C-repeat binding factor proteins integrate cold signaling into the SVP-mediated flowering pathway in Arabidopsis

Abstract

The CBF transcription factors serve as key nodes that integrate cold signaling into the SVP-mediated flowering pathway, which fine-tunes seasonal flowering in response to ambient temperature.

Dear Editor,

The floral transition is critical to survival and reproductive success, yet it is typically delayed by cold exposure in summer annual plants, which impacts fruit quality and crop yield (Amasino 2004; Jung and Müller 2009; Nilsson 2013). The MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) acts as a floral repressor, and its degradation under warm temperature results in early flowering (Lee et al. 2013). A similar early flowering phenotype is observed in the svp mutants under cold stress (Fig. 1, a and b), which is partly attributed to its role in directly suppressing the expression of the floral integrator FLOWERING LOCUS T (FT) in Arabidopsis (Lee et al. 2007). Although cold-induced late flowering has been relatively well studied, the downstream targets of SVP during cold response remain unclear. To elucidate its downstream targets, we performed transcriptome profiling on the Arabidopsis svp-32 null mutant, followed by GO enrichment analysis. This analysis revealed a significant reduction in the transcript levels of two core cold regulator genes, C-REPEAT BINDING FACTOR 1 (CBF1) and CBF2, in the mutant (Fig. 1c and Dataset S1), which indicated their potential role in the SVP-mediated flowering pathway under cold stress. Consistent with this, quantitative real-time polymerase chain reaction (qPCR) data revealed that the cold-induced upregulation of CBF1, CBF2, and CBF3 was strongly attenuated in svp mutants but enhanced by SVP overexpression (Fig. 1, d—f and Figure S1). Since CBFs function as transcriptional activators for a subset of COLD-REGULATED (COR) genes during cold exposure (Thomashow 2010; Medina et al. 2011; Liu et al. 2018), we propose that the SVP-CBF module enhances COR genes transcription. Supporting this, our data displayed that the cold-induced transcription of COR15A, COR47, COR78, and COLD-INDUCED 1 (KIN1) was markedly impaired in svp mutants (Figure S2). These results support a positive regulatory role for the floral repressor SVP in controlling CBF gene expression under cold stress.

Figure 1.

The SVP-CBF module mediates flowering delay under cold stress. (a, b) Flowering time was determined by counting the number of rosette leaf (b) in Col-0, svp-31, and svp-32 plants. The plants were grown under long-day conditions (16 h light/8 h darkness) at either 22 °C or 4 °C for periods of 21 or 90 days. Scale bars, 2 cm. (c) Gene ontology (GO) enrichment analysis of genes downregulated in svp-32 after 2 hours of cold treatment. (d—f) Transcript levels of CBF1, CBF2, and CBF3 in 10-day-old Col-0, svp-31, and svp-32 seedlings after cold exposure for 0, 0.5, 1, 2, 3, 6, 12, or 24 h (n = 3). (g) The binding of SVP to the CArG motifs in the CBF promoters was assessed using reciprocal competitive EMSA. (h, i) IGV snapshot of ChIP-sequencing profiles showing SVP binding to the CBF1 and CBF2 promoters in SVPpro:SVP-3×FLAG (SVP-3×FLAG) and 35S:3×FLAG (3×FLAG) transgenic Arabidopsis under chilling stress. The binding signals were shown as counts per million of mapped reads. The SVP-3×FLAG binding regions were outlined with gray dotted rectangles. Annotated gene structures were shown at the bottom with exons in dark gray boxes, untranslated regions in light gray boxes, and introns as thin lines. (j) Transcriptional activation analysis in tobacco leaves demonstrated that SVP activates the expression of the CBF genes under cold stress, as measured by the ratio of firefly to renilla luciferase activity (n = 5). Gray and blue indicate the effectors 35S:3×FLAG (3×FLAG) and 35S:SVP-3×FLAG (SVP-3×FLAG), respectively. (k—m) Flowering time was measured by recording both the number of rosette leaf (l) and the bolting time (m) in Col-0, svp-32, and svp-32 plants overexpressing CBF1 (CBF1oe), CBF2 (CBF2oe), or CBF3 (CBF3oe) under long-day conditions at 22 °C or 4 °C (n = 24 plants for each genotype). Scale bars, 2 cm. For each genotype, the ratio of leaf number (l) or bolting time (m) between plants grown at 4 °C and 22 °C was shown above the corresponding columns. In (d, e, f, and j), error bars are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test. Detailed experimental procedures were described in Materials and Methods, and primer sequences were listed in Table S1.

MADS-box transcription factors regulate target gene expression by binding to the CArG motif (Wang et al. 2018). To understand how SVP promotes CBF expression, we identified CArG motif variants in their promoters and established that SVP directly binds these motifs in vitro via electrophoretic mobility shift assay (EMSA) (Fig. 1, g and Figure S3). Chromatin immunoprecipitation (ChIP) sequencing further verified in vivo binding of SVP to the promoters of CBF1, CBF2, and other targets (Fig. 1, h and i, Figure S4). Notably, we did not detect in vivo binding of SVP to the CBF3 promoter after either 2 or 3 hours of cold exposure, suggesting that the recruitment of SVP to these three CBF genes may occur in a time-dependent manner during Arabidopsis response to cold stress. In addition, promoter activation analysis announced that SVP clearly activates the expression of CBF genes under cold stress (Fig. 1j). These findings support a model in which the floral repressor SVP activates CBF gene expression in response to cold stress by directly binding to the CArG motifs in their promoters. This transcriptional activation increases CBF protein levels, which regulate downstream target genes to ultimately delay flowering under cold stress.

Given that overexpression of CBF1, CBF2, or CBF3 results in a noticeable late flowering phenotype in Arabidopsis (Gilmour et al. 2004). We speculated that the absence of SVP causes a decrease in the expression of CBF genes, which alleviates late flowering during cold exposure. To test this hypothesis, we generated overexpression lines of CBF1, CBF2, and CBF3 in the svp-32 background mutant (Figure S5). Physiological analysis showed that the early-flowering phenotype of svp-32 mutant plants was fully rescued by overexpressing CBF1 or CBF3, and partially restored by CBF2 overexpression under cold stress (Fig. 1, k—m). These results declared CBF genes as downstream targets of SVP in the regulation of flowering time under cold stress. In contrast to normal conditions, the cbf123 triple mutants exhibited early flowering under cold stress (Figure S6, a—c). Additionally, our analysis revealed a significant increase in FT transcript levels in both svp and cbf123 mutants under cold stress (Figure S6, d and e). We further proved that CBF transcription factors directly bind to the FT promoter and intensely repress its expression (Figure S6, f and g). These results indicated that FT acts as a downstream target of the SVP-CBF regulatory module in mediating cold-induced late flowering. As core regulators of the cold response, the transcription of CBF genes is also known to be governed by INDUCER OF CBF EXPRESSION 1 (ICE1), PHYTOCHROME INTERACTING FACTORs (PIFs), POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), Thioredoxin h2 (Trx-h2) in Arabidopsis (Chinnusamy et al. 2003; Lee and Thomashow 2012; Lee et al. 2022; Gomez-Martinez et al. 2024). These studies established that plants have evolved a sophisticated cold response strategy mediated by the CBF pathway, thus boosting their hardiness and survival.

Here, we identified the SVP-CBF regulatory module as a critical signaling hub that integrates cold signaling into the flowering pathway, which enables plants to fine-tune their seasonal flowering in response to low temperature. Given the stronger flowering impact of the svp mutant compared to the cbf123 triple mutant and the direct FT repression by SVP under cold stress (Lee et al. 2007), we conclude that SVP modifies cold-induced late flowering through both CBF-dependent and -independent pathways. Nevertheless, the molecular mechanism underlying the accumulation and/or activation of SVP protein in response to cold stress still needs further investigation.

Author contributions

J.-K.Z. and Z.W. conceived the project; Z.W. designed the experiments; Z.Q.W., Z.G., Y.G., and Y.H. performed the experiments; Z.W. finalized and revised the manuscript.

Supplementary material

Supplementary material is available at Plant Physiology online.

Funding

This work was supported by the National Natural Science Foundation of China (32570335) and Anhui Provincial Natural Science Foundation (2208085Y08).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.