Heat shock factor binding protein BrHSBP1 regulates seed and pod development in Brassica rapa

Abstract

Plant heat shock factor binding proteins (HSBPs) are well known for their implication in the negative regulation of heat stress response (HSR) pathways. Herein, we report on the hitherto unknown functions of HSBP1 in Brassica rapa (BrHSBP1). BrHBSP1 was found to be predominant in flower buds and young leaves, while its segmental duplicate, BrHSBP1-like, was abundant in green siliques. Exposure to abiotic stress conditions, such as heat, drought, cold, and H₂O₂, and to phytohormones was found to differentially regulate BrHSBP1. The activity of BrHSBP1-GFP fusion proteins revealed their cellular localization in nuclei and cytosols. Transgenic overexpression of BrHSBP1 (BrHSBP1^OX) improved pod and seed sizes, while CRISPR-Cas BrHSBP1 knock-out mutants (Brhsbp1_KO) were associated with aborted seed and pod development. The transcriptomic signatures of BrHSBP1^OX and Brhsbp1_KO lines revealed that 360 and 2381 genes, respectively, were differentially expressed (Log2FC≥2, p_adj<0.05) expressed relative to control lines. In particular, developmental processes, including plant reproductive structure development (RSD)-related genes, were relatively downregulated in Brhsbp1_KO. Furthermore, yeast two-hybrid assays confirmed that BrHSBP1 can physically bind to RSD and other genes. Taking the findings together, it is clear that BrHSBP1 is involved in seed development via the modulation of RSD genes. Our findings represent the addition of a new regulatory player in seed and pod development in B. rapa.

Keywords: BrHBSP1; Brassica rapa; CRISPR-Cas; drought; floral genes; heat stress; seed.

Figure 1

Expression profiling of BrHSBP1 and BrHSBP1-like across different abiotic stresses, exogenous phytohormones, and tissues. (A) Changes in expression of BrHSBP1 and BrHSBP1-like under different temperature regimes (25°C, 37°C at different time points of exposure (0.5, 1, 3, and 6 hr), and 4°C). (B) Changes in expression of BrHSBPs during exogenous application of phytohormones (ABA, ethylene (ethophen), gibberellin, IAA, kinetin, methyl jasmonate, and salicylic acid), 350 mM mannitol, and hydrogen peroxide to eight-day-old seedlings growing under optimal growth conditions. (C) Relative quantification of BrHSBPs in different tissue samples, including the root (primary (PR) and secondary root (SR) tissues), leaf peduncle (LP), shoot (ST), leaves (young (YL) and old leaf (OL) tissues), seeds (SD), green siliques (SQ), flower buds (FB), and flowers (FL). Significant differences (indicated by a, b, and c) were assessed via one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, non-significant.

Figure 2

Expression activity of the GFP-tagged BrHSBP1 signal was localized to the nuclei and cytosol. (A) A schematic representation of the expression cassette designed for overexpression of BrHSBP1. (B) Relative expression levels of BrHSBP1 in BrHSBP1^OX lines. (C) Confocal microscopic images showing the GFP-tagged BrHSBP1 signal in abaxial epidermis peels of BrHSBP1^OX lines. i) The different panels labeled GFP, chlorophyll, and bright field represent the corresponding signals for the same samples. ii) Magnified stomata show the GFP-BrHSBP1 signals in nuclei and cytosol; this was confirmed by nuclei-specific DAPI (4’,6-diamidino-2-phenylindole) staining at a final concentration of 1µg/ml. Scale bar = 25 µm. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

Seed and pod phenotypes of BrHSBP1^OX and Brhsbp-KO lines of Brassica rapa sp. pekinensis. (A) BrHSBP1 gene structure denoting the gRNA target positions. (B) Some of the representative BrHSBP1-KO mutant genotypes. (C) Representative images of matured wild-type (WT), BrHSBP1^OX, and Brhsbp-KO lines. (D) The seeds (20 in number) of WT and BrHSBP1^OX lines. Scale bars = 5 cm.

Figure 4

Expression profiling of raffinose biosynthesis pathway genes in BrHSBP1^OX lines, showing relative expression levels of raffinose biosynthesis pathway genes Bra004474 [GolS1], Bra007842 [GolS6], Bra025579 [RS5], Bra027156 [GolS4], Bra027922 [GolS2], Bra030839 [RS1], Bra031509 [GolS7], Bra031663 [GolS3], and Bra032505 [GolS5] genes in wild (DB) and BrHSBP1^OX lines (83-1-3,4,83-3-1, and 83-6-2). BrACT was used for normalization of gene expression levels. The relative expression profile was derived from qRT-PCR of three independent wild-type lines and BrHSBP1^OX lines. Expression was normalized to the Actin gene. Asterisks represent statistical significance as assessed via one-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

The transcriptomic signatures of BrHSBP1^OX and Brhsbp1-KO reveal differential gene expression of plant reproductive structure developmental genes. (A) Volcano plots representing the differentially expressed genes in comparisons between transcriptomes of DB vs. BrHSBP1^OX, DB vs. Brhsbp1_KO, and rHSBP1^OX vs. Brhsbp1_KO. (B) Bar charts indicating the number of significantly differentially expressed genes (Log₂FC≥2 and p_adj<0.05) in the indicated comparisons. (C) Ridgeline plots depicting the differential regulation of key biological processes identified during comparative transcriptomic analyses of DB, BrHSBP1^OX, and Brhsbp1_KO libraries.

Figure 6

Protein–protein interactions of BrHSBPs with plant reproductive structure development-related genes and others. The figure represents yeast two-hybrid assays with cells harboring the indicated constructs grown on DDO (SD/-L/-T) or QDO supplemented with 15 mM 3-amino-1,2,4-triazole (3-AT) (SD/-A/-L/-T/-H+ 15mM 3AT) medium to verify their interaction. Yeast cells transformed with pGBKT7-P53/pGADT7-T (P53/T) (interactions between p53 and the SV40 large T-antigen) were used as positive controls, and cells transformed with pGBKT7-Lam/pGADT7-T (Lam/T) (lamin and the SV40 large T-antigen) were used as negative controls. Scale bars = 5 cm.