Global analysis of basic leucine zipper transcription factors in trifoliate orange and the function identification of PtbZIP49 in salt tolerance

As one of the most widely distributed and highly conserved transcription factors in eukaryotes, basic leucine zipper proteins (bZIPs) are involved in a variety of biological processes in plants, but they are largely unknown in citrus. In this study, 56 bZIP family members were identified genome-wide from an important citrus rootstock, namely trifoliate orange (Poncirus trifoliata L. Raf.), and these putative bZIPs were named PtbZIP1-PtbZIP56. All PtbZIPs were classified into 13 subgroups by phylogenetic comparison with Arabidopsis thaliana bZIPs (AtbZIPs), and they were randomly distributed on nine known (50 genes) chromosomes and one unknown (6 genes) chromosome. Sequence analysis revealed the detailed characteristics of PtPZIPs, including their amino acid length, isoelectric point (pI), molecular weight (MW), predicted subcellular localization, gene structure, and conserved motifs. Prediction of promoter elements suggested the presence of drought, low-temperature, wound, and defense and stress responsive elements, as well as multiple hormone-responsive cis-acting elements. Spatiotemporal expres-sion analysis showed the transcriptional patterns of PtbZIPs in different tissues and under dehydration, high salt, ABA, and IAA treatments. In addition, 21 PtbZIPs were predicted to have direct or indirect protein-protein interactions. Among these, PtbZIP49 was experimentally proven to interact with PtbZIP1 or PtbZIP11 by using a yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC). Subcellular localization analysis further revealed that PtbZIP1, PtbZIP11, and PtbZIP49 were localized in the nucleus. Moreover, PtbZIP49 was functionally identified as having an important role in salt stress via ectopic expression in A. thaliana and silenced in trifoliate orange using virus-induced gene silencing (VIGS). This study provided comprehensive information on PtbZIP transcription factors in citrus and highlights their potential functions in abiotic stress.