Post-translational regulation of SHORT VEGETATIVE PHASE as a major mechanism for thermoregulation of flowering

Abstract

In contrast to our extensive knowledge of vernalization, we know relatively little about the regulation of ambient temperature-responsive flowering. Recent reports revealed that FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) regulate high ambient temperature-responsive flowering through two different mechanisms: degradation of SVP protein and formation of a non-functional SVP-FLM-δ complex. To investigate further the mechanism of thermoregulation of flowering, we performed real-time quantitative polymerase chain reaction (RT-qPCR) and in vitro pull-down assays. We found that FLM-β and FLM-δ transcripts show similar absolute levels at different temperatures. Also, His-SVP protein bound to the GST-FLM-β or -δ proteins with similar binding intensities. These results suggest that functional SVP-FLM-β and non-functional SVP-FLM-δ complexes form similarly at warmer temperatures, thus indicating that post-translational regulation of SVP functions as a major mechanism for thermoregulation in flowering.

Climate change alters resource availability and growth conditions, essential factors for the survival of all organisms. In evolutionary terms, plants and animals have different survival strategies (plasticity and mobility, respectively). Plants, as sessile organisms, can flexibly adjust their development and thus adapt to continuously fluctuating environments.^1,2 For example, plant reproduction requires the proper seasonal timing of flowering, and plants adjust their flowering time primarily based on day length and temperature.^3-8 Indeed, small changes in ambient growth temperature significantly affect flowering in plants,^9,10 an observation that highlights the potential far-reaching impacts on plant ecosystems due to projected increases in mean global temperature.¹¹ Although numerous studies have revealed various components that regulate flowering in response to a wide range of temperatures,^12-14 our current knowledge about the regulation of ambient temperature-responsive flowering remains limited.

Recently two papers provided insight into the basic mechanisms controlling ambient temperature-responsive flowering in Arabidopsis.^15-17 These reports showed that FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) proteins form a repressor complex to repress flowering at colder temperatures by direct binding to the genomic regions of floral activator genes like FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). However, they also reported different mechanisms for thermoregulation of flowering. We showed that the reduced stability of SVP protein at warmer temperatures leads to decreased levels of the SVP-FLM repressor complex, thereby inducing early flowering at that temperature. By contrast, Posé et al. (2013) showed that increased temperature leads to higher levels of FLM-δ protein; in this model, SVP protein interacts with FLM-δ protein to form a non-functional complex with impaired DNA-binding ability, thereby accelerating flowering.

Here, we examined these two models by measuring the absolute levels of FLM-β and FLM-δ transcripts at different temperatures, and examining the in vitro interaction between SVP and FLM-β or FLM-δ proteins. SVP-like proteins have conserved functions across plant species,^18-21 and our results suggest that plants may preferentially use post-translational regulation of SVP protein levels at warmer temperatures to regulate ambient temperature-responsive flowering.

Alternative splicing of FLM is temperature-dependent, and FLM-β and FLM-δ transcripts are highly expressed at 16 °C and 27 °C, respectively^15,16; therefore, we measured absolute copy numbers of FLM-β and FLM-δ transcripts at different temperatures. We calculated their absolute copy numbers using standard curves of FLM-β and FLM-δ transcripts, as previously reported.²² At 16 °C, the copy numbers of FLM-β transcripts were higher than those of FLM-δ transcripts; by contrast, at 27 °C the copy numbers of FLM-β and FLM-δ transcripts were nearly identical (Fig. 1A). Also, after a shift from 23 °C to 16 °C, the copy numbers of FLM-β transcripts increased within 1 d (Fig. 1B). However, after a shift from 23 °C to 27 °C, the copy numbers of FLM-β and FLM-δ transcripts were almost the same (Fig. 1C). This suggested that the plants produce similar amounts of FLM-β and FLM-δ transcripts at higher temperatures.

Figure 1. Absolute quantification of FLM-β and FLM-δ transcripts at different temperatures. (A) Copy numbers of FLM-β and FLM-δ transcripts in 8-d-old Col seedlings grown at 16 °C, 23 °C, and 27 °C under long-day (LD) conditions. (B and C) Copy numbers of FLM-β and FLM-δ transcripts in temperature-shifted Col seedlings under LD conditions. Error bars indicate standard deviation of one biological replicate with three technical replicates.

Because FLM-β and FLM-δ proteins both interact with SVP protein,^15,16 we also compared the binding affinity of SVP to FLM-β or FLM-δ proteins in vitro. We used affinity-purified histidine (His)-SVP, Glutathione S-transferase (GST)-FLM-β, and GST-FLM-δ proteins expressed in Escherichia coli. To test binding, we incubated His-SVP and GST-FLM-β or GST-FLM-δ proteins, used anti-GST antibody to pull down the FLM proteins, then used anti-His antibody to detect SVP by western blot. This in vitro assay revealed that the SVP protein bound to the GST-FLM-β or GST-FLM-δ proteins with similar intensities (Fig. 2, lanes 2 and 3); however, GST protein did not bind to SVP protein (lane 4). This suggested that the binding strengths of SVP protein to FLM-β or FLM-δ proteins are substantially similar.

Figure 2. In vitro pull-down assay between His-SVP protein and GST-FLM-β or GST-FLM-δ proteins. Anti-GST antibody was used for pull-down. The eluates were separated by 12.5% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with anti-His antibody. About 10% of His-SVP protein was loaded as an input control. The amounts and qualities of the GST-tagged proteins tested are shown below.

Based on these results, we propose that decrease in functional SVP-FLM-β complex caused by rapid degradation of SVP protein at warmer temperatures functions as a more important mechanism for thermoregulation in flowering than alterations in FLM-β and FLM-δ transcripts (Fig. 3). At warmer temperatures, the absolute levels of FLM-β and FLM-δ transcripts are almost identical, and two different FLM proteins produced by two spliced transcripts have similar binding affinities for SVP protein. This results in the formation of functional SVP-FLM-β and non-functional SVP-FLM-δ complexes in similar amounts. However, rapid degradation of SVP proteins at high temperatures reduces the abundance of the functional SVP-FLM-β complex, which can bind to the genomic regions of downstream target genes like FT, TWIN SISTER OF FT (TSF), and SOC1, thereby inducing flowering at that temperature. However, we cannot exclude several possibilities, including that formation of non-functional SVP-FLM-δ complex facilitates the degradation of SVP protein, and that FLM-β or FLM-δ proteins also could be subject to degradation. Thus, it will be interesting to investigate which conditions, such as the formation of SVP-FLM-δ complex, facilitate the degradation of SVP protein, and how degradation of FLM proteins contribute to ambient temperature-responsive flowering.

Figure 3. A proposed model to explain the thermal induction of flowering at warmer temperatures. When temperature increases, the absolute expression levels of two different FLM transcripts are almost identical and two different FLM proteins have similar binding affinities for SVP protein. Ultimately, this leads to formation of similar amounts of functional SVP-FLM-β and non-functional SVP-FLM-δ complexes. Because SVP protein levels are rapidly reduced through post-translational regulation,¹⁵ the amounts of functional SVP-FLM-β complex are also reduced, which would alleviate the repression of the transcription of downstream target genes, thereby accelerating flowering at that temperature.

Glossary

Abbreviations:

FLM

FLOWERING LOCUS M

FT

FLOWERING LOCUS T

GST

Glutathione S-transferase

His

Histidine

SVP

SHORT VEGETATIVE PHASE

SOC1

SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1

TSF

TWIN SISTER OF FT

Disclosure of potential conflicts of interests

No potential conflicts of interests were disclosed.

Acknowledgments

This work was supported by a National Research Foundation of Korea grant funded by the Korea government (Ministry of Science, ICT, and Future Planning) (2008-0061988) to J.H.A.