Genome-wide identification of BpbZIPs
Fifty-one genes with conserved bZIP domains were identified in the B. platyphylla genome and named BpbZIP1~BpbZIP51 sequentially according to their positions on chromosomes. All BpbZIP genes were unevenly distributed on 14 chromosomes, with chromosome 1 containing the most genes (7 BpbZIP genes) and chromosomes 7 containing a single BpbZIP gene, and most of BpbZIPs were distributed at both ends of the chromosomes (Fig.1).
The physicochemical properties of 51 BpbZIP proteins showed that the number of amino acids ranged from138 (BpbZIP25) to 584 (BpbZIP17), molecular weight ranged from 15.71 (BpbZIP25) to 84.26 (BpbZIP26) kD, and isoelectric points ranged from 4.50 (BpbZIP22) to 10.16 (BpbZIP7) (Table 1). The subcellular location predicted that most of the proteins were nuclear proteins. Only BpbZIP2, BpbZIP7, BpbZIP22, BpbZIP29, BpbZIP40, and BpbZIP48 were distributed in the chloroplast.
Phylogenetic analysis of BpbZIPs
In accordance with the classification of A. thaliana bZIP proteins, 51 BpbZIP proteins were divided into 13 subgroups except for BpbZIP33 and BpbZIP38, which were named as Subgroups A, B, C, D, E, F, G, H, I, J, K, M, and S (Fig. 2). The largest subgroup S contained 11 BpbZIP members, followed by the Groups A and I containing 8 and 6 BpbZIP members, respectively. Subgroup J, K, and M all had only one protein.
Evolutionary analysis of BpbZIPs
As shown in Fig.3A, 12 duplication events with 51 BpbZIP genes were detected in the B. platyphylla genome. One pair of genes (BpbZIP40 and BpbZIP41) was found to have undergone a tandem duplication event and five pairs of genes (BpbZIP4 and BpbZIP36, BpbZIP4 and BpbZIP20, BpbZIP6 and BpbZIP20, BpbZIP8 and BpbZIP22, BpbZIP11 and BpbZIP27) underwent a fragment duplication event. This evidence suggested that fragment duplication events were a major driver of BpbZIP gene diversity. The syntenic relationships of the BpbZIP genes showed that 28 orthologs existed between B. platyphylla and A. thaliana (Fig.3B), 83 orthologs existed between B. platyphylla and G. max (Fig.3C), and 73 orthologs existed between B. platyphylla and P. trichocarpa (Fig.3D). The differences in numbers of orthologous pairs were related to the evolutionary distance [22].
Conserved motifs and gene structures
A total of 20 conserved amino acid motifs were identified in the BpbZIP proteins (Fig.4B), and motifs with similar structures and domains were clustered into one group indicating that they had an analogous function (Fig.4A), such as group I (BpbZIP13, BpbZIP28, BpbZIP40, BpbZIP41, BpbZIP5, and BpbZIP9). Motif1 was distributed in all members of the BpbZIPs, which was recognized as bZIP conserved domain sequence. However, some motifs were very rare, such as motif 19 only found in group I, motif 18 in group F.
Exon–intron analysis was performed on the 51 BpbZIP genes (Fig.4C). The results showed that 78.43% (40/51) of the BpbZIPs had introns varying from 1 (BpbZIP29) to 13 (BpbZIP2, 46), and the eleven intron-less genes were BpbZIP3, 4, 11, 14, 17, 20, 24, 27, 30, 36, and 47. In addition, 70.59% (36/51) of the BpbZIPs had untranslated regions (UTRs) varying from 1 (BpbZIP1, 5, 6, 15, 25, 31, 35, 40, 43, 48) to 3 (BpbZIP22, 46), and the 15 genes (BpbZIP3, 4, 11, 14, 17, 20, 24, 27, 29, 30, 33, 36, 37, 38 , 47) had no UTR.
The cis‑elements in the promoter regions of BpbZIPs
The 2.0 kb promoter region located upstream of the transcriptional start site of each BpbZIP gene was used to predict their possible expression regulation patterns (Fig.5). The cis-elements of the BpbZIPs belonging to the same group in phylogenetic analysis did not show the same pattern. The number of responsive elements ranged from 25 (BpbZIP26) to 84 (BpbZIP1), and light, hormone, and stress were three main categories of responsive elements. Light responsive elements were the most prevalent in all the BpbZIPs promoters varying from 4(BpbZIP36, 47) to 34(BpbZIP1), and Gbox and box4 were the top two responsive elements. ABA and JA were the dominant hormone elements. Wound-responsive element was rich in the stress responsive elements. The different types and amounts of cis-elements in the promoters of BpbZIPs suggested that they might had different functions in B. platyphylla growth and development.
Gene expression of BpbZIPs under S-nitrosation treatment
qRT-PCR was used to investigate gene expression patterns of BpbZIPs in response N6022 treatment. The results revealed that all BpbZIP genes expressed in control tissues of B. platyphylla, and 62.7% BpbZIPs (32 genes) in leaves were higher than that of stems and roots (Fig.6). Among them, BpbZIP21, BpbZIP17 and BpbZIP26 highly expressed in leaves, BpbZIP16, BpbZIP26 and BpbZIP21 in stems, BpbZIP16, BpbZIP24 and BpbZIP15 in roots. N6022 treatment changed gene expression levels of BpbZIPs in B. platyphylla seedlings, BpbZIP16, BpbZIP2 and BpbZIP26 highly expressed in leaves, and their increases were 3.96, 2.36, and 2.06 times greater than those of controls, respectively. BpbZIP 25 and BpbZIP26 highly expressed in stems under N6022 treatment, and their increases were 19.53 and 1.70 folds higher than those of controls, respectively. BpbZIP35, BpbZIP33, and BpbZIP26 highly expressed in roots under N6022 treatment, their increases were 27.4, 15.8, and 12.50 folds greater than those of controls, respectively. The above results suggested that BpbZIP26 may play a key role in B. platyphylla responses to S-nitrosation treatment. Hence, we cloned BpbZIP26 via PCR (SFig. 1, 2).
Overexpression of BpbZIP26 enhanced triterpenoid production under S-nitrosation treatment
Our previous study showed that N6022 treatment significantly enhanced triterpenoid content [12, 13], and it also significantly increased gene expression of BpbZIP26. To investigate the function of BpbZIP26 in triterpenoid synthesis, overexpression and silencing vector of BpbZIP26 were constructed to transfer into B. platyphylla calli. After three days of Agrobacterium-mediated transient transformation, the silencing of BpbZIP26 in B. platyphylla calli (0.44 times than that of untransformed calli) significantly decreased the triterpenoid contents (40.46%) and reduced the gene expression of BpCAS, BpLUS, and BpβAS, which are key enzyme genes for triterpenoid synthesis. The overexpression of BpbZIP26 in B. platyphylla calli (812 times than that of untransformed calli) enhanced the triterpenoid contents (26.21%) and increased the gene expression of BpCAS, BpLUS, and BpβAS. N6022 treatment further enhanced triterpenoid contents (90.38% and 65.44%) and gene expression of BpCAS, BpLUS, and BpβAS in control and overexpression of BpbZIP26 calli of B. platyphylla (Fig.7). The above results suggested that BpbZIP26 mediated triterpenoid production under control and S-nitrosation treatment.