Functional Analysis Of TsANC1 In T.Halophila And The Homologous Genes In Zea Mays

Thellungiella halophila(also Thellungiella salsuginea)is a halophyte,and a highly close relative of Arabidopsis,with a relatively high similarity cDNA sequence.NAC proteins constitute one of the largest families of plant-specific transcription factors with 107 members in Arabidopsis at least.NAC transcription factors have important roles in plant development,cell apoptosis,abiotic stress tolerance,secondary cell walls(SCW)formation and the endoplasmic reticulum stress resistance.In our lab Gao et al.identified a VACUOLAR H-PYROPHOSPHATASE(H-PPase)gene TsVPl from T.halophila and transferred the gene into in different crops such as maize,cotton for overexpression.It was interesting that the overexpression of TsVPl could improve the salt and drought tolerance of the transgenic plants,and also increase crop yield under normal cultivation conditions.TsVPl is an orthologue of AVP1 in Arabidopsis thaliana,but its expression is induced by salt stress.Sun et al.identified one 130 bp(-667 to-538)region as the key sequence for salt stress response from the promoter of TsVP1.With the 130 bp key region as bait,one NAC transcription factor TsNAC1 was cloned from T.halophila.In order to understand the function of TsNAC1,Sun et al.constructed the overexpression structure and RNAi structure of TsNAC1,and introduced them into T.halophila to produce transgenic plants.Overexpression of TsNACl could significantly increase the tolerance of plants to abiotic stresses such as high salt,drought,low temperature and reactive oxygen stress,while the growth of plant is retarded and the biomass of overexpressed plants is significantly decreased.On the basis of Sun's work,the functional analysis of transcription factor TsNAC1 in T.halophila and its homologous gene in maize were performed.TsNAC1 plays a key role in the growth and stress resistance in T.halophilaWhen compared with WT,the transgenic homozygous lines with the overexpressed TsNAC1 significantly showed slowed plant growth,and increased plant stress resistance.The analysis of TsNACl sequence and TsNACl protein information showed that TsNAC1 is a homologous gene RD26 in Arabidopsis.The transcriptional activation activities of TsNAC1 and RD26 were compared with the target genes'promoter regions of TsNAC1 and the results showed that the transcriptional activation activity of TsNAC1 was higher in tobacco and yeast.Referenced to the results of ChIP-Seq,TsNAC1 targets to the positive regulators on ion response and transportation,such as TsVPI,MYB HYPOCOTYL ELONGATION-RELATED(MYBH)and HOMEOBOX 12(HB12)transcription factors.In addition,TsNAC1 negatively regulates the expansion of unidimensional cells,including the repression of LIGHT-DEPENDENT SHORT HYPOCOTYLS1(LSH1)and UDP-XYLOSYLTRANSFERASE 2(TXT2)as well as directly control the expression of MULTICOPY SUPPRESSOR OF IRA1 4(MSI4),Furthermore several regulators involved in the response of abiotic stresses are also positively regulated by TsNAC1,such as the expression of DIACYLGLYCEROL KINASE 2(DGK2),ASPARTATE AMINOTRANSFERASE 5(AAT3)and CALMODMLIN-BINDING TRANSCRIPTION ACTIVATOR 1(CAMTA1)are induced by low temperature to enhance plant freezing tolerance,and reactive oxygen species stress response genes such as ACONITASE 3(AC03),POLLEN-PISTIL INCOMPATIBILITY2(POP2),NAD-DEPENDENT MALIC ENZYME 2(NAD-ME2)are induced by the oxidative stress to protect plants.Moreover,TsNAC1 recognize CAT(A)G motifs was ascertained via ChIP-Seq.It is concluded that the overexpression of TsNAC1 could improve abiotic stress resistance,especially in the salt stress tolerance,and retard the growth of the plants.In addition,the induction of DGK2,AAT3,CAMTA1,ACO3,POP2 and NAD-ME2 by TsNAC1 may play important roles in cold stress tolerance or oxidative stress.Based on these results,we propose that TsNAC1 functions as an important upstream regulator of plant abiotic stress responses and vegetative growth.TsHD1 collaborates with TsNACl on plant growth and abiotic stress resistanceA HOMEOBOX PROTEIN(TsHD1)was identified from T.halophila by Co-IP,which is a homeodomain(HD)transcription factor and functions as the collaborator of TsNAC1.TsHD1 could form a homodimer through its zinc finger domain and its core recognition site is T(A/T)AATT.TsHD1 could form heterodimers with TsNAC1 via the interaction between its Zinc finger(ZF)domain and the A subdomain of TsNAC1.The co-overexpression of TsHD1 and TsNAC1 improved the heat stress resistance of T.halophila and retarded its vegetative growth significantly.The co-overexpression of TsHDl and TsNACl highly improved the heat and drought stress tolerance by increasing the accumulation of heat shock proteins(HSPs)and enhancing the expression level of drought stress genes,such as MYB DOMAIN PROTEIN 77&96(MYB77&96)and SALT TOLERANCE ZINC FINGER 10&18(ZAT10&18),but seriously retarded the vegetative growth of T.halophila by restraining cell expansion.The heterodimer of TsHD1 and TsNAC1 shows higher transcriptional activation activity and higher stability compared to the homodimer of TsHD1 or TsNAC1.The binding sites of the TsHD1 and TsNAC1 heterodimers were found to exist in the promoters of most upregulated expression genes in the co-overexpression lines of TsHD1 and TsNACl compared to the WT using RNA-Seq and genomic data analyses.In addition,the binding sites in the promoter region of the most down-regulated expressed genes are located in the vicinity of the TATA-box.This study reveals that TsNAC1 and TsHD1 have synergistic effects on the regulation of plant growth and abiotic stress resistance.Orthologues of TsNACl in maize and phenotypes of their mutantsThe NAC family tree of maize was established,and a sub-clan consist of six candidate genes was found to be orthologues of TsNAC1.The expression of GRMZM2G068973/ZmNAC23 and GRMZM2G347043/ZmSNACl could be highly induced by high salt,drought stress and ABA treatments,and the expression of GRMZM2G180328/ZmNAC20 is also induced by high salt and ABA treatments.While other three genes are not induced by abiotic stress obvirously.It is supposed that these three genes were functionally similar to TsNAC1.The proteins coded by them are located in the nucleus,and shows strong transcriptional activation activity.Under normal conditions the expression abundances of ZmNAC20,ZmNAC23 and ZmSNACl were the highest in the root tip(VE)and the undeveloped leaves(V1),and were higher in the kernels on 10th day after pollination.To understand the functions of ZmNAC20,ZmNAC23 and ZmSNAC1,transgenic lines with the overexpression of ZmNAC20,ZmNAC23,ZmSNAC1 or ZmNAC20-GFP,ZmNAC23-GFP,ZmSNAC1-GFP were respectively produced from maize line Qi319.At the same time the homozygous mutants of ZmNAC20,ZmNAC23 and ZmSNAC1 were isolated from the Mu transposon insertion mutants of maize W22.The differences of morphological characteristics and stress resistance between these two types of lines contrast to their WT control were analyzed.The results showed that the vegetative growth rate of overexpressed ZmNAC20,ZmNAC23 or ZmSNACl plants was reduced,and the tolerance to high salt environment and osmotic stress was significantly increased.The corresponding mutants were more sensitive to high salt stress at seedling stage,and had higher biomass than that of W22 under normal conditions.By two-hybrid yeast screening,three transcription factors which could interact with ZmNAC20,ZmNAC23,ZmSNAC1 were found,they were Zmbhlh168,ZmSBP9,Zmfarl13.The expression of these three genes showed higher levels in the kernels on the 10th days after pollination,leaves(V1)and roots(VE),and also were induced by high salt stress or ABA treatment,which meant the expression pattern was very similar to that of ZmSNACl,ZmNAC23 and ZmNAC20.According to the functions of orthologous genes in other species,these three genes involved in the plant heat stress response,light and oxidative stress response,and negatively regulated the cell expansion process,which retarded plant growth.Downstream genes regulated by ZmNACsThe binding sites of ZmSNAC1,ZmNAC23 and ZmNAC20 were identified as CA(T/A)G(T/G)G,CA(T/A)G(T/C)G and(C/A/T)(T/A)G(T/G)G by means of ChIP-Seq and EMSA assay,respectively.The enriched candidate target genes are mainly found in the regulatory biological process categories.Further GO classification showed that the candidate target genes were mainly enriched in response to abiotic stimulus,cell growth,macromolecule metabolic process and homeostatic items.Among them,genes with enrichment fold>10,with binding motif of the TFs in their promoter regions,and the expression was induced by ABA or salt treatment were seclected for 16,14 and 17 members,respectively.From them,five,five,six of genes with expression levels regulated by ZmSNAC1,ZmNAC23,ZmNAC20,respectively,were verified by one-hybrid yeast assay,and the target genes of ZmNACs were identified.ZmSNAC1 positively regulated the expression level of auxin responsive genes AAR3,PHR1,OX3,ZmbZIP73/GRMZM2G175870(latter two involved in stress resistance),and negatively regulated the expression of AXR1(also an auxin responsive gene).The overexpressed lines or mutants of those orthologous genes in Arabidopsis showed significat differences in growth and resistance compairing with WTs,which suggested ZmSNAC1 is involved in the regulation of maize growth and development and response to abiotic stress with a mechanism to be revealed.ZmNAC23 negatively regulated the expression of PRR1,the key enzyme of lignin synthesis,and restricted the development of cell wall to slow down the growth of plants.The down-regulated expression of NOX1 in ZmNAC23 overexpressed line inhibited auxin efflux carrier PIN 1 mediated auxin transportation and remarkably decreased the root growth.The expression of auxin response gene SAGT1 encoded a salicylate glucosyltransferase,was also down-regulated in ZmNAC23 overexpressed line.And the expression of RABGAP was positively regulated by ZmNAC23,and the gene encoded gyplp protein that specifically expressed in guard cells,when the mutant in Arabidopsis was treated with ABA,the plants showed the much larger opening of stoma and increased biomass compared to WT lines.It was concluded that ZmNAC23 played an important role in the regulation of maize plant growth.In ZmNAC20 overexpressed lines,the expression of multiple growth regulators was significantly down-regulated,such as PGX3.As a member of pectate lyases superfamily that played an important role in the formation of secondary cell wall,an ortholog mutant pgx3-1 in Arabidopsis showed about 60%leaf area than that of WT,with improved adaptability to drought and high temperature.It was implied that ZmNAC20 has distinctive function in the regulation of abiotic stress resistance and plant growth in maize.By comparing the regulatory networks of TsNAC1 in T.halophila and its orthologues genes in maize,it could be concluded that TsNACl and its orthologues genes play an important role in the regulation of plant growth and abiotic stress resistance,and the regulatory mechanism is relatively conserved between dicotyledonous and monocotyledonous plants.