Dynamic subnuclear relocalization of WRKY40, a potential new mechanism of ABA-dependent transcription factor regulation

Abstract

The phytohormone ABA plays a major role during plant development, e.g. seed maturation and seed germination, and during adaptation to abiotic stresses like stomatal aperture regulation. The three closely related WRKY transcription factors WRKY18, WRKY40 and WRKY60 function in ABA signal transduction. We recently demonstrated that WRKY18 and WRKY40 but not WRKY60 localize to nuclear bodies in A. thaliana mesophyll protoplasts. WRKY40, a negative regulator of ABA-dependent inhibition of seed germination, relocalizes from PNBs to the nucleoplasm in the presence of ABA in a dynamic and phosphorylation-dependent manner. We propose that subnuclear relocalization of WRKY40 might constitute a new regulatory mechanism of ABA-dependent modulation of transcription factor activity.

Abbreviations

ABA

Abscisic acid

PHYB

PHYTOCHROME B

PIF

PHYTOCHROME INTERACTING FACTOR

PNB

PHYTOCHROME B containing nuclear body

The developmental process of seed germination is tightly regulated by the plant hormone ABA to prevent germination under unfavorable conditions.¹ WRKY40 is transcriptionally suppressed by ABA and negatively regulates ABA-dependent inhibition of seed germination by direct repression of ABA INSENSITIVE 4 (ABI4) and ABI5 gene expression^2-6 (Fig. 1).

Figure 1.

WRKY40 may connect light- and ABA signaling at the level of ABI5. (A) WRKY40 expression in A. thaliana protoplasts is light-dependent. Merged confocal fluorescence microscopy images of A. thaliana Col-0 protoplasts, transformed with YFP-WRKY40 and incubated over-night in light (plant growth chamber; upper panel) or in dark (lower panel) (scale bar 25 µm). (B) Schematic model of light- and ABA-signaling at the level of WRKY40 and ABI5 transcription factors during seed germination. The bZIP transcription factor ABI5 is a central positive regulator of ABA-dependent inhibition of seed germination, downstream of the ABA core signaling cascade.^1,29 Red-light induced PHYB signaling targets ABI5 and ABA biosynthesis via the transcription factor PIF1/PIL5 to induce seed germination in the light.^26-28 During far-red light and PHYA-dependent signaling, seed germination is inhibited via expression of ABI5 by FHY3 and FAR1.²³ The transcription factor WRKY40, which is transcriptionally suppressed and relocalizes from PNBs to the nucleoplasm in response to ABA, negatively mediates ABA-dependent inhibition of seed germination by repressing ABI5 transcription.^2,3,7 Red light-induced PHYB signaling positively regulates WRKY40 expression through inhibition of PIF3.²²

We recently showed that after transient expression in A. thaliana protoplasts, WRKY18 and WRKY40 co-localize with PHYTOCHROME INTERACTING FACTOR 3 (PIF3), PIF4 and PHYTOCHROME B (PHYB) to PHYTOCHROME B-containing nuclear bodies (PNBs).⁷ Subsequent ABA treatment induces relocalization of WRKY40 from PNBs to the nucleoplasm in an OPEN STOMATA 1 (OST1)-dependent manner. The reversible localization of WRKY40 in PNBs within 15 minutes after removal of ABA by buffer exchange points toward a highly dynamic process.⁷ So far this stimulus-dependent relocalization was only shown to be evoked by ABA, since other abiotic and biotic stimuli we tested, including flg22 and high salinity, do not induce subnuclear relocalization. Treatment with the general Ser/Thr-kinase-inhibitor Staurosporine, however, induces a comparable increase in nucleoplasmic localization of WRKY40 as ABA, indicating that PNB-localization is phosphorylation-dependent. It remains to be tested if Staurosporine prevents PNB localization or whether it induces dynamic relocalization to the nucleoplasm.

ABA-dependent subnuclear relocalization has also been reported for Arabidopsis UBP1-ASSOCIATED PROTEIN 2 A (UBA2a) and for the Vicia faba homolog AAPK INTERACTING PROTEIN (AKIP1), which in contrast to WRKY40 both display an increased nuclear body localization in response to ABA.^8-10 The identity of those nuclear bodies has not been determined yet. Nuclear body formation and dynamic relocalization also occur for SR splicing factors during RNA processing in nuclear speckles.¹¹ Moreover, the Xanthomonas Type III effector protein XopD targets and translocates the Arabidopsis transcription factor MYB30 into nuclear bodies, which leads to MYB30 inactivation and suppression of plant defense.¹² Dynamic nuclear body localization might therefore be a regulatory mechanism of various plant responses.

The best studied protein that is dynamically localized to nuclear bodies is the photoreceptor PHYB, which upon light exposure relocalizes from the cytoplasm and the nucleoplasm toward PNBs, to interact with PIF transcription factors, which triggers light signaling.^13-15 This mechanism is phosphorylation-dependent, since PHYB itself is phosphorylated at the N-terminus which affects the reversion from the active P_fr form that is localized in PNBs, to the inactive P_r form that is localized in the cytoplasm.^15,16

The negative role of WRKY40 during ABA-dependent seed germination and the ABA-induced subnuclear relocalization of WRKY40^2,3 raise the question of the physiological function of PNB localization and ABA-dependent relocalization. For PHYB and PIF localization to PNBs different models are conceivable.¹⁷ In a widely proposed model, PNBs are characterized as degradation sites for distinct transcription factors, as it has been reported for PIF3, which is rapidly phosphorylated and degraded upon interaction with PHYB within PNBs in the presence of light.^13,18 The idea of nuclear bodies as degradation sites is further supported by the ABI FIVE BINDING PROTEIN (AFP), which recruits ABI5 into nuclear bodies while enhancing ABI5 degradation.¹⁹ On the other hand, PIF7 remains light-stable upon interaction with PHYB in PNBs.²⁰ Concerning WRKY40, localization within PNBs as well as ABA-dependent relocalization were not affected by treatment with the proteasome inhibitor MG132.⁷

A further model describes PNBs as storage depots to regulate transcription factor and photoreceptor activity during light signaling, whereby transcription factors are stored in nuclear bodies to either inhibit or to induce transcription of responsive genes.¹⁷

Localization of WRKY40 within PNBs may indeed suggest that WRKY40 is part of PHYB-dependent light signaling, although no direct interactions between PHYB, PIFs and WRKY40 were detected.⁷ Interestingly, WRKY40 expression in and/or transformation of A. thaliana protoplasts is highly light dependent. Transformation of protoplasts with YFP-WRKY40 and subsequent overnight-incubation in the dark did not result in detectable WRKY40 expression (Fig. 1A). Incubation in the light, however, resulted in WRKY40 expression.

Seed germination is controlled by ABA levels as well as by the ratio of red to far-red light (R:FR).^1,21,22 Thereby, PHYA mediates far-red light signaling via FAR-RED ELONGATED HYPOCOTYLS 3 (FHY3) and FAR-RED IMPAIRED RESPONSE 1 (FAR1), which directly regulates ABI5 gene expression.^22-25 PHYB-dependent regulation of seed germination in red light is mediated by PIF1/PIL5, which is destabilized upon interaction with PHYB.^22,26-28 PIF1 stimulates the expression of ABA synthesis genes and targets ABI5 directly.^26-28 WRKY40 is transcriptionally suppressed by ABA, and itself negatively mediates ABI5 expression. WRKY40 gene expression is upregulated in pif3–3 mutant seedlings upon light induction,²² which potentially connects red light and ABA signaling via PHYB, PIF3 and WRKY40 (Fig. 1B).

A detailed expression analysis of WRKY40 target genes, e.g., ABI5, in protoplasts with PNB localization versus protoplasts with nucleoplasmic localization will be required to further study the role of WRKY40 in ABA - and light signaling.

Funding

This research was supported by a return fellowship and a research grant from the Deutsche Forschungsgemeinschaft (BO3155/2–1; BO3155/3–1 to M.B.) and start-up funds from the Westfälische Wilhelms-University of Münster.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank František Baluska for the invitation for this addendum.