Mechanism Of MYB6 Regulating The Size Of Shoot Apical Meristem In Arabidopsis Thaliana

In plants,stem cells gather together to form specialized tissues called meristems.The regular activities of meristems enable plants to continuously produce new tissues and organs throughout their life cycle.In the aerial part of plants,stem cells are mainly concentrated on the shoot apical meristem,producing all organs of the aerial parts of the plant,including leaves and flowers,and play a key role in the growth and propagation of the plants.In the shoot apical meristem,the division and differentiation of stem cells are precisely regulated by a complex molecular network,allowing the shoots to form new organs in a precise spatiotemporal pattern.At present,the molecular regulatory network of the shoot apical meristem has been basically constructed,but some of the regulatory factors,especially the regulators that play an important role in the regeneration process in vitro,are still incomplete in the apical meristem.The MYB transcription factor family is large and versatile and is ubiquitous in all eukaryotic organisms.As one of the largest transcription factor families in Arabidopsis,MYB family members play important roles in a variety of biological processes of plants,including biotic and abiotic stress responses,cell differentiation,hormone response,plant defense response,regulation of circadian rhythms and other aspects.So far,no studies have reported that the MYB family members play a role in the development of the shoot apical meristem.In the previous work of our laboratory,a MYB family transcription factor MYB6,which plays an important role in the regeneration process,was obtained by analyzing the results of gene expression microarray of callus with different regenerative capacities.Based on this,we analyzed the phenotype of MYB6 mutant plants and found that the overexpressing mutant MYB6-OX could not bolting,suggesting the abnormally development of its shoot apical meristem.In this study,we first performed the overall phenotype of the MYB6 deletion mutant myb6,Its homologous gene MYB4 deletion mutant myb4,double mutant myb4/6 and the MYB6 overexpressing mutant MYB6-OX.It was found that the main branch of MYB6-OX could not develop normally,and the lateral branch was bolted after 2-3 months of germination,and the bolting was highly random.In addition,the other three mutants showed no significant difference compared to the wild type,so the paraffin sections were taken on the shoots of all mutants to observe the shoots at cytological level.The results of paraffin section showed that the average shoot width of myb4,myb6 and myb4/6 mutants increased by 6.02%,26.00%and 28.12%,while the average shoot width of MYB6-OX mutant was 19.52%lowers than that of wild type.At the same time,the expression pattern of these two genes in the shoots was observed by GUS reporter gene,and it was found that both MYB4 and MYB6 were expressed in the shoot range.The expression of MYB4 was concentrated on the leaf primordium far from the SAM,and the expression position of MYB6 was concentrated on the surface cells of the leaf primordia the and the peripheral zone of the SAM.It is concluded that both MYB4 and MYB6 are negative regulators of the SAM,which is expressed in the shoots to regulate the growth of the SAM,and the effect of MYB6 is significantly greater than that of MYB4.For further experiments,we crossed the WUSpro:GUS and CL V3pro:GUS plants,respectively,with different mutants to analyze if the expression patterns of these two important stem cell regulatory factors in different mutants were changed.The results showed that the expression range and expression level of WUS and CL V3 were significantly increased in the deletion mutant.Among them,the change of expression in myb4/6 was the most obvious,a little more significant than the change in myb6.At the same time,the expression level and expression range of WUS and CLV3 in MYB6-OX mutant decreased.Since the expression patterns of WUS and CL V3 in the mutants changed significantly,the clv3-7 mutant was hybridized with MYB6-OX,and the genetic relationship between CLV3 and MYB6 was demonstrated by the phenotype of the hybrid line.The results showed that clv3-7 could not rescue the phenotype of MYB6-OX.The results indicate that although the changes in the expression of MYB4 and MYB6 caused changes in the expression of WUS and CLV3 in the SAM,MYB4 and MYB6 did not regulate the growth of the SAM through WUS and CL V3.To investigate how MYB6 regulates the development of SAM,the RNA from myb6 SAM was extracted from transcriptome sequencing.By analyzing the results of transcriptome sequencing,it was found that the expression level of some auxin-related genes and some ABA-realated genes was changed in the myb6 mutant.At the same time,the expression levels of A UX/IAA family and ARF family genes in the shoots of myb6 mutant were detected.The results showed that the expression level of most A UX/IAA family and ARF family genes are significant changed in myb6 mutant.The DR5:GUS vector was constructed to transform different mutants to observe the content of auxin in the shoots of myb6 mutant.The results showed that the auxin content in the myb6 mutant was significantly lower than that in the wild type.EMSA and LUC experiments were used to detect whether MYB6 can directly bind to the predicted auxin pathway gene DNA.The results showed that MYB6 can directly bind to the DNA of these genes and regulate its expression of the SAM.The results indicate that MYB6 regulates the expression of auxin-related genes directly or indirectly,and regulates the content of auxin in the shoots,thereby regulates the growth of SAM.To verify whether MYB6 regulates the growth of the SAM via the auxin pathway,the auxin response factor mutant mARFx was hybridized with MYB6-OX.The results showed that the mARFx×MYB6-OX hybrid lines were able to bolt normally.In summary,MYB6 regulates the content of auxin in the SAM by regulating the expression of the auxin pathway gene directly or indirectly and regulates the growth of the SAM,while responsive to external stimuli by modulating the ABA response gene.These two effects combine to regulate the development of the SAM.