The Functional Analysis For Transcription Factors HvATF1 And HvATF2 In Aluminum Tolerance And Identification Of MiRNAs Responding To Aluminum Stress In Barley

Aluminum(Al)toxicity is a major factor reducing crop yield in acid soils.Understanding molecular mechanisms for Al tolerance in plants is imperative for developing Al-tolerant cultivars and improving the agronomic practices against Al toxicity.Barley is a widely planted cereal crop in the world,but quite sensitive to Al stress.Therefore,it is necessary to identify the Al-tolerant germplasm and elite genes for breeding of Al-tolerant barley cultivars.Up to date,the Al-tolerant mechanism in barley is only well-known Al exclusion mediated by a citrate transporter HvAACT1.In this study,we cloned the two genes,HvATF1(Al-tolerance Transcription Factor 1)and HvATF2.The first gene is the closest homolog of AtSTOP1 and OsART1 in barley and the second is homologues with HvATF1 in barley genome.Gene knockdown(RNAi)or overexpression lines were obtained using transformation technology and then the functions of these two genes in Al stress tolerance were investigated.In addition,in view of the results in the previous study that Tibetan wild barley accessions with high Al toxicity tolerance were identified and secretion of organic acids or expression of HvAACT1 could not explain Al tolerance in the wild barley,the expression profiles of miRNA for a wild barley accession XZ29 and a cultivar Golden Promise were comparatively analyzed using small RNA sequencing,so as to understand the molecular mechanisms of higher Al tolerance underlying the Tibetan wild barley.The main results are as follows: 1.Cloning and functional analysis of Al-tolerant gene HvATF1.The gene HvATF1,homologues with AtSTOP1 and OsART1,was cloned.It was equally expressed in various barley tissues,and little affected by Al stress.Subcellular localization assays and transcriptional activation test showed that HvATF1 is localized in nucleus and works as a transcription factor with transcriptional activation.Furthermore,knockdown(RNAi)of HvATF1 resulted in an increased Al sensitivity of the RNAi lines,but did not change the expression of Al tolerant gene HvAACT1.On the other hand,RNA-sequencing was used to compare transcriptomics between the RNAi lines and WT.Totally 806 differently expressed genes were identified in the RNAi lines.Among them,64 genes were differently expressed in both control and Al stress,which were considered as downstream candidate genes regulated by HvATF1.These genes were partially similar to downstream genes controlled by homologs of HvATF1 in other plants,indicating its potential role involved in Al tolerance.2.Cloning and functional analysis of HvATF2HvATF2,a HvATF1 homologous gene in barley was cloned.There was no difference in the expression of HvATF2 in various tissues of barley under the normal condition(control).However,the gene was induced by Al stress in both root tip and basal root.Compared to HvATF1,the expression of HvATF2 was much lower.Analysis of subcellular localization showed that HvATF2 is located in the nucleus and transcriptional activation test confirmed that it has transcriptional activation.There was no significant difference in hematoxylin staining of root tips and Al content in root cell wall and cell sap between HvATF2 overexpression lines and the WT exposed to Al stress.In addition,Al-tolerant key genes HvAACT1,HvATF1 and its downstream genes were not differentially expressed in the HvATF2 overexpression lines.Therefore,it may be concluded that HvATF2 could not alleviate Al toxicity in barley.3.Identification of miRNAs in response to Al stress in the roots of Tibetan wild barley and cultivated barleyTibetan wild barley accession XZ29 showed higher Al tolerance than cultivated barley Golden Promise.However,the expression of HvAACT1 and secretion of citrate did not explain the difference of Al tolerance between these two genotypes.Small RNA sequencing was performed on roots of XZ29 and Golden Promise with presence or absence of Al stress.Target genes of miRNAs were obtained through bioinformatics prediction or degradome identification.Comparative analysis detected 50 miRNAs responding to Al stress,including 41 and 29 differently expressed miRNAs in XZ29 and Golden Promise,respectively,which 20 miRNAs were commonly shared.The comparison of miRNA profiles between two genotypes showed miR319 and PC-miR1 were up-regulated in XZ29,leading to higher activity of root cell proliferation and cell wall modification.It may be assumed that the up-regulated expression of miR319 and PC-miR1 is attributed to high Al tolerance in XZ29.