Mechanism Of ALA Relieving ABA-Inhibited Strawberry Root Growth

The root is the mainstay of the plant's rooted soil,and it is also the main organ that absorbs and transports water or nutrients.Roots also stores photosynthetic products and regulates the growth and development of the above-ground parts of plants through synthetic auxins,cytokinins,abscisic acid and other plant hormones.So a well-developed root is an important guarantee for healthy growth.However,stress conditions such as salt damage,drought,leeches,and heavy metal pollution often inhibit root growth,and the increase in endogenous ABA content is an important limitation to root growth.5-aminolevulinic acid(ALA)is a synthetic precursor of all tetrapyrrole(porphyrin)compounds and is also a novel plant growth regulator with various physiological functions.It can relieve the inhibition of plant root growth by a variety of stress(such as salt,drought,leeches).However,it has not been reported that ALA regulates plant root growth,especially ABA-inhibited root growth mechanisms.Therefore,this study used ABA as a stress factor and strawberry(Fragaria x ananassa Duch.cv.'Benihoppe')as the main test material,using several different genotypes of Arabidopsis thaliana(Col-0,tt4,PIN1::GFP,PIN2::GFP,PIN7 GFP,DR5::GFP were used as auxiliary materials to observe the antagonistic effects of ALA and ABA on root growth.It was found that flavonoids may be involved in ALA to relieve ABA-induced inhibition of root growth.The main findings are as follows The effect of ALA on the accumulation of flavonol in root system was observed under fluorescence microscope,the content of flavonoids in root system was also determined and the key gene that regulation of flavonoid metabolism expression level was analyzed in root by ALA.In strawberry,the plant hormone content in the root after treatment was tested.The effects of ALA and flavonoids on the transtransporter proteins of Arabidopsis were observed by laser confocal microscopy with various Arabidopsis thaliana,to explore whether ALA and flavonoids can alleviate the inhibition of ABA on plant root auxin polarity transport.The main results are as follows:1.high concentration of exogenous ABA can significantly inhibit the growth of strawberry roots,while exogenous ALA can relieve the inhibitory effect of ABA on root growth.In the 2 ?M ABA and(or)2.5 mg/L ALA test systems,(1)ABA treatment significantly decreased the length of adventitious roots in strawberry,while ALA could antagonize the effects of ABA and partially restore adventitious root growth;(2)ALA and ABA mainly affected the length of strawberry root apical meristem;(3)ALA could relieve the decrease of strawberry root activity caused by ABA.The expression of auxin synthesis gene YUC,input gene AUX1 and output gene PIN1 in strawberry roots were analyzed after different treatments.The results showed that ALA and ABA mainly affected the expression of PIN1,but had little effect on other genes.HPLC assay showed that ABA treatment resulted in a decrease in IAA content in strawberry roots,while ALA treatment reversed the decrease in IAA content caused by ABA.These results suggest that in the presence of higher concentrations of ABA,ALA may relieve ABA-inhibited strawberry root growth by promoting IAA transport.2.Using DPBA dyes and fluorescence microscopy studies,it was found that exogenous ALA promoted flavonol accumulation in strawberry root tips;It also confirmed that ALA promoted flavonoid content in strawberry roots,while ABA treatment reduced root flavonoid content by spectrophotometry.The results of qRT-PCR showed that ALA promoted the up-regulation of genes such as CHS,CHI and FLS in flavonoid metabolic pathways,while ABA down-regulated the expression of these genes.Both exogenous flavonoids(including quercetin and kaempferol)significantly reduced the inhibition of ABA on strawberry root growth and the development of the root appressorium area,and also promoted PIN gene expression and increased IAA content.These results indicated that ALA may promote auxin transport to antagonize the inhibitory effect of ABA on root tip growth by inducing flavonoids accumulation.3.In order to validate the above work hypothesis,the authors used different genotypes of Arabidopsis[including tt4 mutants that could not synthesize flavonoids,transgenic lines with green fluorescence protein(GFP)that were involved in auxin synthesis and transport.The PIN1::GFP,PIN2::GFP,PIN7::GFP,DR5::GFP]seedlings were used as materials in order to more intuitively determine the effects of flavonoids and auxin.The results showed that exogenous ALA and flavonoids had similar mitigating effects on ABA-inhibited wild-type(Col-0)Arabidopsis root growth,but flavonoids still had antagonistic effects on tt4 mutants and ALA had lost.The alleviation effect suggests that the effect of ALA may depend on the accumulation of flavonoids.In the root,ALA can promote the up-regulation of key genes of flavonoid biosynthesis(including CHS,CHI and FLS)and also induce the accumulation of root flavonoids.Direct observation of the root tips of transgenic seedlings associated with auxin transport with GFP by laser scanning confocal microscope(LSCM)revealed that both ALA and flavonoids reversed ABA-inhibited PIN1 and PIN7 protein accumulation and DR5 accumulation.While PIN2 did not respond,indicating that ABA inhibited the extracellular transport of IAA from the root tip and reduced its content,but did not affect lateral transport.Both ALA and flavonoids could slow down the inhibitory effect of ABA on IAA.In summary,we used strawberry tissue culture plants to demonstrate that exogenous ALA can relieve ABA-inhibited root growth,and this process may depend on flavonoid accumulation and IAA transport.We demonstrated that ALA also has similar regulatory activity in Arabidopsis with the transgenic plants and a deletion mutant Arabidopsis thaliana.We also observed the physiological effects of ALA and flavonoids directly under LSCM.Therefore,we believe that flavonoid accumulation and IAA transport are involved in the regulation of ALA-ABA root growth in strawberry and Arabidopsis.