Genome-wide identification and expression analysis of WOX gene family in Trifoliate orange

Abstract: 【Objective】Abiotic stresses such as drought, low temperature and high salt are important environmental factors that affect the growth, development, yield and quality of citrus crops. Trifoliate orange(Poncirus trifoliata) has strong stress resistance and is a common rootstock for improving the stress resistance of citrus. WOX gene family is a gene family unique to plants that encodes transcription factors, which play an important role in regulating plant growth and development and response to abiotic stress. However, the potential role of WOX gene family in trifoliate orange is still unknown. The objective of this study is to investigate the phylogeny, protein structure and expression patterns of WOX gene family in trifoliate orange under abiotic stress.【Methods】Based on genome database of trifoliate orange, the trifoliate orange WOX family genes(PtrWOXs) were screened out to analyze their physical and chemical properties, phylogenetic relationship, chromosomal localization, gene structure, cis-acting elements, synteny and codon preference using bioinformatics tools. The expression levels of PtrWOXs in different tissues of trifoliate orange were analyzed by qRT-PCR. Meanwhile, the expression patterns of PtrWOXs in the roots, stems and leaves of trifoliate orange under cold, drought, salt and ABA stresses were also analyzed by qRT-PCR.【Results】A total of 10 WOX family genes were screened out from genomic database of trifoliate orange. These genes were unequally distributed on 7 chromosomes, of which PtrWOX1 was located on chromosome number 1, PtrWOX6 and PtrWOX9 were located on chromosome number 2, PtrWOX11 was located on chromosome number 3, PtrWOX5 was located on chromosome number 4, PtrWOX4 and PtrWOX10 were located on chromosome number 5, PtrWUS and PtrWOX13 were located on chromosome number 7, and PtrWOX3 was located on chromosome number8. All of the PtrWOX proteins contained the conserved homeotic domain of‘helix-loop-helix-turn-helix'. The results of the physical and chemical properties analysis showed that the PtrWOX proteins were all hydrophilic proteins. Subcellular localization prediction analysis showed that PtrWOX proteins were mainly localized in the nucleus. Phylogenetic analysis showed that the 10 PtrWOX proteins could be divided into modern clade(PtrWUS, PtrWOX1, PtrWOX3 to PtrWOX6), intermediate clade(PtrWOX9 and PtrWOX11) and ancient clade(PtrWOX10 and PtrWOX13). Gene structure analysis revealed that the number of introns of the WOX family genes was 1-5, and the number of exons was 2-6.A large number of cis-acting elements associated with growth and development, phytohormone and stress responses were found in the promoters of PtrWOXs. These cis-acting elements included the meristem expression element(CAT-box) related to the regulation of plant growth and development, circadian element involved in the regulation of circadian rhythms, abscisic acid response elements(ABRE) involved in ABA and drought response, drought stress response element(MBS), drought and salt stress response element(DRE1), low temperature stress response element(TCA, LTR and MYC), stress response element(STRE), damage response element(WRE3 and WUN-motif)), as well as defense and stress response elements(TC-rich repeats). In addition to PtrWOX5 and PtrWOX11, the promoters of other PtrWOXs contained ABRE. All PtrWOXs contained at least one MYC. These results suggested that PtrWOXs might be involved in regulating the growth and development of trifoliate orange, and also might play an important role in regulating trifoliate orange response to hormone and abiotic stress. A number of miRNAs that might be involved in response to abiotic stress and in regulating the expression of PtrWOXs were identified from trifoliate orange. In these miRNAs, miR394 was involved in response to drought stress. miR403 was involved in regulating plant response to drought and cold stresses.miR408 played an important role in regulating drought, cold and salt stresses. Synteny analysis showed that only two fragment repeat events, including PtrWOX1, PtrWOX6 and PtrWOX10, PtrWOX13, occurred among the 10 PtrWOXs. Synteny analysis of WOX family genes in multiple plants by MCScanX showed that PtrWOXs had a closer relationship with dicotyledonous plants, which was consistent with the results of phylogenetic analysis. Weak codon preference was found for the PtrWOXs. The results of tissue expression analysis indicated that PtrWUS, PtrWOX1, PtrWOX9 and PtrWOX11 were expressed at the highest level in the roots, PtrWOX4, PtrWOX10 and PtrWOX13 were expressed at the highest level in stems, and PtrWOX6 was expressed at the highest level in leaves. Further analysis showed that the expression of most PtrWOXs in leaves, stems and roots was induced by several abiotic stresses. The expression changes of most PtrWOXs in roots were more dramatic than those in leaves and stems. Furthermore, the expression levels of PtrWOX1, PtrWOX3, PtrWOX6, PtrWOX9 and PtrWOX11 were induced by cold, drought, salt and ABA in roots, suggesting these genes might play important roles in trifoliate orange root in responses to abiotic stresses.【Conclusion】A total of ten WOX family genes in trifoliate orange were identified in this paper, which might play important roles in various abiotic stress tolerance in trifoliate orange.