PfbZIP85 Transcription Factor Mediates ω-3 Fatty Acid-Enriched Oil Biosynthesis by Down-Regulating PfLPAT1B Gene Expression in Plant Tissues

Abstract

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the biggest TF families identified so far in the plant kingdom, functioning in diverse biological processes including plant growth and development, signal transduction, and stress responses. For Perilla frutescens, a novel oilseed crop abundant in polyunsaturated fatty acids (PUFAs) (especially α-linolenic acid, ALA), the identification and biological functions of bZIP members remain limited. In this study, 101 PfbZIPs were identified in the perilla genome and classified into eleven distinct groups (Groups A, B, C, D, E, F, G, H, I, S, and UC) based on their phylogenetic relationships and gene structures. These PfbZIP genes were distributed unevenly across 18 chromosomes, with 83 pairs of them being segmental duplication genes. Moreover, 78 and 148 pairs of orthologous bZIP genes were detected between perilla and Arabidopsis or sesame, respectively. PfbZIP members belonging to the same subgroup exhibited highly conserved gene structures and functional domains, although significant differences were detected between groups. RNA-seq and RT-qPCR analysis revealed differential expressions of 101 PfbZIP genes during perilla seed development, with several PfbZIPs exhibiting significant correlations with the key oil-related genes. Y1H and GUS activity assays evidenced that PfbZIP85 downregulated the expression of the PfLPAT1B gene by physical interaction with the promoter. PfLPAT1B encodes a lysophosphatidate acyltransferase (LPAT), one of the key enzymes for triacylglycerol (TAG) assembly. Heterogeneous expression of PfbZIP85 significantly reduced the levels of TAG and UFAs (mainly C18:1 and C18:2) but enhanced C18:3 accumulation in both seeds and non-seed tissues in the transgenic tobacco lines. Furthermore, these transgenic tobacco plants showed no significantly adverse phenotype for other agronomic traits such as plant growth, thousand seed weight, and seed germination rate. Collectively, these findings offer valuable perspectives for understanding the functions of PfbZIPs in perilla, particularly in lipid metabolism, showing PfbZIP85 as a suitable target in plant genetic improvement for high-value vegetable oil production.

Keywords: bZIP transcription factors; expression analysis; genome-wide identification; lysophosphatidate acyltransferase (LPAT); oil biosynthesis and regulation; perilla (Perilla frutescens).

Figure 1

Phylogenetic tree of bZIP members from P. frutescens and Arabidopsis. The PfbZIP proteins (blue solid circle) and AtbZIP proteins (red solid circle) were classified into distinct groups in the phylogenetic tree. Different colors on the periphery indicate the distinct subgroups of bZIP proteins.

Figure 2

Multiple sequence alignments of the PfbZIP protein domains from P. frutescens. The basic region (BR) and leucine zipper (LZ) are shown by solid and dotted lines, respectively.

Figure 3

Synteny analysis of bZIP members between P. frutescens and the other two plant species. (A) Synteny examination of inter-chromosomal associations of PfbZIP genes. All PfbZIP gene pairs in the P. frutescens genome were indicated by red lines. (B) Synteny of bZIP members between P. frutescens, A. thaliana, and S. indicum. The synteny bZIP gene pairs between P. frutescens, A. thaliana, or S. indicum were emphasized using red lines. The collinear blocks of other genes between P. frutescens and A. thaliana, or S. indicum, were shown with gray lines.

Figure 4

GO and KEGG enrichment analysis and expression profiles of PfbZIP genes during perilla seed development. (A) The distinct seed developmental stages of the perilla variety ‘Jinzisu 1’. (B) GO enrichment analysis of PfbZIP gene family members. (C) KEGG enrichment analysis of PfbZIP gene family members. (D) Heat map of the PfbZIP gene expression patterns in developing seeds of P. frutescens. (E) The relative expression levels of the selected PfbZIP genes were detected by RT-qPCR in seed development. Values are presented as mean ± SD (n = 3).

Figure 5

Prediction and interaction analysis of candidate PfbZIP TF-regulated target genes involved in lipid biosynthesis. (A) Total lipid dynamic accumulation in the developing seed of perilla. The total lipid contents are expressed by the mass of esterified fatty acids. (B) Heat map of expression correlation between eight PfbZIP TFs and key lipid-related enzyme genes. Red indicates a positive correlation, while blue indicates a negative correlation. * represents a significant difference at p < 0.05. ** represents a significant difference at p < 0.01. *** represents a significant difference at p < 0.001. (C) Yeast one-hybrid assay of PfbZIP52 and PfbZIP85 with promoters of the PfGPAT1 and PfLPAT1B genes, respectively.

Figure 6

(A) Subcellular localization of PfbZIP85 in tobacco leaf cells. The fluorescence signals were observed using a laser-scanning confocal microscope (Leica TCS SP8). Scale bars = 50 μm. (B) GUS staining in the infiltrated tobacco leaves. Left, tobacco leaves infiltrated with the proPfLPAT1B::GUS plasmid alone. Right, tobacco leaves co-infiltrated with 35S::PfbZIP85 and proPfLPAT1B::GUS plasmids. (C) Expression analysis of the GUS gene in the transiently infected tobacco leaves. Values are presented as mean ± SD (n = 3). ‘a’ and ‘b’ indicate significant differences at p < 0.05.

Figure 7

Analysis of TAG content and major FAs in TAG in the transgenic tobacco lines. (A) Transgenic tobacco plants. (B,C) Contents of total TAG and key TAG-associated FAs in PfbZIP85-expressing tobacco leaves. (D) Transgenic tobacco seeds. (E,F) Contents of total TAG and key TAG-associated FAs in PfbZIP85-expressing tobacco seeds. LDW, the dry weight of tobacco leaves. WT, untransformed tobacco plant. OE-1/-2/-3, the transgenic tobacco lines overexpressing the PfbZIP85 gene. The TAG contents are expressed by the mass of esterified fatty acids. Values are presented as mean ± SD (n = 3). Different lowercase letters indicate significant differences at p < 0.05.

Figure 8

Other agronomic traits in tobacco plants overexpressing the PfbZIP85 gene. (A) Starch content. (B) Soluble sugar content. (C) Protein content. (D) Leaf photosynthesis (Pn). (E) Leaf dry weight. (F) Seed germination rate. WT, untransformed tobacco plant. OE-1/-2/-3, the transgenic tobacco lines overexpressing the PfbZIP85 gene. Values are presented as mean ± SD (n = 3). Different lowercase letters indicate significant differences between different samples at p < 0.05.

Figure 9

Expression analysis of genes associated with FA/oil biosynthesis in the transgenic tobacco plant overexpressing PfbZIP85. The N. tabacum actin gene was used as the internal control gene. WT, untransformed tobacco plant. OE-1/-2/-3, the PfbZIP85-expressing tobacco lines. Values are presented as mean ± SD (n = 3). Different lowercase letters indicate significant differences between different samples at p < 0.05.

Figure 10

Mechanistic model of FA/TAG biosynthesis mediated by the PfbZIP85 TF. For simplicity, only one TAG molecule is displayed, and certain intermediates or reactions are omitted. The blue downward arrow indicates the reduction in oil content.