A hub for ABA signaling to the nucleus: Significance of a cytosolic and nuclear dual-localized PPR protein SOAR1 acting downstream of Mg-chelatase H subunit

Abstract

SOAR1 is a cytosol-nucleus dual-localized pentatricopeptide repeat (PPR) protein, which we indentified recently as a crucial regulator in the CHLH/ABAR (Mg-chelatase H subunit /putative ABA receptor)-mediated signaling pathway, acting downstream of CHLH/ABAR and upstream of a nuclear ABA-responsive bZIP transcription factor ABI5. Downregulation and upregulation of SOAR1 expression alter dramatically both ABA sensitivity and expression of a subset of key, nuclear ABA-responsive genes, suggesting that SOAR1 is a hub for ABA signaling to the nucleus, and CHLH/ABAR mediates a central signaling pathway to regulate downstream gene expression through SOAR1.

Abbreviations

ABA

abscisic acid

ABAR

putative ABA receptor

CHLH

Mg-chelatase H subunit

CPN20

cochaperonin 20

PPR

pentatricopeptide repeat

SOAR1

suppressor of the ABAR-overexpressor 1

WRKY

WRKY-domain transcription factor

The pentatricopeptide repeat (PPR) proteins include a typical PPR motif characterized by a degenerate 35-amino acid sequence repeated in tandem 2 to 50 times, which is considered to be RNA-binding motif.^1-3 PPR proteins are found in all eukaryotes and function universally in organellar gene expression, but the PPR family in plants is notable for its enormous size. The Arabidopsis thaliana genome encodes 450 putative PPR proteins and the rice genome encodes more than 600 putative PPR proteins.^2-5 Most of the PPR proteins are localized to mitochondria and/or chloroplasts in plants, and involved in many aspects of RNA processing in these 2 organelles.^3,6,7 Only were 2 nucleus-localized PPR proteins identified thus far, which were suggested to regulate nuclear gene expression.^8,9 Currently, it remains largely unclear whether and how the PPR proteins regulate nuclear gene expression.

Abscisic acid (ABA) is a phytohormone that plays vital roles in many aspects of plant growth and development as well as in plant responses to a diversity of environmental stresses.^10-12 It has been well established that the chloroplast magnesium-protoporphyrin IX chelatase large subunit CHLH/ABAR (Mg-chelatase H subunit /putative ABA receptor) positively regulate ABA signaling likely as a candidate receptor for ABA in Arabidopsis. In the CHLH/ABAR-mediated signaling pathway, CHLH/ABAR interacts with, and antagonizes, a group of transcription factors WRKY40/18/60, to relieve ABA responsive genes, such as ABI4 and ABI5, of inhibition; however, a chloroplast protein cochaperonin CPN20 antagonizes in turn CHLH/ABAR to derepress ABA-responsive WRKY40 transcription repressor.^13-20 Interestingly, in a most recent report, we identified a cytosol-nucleus dual-localized PPR protein, SOAR1, as a crucial regulator of the ABA signaling pathway, which acts downstream of CHLH/ABAR and upstream of a nuclear ABA-responsive bZIP transcription factor ABA-INSENSITIVE5 (ABI5).²¹ Identification of SOAR1 supplements the CHLH/ABAR-mediated signaling pathway with a critical component, and suggesting high complexity of ABA signaling network (Fig. 1A and B). Although previously there were 5 mitochondrion-localized Arabidopsis PPR proteins identified as players of ABA signaling,^22-26 no cytosol- or nucleus-localized PPR protein was found to be involved in ABA signaling. Therefore, the discovery of the SOAR1 PPR protein supports the idea that, in addition to their roles in ABA signaling by regulating the mitochondrion gene expression, PPR proteins may also be involved in ABA signaling by regulating nuclear gene expression.

Figure 1.

(See previous page). SOAR1 is a crucial player in ABA signaling. (A) Overexpression of SOAR1 abolishes ABA responses of seeds and young seedlings. Seeds of the wild-type Col-0 plants and 2 SOAR1-overexpression lines (OE16 and OE17) were directly sowed in the MS medium supplemented with 3 (top), 100 (below the top), 200 (above the bottom) and 500 μM (±)ABA (bottom), and the growth was investigated 14 d or 20 d after stratification (as indicated). (B) A model proposed for the ABAR-mediated signaling. ABAR antagonizes WRKY40/18/60 transcription repressors to relieve ABA responsive genes of inhibition. The WRKYs negatively regulate ABA signaling and inhibit expression of ABA-responsive genes, such as ABA-responsive transcription factors including ABI4 and ABI5. High level of ABA promotes ABAR-WRKYs interaction, which downregulates WRKYs expression and relieves ABI4 and ABI5 genes of inhibition. The ABAR-WRKYs interaction is in turn inhibited by CPN20 that competitively interacts with ABAR. High level of ABA inhibits the ABAR-CPN20 interaction, promoting thus the ABAR-WRKY40 interaction to trigger the downstream signaling to repress WRKY40 expression. In addition to the ABAR/CPN20-WRKY40-ABI5 linked signaling pathway, SOAR1functions downstream of ABAR and upstream of ABI5 as a hub of ABA signaling to the nucleus. Arrows denote positive regulation or activation, and bars negative regulation or repression. The solid lines indicate direct effect, and dotted lines indicate indirect effect or yet unknown mechanism. Question mark indicates unconfirmed link.

We observed that downregulation and upregulation of SOAR1 expression alter expression of a subset of important ABA-responsive genes such as ABI1, ABI2, ABI3, ABI4, ABI5, ABF4, DREB2A, PYR1/RCAR11, PYL7/RCAR2, PYL9/RCAR1, RAB18, RD29A, RD29B, SnRK2.2 and SnRK2.3,²¹ among which ABI1, ABI2, ABI5, ABF4, PYR1/RCAR11, PYL7/RCAR2, PYL9/RCAR1, SnRK2.2 and SnRK2.3 are key components of the PYR/PYL/RCAR-mediated ABA signaling pathway, a well-characterized core ABA signaling pathway.^12,27-30 This suggests that SOAR1 may function as a node of crosstalk between PYR/PYL/RCAR- and CHLH/ABAR-mediated signaling pathways by regulating expression of the nuclear, key ABA-responsive genes.

Downregulation and upregulation of SOAR1 expression alter dramatically ABA sensitivity.²¹ Particularly and surprisingly, overexpression of SOAR1 almost completely impairs ABA responses in seed germination and seedling growth (Fig. 1A).²¹ Figure 1A shows ABA insensitive phenotypes of 2 SOAR1-overexpression lines (OE16 and OE17) in ABA-induced inhibition of seed germination and postgermination growth arrest. The OE16 and OE17 lines were generated with the same procedures as described previously by introducing the cauliflower mosaic virus (CaMV) 35S promoter-driven full length SOAR1 cDNA linked to GFP;²¹ immunoblot analysis detected the SOAR1-GFP fusion protein expressed in the 2 lines (data not shown). The wild type seeds can germinate but cannot continue to grow in 3-μM (±)ABA-containing medium, whereas the seeds of the SOAR1-overexpression lines can germinate and continue to grow in 100- and 200-μM (±)ABA-containing medium (Fig. 1A),²¹ and even germinate in 500-μM (±)ABA-containing medium (Fig. 1A). This intensity of ABA insensitivity greatly exceeds that of the previously-reported mutants or transgenic lines including abi1-1 dominant mutant, abi4, abi5 loss-of-function mutants, ABI2-overexpressor and triple knockout mutant of 3 SnRK2 members srk2dei. The srk2dei mutant seeds can germinate and continue to grow in the presence of 50 or 100 μM exogenous ABA, which is believed to completely abolish ABA response.^31-33

Such strong ABA insensitive phenotypes of the SOAR1-overexpression lines suggest that CHLH/ABAR mediates a central signaling pathway to regulate downstream gene expression through SOAR1, and that SOAR1, as a major player to modulate expression of nuclear, key ABA-responsive genes, functions as a hub for ABA signaling to the nucleus, likely linking PYR/PYL/RCAR- and CHLH/ABAR-mediated signaling pathways together. Further exploration of the detailed mechanisms of SOAR1 protein by which it regulates the nuclear events, will be of particular importance to elucidate functional mechanism of PPR proteins and highly complicated ABA signaling network.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed

Funding

This research was supported by the National Key Basic Research Program of China (2012CB114300-002), National Natural Science Foundation of China (grant nos. 31200213 and 31170268), and the Ministry of Agriculture of China (grant 2013ZX08009003).