Bioinformatics Analysis Of ZxArf Gene Family And Function Identification Of ZxArf7c And ZxArf7d In Zygophyllum Xanthoxylum

As a super-xerophyte,Zygophyllum xanthoxylum is a major species in the northwest desert of China.During the long-term evolution,Z.xanthoxylum has formed highly developed root system network dominated by lateral roots,which can help fully absorbing water and nutrients from the arid and barren desert to survival.However,the molecular mechanism of its lateral root formation remains elusive.Auxin plays a key role in the growth and development of plant roots,and ARF is the core transcription factor of nuclear auxin signaling pathway.Particularly,A-At ARFs play vital roles in every stage of lateral roots development in Arabidopsis.Therefore,we suspecte that there is close relationship between A-ZxARFs and the developed root system of Z.xanthoxylum.In this study,ZxARF gene family were isolated and identified from the Z.xanthoxylum genome.Meanwhile,we preliminarily predicted the relationship between A-ZxARFs and the formation of developed root system,and the drought adaptation of Z.xanthoxylum by bioinformatics analysis.In order to verify the prediction,ZxARF7 c and ZxARF7 d were cloned,and their functions in plant lateral root development and drought tolerance of plants were analyzed.The main results are as follows:1.1,14,30,31 and 25 ARF protein coding genes from Chara braurii,Amborella trichopoda,Z.xanthoxylum,Pugionium dolabratum and Camellia sinensis were isolated and identified,respectively.2.By constructing the phylogenetic tree of ARF proteins from the representative plants at each evolutionary stages,it was found that ARFs are splited into A,B,and C3 clades.The ancestor of plant ARFs may be a C-ARF.By constructing phylogenetic tree of ARFs from xerophyte(Z.xanthoxylum and P.dolabratum)and mesophyte(Arabidopsis,Citrus sinensis and Ca.sinensis)plants at the same evolutionary stage,we found that the massive expansion of A-ZxARFs may be the main mechanism for the formation of the developed root system of Z.xanthoxylum.The chromosome localization analysis revealed that ZxARFs are randomly distributed on the 11 chromosomes of Z.xanthoxylum.The synteny and Ks values analysis indicated that ZxARFs were mainly generated by whole-genome duplication and chromosome fragment replication events.3.Analysis of promoter sequences indicated that the number of abiotic stress-related cis-acting elements in p A-ARFs of Z.xanthoxylum and P.dolabratum were significantly more than that of Arabidopsis,Citrus sinensis and Ca.Sinensis.RT-q PCR results showed that all A-ZxARFs genes were expressed in high abundance in the roots of Z.xanthoxylum,and they were significantly induced by osmotic stress.The results suggest that A-ZxARFs may play important roles in root development and drought tolerance of Z.xanthoxylum.4.Prediction of protein domain indicated that all ZxARFs proteins contain a conserved ARF domain,consistented with the characteristic of ARF transcription factors.Among them,ZxARF5 b and ZxARF8b(A-ZxARFs)have lost the important B3 DNA-binding domain partly and completely,whereas they retain the PB1(phox and beml)domain.We infer that ZxARF5 b and ZxARF8 b may compete with other A-ZxARFs to bind the transcriptional repressor Zx Aux/IAAs and help release full-length A-ZxARFs to activate transcription of downstream genes.Therefore,they probably play positive role in the lateral root development.It could be an important mechanism for the formation of the large root system of Z.xanthoxylum.5.ZxARF7 c and ZxARF7 d genes were cloned from the Z.xanthoxylum genome.The length of ZxARF7 c coding sequence is 3072 bp,encoding 1023 amino acids.The length of ZxARF7 d coding sequence is 3246 bp,encoding 1081 amino acids.Then,ZxARF7 c and ZxARF7 d were overexpressed in Arabidopsis wild-type Col-0 and arf7/19 mutant,respectively.The transgenic lines were generated via BASTA-resistance screening and RT-PCR analysis.6.Under normal conditions,the overexpression of ZxARF7 c and ZxARF7 d promoted the lateral roots growth and development of Col-0 and significantly rescued the defect phenotype of lateral root development of arf7/19,respectively.The results demonstrated that both ZxARF7 c and ZxARF7 d played positive roles in lateral root growth and development.7.Under osmotic stress(simulated drought treatment)or drought stress,the lateral roots growth and development,the wilting degree of rosette leaves,photosynthetic net rate and root activity of ZxARF7 c and ZxARF7 d overexpression plants were better than the corresponding background plants.The results suggest that overexpression of ZxARF7 c and ZxARF7 d may improved the water and nutrient uptaking of plants by promoting the growth of lateral roots,leading to the bettter growth and development of plants either in normal condition or in drought environment.In conclusion,this study preliminarily revealed that the expansion of A-ZxARFs,their high expression levels in the roots and being induced by osmotic stress in transcriptional levels,and changes of the A-ZxARFs domains may be important reasons for the formation of highly developed roots of Z.xanthoxylum.Meanwhile,we showed that ZxARF7 c and ZxARF7 d play positive roles in plant lateral root development and resistance to drought stress.These results will help to reveal the molecular mechanisms of root development and drought adaptation of Z.xanthoxylum,and will provide a theoretical basis for genetic improvement of drought tolerance in crops and forages.