of stem cell transcriptome sequencing and the exploration of related functional genes.Cytokinin promotes in vitro shoot regeneration.Studies have shown that A-type ARABIDOPSIS RESPONSE REGULATORS(ARRs)inhibit shoot regeneration,while B-type ARRs(ARR1/10/12)promote shoot regeneration.However,our lab's preliminary research found that ARR1 inhibits in vitro shoot regeneration,so this study focus on the comparative study on the interaction between ARR1 and ARR12 in regulating the reestablishment of stem cell niche and SAM in vitro.External environmental factors,such as light,also regulates in vitro shoot regeneration.Several genes related to light signal pathway(such as bZIP transcription factor HY5)are involved in in vitro shoot regeneration.However,the mechanism of HY5 in regulating reestablishment of the stem cell niche and SAM in vitro remains unclear.In this work,studies about the function of genes that involved in stem cell center and SAM reestablishment and the related molecular mechanisms were carried out using stem cell sorting in vitro and transcriptome sequencing of them.The results of this study laid a foundation for understanding the mechanism underlying in vitro shoot regeneration.The main course of the study and the results were as follows:1.Screening of functional genes involved in in vitro stem cells proliferation and differentiationProtoplasts were isolated and cultured using the embryonic callus suspension system of pCLV3::GFP:GUS.It was found that cells marked by CLV3 could divide into 2 cells,4 cells and embryonal cell groups,and then differentiate into regenerated plants,suggesting that CLV3 is involved in somatic embryogenesis formation,and cells marked by CLV3 have the ability to proliferate and differentiate,which is the characteristics of stem cells.CLV3 marked stem cells and non-marked cells with a sorting rate of 100%were obtained from pCLV3::GFP::GUS callus suspension using FACS(Fluorescence activated Cell Sorting),and transcriptome sequencing of them was performed.It was found that there were 4,259 DEGs(Differentially Expressed Genes)including 886 up-regulated genes and 3,363 down-regulated genes in the CLV3 marked cells compared with non-marked cells.GO and KEGG enrichment analysis of the DEGs showed that genes related to plant hormones,light signals,ribosome synthesis,oxidative phosphorylation and other pathways were significantly enriched,suggesting that they were involved in in vitro stem cell division and differentiation.157 genes that may be associated with stem cell maintenance and differentiation were identified,including 17 genes associated with cytokinin and 14 genes associated with the light signaling pathway.The expression of some randomly selected genes was verified by qRT-PCR,which proved the reliability of transcriptome data.Using RNA in situ hybridization,some candidate genes including ARR1,ARR12,ARR2,CKX3,CKX7,LOG3,HY5,HYH,PHYA and PHYB were found to be expressed in stem cell areas in Arabidopsis SAM,which overlapped or partially overlapped with the expression of CLV3,indicating that they may also be involved in stem cell maintenance and differentiation.The functions of candidate genes ARR1/ARR12 and HY5 in in vitro shoot regeneration were identified using their mutants,and the mechanism of them in regulating SAM reestablishment was also studied.2.Mechanism of ARR1 and ARR12 in regulating the reestablishment of in vitro stem cell centers and SAMIn order to elucidate the molecular mechanism of candidate genes in regulating in vitro stem cell center and SAM reestablishment,the root explants of pCLV3::mCherry-NLS;pWUS::mCherry-NLS,pWUS::sGFP-N7,pWUS::GFP-GUS and pCLV3::GFP-GUS transgenic plants were used to analyze the spatial and temporal expression patterns of WUS and CLV3 during shoot regeneration.It was found that the CLV3 expression initiated at 2 to 3 days on CIM(callus induced medium)and was distributed in the pericardium and adjacent callus.WUS expression initiated at 2 to 3 days on SIM(shoot induced medium)and was dispersed in the 2 to 3 layers outside the pericycle of callus.When incubated on SIM for 3 to 4 days,WUS and CLV3 were expressed in the callus in a disordered and scattered manner,and no specific positional of them was formed When incubated on SIM for 4 to 5 days,the expressions of WUS and CLV3 were partially overlapped in some specific cell populations,indicating the stem cell niche initiation;when incubated on SIM for 6 to 7 days,WUS and CLV3 were co-located in a specific cell population,indicating the formation of the stem cell niche.When incubated on SIM for 8 to 9 days,the expression of CLV3 moved up to the outer layer of the stem cell center,and WUS was expressed in the central area,indicating the formation of the original SAM.When incubated on SIM for 8 to 9 days,the primary SAM developed into mature SAM,and CLV3 was expressed at the top of SAM and WUS was expressed in the OC of SAM.In conclusion,the expression pattern of WUS and CLV3 were used to characterize the molecular feature of different stages of in vitro shoot regeneration.The in vitro shoot regeneration capacity of arrl(arr1 loss-of-function mutant),arr12(arr12 loss-of-function mutant),arrl arr12(arr1 and arr12 double mutant)and Col-0 were identified.The results showed that the shoot number of arr1 root explants increased and arr12 decreased compared to that of Col-0,and the regeneration capacity of arr1 arr12 was seriously deficient.The number of reconstructed SAM was also in the following order:arr1>Col-0>arr12>arr1 arr12.The conclusion was that ARR1 inhibits in vitro SAM reestablishment and shoot regeneration,and ARR12 promotes in vitro SAM reestablishment and shoot regeneration.Further analysis showed that the shoot regeneration ability of p35S::ARR1 in Col-0 background(p35S::ARR1 vector was transferred into wild type plants)was less than that of Col-0,and shoot regeneration ability of p35S::ARR1 in arr1 arr12 background was stronger than that of arr1 arr12.These results indicate that ARR1 overexpression inhibits the shoot regeneration ability of Col-0 and promotes the shoot regeneration ability of arr1 arr12,which confirm that the inhibition effect of ARR1 on in vitro shoot regeneration depends on the existence of ARR12.The expression pattern analysis of ARR1 and ARR12 showed that ARR1 and ARR12 were strongly expressed in callus of root explant on CIM and SIM,but the expression level of ARR1 exhibited a trend of decreasing from inside cell layers to outside cell layers in callus and no significant specificity in the central region of stem cells,while ARR12 showed a trend of increasing from inside cell layers to outside cell layers and more concentrated in the central region of stem cells,indicating that the expressions of ARR1 and ARR12 are different during shoot regeneration.Results from ChIP and LUC experiments showed that ARR12 promoted the reestablishment of in vitro stem cell niche by directly promoting the expression of CLV3,while ARR1 negatively regulated the reestablishment of in vitro stem cell niche by directly inhibiting the expression of CLV3 in an ARR12-dependent manner.On the other hand,both ARR1 and ARR12 could directly bind to the same target sites of the WUS promoter.ARR1 competitively bind to the WUS promoter with ARR12 to inhibit the promotion effect of ARR12 on WUS expression,and thus inhibited the in vitro stem cell niches,SAM reestablishment and shoot regeneration.3.Mechanism of HY5 in in vitro stem cell niche and SAM reestablishment in Arabidopsis thalianaUsing in vitro shoot regeneration experiment,it was found that the callus formation and shoot regeneration ability of hy5 and hy5-215(HY5 loss-of-function mutant)root explants were stronger than that of Col-0,while the callus formation and shoot regeneration ability of p35S::HY5-GFP were significantly weakened,indicating that HY5 inhibits the callus formation and in vitro shoot regeneration.By detecting the GFP signals in the root explants of pHY5::HY5-GFP transgenic plant,it was found that HY5 was strongly expressed in the whole callus of root explants cultured on CIM and SIM,suggesting that HY5 may be involved in the callus formation and in vitro shoot regeneration.The GUS signal in root explants of pCLV3::GFP-GUS,hy5 ×pCLV3::GFP-GUS,pWUS::GFP-GUS and hy5 × pWUS::GFP-GUS were detected.It was found that the expression of WUS in hy5 background initiated in the callus of root explant incubated on CIM for 5 days,and was enhanced after incubated on SIM for one day,which was significantly earlier than that of Col-0 background.The expression of WUS and CLV3 under hy5 background were apparently stronger than that under Col-0 background,and the time when concentrated expression of WUS and CLV3 in specific cell groups,stem cell centers and SAM appeared was earlier than that under Col-0 background.The results indicates that HY5 represses WUS and CLV3 expression during callus formation and in vitro shoot regeneration,thereby inhibiting callus formation,stem cell center and SAM reestablishment.Results from ChIP,EMSA and LUC experiments showed that HY5 directly bind to the promoters of WUS and CLV3 to repress their expressions,and thus negatively regulated the reestablishment of stem cell niches and SAM.Genetic complementation experiment showed that the shoot regeneration ability of hy5-215 × wus-101 was the same as that of wus-101,and the shoot regeneration ability of hy5-215 × clv3-7 partially recovered to the level of clv3-7,indicating that HY5 acts upstream of WUS and CLV3 during in vitro shoot regeneration.Root explants of pTCSn::GFP and hy5 × pTCSn::GFP were used for GFP observation,and it was found that the GFP intensity under hy5 background was stronger than that under Col-0 background,indicating that HY5 inhibits the cytokinin response in root explants.Further analysis of B-type ARRs expression levels in Col-0 and hy5 root explants revealed that the expression of ARR12 was up-regulated in hy5,suggesting that HY5 may inhibit in vitro shoot regeneration by repressing ARR12 expression.Results from ChIP,EMSA and LUC experiments showed that HY5 inhibit ARR12 expression via directly binding to its promoter,and thus negatively regulate in vitro shoot regeneration.Genetic complementation experiment showed that the shoot regeneration ability of hy5-215 × arr12 was the same as that of arr12,and the shoot regeneration ability of hy5-215 × arrl arr12 was the same as that of arr1 arr12,indicating that HY5 functions upstream of ARR12 during in vitro shoot regeneration.In conclusion,the functional genes ARR1/12 and HY5 which regulate the in vitro reestablishment of stem cell niche and SAM were identified in this study.The interplay between ARR1/ARR12/HY5 and stem cell marker genes WUS and CLV3 were demonstrated,and related molecular mechanisms in regulating in vitro shoot regeneration(especially in stem cell niche and SAM reestablishment)were analyzed.The results provide valuable clues for the comprehensive understanding of plant cell totipotency.