Study On The Roles Of Two NAC Transcription Factors In Response To Aluminum Stress In Tomato

Aluminum(Al)toxicity is one of the major limiting factors for crop production on acidic soils,which occupy about 50%of potential arable land worldwide.Tomato is one of the most important vegetable crops worldwide and is highly sensitive to acid and aluminum.Therefore,elucidation of its response mechanism and transcriptional regulation mechanism to acid-aluminum stress can not only reveal the mechanism of acid-aluminum toxicity in plants,but also provide a theoretical basis for genetic improvement and breeding new acid-and aluminum-tolerant varieties of tomato.In this study,through the screening of the ability of tomato acid aluminum tolerance and RNA-Seq,we identified tomato root tip early aluminum stress response genes,and through bioinformatics analysis,we studied the role of two tomato NAC family members,NAC063 and NAC064,in regulating the mechanism of tomato aluminum tolerance.The main results are as follows:Mechanisms of early Al sensitivity and late root elongation recovery in tomato.To investigate the mechanisms of aluminum toxicity and aluminum stress response in tomato,we first screened seven different genotypes of tomato for aluminum tolerance.The aluminum-induced root elongation inhibition study revealed no significant differences among the genotypes in response to aluminum stress.Therefore,we used cultivar tomato AC(Ailsa Craig)for all subsequent experiments.The pH gradient and Al concentration gradient tests determined that 5 ?M Al and pH 5.0 were the most suitable conditions for aluminum treatment of tomato seedlings.We found that the root elongation of tomato was inhibited by 40%after 6 h of treatment under this treatment condition.However,after 24 h of treatment,root elongation was inhibited by only 10%,suggesting that the Al tolerance mechanism was activated or a new Al tolerance mechanism emerged in tomato at a later stage of Al stress.Further studies revealed that the root elongation recovery mechanism was not related to Al activity and total Al content in root tips,but to Al distribution and translocation in root tips.Phylogenetic and gene expression analyses indicated that aluminum translocation and distribution in root tips might be associated with the synergistic effect of SlNIP1;2 and SIALMT10.Identification and analysis of Al-responsive genes in tomato root tips.Through transcriptome analysis,we identified an average of 20,931 and 20,785 genes in the control(0 ?M Al)and treatment(5 ?M Al)groups,respectively.A total of 2409 differentially expressed genes(DEGs)were identified,including 1620 up-regulated genes and 789 down-regulated genes.The differentially expressed genes were analyzed by GO biological function enrichment and KEGG metabolic pathway enrichment,which revealed that Al stress mainly affected the expression of genes related to metabolic pathways and secondary metabolism in tomato,thereby inhibiting root growth.Expression analysis of tomato homologs of known aluminum tolerance genes revealed that all of these aluminum tolerance homologs were induced to be up-regulated by aluminum stress.Known Al tolerance homologous genes were all induced to be up-regulated by Al stress in tomato.We identified 89 differentially expressed transporter protein genes,mainly involved in Al uptake and accumulation,organic acid secretion,and cell wall modification.It was also found that most of the transcription factors whose expression was up-regulated by Al stress belonged to the MYB and NAC families.These results lay the foundation for the construction of a regulatory network for Al tolerance in tomato.Identification and analysis of tomato NAC family members.Using bioinformatics analysis,we identified a total of 93 NAC family members in the tomato genome.Phylogenetic analysis showed that the 93 NAC genes could be divided into five subfamilies.We also renamed all tomato NAC transcription factors according to the distribution of NAC genes on tomato chromosomes,solving the reported confusion of tomato NAC nomenclature.Expression profiling revealed that 93 NACs had different expression patterns in different tissues and at different fruit developmental stages.Combined with the tomato root tip transcriptome and qRT-PCR analysis after cycloheximide treatment,we found that NAC genes are early genes involved in the corresponding aluminum stress in tomato root tips.This study provides a basis for further studies on the biological functions of the tomato NAC gene,which will contribute to the improvement of tomato agricultural traits in the future.SlNAC063 transcription factor is involved in the regulation of aluminum tolerance in tomato.Transcriptional regulation plays an important role in the mechanisms of plant adaptation to aluminum toxicity However,there are still very limited studies on the identification of transcription factors involved in plant aluminum stress response.Here,we identified a tomato NAC transcription factor gene,SlNAC063,whose expression is up-regulated by Al stress.Further studies revealed that SlNAC063 is a nuclear-localized transcriptional activator whose transcriptional activation activity is dependent on the C-terminal C2 structural domain.Expression pattern analysis revealed that SINAC063 was specifically up-regulated by aluminum in tomato roots under Al treatment conditions and was particularly significant at the root tip.In addition,the expression level of SlNAC063 induced up-regulation by Al stress increased with increasing Al treatment concentration and time.We obtained loss-of-function mutants of SlNAC063,nac063#5-5 and nac063#5-7,by CRISPR/Cas9 gene editing technology.Phenotypic analysis showed that under normal conditions,root elongation of nac063#5-5 and nac063#5-7 was smaller than that of wild type(WT).Under Al stress,tthere was no significant difference in root elongation between the mutants and WT,but the total Al content of the mutants was lower than WT.SlNAC063 negatively regulates aluminum tolerance and affects root development in tomato.SlNAC064 is involved in tomato aluminum stress response through regulation of SlAAE3-1 The Acyl-Activating Enzyme3(AAE3)gene encodes an oxalyl-CoA synthase.Oxalate metabolic mediated by AAE3 plays an important role in enhancing tolerance to biotic and abiotic stresses(Al and Cd).However,the transcriptional regulatory mechanism of AAE3 remains unclear.Combining the tomato root tips transcriptome and the TomExpress platform tomato transcriptome database,we found six possible transcription factors,including HSFA3,JA2(SlNAC090),C6HC-type,C3HC4 RING-type,Homebox-WOX,and NOR-likel(SlNAC064),regulating SlAAE3-1 by analyzing the co-expression correlation coefficients between SlAAE3-1 and all tomato transcriptional regulators.Analysis of the dual luciferase transient expression system showed that all six transcription factors could regulate SIAAE3-1.Interestingly,by analyzing the expression of SlAAE3-1 in the nac064 mutant,the regulation of SlAAE3-1 by SlNAC064 was found to be tissue-specific,i.e.,positive regulation of SlAAE3-1 in leaves and negative regulation of SlAAE3-1 in roots.In addition,we identified and analyzed 53 AAE family members in tomato and found that a total of nine member gene expressions were regulated by aluminum stress,among which three AAE genes(SlAAE3-1,SILACS3,and SlAAE1-6)expressions were most significantly up-regulated.It was also found that these three AAE genes responded to abiotic stresses such as different heavy metals,phytohormones and salinity.In summary,we identified that SlNAC063 and SlNAC064 are involved in Al tolerance in tomato and initially clarified that the Al tolerance gene SlAAE3-1 is regulated by SlNAC064,which enriches the Al-tolerance mechanisms in tomato and provides a theoretical basis for future genetic improvement and molecular breeding of tomato,and ultimately improves the yield of tomato grown in acidic soil.