Mechanisms Of ?-Aminobutyric Acid On Regulating The Formation Of Poplar Adventitious Roots

Vegetative propagation,which can transmit desirable traits quickly and effectively,is widely used in horticulture,agriculture,and forestry.Of which,the formation of adventitious roots(ARs)is a crucial stage.Therefore,it is very important to understand the regulatory mechanism of AR formation.The formation of ARs is regulated by many exogenous and endogenous factors,including carbon and nitrogen metabolism in plants.The ?-aminobutyric acid(GABA)shunt has been suggested as a central part of carbon and nitrogen metabolism,and affects primary root growth.Moreover,we recently revealed the poplar ARs were significantly inhibited by blocking ?-ketoglutarate dehydrogenase(?-KGDH),accompanying the increased endogenous GABA content.Our finding suggested a close relationship between GABA and ARs,but there is no direct evidence and the underlying mechanism will need to be characterized.Thus,based on our recent research,we investigated the effects of GABA on poplar ARs by increasing endogenous GABA levels using the overexpression of Pag GAD2 and the application of GABA or VGB(a specific inhibitor of GABA transaminase,aminohexenoic acid/Vigabatrin;VGB),with 84 K poplar(Populus alba × Populus glandulosa).The objective of this study was to further decipher the mechanism responsible for mediating the effects of GABA on AR formation and growth.The main results were as follows:1.The leafy stem segments of overexpressed Pag GAD2 lines and wild poplar treated by exogenous GABA and VGB were cultured in 1/2 MS medium for 12 days.The results showed that overexpression of Pag GAD2 inhibited the formation of ARs;AR growth was induced to increase at the lower GABA concentrations(0.01 and 0.1 m M),while inhibited at the highest GABA concentration(10 m M);the application of increasing concentrations of VGB significantly inhibited AR growth.All three treatment methods also increased the endogenous GABA contents when inhibiting ARs.Further biochemical analysis showed that,accompanying to the increase of endogenous GABA,the contents of soluble sugars in ARs decreased;glycine increased but the histidine decresed,which are closely related to rooting;IAA was increased,GA3 was decreased under GABA treatment,but incresed under VGB treatment.These results indicated that changes in endogenous GABA affected carbon and nitrogen metabolism as well as hormone balance,which affected the growth of poplar adventitious roots.2.Based on the above mentioned,the poplar tissue-cultured seedlings were treated with the GABA(10 m M)or VGB(100 ?M)as well as the control for 6 days.The GABA and VGB treatments delayed the formation of AR primordia and the appearance of ARs coupled with the increased endogenous GABA contents.Further biochemical analysis showed that soluble sugars in the base of the stems increased,and malate decreased first and then increased.Valine,proline,glycine and tyrosine,which are negatively correlated with rooting,were up-regulated;IAA,negatively with the later stage of AR formation,was up-regulated,while ethylene and gibberellin,which are positively correlated with rooting,were down-regulated,and moreover,the corresponding genes involved in IAA and ethylene signaling pathway were also significantly affected.These results demonstrated that the accumulation of endogenous GABA affected carbon and nitrogen metabolism as well as hormone signal pathways,and ultimately affected the formation of poplar ARs.In summary,this study verified the negative correlation between the accumulation of GABA and AR formation as well as growth of poplar by three methods,and deciphered the reasons from carbon and nitrogen metabolism as well as hormone signal pathway.Our finding was the first time to demonstrate the roles of GABA on AR development,and laid the foundation for further study on the mechanism of GABA in plant growth and adaptation.