The E3 ubiquitin ligase WVIP2 highlights the versatility of protein ubiquitination

Abstract

Plant cells regulate many cellular processes controlling the half-life of critical proteins through ubiquitination. Previously, we characterized two interacting RING-type E3 ubiquitin ligases of Triticum durum, TdRF1 and WVIP2. We revealed their role in tolerance to dehydration, and existing knowledge about their partners also indicated their involvement in the regulation of some aspects of plant development. Here we located WVIP2 in the regulation of the ABA signaling, based on sequence similarities. Further we acquired general evidence about the versatility of ubiquitination in plant cells. A protein can be target of different E3 ligases for a perfect tuning of its abundance as well as the same E3 ligase can ubiquitinate different and unrelated proteins, thus representing a cross-connections between different signaling pathways for a global coordination of cellular processes.

Determining the half-life of proteins, ubiquitination plays a fundamental role in the regulation of cellular processes through the proteasome–mediated protein degradation. Main actors of this theme are the E3 ubiquitin ligases which specifically recruit the protein targets for ubiquitination.¹ Further, ubiquitination enzymes can crosstalk, providing an additional layer of ubiquitin-dependent control on cellular activities.

The ubiquitin proteasome system strongly impacts on the signaling pathways of abiotic stress tolerance, including those dependent by ABA.² The control of the E3 ligases on stress response can be exerted through the suppression of stress signaling pathways during favorable growth conditions, the degradation of negative regulators of stress response upon stress exposition, or the attenuation of stress signaling pathways for further growth once environmental conditions have improved.²

In a previous paper we showed that WVIP2³ and TdRF1 are two RING finger proteins of durum wheat (Triticum durum) with a demonstrated ubiquitin ligases activity.⁴ The corresponding genes are stress responsive, both upregulated upon cold treatment and downregulated by ABA, and with contrasting behavior in response to dehydration, with TdRF1 upregulated and WVIP2 downregulated. Further, transient induced gene silencing experiments suggested a positive and negative effect for TdRF1 and WVIP2, respectively, on tolerance to dehydration in vitro.

TdRF1 guides to degradation the transcription factor WBLH1 whose barley homolog HvJuBEL1 controls leaf development.^5,6 TdRF1 is phosphorylated by TdWNK5 which belongs to the WNK family of kinases known to have a role in the control of flowering time.⁷ For WVIP2 an interaction with the master regulator of embryo dormancy VP1 can be hypothesized based on its extensive homology with the Avena fatua AfVIP2.⁴ Indeed, it has been demonstrated that AfVIP2 interacts with AfVP1,⁸ thus VIP2 could target VP1 to degradation in monocot species.

The Arabidopsis homolog of VP1 is ABI3, although with an amino acid sequence similarity of 47.6% only (Table 1). Indeed VP1 can complement the abi3 mutant,⁹ and AfVIP2 can interact with ABI3, besides AfVP1.⁸ In Arabidopsis, ABI3 is guided to degradation in response to abiotic stress by AIP2, a RING type E3 ligase.¹⁰ Thus the overall evidence, that AIP2 and VIP2 are both RING ubiquitin ligases interacting with the homolog proteins ABI3/VP1, suggests that VIP2 could represent the monocot AIP2.

Table 1. Sequence similarity between homolog proteins of monocot species (maize and bread wheat) and Arabidopsis.

		Arabidopsis
		ABI3	AIP2	VIP2-like
				At3g05545	RFI2, at2g47700	At4g13490
Maize	VP1	47.6
Wheat	AIP2-like (Ta.6216)		68.1	5.4	7.7	11.2
	WVIP2		6	48	44	31

To assess the extent of conservation of the regulatory mechanisms based on ABI3/VP1 among monot/dicot species we have run a Blast session of sequence similarity searches at the molecular database for Arabidopsis (The Arabidopsis Information Resource, www.arabidopsis.org) and for Triticum aestivum (TIGR Plant Transcript Assemblies, http://plantta.jcvi.org/index.shtml). Then, we did pairwise alignments through EMBOSS Needle to evaluate global sequence similarity (http://www.ebi.ac.uk/Tools/psa/). We found that VIP2 and AIP2 are, unexpectedly, not homolog, being not similar at all (Table 1). Notwithstanding, wheat has an AIP2-like protein with 68% similarity with the corresponding Arabidopsis protein, while Arabidopsis has putative WVIP2 homologs with 48–44% similarity (Table 1). One of the closest homolog of WVIP2 in Arabidopsis is RFI2 (Red and Far-red Insensitive2), a RING finger protein controlling both deetiolation and flowering responses.^11,12 Considering these findings and the general conservation of the ubiquitination mechanisms across plant species, we suggest that VP1/ABI3 is targeted by two E3 ligases that coordinately regulate its amount (Fig. 1).

Figure 1. ABA signaling pathway in Triticum durum/Avena fatua and Arabidopsis based on experimental data and protein homology. Full lines represent experimentally demonstrated relationships, dotted lines are inferred interactions. Colored box (gray, light gray and white) contain homolog proteins.

More E3 ligases for a target

On the bases of the observations illustrated above, some additional regulative components could be added to the currently known ABA signaling pathway.^13,14 WVIP2 and AIP2 could both address the control of ABI3/VP1 amount which is known to accumulate only during specific developmental stages. It has been demonstrated that AIP2 is upregulated by ABA during drought response leading to an attenuation of the ABA signaling.¹⁰ Instead a suppressive function under favorable growth conditions or a specific developmental stage can be hypothesized for WVIP2. Indeed, WVIP2 transcripts are downregulated upon ABA treatment or dehydration and its knockout by RNAi increased the dehydration tolerance in vitro.⁴ The Arabidopsis ABA signaling offers another example of two E3 ligases regulating the same transcription factor. Indeed ABI5 is guided to degradation by KEG under low or no ABA conditions,^15,16 and by DWA1 and DWA2 during stress conditions with high ABA level.¹⁷ The final effect is a perfect fine tuning of the abundance of substrates of the E3 ligases based on the cellular conditions and in response to different stimuli.

More targets for an E3 ligase

E3 ligases specifically recruit protein targets. Recently it has been reported that one ligase can targets more substrates, without any sequence similarity. Indeed the ubiquitin ligase HOS1 leads to degradation of two transcription factors, ICE1, a master regulator of the cold response, and CONSTANS, the central component of the flowering pathways.^18,19 A multi target action has been hypothesized for AIP2, since the corresponding gene has a wider expression than its known target ABI3.¹⁰ Similarly, multi-targets have been suggested also for TdRF1 and WVIP2, according to different scenarios about their interaction.⁴ They can regulate each other’s, that is TdRF1 can guide WVIP2 to degradation or WVIP2 can ubiquitinate TdRF1, besides their own protein targets, the transcription factor WBLH1 and VP1, respectively (Fig. 1).

E3 ligases, a connecting point between signaling pathways

In all these systems, the E3 enzyme represents the point of connection between regulatory proteins of different cellular processes, e.g., the response to abiotic stress and various aspects of plant development. Different E3 ligases can target the same substrates, their activities can be coordinated to regulate the same process, they can regulate each other's besides other targets. All these scenarios create cross-talking between different signaling pathways or cellular processes. Thus plants can exploit ubiquitination to coordinate the functioning of different processes according to environmental and cellular conditions.

Glossary

Abbreviations:

ABA

Abscissic Acid

WVIP2

Wheat Viviparus1 Interacting Protein 2

TdRF1

Triticum durum Ring Finger 1

WBLH1

Wheat Bel1-type Homeodomain1

TdWNK5

Triticum durum With No Lysine5

VP1

Viviparous1

ABI3

Abscissic acid Insensitive3

AIP2

ABI3-Interacting Protein2

RFI2

Red and Far-red Insensitive2

KEG

Keep on Going

DWA1

DWD hypersensitive to ABA1

HOS1

High expression of Osmotically responsive genes1

ICE1

Inducer of CBF Expression 1

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.