Regulation Of Root Hair Development By ?-aminobutyric Acid Metabolism In Arabidopsis Thaliana

Root hair are tubular-shaped outgrowths from root epidermal cells that are important for acquisition of nutrients,microbe interactions,and plant anchorage.root hair development is regulated by the transcription factors such as CAPRICE(CPC)and WEREWOLF(WER),plant hormones including auxin and ethylene,and ROS so on.?-Aminobutyric acid(GABA)is a four-carbon non-protein amino acid,it rapidly accumulates under stress condition as a metabolite scavenging ROS,improve plant tolerance to oxidative stress,can also as a signaling molecule influencing plant development.In this study,an endogenous GABA synthetic defective mutant gad1 of model plant Arabidopsis thaliana as materials.a preliminary study on the molecular mechanism of gad1 mutants regulating root hair development by determination of root hair length and density,staining of ROS,enzyme activity and expression level of related genes.the experimental results are as followed:1.The measurement of root hair length and density results showed that those in gad1 mutant were significantly higher than wild-type.Both the density or length of root hair in gad1 mutant and wild-type were significantly suppressed by exogenous GABA,but the length of trichoblast cell not be affected.Indicating that formation of root hair was affected in gad1 mutant.2.In order to clarify the correlation between root hair growth and ROS i-n mutant background,subsequently the whole level of ROS in root was staine-d by fluorescence dye(H₂DCFDA),using chemical dye NBT(nitro-blue tetrazo-lium)and DAB(3,3'-diaminobenzidine)to dye O₂^-and H₂O₂respectively.the results showed that the fluorescence intensity and chemical staining in gad1 m-utant were higher than wild type,but decreased under exogenous GABA treat-ment.The contents of total ROS and H₂O₂were measured respectively and the results were consistent with staining.this suggests that gad1 mutations can affe-ct levels of ROS in the root tip and root hair regions,including O₂^-and H₂O₂levels.3.Measurement of production capacity of·OH present downstream of O₂^-and H₂O₂,the ability to produce·OH in gad1 mutants was higher than wild-type and decreased under exogenous GABA treatment.Therefore,the increase in root hair length in gad1 mutants may be due to the increase in·OH content.4.To further explore the reasons in the altered H₂O₂level in gad1 mutant,we found that the activity of scavenging H₂O₂enzyme APX and CAT in gad1 mutants was lower than wild-type,and exogenous GABA could induce increased activity of APX and CAT.suggesting that higher H₂O₂levels in gad1 mutants may be associated with decreased scavenging capacity APX and CAT involved of ROS.5.The expression levels of ROS related genes were evaluated by qRT-PCR experiments,the results show that the expression of ROS synthesis related genes was not affected,but the expression of superoxide dismutase gene MSD1?CSD1 and CSD2 was inhibited,and exogenous GABA could increase its expression.indicating that the increase of O₂^-content in gad1 mutants is due to the inhibition of the expression of scavenging genes.6.The expression of PHYA and PHYB of phytochrome family and root hair differentiation related genes SCM1?TRY?ETC1 in gad1 mutant was obviously induced.suggesting that gad1 mutation may also affect the process of root hair fate determination and light stimulation root hair formation,finally,causes more trichoblasts to form root hairs,which increased the density of root hairs.