Functional Analysis Of MLO7 And Its Homologous Genes In Plant Response To Salt Stress

Zygophyllum xanthoxylum,as a major planting species in the northwest desert region of China,has strong drought and salt tolerance,and contains unique resistance mechanisms and rich resistance genetic resources.Previously,we found that several Arabidopsis MLO7 homologs,ZxMLOs,were strongly induced by 50 m M Na Cl in the root of Z.xanthoxylum through the transcriptome analysis,thus we hypothesized that MLO7 and its homologs might play important functions in plant response to salt stress.However,up to now,studies on plant MLO genes have mainly focused on their regulation of plant powdery mildew resistance and the molecular mechanism,while little has been reported on the function of MLO in abiotic stress processes.Accordingly,to explore their functional origins of MLO,we screened and identified the MLO genes in the Z.xanthoxylum genome and performed bioinformatics analysis of this family.In addition,At MLO7 and At MLO2 T-DNA insertion deletion mutant plants were used to analyze their function in plant salt tolerance.The main results are as follows:1.A total of 20 ZxMLOs gene family members were identified based on the Z.xanthoxylum genome.Protein sequence analysis revealed that all ZxMLOs have conserved motifs with seven transmembrane domains,which is consistent with the structural features of MLO proteins,indicating that ZxMLO proteins are highly conserved in amino acids.Gene structure analysis revealed that the ZxMLO gene family mainly consisted of 13-16 exons and 12-15 introns,and each subclade had a similar number of exons and introns.Chromosome localization analysis revealed that20 ZxMLOs were randomly distributed on the nine chromosomes of Z.xanthoxylum.The MLO genes were identified separately in representative species during plant evolution,and it was found that MLO originated from aquatic unicellular algae;and the number of MLO genes in the salt-water aquatic plant Zostera marina was three times higher than that in the freshwater aquatic plant Chara brucei,so it was hypothesized that the functional origin of MLO genes was closely related to plant adaptation to salt environment.2.Analysis of promoter cis-acting elements indicated that the ZxMLO gene family contains multiple cis-acting elements in response to stress and hormones.Validation of ZxMLOs by RT-q PCR at the transcriptional level revealed that several ZxMLOs in Z.xanthoxylum root were up-and down-regulated to different degrees in response to salt treatment,consistent with the transcriptome data of Z.xanthoxylum root in response to salt treatment.Among them,Zygxa0015281.1,Zygxa0022307.1and Zygxa0008672.1 were the most significantly up-regulated,and the sequence alignment results showed that they were all homologs of Arabidopsis MLO7,so they were named ZxMLO7.1,ZxMLO7.2 and ZxMLO7.3;it is also hypothesized that ZxMLO7 plays an important function in the adaptation of Z.xanthoxylum to salt stress.3.Expression pattern analysis of At MLO7 and its homolog At MLO2 revealed that both At MLO7 and At MLO2 were induced by 100 m M Na Cl,indicating that At MLO7 and At MLO2 may play an important role in A.thaliana in response to salt stress.The subcellular localization analysis revealed that At MLO7 and At MLO2 were both localized in the cytoplasmic membrane,which was consistent with the localization characteristics of MLO proteins.4.The 3-week-old seedlings of wild-type Col-0,Atmlo7,and Atmlo2 were further treated with 50 m M Na Cl and the phenotypes were observed,and it was found that the wild-type Col-0 showed yellowing and wilting phenotypes earlier,while the Atmlo7 and Atmlo2 strains grew well compared with the wild-type.The higher chlorophyll content,lower cellular osmotic potential,good cell membrane stability and higher root vigor of Atmlo7 and Atmlo2 strains under salt stress conditions compared with wild-type Col-0;The Na⁺concentration in Col-0,Atmlo7 and Atmlo2plants increased significantly under 50 m M Na Cl treatment,while the K⁺concentration decreased significantly;and the Na⁺concentration in Atmlo7 and Atmlo2 plants was significantly lower than that in the wild type,while the K⁺concentration was higher than that in the wild type,indicating that At MLO7 and At MLO2 are involved in regulating the uptake of Na⁺by plants and the balance of K⁺in their bodies.In conclusion,the present study initially revealed the important functions of MLO7 and MLO2 in the response of plants to salt stress.The above findings will help to further reveal the molecular mechanisms of Z.xanthoxylum adapting the desert habitats and plant adaptation to salt stress,and provide a theoretical basis for molecular design breeding of agricultural crops and forage grasses.