B-BOX-DOMAIN PROTEIN32 modulates seed oil biosynthesis in Arabidopsis by interacting with WRINKLED1

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The Arabidopsis (Arabidopsis thaliana) genome encodes for more than 1,500 transcription factors (TFs) of which 45% are plant specific (Riechmann et al. 2000). B-box (BBX) proteins are a subfamily of zinc finger TFs (Khanna et al. 2009) characterized by B-box domains at the N-termini. Some also contain the CCT (CONSTANS, CONSTANS-LIKE, and TOC1) domain near their C-termini. In Arabidopsis, the BBX TF family is divided into 5 subgroups based on the presence of functional domains. Subgroups I, II, and IV contain 2 BBX domains while subgroups III and V contain 1 (Khanna et al. 2009; Gangappa and Botto 2014). BBX TFs play roles in growth, development, and responses to environmental stimuli (Khanna et al. 2009; Gangappa and Botto 2014; Song et al. 2020); however, their involvement in plant oil biosynthesis is unknown.

Plant seed oils are stored as an energy source for seedling development, and they are crucial in human diets and have diverse industrial applications. WRINKLED1 (WRI1) is an APETALA2 (AP2) TF that regulates oil biosynthesis (Cernac and Benning 2004; Baud et al. 2007; Maeo et al. 2009; Kong et al. 2020a; Yang et al. 2022). WRI1 is a transcriptional activator of genes encoding enzymes involved in glycolysis and fatty acid biosynthesis (Baud et al. 2007; Maeo et al. 2009; Kong et al. 2020b). Protein–protein interactions between transcriptional regulators form regulatory modules that regulate plant development (Bemer et al. 2017; Tripathi et al. 2017; Alizadeh et al. 2021). However, WRI1-interacting factors and their effects on WRI1 activity and oil biosynthesis remain poorly understood. We aimed to identify WRI1-interacting regulators to understand the regulatory mechanisms controlling seed oil biosynthesis.

We conducted yeast-two-hybrid (Y2H) assays using a truncated WRI1 form (WRI1^1–306) due to high transactivation activity of the C-terminal region (Ma et al. 2015) to investigate WRI1 interactions. Results showed WRI1–BBX32 interaction (Fig. 1A). An in vitro pull-down experiment using GST–agarose-coupled WRI1 and His-MBP–fused BBX32 further showed WRI1–BBX32 physical interaction (Fig. 1B). We also investigated the subcellular location of WRI1–BBX32 interaction in planta. The fluorescent fusions YFP–BBX32 and WRI1–CFP were transiently coproduced in Nicotiana benthamiana leaves. Confocal microscopy results indicated that YFP-BBX32 and WRI1-CFP were colocalized in the nucleus (Supplemental Fig. S1). WRI1–BBX32 interaction in planta was corroborated using bimolecular fluorescence complementation (BiFC) assays in N. benthamiana. The BiFC constructs producing WRI1 fusing with N-terminal half of YFP (nYFP–WRI1) and C-terminal half of YFP (cYFP–BBX32) were cotransformed into N. benthamiana leaves, which resulted in YFP fluorescence signal in the nucleus (Fig. 1C). We used reverse transcription quantitative PCR (RT-qPCR) to measure WRI1 and BBX32 expression in seeds. Results showed that WRI1 expression was increased during seed development, reaching its peak at 6 to 8 d after flowering (DAF). BBX32 expression remained high at 10 DAF and coincided with WRI1 expression (Supplemental Fig. S2). The shared expression pattern and interaction suggest that WRI1 and BBX32 potentially act together to regulate fatty acid biosynthesis.

Figure 1.

BBX32 physically interacts with WRI1. A) Yeast-two-hybrid (Y2H) assay showing the interaction between BBX32 and WRI1^1–306. Yeast growth on either permissive (-Leu/-Trp) or stringent selective (-Leu/-Trp/-His with 7.5 mM 3-AT) media was shown. Empty vector (EV) was used as a negative control. B) In vitro pull-down assay showing the interaction between BBX32 and WRI1. Glutathione S-transferase (GST) and GST–WRI1 were used as baits. The top panel showed the immunoblot using anti-His antibody. The bottom panel showed Coomassie stained SDS–PAGE gel of GST and GST–WRI1 pull-down with Escherichia coli expressed proteins (histidine-tagged maltose binding protein [His-MBP] and His-MBP–BBX32). C) BiFC assay in N. benthamiana leaves showing the interaction between BBX32 and WRI1 in plant cells. N. benthamiana epidermal cells were transiently coexpressing nYFP-WRI1 and cYFP-BBX32 or nYFP-WRI1 and cYFP, or nYFP and cYFP-BBX32. Scale bar is 20 μm.

We evaluated the effect of WRI1–BBX32 interaction on the transcriptional activity of WRI1 using a dual-luciferase (LUC) reporter assay (Kong et al. 2022; Lim et al. 2022). Promoters (proBCCP2 and proPKP-β1) of two WRI1 target genes that are involved in de novo fatty acid biosynthesis were used to drive LUC reporter gene expression (Fig. 2A). WRI1 and BBX32 served as effectors under the control of the Cauliflower mosaic virus (CaMV) 35S promoter (Fig. 2A). WRI1 significantly activated LUC activity of the promoters, and LUC activity was significantly enhanced when BBX32 and WRI1 were coproduced (Fig. 2B). Our data suggest that BBX32–WRI1 interaction enhances WRI1 transcriptional activity.

Figure 2.

BBX32 effect on the activity of WRI1. A) Schematic diagram of constructs used in the dual-LUC assay. B) Coexpression of WRI1 with BBX32 increased the transactivation activity of WRI1 on promoters of BCCP2 and PKP-β1 (proBCCP2 and proPKP-β1). The activity of firefly LUC was normalized to the activity of Renilla (REN) LUC. Results are shown as means ± Se (n = 5 to 6). “**” and “***” indicate significant differences (P < 0.01 and P < 0.001, respectively, Student's t-test) between expression of WRI1 alone and coexpression of WRI1 and BBX32. C) Total fatty acid content in seeds of WT and transgenic Arabidopsis overexpressing BBX32 (BBX32-OE). Results are shown as means ± Se (n = 3 to 5). “**” and “***” indicate significant differences (P ≤ 0.01 and P < 0.001, respectively, one-way ANOVA) between WT and BBX32-OE lines. D) EMSA shows WRI1^1–302 binding to the proBCCP2 fragment (a DNA probe containing AW-box) in the presence of an increasing amount of GST–BBX32. For GST and GST–BBX32, “+” and “++” indicate the addition of 0.3 and 0.4 pmol of protein, respectively. An arrow indicates the increasing amount of the protein–DNA complex in the image of EMSA. E) A proposed model of BBX32-mediated WRI1 activity. The interaction of BBX32 with WRI1 enhances the binding of WRI1 to the AW-box and hence leads to an increased WRI1 activation (represented by a thicker arrow).

We generated Arabidopsis transgenic lines overexpressing FLAG–BBX32 (BBX32-OE). Multiple homozygous BBX32-OE lines were used to validate BBX32 expression using RT-qPCR (Supplemental Fig. S3). BBX32-OE lines displayed enhanced oil content compared to wild-type (WT) (Fig. 2C), and their seeds were larger, with increased weight, length, width, area, and perimeter (Supplemental Fig. S4). We used RT-qPCR to examine the expression of WRI1 target genes involved in de novo fatty acid biosynthesis and observed a significant increase in their expression in BBX32-OE compared to WT (Supplemental Fig. S5). To determine whether BBX32 regulates WRI1, we measured transactivation activity of the WRI1 promoter (proWRI1) in the presence of BBX32 using the dual-LUC assay in N. benthamiana leaves (Supplemental Fig. S6A). BBX32 did not alter basal LUC activity conferred by proWRI1 (Supplemental Fig. S6B), suggesting that BBX32 does not directly regulate WRI1. Characterization of a homozygous Arabidopsis bbx32 T-DNA insertional mutant line [designated as bbx32-1 (SALK_059534)] and a BBX32 microRNA line (BBX32-AMI #3) (Holtan et al. 2011; Tripathi et al. 2017) showed significant reduction in seed oil content compared to WT and reductions in seed weight, length, width, area, and perimeter (Supplemental Fig. S7).

The WRI1–BBX32 complex substantially enhanced transcriptional activities of BCCP2 and PKP-β1 promoters. Therefore, we conducted electrophoretic mobility shift assay (EMSA) to examine the DNA binding ability of WRI1 in the presence of BBX32. We designed a probe based on the proBCCP2 sequence. WRI1^1–302 bound to proBCCP2 probe (Fig. 2D), similar to previous findings (Maeo et al. 2009), while BBX32 did not. We then mixed WRI1^1–302 and BBX32 in the EMSA reaction. With a higher amount of BBX32 in the reaction mixture, the binding of WRI1^1–302 to the proBCCP2 probe increased (Fig. 2D and Supplemental Fig. S8), suggesting that BBX32–WRI1 interaction enhanced WRI1 binding to proBCCP2. Therefore, we proposed a model to summarize our findings (Fig. 2E). BBX32–WRI1 interaction enables stronger binding of WRI1 with its target promoters, leading to enhanced expression of target genes (Fig. 2E). Our proposed molecular mechanism allows an effective modulation of WRI1 activity.

BBX32 participates in various physiological processes (Holtan et al. 2011; Tripathi et al. 2017; Yeh et al. 2019; Ravindran et al. 2021). From an evolutionary perspective, B-box proteins are used for other processes in photosynthetic organisms (Crocco and Botto 2013) but may have later co-opted to regulate oil biosynthesis in land plants. Results highlight that BBX32 enhances WRI1 activity and modulates seed oil accumulation in Arabidopsis. The WRI1–BBX32 regulatory module shows the previously unidentified function of BBX32 and expands the WRI1 regulatory network in Arabidopsis seed oil biosynthesis.

Acknowledgments

We thank Dr. Steve Kay (University of Southern California) for the seeds of the BBX32 microRNA line (BBX32-AMI #3), Dr. Yansong Miao (Nanyang Technological University), and Dr. Zhiming Ma (Nanyang Technological University) for the help with confocal microscopy experiments, and NTU Protein Production Platform for His-WRI1^1–302 protein expression and purification.

Author contributions

Q.K. and W.M. conceived and designed the experiments. A.R.Q.L., Q.K., A.N., and Y.Q.S. performed the experiments. A.R.Q.L., Q.K., A.N., Y.Q.S., S.P., L.Y., and W.M. analyzed the data. A.R.Q.L., Q.K., S.P., L.Y., and W.M. wrote the manuscript.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Methods.

Supplemental Figure S1. Subcellular location of WRI1 and BBX32.

Supplemental Figure S2.WRI1 and BBX32 expression analysis during seed development in wild-type (WT) Arabidopsis.

Supplemental Figure S3. Characterization of BBX32-OE transgenic lines.

Supplemental Figure S4. Analysis of seeds of WT and BBX32-OE lines.

Supplemental Figure S5. Gene expression analysis of the transcript level in BBX32-OE.

Supplemental Figure S6. Transactivation activity of BBX32 on the promoter of WRI1 (proWRI1) in plant cells.

Supplemental Figure S7. Functional analysis of WT and BBX32 loss-of-function mutants.

Supplemental Figure S8. Effect of BBX32 on WRI1 binding to proBCCP2.

Supplemental Table S1. Primers used in this study.

Funding

This work was supported by the Ministry of Education (MOE) of Singapore Tier 2 (grant no. MOE-T2EP30220-0011 to W.M.) and MOE of Singapore Tier 1 (grant no. RG89/21 to W.M.).

Data availability

All data that supports the findings of this study are available in the main paper and supplementary data.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• BCCP2 Gramene: AT5G15530

• BCCP2 Araport: AT5G15530

• WRI1 Gramene: AT3G54320

• WRI1 Araport: AT3G54320

• BBX32 Gramene: AT3G21150

• BBX32 Araport: AT3G21150

Author notes

A.R.Q.L. and Q.K. contributed equally to this work.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is Wei Ma ([email protected]).

Conflict of interest statement. None declared.