ARF4 Regulates Shoot Regeneration Through Interaction With IAA12 In Arabidopsis

Under appropriate culture conditions,the plant has the ability to regenerate the shoot apical meristem and then regenerate the whole plant.This process is a prerequisite for plant propagation in vitro,cultivation of virus-free seedlings,and genetic engineering.It also provides an ideal research system for answering basic biological questions such as the mechanism of plant hormone signal transduction and cell fate determination.Previous studies have found that auxin plays an important role in the regulation of shoot apical meristem regeneration.Auxin regulates plant development through its signal transduction pathway,and the Auxin Response Factor(ARF)family are an important link responses for regulating downstream gene transcription.The ARF family can be divided into two categories: transcription activators and transcription repressors.At present,the mechanism of transcription activator-mediated auxin signal transduction has been deeply studied,but the mechanism of action of transcription repressor is still unclear.Previous studies have shown that ARF4 is highly expressed in shoot apical meristem.In this study,the molecular mechanism of ARF4 regulating Arabidopsis shoot regeneration was analyzed by means of cell biology,biochemistry and genetics.We found that the expression signal of ARF4 showed a specific expression pattern during the development of the shoot apical meristem and gradually concentrated to the surrounding area of the meristem.ARF4 mutates leads to a lower frequency of shoot regeneration.In order to further analyze the period of ARF4 regulation,we constructed am-ARF4 transgenic plants by constructing ethanol induction vectors.Adding ethanol to the shoot induction medium reduced the shoot regeneration ability,indicating that ARF4 regulated the regeneration of shoot meristem.Yeast two-hybrid analysis revealed that ARF4 interacted with IAA5,IAA7,IAA12,and IAA34 in the Aux/IAA protein family.The results of expression pattern analysis showed that among the above four Aux/IAA proteins,only IAA12 expressed signals in shoot apcial meristem.Further Pull-down,BiFC and Co-IP analysis confirmed the interaction between ARF4 and IAA12.Previous studies have shown that ARF5 mediates auxin signaling through interaction with IAA12.Cytological analysis found that ARF5 and IAA12 showed similar expression patterns to ARF4 during shoot apcial meristem regeneration,suggesting that ARF4's regulation may be related to ARF5 and IAA12.ARF5 mutating,overexpression of IAA12,and specific induction of IAA12 expression during the regeneration of shoot meristems all inhibited shoot regeneration.Further analysis found that induced expression of ARF4 can increase the frequency of shoot regeneration,and can also partially restore the regeneration defect phenotype induced by ARF5 mutation and IAA12 overexpression.Yeast three-hybrid and Pulldown analysis proved that ARF4 can be combined with IAA12 by competing with ARF5.The above results indicate that during the regeneration of shoot meristem,ARF4 can regulate the regeneration process of Arabidopsis by competing ARF5 to interact with IAA12.The research results provide an understanding of the mechanism of shoot apical meristem regeneration and the new mechanism of transcriptional repressive ARF.