Functional Analysis Of Arabidopsis Glycosyltransferase Gene UGT86A1 In Plant Adaptation To Abiotic Stresses

Under unfavorable environments,plants can quickly perceive thoses cues and produce a series of physiological and biochemical reactions,mainly including changes in physiological metabolism,hormones and other molecules production,and the expression of stress-related genes.Glycosyltransferases are enzymes which are responsible for the glycosylation modification of molecules.By glycosylation,plant molecules can change their water solubility and biological activities,and thus affecting the physiological activities of plants.According to the publicly available microarray data,the expression of the Arabidopsis glycosyltransferase gene UGT86A1 can respond to abiotic stresses,suggesting that this gene may be involved in the plant adaptation to abiotic stresses.In order to clarify the role of glycosyltransferase gene UGT86A1 in plant response to stress,this study was carried out.Firstly,we conducted the stress-treatment of NaCl and Mannitol for wild-type Arabidopsis thaliana,and then we tested the expression level of UGT86A1 with qPCR.The results showed that UGT86A1 was up-regulated by those abiotic stresses.We constructed the CRISPR/Cas9 knockout vector of UGT86A1,plant overexpression vector of UGT86A1,UGT86A1promoter:GUS reporter vector and prokaryotic expression vector of UGT86A1.We successfully obtained UGT86A1 CRISPR/Cas9 knockout lines,overexpression lines,GUS transgenic lines and prokaryotic expression bacteria through transgenic technology.In order to explore the role of UGT86A1 in plant response to abiotic stress,GUS reporter lines was stained under stresss treatment.At the same time,a series of stress tolerance analyses were carried out for UGT86A1 knockout lines and overexpression lines.The results were obtained as follows.Histochemical staining of UGT86A1 promoter:GUS transgenic lines showed that GUS activity was significantly enhanced under treatment of NaCl and Mannitol than the control treatment,further demonstrating that UGT86A1 expression was induced by abiotic stress.After a series of stress challenge experiments,we found that UGT86A1 knockout lines were more sensitive while overexpression lines were more tolerant than wild-type in the terms of seed germination,cotyledon greening and root elongation under the stress treatments with NaCl or Mannitol.Salt tolerance and drought tolerance experiments were also carried out under soil culture conditions.The experimental results showed that the survival rate of UGT86A1 knockout lines were lower while overexpression lines were higher than wild-type under the conditions of salt and drought treatments.These results indicated that UGT86A1 may be involved in the adaptation of plants to adversity in both seed germination and subsequent post-germinationm growth.In order to explore the functional mechanism of UGT86A1 in plant coping with adverse stress,the physiological changes of UGT86A1 knockout lines and overexpression lines under adverse conditions were determined.Firstly,the water loss rate of the detached leaves of UGT86A1 knockout lines and overexpression lines was investigated,and the stomatal aperture of the leaves under drought condition was observed.It was found that the water loss rate of the detached leaves of UGT86A1 knockout lines was higher,while the water loss rate of UGT86A1 overexpression lines was relatively lower than wild type.It was found that under drought conditions,the stomatal aperture of the knockout lines was larger,and the stomatal aperture of the overexpression lines was smaller than wild type under drought condition.This suggested that the physiological function of UGT86A1 may have some connection with the stomatal movement,and its loss of function resulted in the rapid loss of water in the leaves under drought condition.Our experimental results also showed that the content of soluble sugar of UGT86A1 knockout lines was lower while the content of malondialdehyde was higher than that of wild-type.In UGT86A1 overexpression lines,the content of soluble sugar is higher while the content of malondialdehyde is lower than that of wild-type.The measurements of reactive oxide species(ROS)showed that UGT86A1 overexpression lines had lower ROS level than wild type.These experimental data indicated that the role of UGT86A1 may also be related to osmotic protection,membrane stability and reactive oxygen scavenging.Finally,we examined the stress-related genes expression of UGT86A1 knockout lines and overexpression lines,including DREB2A,RD29B,AIL1,RD22,COR47 and CAT1,It was found that the expression levels of these stress-related genes were decreased in UGT86A1 knockout lines and were increased in overexpression lines than wild type,respectively.We proposed that UGT86A1 may affect the expression of these stress-related genes in some unknown mechanism,and then cause changes in the abovementioned physiological indexes to enhance the plant tolerance to stress environments.In this study,we attempted to further explore the molecular mechanism of UGT86A1 function from the aspect of its substrate.However,due to the difficulty of enzyme protein expression and purification,no progress has been made in the identification of substrate.In summary,through a series of studies on UGT86A1 knockout lines and overexpression lines,it is clear that the glycosyltransferase gene UGT86A1 plays an important role in enhancing the adaptation of plants to abiotic stress.At the same time,it is found that UGT86A1 gene may affect the stress related physiological processes by affecting the expression of stress-related genes under adverse circumstances.The substrate identification of glycosyltransferase UGT86A1 and the deep molecular mechanism of UGT86A1 function remain to be further studied.