Molecular Mechanism Of Cytokinin Regulation Of Root Development Via Salicylic Acid Signaling In Arabidopsis

Root system plays an indispensable role in the growth and development of plants.It not only holds plants,but also is an important organ for synthesizing hormones and absorbing water and nutrients.Cytokinin(CK)inhibits primary root elongation by promoting the differentiation of transitional proliferating cells in the transition zone.AtABCG14 is the core transporter of CK to the top,and t Z can not be transported to the above-ground tissues and accumulate excessively in the root,resulting in the significantly shorter root of atabcg14 mutants.Salicylic acid(SA)regulates root development in a concentration-dependent manner.Reactive oxygen species(ROS)play an important role in maintaining the balance of cell proliferation and differentiation.CK can promote stomatal and plant defense responses,which is consistent with the pathway of SA regulating defense immune signals.CK signals regulate PRX-mediated ROS production in guard cells to control the perception of stomatal movement to PAMP.Studies have shown that CK can promote the TGA3/NPR1-dependent SA signaling pathway by activating Type B Arabidopsis Responses Regulator 2(ARR2),thus affecting plant resistance to pathogens.However,whether CK regulates root development through SA and ROS has not been reported.Previous analysis of root transcriptome data of Arabidopsis atabcg14 in our laboratory showed that 247 genes were up-regulated and 60 genes were down-regulated.The expression of SA synthesis gene ICS2 was significantly increased.This suggests that there may be a mechanism by which CK and SA interact to co-regulate root development.By means of biochemical chemistry,molecular biology,physiology and genetics,this study studied the mechanism of whether CK regulates root development through SA and ROS,and obtained the following research results:(1)Detection of root hormone-related gene expression.Based on the results of previous transcriptome analysis,we detected the expression levels of hormone-related genes such as auxin,CK,gibberellin,SA,ethylene,ABA and brassinolide in the roots of atabcg14.The results showed that the expression levels of ARR15,ARR17 and cytokinin oxidase CKX4 in class A ARR genes were increased,while the expression level of SA synthesis gene ICS2 was increased 13-fold in the root of atabcg14.The expression changes of hormone related genes such as auxin,gibberellin,ethylene and brassinolide were consistent with the results of transcriptome analysis.The increased expression of the SA synthesizing gene ICS2 in the roots of atabcg14 may lead to the corresponding increase of SA content in the roots.(2)Determination of root hormone content.Mass spectrometry analysis of SA content in atabcg14 roots cultured under 24 h and 16 h/8 h light conditions showed that SA content in atabcg14 roots increased by 5 times under 16 h/8 h light conditions,and increased by 3 times under 24 h light conditions.The contents of CKs in different types and ACC also increased,but the contents of JA,JA-Ile,IAA and ABA decreased.(3)CK and ROS regulate root development.The increase of SA content often leads to the increase of ROS content.Therefore,DAB staining and DCFH-DA staining were used to detect the ROS content in the roots,and it was observed that the root of atabcg14 was darker than that of the wild type.The root length of atabcg14was recovered after treatment with the peroxide inhibitor SHAM,suggesting that ROS was involved in CK inhibition of root development.(4)Root phenotypes of ics2 atabcg14,npr1 atabcg14,pad4 atabcg14 and Nah Gatabcg14.The sid2 and ics2 mutants of SA synthesis genes and the npr1,pad4and Nah G transgenic plants of SA signal transduction pathway were hybridified with atabcg14 to study the effect of SA synthesis and blocking of signal pathway on the inhibition of CK root.Compared with atabcg14,the root length of ics2 atabcg14recovered to a certain extent,while the root length of sid2 atabcg14 and ics2atabcg14had no difference with that of atabcg14.The root length of npr1 atabcg14,pad4atabcg14 and Nah G atabcg14 was almost completely recovered compared with atabcg14.The root apical meristem length of npr1atabcg14,Nah Gatabcg14 and pad4atabcg14 was longer than that of atabcg14.However,the number of QC cells in the root tip meristem area did not change,indicating that SA inhibition of root development is through regulation of meristem development without affecting QC development.(5)Analysis of SA and ROS content in roots of ics2 atabcg14.The SA content in the root of ics2 atabcg14 was recovered to the level of wild type.At the same time,ROS content in roots of ics2 atabcg14 was detected to decrease.In addition,the contents of H₂O₂,CAT and POD in roots of ics2 atabcg14 were detected,and the contents of H₂O₂ and CAT in roots of ics2 atabcg14 were decreased compared with that in roots of atabcg14,while the content of POD was increased compared with that in roots of atabcg14.(6)Single hybrid screening of yeast.Two ICS2 promoters of different lengths,1199 bp and 2600 bp,were analyzed with GUS as the reporter gene to determine which length of ICS2 promoter had promoter activity.GUS staining results of stable transgenic lines showed that the two fragments of different lengths were both active and induced by t Z.Therefore,a 1199 bp fragment was selected as the"bait"to screen four transcription factors that may regulate the expression of ICS2 gene through yeast single hybrid screening c DNA library.In conclusion,the inhibition of CK on root development was affected by the blocking of SA synthesis and signaling pathway.CK could partially restore or restore the root length of atabcg14,the root tip meristem length was also restored,and the content of SA and ROS was restored.Therefore,this study preliminarily elucidates the molecular mechanism by which CK regulates root development through SA and ROS.