Type-B Arabidopsis Response Regulators Regulate Regeneration And Maintanence Of Shoot Apical Meristem

The shoot apical meristem?SAM?gives rise to all above-ground tissues of a plant.Under proper cultural conditions,somatic tissues can form callus,through which SAM is regenerated.SAM regeneration is the foundation for in vitro propagation and genetic transformation and provides idea systems for investigating basic questions,such as stem cell specification,cell fate determination and hormonal signaling.Exogenous cytokinin and auxin determine cell fate for the establishment of the stem cell niche,which is the vital step of shoot regeneration.Our results reveal a long-standing missing link between cytokinin signaling and WUS regulator,and the findings provide critical information for understanding cell fate specification.The main results are shown as follows:?1?Type-B ARRs display dynamic expression patterns during shoot regenerationWe observe the relative spatio-temporal expression patterns of ARR1,ARR10,ARR12and WUS by using the double reporter lines ProWUS:dsRED with ProARR1:ARR1-GFP,ProARR10:ARR10-GFP,ProARR12:ARR12-GFP during the SAM regeneration.The expression signals of ARRs were gradually enhanced and constitutive distributed at the beginning of shoot induction.Signals concentrated at the specific region when the explants incubated on shoot induced medium?SIM6D?and at this time WUS signals were first detected in a few cells of type-B ARRs signals distributed region.After that,type-B ARRs signals were restricted to the central region of SAM which is under development,overlapping with that of WUS.When the SAMs were established at SIM12D,type-B ARRs signals were detected in the central zone of the SAM and overlapped with WUS signals.?2?The function of ARR1,ARR10 and ARR12 were required in the reestablishment of SAMWe used artificial microRNAs?am?driven by an ethanol-inducible promoter to simultaneously silence ARR1,ARR10 and ARR12 at different stages of SAM regeneration,respectively.As a result,ethanol induction before restricted WUS expression at SIM4D largely abolished shoot regeneration.However,when ethanol induction started after the specification of shoot stem cell niche at SIM8 or the establishment of the SAM at SIM12D,the frequency of shoot regeneration was similar to those of control.The results show that ARR1,ARR10 and ARR12 play a role in the SAM regeneration,and once the SAM formed,low transcriptional levels of ARR1,ARR10 and ARR12 are enough to maintain shoot development.?3?Type-B ARRs directly activate WUS transcriptionThe transcript level of WUS was significantly decreased during shoot regeneration in the arr1/10,arr1/12,arr10/12 double mutants and transgenic plant am-ARR1/10/12,in which the expression of ARR1,ARR10,ARR12 were significantly repressed.The expression of WUS marked by gWUS-GFP₃ reporter lines was significantly decreased after the repression of ARR1,ARR10,ARR12.Overexpressing WUS was able to fully complement the decreased shoot regeneration phenotype of arr1/12 double mutants.Type-B ARRs directly binding to the WUS promoter and directly activates its transcription through performing the ChIP,EMSA,yeast one-hybrid system and in vitro transient activation experiments.All the results confirm that ARR1,ARR10 and ARR12 positively regulate the transcription of WUS during SAM regeneration.?4?ARR1,ARR10 and ARR12 coordinately confer the proper expression of YUC1 and YUC4In arr1/10,arr1/12 and arr10/12 double mutants,transcript levels of the auxin biosynthetic genes YUC1 and YUC4,but not YUC2 and YUC6,were significantly higher than in the wild type.And the co-expression patterns of ARR10 and YUC4 were analyzed during the shoot regeneration.At SIM2D and SIM4D,the expression patterns of YUC4 were similar to ARR10.Both of them were expressed in the whole explants,and ARR10 signals were stronger than YUC4.At SIM6D,ARR10 gradually concentrated in a particular area and YUC4signals reduced in this area.At SIM8D,ARR10 was expressed in the central region of the SAM which was under development,while YUC4 was expressed on both sides of ARR10.After the formation of the SAM?SIM12D?,ARR10 was expressed in the central region of the SAM and YUC4 was expressed in the outermost cells of the SAM.In arr10/12 double mutants,the expression patterns of YUC4 and WUS had been changed.The expression of YUC4 was enhanced and the specific patterns were disrupted,and WUS expression was not detected.The observation to the expression patterns of YUC1 by using the transgenic lines Pro YUC1:GUS in arr double mutants showed that the transcript levels of YUC1 were elevated.Transgenic plant ProDR5:GFP was used to examine the auxin response in arr10/12double mutant.As expected,GFP signals in the mutant exhibited patterns similar to those of YUC1 and YUC4.These results indicate that ARR1,ARR10 and ARR12 suppress the expression of YUC1 and YUC4 in the organizing center?OC?region,thereby limiting YUC1and YUC4 to the surrounding area during the SAM regeneration and maintenance.?5?Spatio-temporal expression patterns of YUC1 and YUC4 mediated by type-B ARRs are essential for shoot regenerationTo further test the essential role of YUCs proper expression in SAM for shoot regeneration,we studied the shoot regeneration phenotype in YUC overexpressing plant.Overexpression of YUC1 or YUC4 attenuated shoot regeneration significantly.Ectopic expressed YUC4 driven by the ARR10 or WUS promoter,shoot regeneration decreased obviously and the latter decreased much more.All those results illustrate that suppressed YUC4 in the type-B ARRs expressed region,especially in the WUS expressed region,is very important for shoot regeneration.The ChIP,EMSA,yeast-one hybridization and analysis of type-B ARRs binding elements mutation experiments all prove that type-B ARRs directly suppress the transcription of YUC4.The results indicate that the suppression of YUCs transcription directly regulated by ARR1,ARR10 and ARR12 plays a vital role in the SAM regeneration.?6?Type-B ARRs regulate SAM maintenance by regulating the transcription of WUSARR1,ARR10 and ARR12 were expressed in the central zone and overlapped with WUS in the OC of SAM,while YUC4 was expressed in the L1 cell layer.The WUS signals marked by gWUS-GFP₃ in arr1/10/12 triple mutants and am-ARR1/10/12,in which the transcript levels of ARR1,ARR10,ARR12 were significantly reduced after ethanol induction for 24h,and were obviously declined compared with gWUS-GFP₃.In arr10/12 and arr1/10/12,YUC4signals expanded from L1 cell layer to the central region of the SAM,and the latter expanded more obviously.By histological analysis of SAM in the arr1/10/12 triple mutants and the overexpressing YUC1 or YUC4 plants,the SAM size decreased and resulted in a reduced cell number.The above results suggest that ARR1,ARR10 and ARR12 mediate the SAM maintenance through their regulation of the WUS,YUC1 and YUC4 transcription.In conclusion,ARR1,ARR10 and ARR12 expression overlapped with YUC1 and YUC4were constitutively expressed at the early stage of the SAM regeneration;after that,type-B ARRs signals concentrated to the special region.In this region,they directly bound to the promoter region of WUS and activated its expression.At the same time,they also repressed the auxin biosynthetic genes YUC1 and YUC4 expression,and thus,repressed the auxin accumulation in the central zone of the SAM,in which auxin indirectly repressed the expression of WUS.The dual role of type-B ARRs to WUS play a vital role in the regeneration and maintenance of the SAM.This founding provides new information to understand the molecular mechanism of hormones regulating the maintenance of meristem,and the in vitro propagation and the establishment of genetic transformation systems in plants.