The RING Finger Ubiquitin E3 Ligase SDIR1 Targets SDIR1-INTERACTING PROTEIN1 for Degradation to Modulate theSalt Stress Response and ABA Signaling in Arabidopsis

Huawei Zhang, Feng Cui, Yaorong Wu, Lijuan Lou, Lijing Liu, Miaomiao Tian, Yuese Ning, Kai Shu, Sanyuan Tang,and Qi Xie1

State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology,Chinese Academy of Sciences, Beijing 100101, China

The plant hormone abscisic acid (ABA) regulates many aspects of plant development and the stress response. Theintracellular E3 ligase SDIR1 (SALT- AND DROUGHT-INDUCED REALLY INTERESTING NEW GENE FINGER1) plays a key rolein ABA signaling, regulating ABA-related seed germination and the stress response. In this study, we found that SDIR1 islocalized on the endoplasmic reticulum membrane in Arabidopsis thaliana. Using cell biology, molecular biology, andbiochemistry approaches, we demonstrated that SDIR1 interacts with and ubiquitinates its substrate, SDIRIP1 (SDIR1-INTERACTING PROTEIN1), to modulate SDIRIP1 stability through the 26S proteasome pathway. SDIRIP1 acts geneticallydownstream of SDIR1 in ABA and salt stress signaling. In detail, SDIRIP1 selectively regulates the expression of thedownstream basic region/leucine zipper motif transcription factor gene ABA-INSENSITIVE5, rather than ABA-RESPONSIVEELEMENTS BINDING FACTOR3 (ABF3) or ABF4, to regulate ABA-mediated seed germination and the plant salt response.Overall, the SDIR1/SDIRIP1 complex plays a vital role in ABA signaling through the ubiquitination pathway.

INTRODUCTION

As sessile organisms, plants differ from animals as they mightencounter various environmental challenges, including bioticand abiotic stresses. Abiotic stresses, such as drought and highsalinity, severely affect plant growth and development and canimpair crop production worldwide. Plants have evolved severalstrategies to adapt to such challenges. Stress signals are trans-duced to activate stress response genes and alter metabolic ratesand ion channel permeability (Hasegawa et al., 2000; Zhu, 2002).Among these strategies, abscisic acid (ABA) plays a major roleby retarding seed germination, cotyledon greening, and plantgrowth, reducing the transpiration rate, and modulating cellularwater potential (Zhu, 2002; Tuteja, 2007).

Since the identification of ABA receptors, considerable prog-ress has been made in understanding ABA perception and thesignal transduction pathway (Raghavendra et al., 2010; Umezawaet al., 2010; Guo et al., 2011). Under normal conditions, PP2Cs(PROTEIN PHOSPHATASES TYPE 2C) keep SnRKs (SUCROSENONFERMENTING 1-RELATED PROTEIN KINASEs) in the in-active form via dephosphorylation (Guo et al., 2011). A high levelof ABA builds up under stress conditions (Nambara and Marion-Poll, 2005) and binds to the PYR/PYL/RCARs (PYRABACTINRESISTANCE 1/PYR-LIKE/REGULATORY COMPONENTS OFABA RECEPTORs), changing their conformational structures tofacilitate their interaction with PP2Cs. This interaction can inhibit

the phosphorylation activity of PP2Cs and release SnRKs fromPP2C inhibition (Ma et al., 2009; Park et al., 2009; Santiagoet al., 2009). In turn, SnRKs phosphorylate downstream ionchannels (Geiger et al., 2009; Lee et al., 2009; Sato et al., 2009)and transcription factors (Fujii et al., 2009) to trigger the ABAresponse.The ubiquitin/26S proteasome pathway plays a very important

role in hormone signaling, including the perception of auxin(Dharmasiri et al., 2005; Kepinski and Leyser, 2005), jasmonates(Sheard et al., 2010), and gibberellins (Murase et al., 2008), and insignal transduction in the ABA and ethylene pathways (Santnerand Estelle, 2009).E3 (ubiquitin ligase), which assists in the transfer of ubiquitin

from the E2 (ubiquitin-conjugating enzyme) ubiquitin interme-diate to the target protein, is the key factor that defines substratespecificity (Hare et al., 2003; Vierstra, 2003; Moon et al., 2004;Smalle and Vierstra, 2004; Vierstra, 2009). The Arabidopsis thalianagenome encodes over 1400 different E3s, including ;470 REALLYINTERESTING NEW GENE (RING)-type E3s (Stone et al., 2005;Vierstra, 2009). Increasing evidence has shown that RING-typeE3-mediated protein degradation by the ubiquitin-26S proteasomesystem plays an essential role in ABA pathways (Lee and Kim,2011). AIP2 (ABA-INSENSITIVE3 [ABI3] INTERACTION PROTEIN2)serves as a negative regulator of ABA on germination by promotingthe B3 domain transcription factor ABI3 for proteolysis (Zhanget al., 2005). KEEP ON GOING is a bifunctional E3 ligase, mediatingboth ABI5 ubiquitination and autoubiquitination of itself (Liu andStone, 2010).In previous studies, we isolated a RING-type E3, SDIR1

(SALT- AND DROUGHT-INDUCED RING FINGER1), which isinvolved in ABA-related high salinity and drought response (Zhanget al., 2007, 2008). SDIR1 expression is upregulated by high salinity

1 Address correspondence to qxie@genetics.ac.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Qi Xie (qxie@genetics.ac.cn).www.plantcell.org/cgi/doi/10.1105/tpc.114.134163

The Plant Cell, Vol. 27: 214–227, January 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

and drought. SDIR1 overexpression leads to hypersensitivity toABA and high salinity and ABA-related hyposensitivity to droughtby limiting the rate of transpiration. Biochemical and genetic evi-dence indicates that SDIR1 acts upstream of the basic region/leucine zipper motif (bZIP)-type transcription factors ABF3 (ABA-RESPONSIVE ELEMENTS BINDING FACTOR3), ABF4, and ABI5,which are key regulators in the ABA pathway (Zhang et al., 2007).

Here, we show that SDIR1 is an endoplasmic reticulum (ER)membrane-localized protein, with its C terminus facing the cy-toplasm. We identified a substrate of SDIR1, SDIRIP1 (SDIR1-INTERACTING PROTEIN1). Complementary to a previous report(Naponelli et al., 2008), we found that SDIRIP1 is localized notonly in the chloroplast but also in the cell periphery and nucleus.SDIRIP1 interacts with the C terminus of SDIR1 in the cytoplasm.SDIR1 acts upstream of ABI5, ABF3, and ABF4 by promotingtheir gene expression (Zhang et al., 2007). However, SDIRIP1selectively regulates the expression of ABI5 only. Collectively, wefound that SDIR1 regulates ABA and salt stress responses bynegatively affecting SDIRIP1 stability.

RESULTS

Identification of SDIRIP1 as an SDIR1 Interaction Protein

The RING-type E3 ligase SDIR1 plays important roles in the ABAsignaling pathway (Zhang et al., 2007). To identify targets andother interacting proteins of SDIR1, a yeast two-hybrid screenwas performed with mutated SDIR1H234YΔTM as the bait, inwhich two transmembrane domains were deleted and His-234was changed to Tyr-234 (Figure 1A). The H234Y point mutation,which disrupts the RING domain and ubiquitin ligase activity ofSDIR1, prevents SDIR1-mediated degradation of its substratesand facilitates substrate isolation. The transmembrane domaindeletion ensures that the GAL4 fusion protein is localized to thenucleus of the yeast. Among 45 positive clones, seven were char-acterized as containing the same protein (encoded by At5g51110),designated SDIRIP1 (Figure 1B).

To verify the interaction between SDIRIP1 and the full-lengthSDIR1, an in vitro pull-down assay was performed. [35S]Methionine-labeled SDIRIP1 was generated through in vitro transcription andtranslation. MBP (MALTOSE BINDING PROTEIN)-SDIR1 expressedin Escherichia coli was used as the bait, with MBP as the negativecontrol. MBP-SDIR1 was able to pull down [35S]methionine-labeledSDIRIP1, but not MBP, suggesting that SDIR1 might interactdirectly with SDIRIP1 (Figure 1C). We next assessed this in-teraction in plants by coimmunoprecipitation (Co-IP). As SDIR1could not be easily detected in transgenic Arabidopsis plants,we performed a Co-IP assay in Nicotiana benthamiana. 35S:GFP(green fluorescent protein)-SDIR1 and 35S:SDIRIP1-MYC weretransiently expressed in N. benthamiana leaves by agroinfiltration.Leaves infiltrated with Agrobacterium tumefaciens suspensionbuffer (10 mM MgCl2) were used as the negative control (mocksamples) (Liu et al., 2010). Total protein was extracted from leaftissues, and equal amounts of proteins from GFP-SDIR1 sampleswere mixed with the proteins extracted from the SDIRIP1-MYCsamples and mock samples. Co-IP assays were performed,and the anti-MYC antibody was able to coimmunoprecipitate

GFP-SDIR1 (Figure 1D). These results indicated that SDIR1could interact with SDIRIP1 in plants.

SDIRIP1 Interacts with SDIR1 in Vivo

The SDIRIP1 gene encodes a plant-specific type 2 PTERIN-4a-CARBINOLAMINE DEHYDRATASE (PCD), but without the PCDactivity (Naponelli et al., 2008). The function of SDIRIP1 re-mained unknown. In mammals, the single copy of PCD functionsas a bifunctional protein, as both a metabolic enzyme and a nuclearcofactor in transcriptional regulation (Suck and Ficner, 1996). It hasbeen reported that in Arabidopsis protoplasts, SDIRIP1-GFP waslocalized exclusively to the chloroplast (Naponelli et al., 2008). Asthe environment of protoplasts might affect protein localization, weaimed to determine the localization of SDIRIP1 in intact plant cells.35S:SDIRIP1-GFPwas expressed transiently inN. benthamiana. Toour surprise, in addition to the chloroplast, SDIRIP1-GFP also ex-isted in the cell periphery and nucleus-like structures (Figure 2A).Coexpression of 35S:SDIRIP1-GFP with 35S:HY5-RFP (red fluo-rescent protein), encoding the nucleus-localized transcription factorHY5, demonstrated that part of SDIRIP1 was also localized to thenucleus (Figure 2B). To confirm this result, we isolated nuclear andchloroplastic fractions from total protein samples. SDIRIP1-GFPcould be detected in the nuclear fraction, which was indicated bythe anti-histone H3 antibody, and in the chloroplastic fraction,which was indicated by the anti-PsbA (Photosystem II ReactionCenter Protein A) antibody (Supplemental Figure 1). These resultsshowed that SDIRIP1 was localized not only to the chloroplast butalso to the cell periphery and nucleus.The subcellular localization of SDIRIP1 prompted us to examine

the SDIR1-SDIRIP1 interaction at the intracellular level. It hasbeen reported that SDIR1 is an intracellular membrane-localizedprotein (Zhang et al., 2007). We first examined the subcellularlocalization of SDIR1 in detail. 35S:GFP-SDIR1 was transientlyexpressed in N. benthamiana leaves and Arabidopsis leaf proto-plasts. Green fluorescence was detected in a net-like compart-ment, which is similar to the ER. A coexpression assay showedthat GFP-SDIR1 was colocalized with RFP-HDEL, an ER locali-zation marker, demonstrating that GFP-SDIR1 is at least locatedon the ER membrane, although SDIR1 localization on othertypes of intracellular membranes cannot be excluded (Figures2C and 2D).According to the yeast two-hybrid results, the C terminus of

SDIR1 behind the second transmembrane domain can interactwith SDIRIP1 (Figures 1A and 1B). To deeply investigate thesubcellular localization of this interaction, it is essential to checkthe orientation of SDIR1 across the membrane. As shown inSupplemental Figure 2, there are two possibilities. The C terminusof SDIR1 exists in the cytoplasm or ER lumen. To address this,we utilized a fluorescence protease protection (FPP) assay(Lorenz et al., 2006a, 2006b, 2008). We transiently coexpressed35S:RFP-HDEL and 35S:SDIR1-GFP in Arabidopsis protoplasts.RFP-HEDL, which resides in the ER lumen, was used as a con-trol. We treated these protoplasts with 50 mM digitonin for 5 min;digitonin can permeabilize the plasma membrane but not the ERmembrane. Subsequently, protoplasts were exposed to 4 mMtrypsin. The ER membrane can protect RFP-HDEL from di-gestion by trypsin. However, proteins in the cytoplasm would be

SDIR1 Destabilizes SDIRIP1 in ABA Signaling 215

digested. Images were taken before and after trypsin treatment.After trypsin treatment, the fluorescence from SDIR1-GFP quicklyvanished, while the fluorescence from RFP-HDEL did not change(Figure 2E). This result indicated that the C terminus of SDIR1faces the cytoplasm.

Next, we performed a bimolecular fluorescence complemen-tation (BiFC) assay (Waadt et al., 2008) to check the interactionbetween SDIRIP1 and SDIR1. The fluorescent protein VENUSwas spliced into the N-terminal fraction (VYNE) and the C-terminalfraction (VYCE). VYNE was fused to the N terminus of SDIR1 to

Figure 1. SDIRIP1 Interacts with SDIR1.

(A) Schematic illustration of the structures of SDIR1 and SDIR1ΔTM (containing an 81-amino acid deletion on the N terminus). TM, transmembranedomain.(B) Interaction between SDIRIP1 and SDIR1H234YΔTM verified by yeast two-hybrid assays. In SDIR1H234YΔTM, His-234 of SDIR1 was mutated to Tyr-234 and the transmembrane domains of SDIR1 were deleted. BD, bait vector pGBKT7; AD, prey vector pGADT7; BD/SDIR1H234YΔTM, SDIR1H234YΔTMfused to pGBKT7 as the bait; AD/SDIRIP1, SDIRIP1 fused to pGADT7 as the prey. Yeast was grown on synthetic dropout medium lacking leucine andtryptophan (SD-L-W) and synthetic dropout medium lacking leucine, tryptophan, and histidine and containing 2 mM 3-aminotriazole (SD-L-W-H + 2 mM3-AT) (selective medium).(C) Pull-down assays for analyzing the interaction between SDIRIP1 and SDIR1 in vitro. MBP-SDIR1 was used as the bait to pull down [35S]methionine-labeled SDIRIP1. The MBP tag was used as the negative control. An equal amount of SDIRIP1 was used in pull-down assays and as the input control.Input and bound forms of SDIRIP1 were detected by autoradiography. The relative amount of bound SDIRIP1 was normalized by using input asa control. Immobilized MBP and MBP-SDIR1 were detected by Coomassie blue straining.(D) Co-IP assays of SDIR1 with SDIRIP1. SDIRIP1-MYC and GFP-SDIR1 were transiently expressed in N. benthamiana leaves. Co-IP assays wereperformed at 4°C, and protein gel blot analysis was performed using anti-MYC antibody. The asterisk indicates the modified forms of SDIRIP1-MYC.The top panel shows the input control.

216 The Plant Cell

generate VN(R)-SDIR1, and VYCE was fused to the C terminus ofSDIRIP1 to generate SDIRIP1-VC. However, no reconstitutedfluorescence was detected (Figure 3A; Supplemental Figures3A and 3B). It is possible that SDIRIP1 was degraded whenassociated with SDIR1. To prove this hypothesis, we treated

the VN(R)-SDIR1/SDIRIP1-VC samples with the 26S proteasomeinhibitor MG132. After MG132 treatment, the reconstituted fluo-rescence was observed to be colocalized with RFP-HDEL (Figure3B). We previously reported that the SDIR1 E3 ligase activity wascompletely abolished in the H234Y mutant of SDIR1 (Zhang et al.,2007). This mutation did not disrupt the SDIR1-SDIRIP1 interactionin the yeast two-hybrid assays (Figures 1A and 1B). Meanwhile,GFP-SDIR1H234Y was still localized to the ER (Figure 2D). In ad-dition, the BiFC assays showed that reconstituted fluorescencewas only detected in the VN(R)-SDIR1H234Y/SDIRIP1-VC samples(Figures 3C and 3D; Supplemental Figure 3C), indicating thatSDIR1H234Y can interact with SDIRIP1 in vivo. Importantly, thereconstituted fluorescence was also not colocalized with thechloroplast but with RFP-HDEL (Figures 3C and 3D).Meanwhile, two other assays were performed. First, 35S:GFP-

SDIR1 or pVIP (vector control) was coexpressed with 35S:SDIRIP1-MYC and 35S:RFP (internal control). SDIRIP1-MYCcould not be detected at all when coexpressed with 35S:GFP-SDIR1, while MG132 treatment increased the protein level ofSDIRIP1. No significant difference was observed between theSDIRIP1 RNA levels of the three samples (Figure 3E). Togetherwith the yeast two-hybrid results, we came to the conclusionthat the C terminus of SDIR1 in the cytoplasm interacts withSDIRIP1. Because SDIR1 is localized on the ER membrane, theBiFC fluorescence was colocalized with the ER marker RFP-HDEL. Therefore, SDIR1 and SDIRIP1 interacted in the cytosolbut appeared to be associated with the ER.

SDIRIP1 Is Unstable and Degraded Partially by the26S Proteasome

Because ATP is essential for ubiquitination and 26S proteasome-dependent degradation (Smalle and Vierstra, 2004), we examinedthe influence of ATP on the stability of SDIRIP1. Total proteinwas extracted from a N. benthamiana sample transiently ex-pressing 35S:SDIRIP1-MYC and incubated at room temper-ature with or without ATP. During incubation, SDIRIP1 shifted tohigher molecular weight forms (Supplemental Figure 4A), and addingATP promoted the degradation of SDIRIP1 (Supplemental Figure4A). To further demonstrate the function of the 26S proteasomeon the degradation of SDIRIP1, we checked whether MG132can attenuate the degradation of SDIRIP1. Because the SDIRIP1level in the former sample was too high, we chose another samplewith a lower SDIRIP1 level. Total protein was extracted from thisN. benthamiana sample, which transiently expressed 35S:SDIRIP1-MYC, and the protein was incubated at room temperature withMG132 or its solvent DMSO as the control. Nearly all SDIRIP1-MYCshifted to higher molecular weight forms after a 15-min incubationand then disappeared quickly after a 30/60-min incubation. MG132could delay the rate of SDIRIP1-MYC degradation, suggesting thatSDIRIP1 is degraded at least partially by the 26S proteasome(Supplemental Figure 4B). To identify the features of the modifiedform of SDIRIP1-MYC, we immunoprecipitated SDIRIP1-MYC andMYC-GFP from transiently expressed samples using anti-MYCantibody. Using anti-ubiquitin antibody, we were able to detect theubiquitinated form of SDIRIP1-MYC but not MYC-GFP in theimmunoprecipitates (Supplemental Figure 4C), indicating thatSDIP-MYC could be ubiquitinated in cell extracts.

Figure 2. Subcellular Localization of SDIRIP1 and SDIR1.

(A) to (D) Different GFP fusion proteins expressed in N. benthamianaleaves. Red autofluorescence, chloroplast marker; HY5-RFP, nucleusmarker; RFP-HDEL, ER marker. In (A) and (B), the red arrows indicate thenucleus and the blue arrows indicate the cell periphery. In (C) and (D),images in the left bottom boxes are magnifications of the selected areas.Shown are SDIRIP1-GFP and chloroplast autofluorescence (A), SDIR-IP1-GFP and HY5-RFP (B), GFP-SDIR1and RFP-HDEL (C), and GFP-SDIR1H234Y and RFP-HDEL (D). Bars = 50 mm.(E) FPP assays. Arabidopsis protoplasts coexpressing GFP-SDIR1 andRFP-HDEL were treated with 50 mM digitonin for 5 min and then exposedto 4 mM trypsin for 1 min. Images were taken before and after trypsintreatment. Bars = 50 mm.

SDIR1 Destabilizes SDIRIP1 in ABA Signaling 217

To investigate the stability of SDIRIP1 in Arabidopsis, wegenerated 35S:SDIRIP1-MYC transgenic Arabidopsis plants inboth Columbia-0 (Col-0) and sdir1-1 backgrounds. As SDIR1expression levels are very low under normal growth conditionsand this gene can be induced by NaCl treatment (Zhang et al.,2007), we then checked the stability of SDIRIP1 with or withoutNaCl treatment in the presence of cycloheximide (CHX), whichcan block new protein synthesis. Figure 4A shows that SDIRIP1was slightly unstable under normal growth conditions and thatNaCl treatment can dramatically increase the degradation ofSDIRIP1.

Meanwhile, MG132 could delay but not completely inhibit thedegradation of SDIRIP1 (Figure 4B). This result indicates that the26S proteasome is one but not the only pathway for SDIRIP1degradation. It has been reported that autophagy plays an im-portant role in the degradation of chloroplast proteins (Wadaet al., 2009; Dong and Chen, 2013). E64d, an inhibitor of lysosomal/vacuolar hydrolases, can dramatically inhibit autophagy (Bassham,2007). Therefore, we also examined the influence of E64d on thestability of SDIRIP1 under NaCl treatment. Figure 4C shows thatE64d can efficiently decrease the degradation rate of SDIRIP1,indicating that SDIRIP1 might also be degraded through theautophagy pathway.

As SDIR1 also participates in the ABA signaling pathway, wethen checked the stability of SDIRIP1 with or without ABA

treatment in the presence of CHX. To our surprise, ABA treat-ment could inhibit the degradation of SDIRIP1 (SupplementalFigure 5A). However, the mechanism remains unclear.

SDIRIP1 Is a Substrate of SDIR1

As SDIR1 is an active RING finger ligase and SDIRIP1 degra-dation is partially dependent on the 26S proteasome pathway,we investigated whether SDIR1 affected the stability of SDIRIP1in Arabidopsis. We selected one Col-0/35S:SDIRIP1-MYC lineand one sdir1-1/35S:SDIRIP1-MYC line with comparable SDIRIP1-MYC protein levels for a half-life study of SDIRIP1 in the pres-ence of CHX. Figure 4D and Supplemental Figure 5B show thatunder both normal growth conditions and NaCl treatment, theSDIR1 null mutation could efficiently delay SDIRIP1 degradation.These data suggested that SDIR1 negatively regulated the sta-bility of SDIRIP1. Together with the coexpression assay resultsin N. benthamianamentioned above, we come to the conclusionthat SDIR1 can promote the degradation of SDIRIP1 by the 26Sproteasome.To verify our speculation that SDIRIP1 is a substrate of SDIR1,

we conducted an in vitro ubiquitination assay. SDIRIP1 wasexpressed in E. coli as a fusion protein with MYC and GST(GLUTATHIONE S-TRANSFERASE) tags, and SDIR1 was ex-pressed with an MBP tag. Figure 4E and Supplemental Figure 6

Figure 3. Coinfiltration Assays and Interaction between SDIRIP1 and SDIR1 in Cells.

(A) to (D) BiFC assays were performed on N. benthamiana leaves by agroinfiltration and transfected with different constructs. Images show thecolocalization of RFP-HDEL, an ER marker, with reconstituted fluorescent protein. In (B) and (C), images in the left bottom boxes are magnifications ofthe selected areas. In (B), MG132 was injected into the leaf tissues 12 h before observation. Bars = 50 mm.(E) SDIR1 promoted SDIRIP degradation. Coinfiltration expression of SDIR1 and SDIRIP1 was performed on N. benthamiana leaves. Top three panels,protein accumulation of SDIRIP1-MYC, GFP-SDIR1, and RFP. Bottom panels, RNA levels of SDIRIP1 and RFP.

218 The Plant Cell

Figure 4. SDIRIP1 Is a Substrate of SDIR1.

(A) to (D) Protein gel blot results of one experiment. Anti-PAG1 (20S proteasome a-subunit G1) was used as the loading control; SDIRIP1-MYC proteinlevel was normalized. Three independent biological repeats were performed and analyzed. Fourteen-day-old seedlings were used in these assays. Errorbars represent SD.

SDIR1 Destabilizes SDIRIP1 in ABA Signaling 219

show that SDIRIP1 was ubiquitinated by SDIR1 in the presenceof E1, E2, and ubiquitin. When any of these proteins in the re-action was omitted, the ubiquitinated form of SDIRIP1 was notfound. These data directly demonstrated that SDIRIP1 is a substrateof SDIR1.

SDIRIP1 Is Involved in the Plant Response to Salt Stress andABA Signaling

In a previous study, we demonstrated that the sdir1 mutant al-leles are less sensitive to salt stress and ABA treatment than thewild type, whereas higher sensitivity to drought stress andSDIR1 overexpression can render opposite phenotypes (Zhanget al., 2007). To answer whether SDIRIP1 works with SDIR1 inthe same physiological regulation pathway, we used a reversegenetic approach to study the function of SDIRIP1. We gene-rated 35S:SDIRIP1 and 35S:SDIRIP1-RNAi transgenic plants ina Col-0 ecotype background due to the lack of SDIRIP1 mutantlines among all available stocks. Two 35S:SDIRIP1 lines (Col-0/35S:SDIRIP1 20.1 and Col-0/35S:SDIRIP1 12.3) and two 35S:SDIRIP1-RNAi lines (Col-0/35S:SDIRIP1-RNAi 4.8 and Col-0/35S:SDIRIP1-RNAi 1.5) were selected for further study. TheSDIRIP1 transcript level in the transgenic lines was much higherin the overexpression lines and lower in the RNA interference(RNAi) lines than in wild-type plants (Supplemental Figure 7A).The seeds of wild-type and transgenic plants were germinatedon half-strength Murashige and Skoog (MS) medium containing0, 100, 125, or 150 mM NaCl or 1 or 2 mM ABA.

In the presence of 100 mM NaCl, none of the Col-0/35S:SDIRIP1-RNAi plants developed true leaves at 14 d, whereas allof the Col-0/35S:SDIRIP1 plants developed true leaves (Figure5A). In the presence of 100 and 125 mM NaCl, germination ofCol-0/35S:SDIRIP1 plants was less sensitive than that of wild-type plants. In the presence of 100 mM NaCl, after 2 d, only7.8% of Col-0/35S:SDIRIP1-RNAi 4.8 and 12.6% of Col-0/35S:SDIRIP1-RNAi 1.5 seeds germinated, but 19.6% of wild-type,23.1% of Col-0/35S:SDIRIP1 20.1, and 40.1% of Col-0/35S:SDIRIP1 12.3 seeds germinated (Supplemental Figure 7B). In thepresence of 150 mM NaCl, after 3 d, only 31.4% of Col-0/35S:SDIRIP1-RNAi 4.8 and 34.8% of Col-0/35S:SDIRIP1-RNAi 1.5seeds germinated, but 64.4% of wild-type, 65.7% of Col-0/35S:SDIRIP1 20.1, and 69.4% of Col-0/35S:SDIRIP1 12.3 seedsgerminated (Supplemental Figure 7B). At this concentration,the germination rate of Col-0/35S:SDIRIP1 plants was similar tothat of wild-type plants. The reason could be that high salt

concentrations might promote SDIRIP1 degradation by inducingthe high expression of SDIR1 in plants. To examine the saltsensitivity of the plants in soil, one RNAi line and one over-expression line were chosen. Seeds were stratified in darknessfor 4 d at 4°C and then sown in soil with or without differentconcentrations of NaCl. After 24 d of growth in soil, photographswere taken (Supplemental Figure 8A). NaCl treatment can dra-matically inhibit plant growth, and SDIRIP1 RNAi plants weremore sensitive to salt stress than wild-type and SDIRIP1 over-expression plants (Supplemental Figure 8B). These results in-dicated that SDIRIP1 could negatively regulate the sensitivity ofplants to salt stress.We also examined the ABA sensitivity of these plants. Com-

pared with wild-type plants, Col-0/35S:SDIRIP1 plants wereless sensitive to ABA, whereas Col-0/35S:SDIRIP1-RNAi plantswere more sensitive (Supplemental Figure 7). In the presenceof 1 mM ABA, after 4 d, only 16.8% of Col-0/35S:SDIRIP1-RNAi4.8 and 16.9% of Col-0/35S:SDIRIP1-RNAi 1.5 seeds germi-nated, but 39.1% of wild-type, 62.3% of Col-0/35S:SDIRIP120.1, and 68.8% Col-0/35S:SDIRIP1 12.3 seeds germinated(Supplemental Figure 7B). In the presence of 1 mM ABA, after10 d, only 43.6% of Col-0/35S:SDIRIP1-RNAi 4.8 and 57.3%of Col-0/35S:SDIRIP1-RNAi 1.5 plants had small green cotyle-dons, but 67.4% of wild-type, 87.1% of Col-0/35S:SDIRIP120.1, and 95.7% of Col-0/35S:SDIRIP1 12.3 plants had smallgreen cotyledons (Supplemental Figure 7C). These results in-dicated that SDIRIP1 could negatively regulate the sensitivity ofplants to ABA.

SDIRIP1 Acts Genetically Downstream of SDIR1 in ABA andSalt Stress Responses

To analyze the genetic relationship between SDIR1 and SDIRIP1,we generated sdir1-1/35S:SDIRIP1-RNAi transgenic plants andselected two SDIRIP1 knockdown lines for further studies (sdir1-1/35S:SDIRIP1-RNAi 8.3 and sdir1-1/35S:SDIRIP1-RNAi 9.10)(Supplemental Figure 9A). Col-0, sdir1-1, and sdir1-1/35S:SDIRIP1-RNAi plants were germinated on half-strength MS medium con-taining 0, 100, 125, or 150 mM NaCl or 1 or 2 mM ABA. Figure 5Band Supplemental Figure 9B show that the knockdown of SDIRIP1expression could rescue the ABA- and salt-insensitive phenotypeof Col-0, sdir1-1, and sdir1-1/35S:SDIRIP1-RNAi plants germinatedunder ABA or NaCl treatment. Compared with wild-type plants,sdir1-1 was less sensitive to ABA and salt stress (Figure 5B;Supplemental Figures 9B and 9C). In the presence of 100 mM

Figure 4. (continued).

(A) Seedlings were pretreated in half-strength MS medium with or without 300 mM NaCl for 3 h and then incubated in the same medium with 50 mMCHX. Samples were collected 0, 2, 4, and 6 h after treatment.(B) Seedlings were pretreated in half-strength MS medium with 300 mM NaCl for 3 h and then incubated in the same medium with or without 75 mMMG132 in the presence of 300 mM NaCl and 50 mM CHX. Samples were collected 0, 2, 4, and 6 h after treatment.(C) Seedlings were pretreated in half-strength MS medium with 300 mM NaCl for 3 h and then incubated in the same medium with or without 10 mME64d in the presence of 300 mM NaCl and 50 mM CHX. Samples were collected 0, 2, 4, and 6 h after treatment.(D) Col-0/35S:SDIRIP1-MYC and sdir1-1/35S:SDIRIP1-MYC seedlings were pretreated in half-strength MS medium with 300 mM NaCl and thenincubated in the same medium with 50 mM CHX. Samples were collected 0, 3, and 6 h after treatment.(E) Ubiquitination of SDIRIP1 by SDIR1 in vitro. The protein gel blot was analyzed using anti-MYC antibody.

220 The Plant Cell

NaCl, after 2 d, 98.9% of sdir1-1 seeds germinated, but 69.2%of wild-type, 72.2% of sdir1-1/35S:SDIRIP1-RNAi 9.10, and32.9% of sdir1-1/35S:SDIRIP1-RNAi 8.3 seeds germinated(Supplemental Figure 9B). In the presence of 1 mM ABA, after 3 d,97.8% of sdir1-1 seeds germinated, but 66.7% of wild-type,70.8% of sdir1-1/35S:SDIRIP1-RNAi 9.10, and 4.3% of sdir1-1/35S:SDIRIP1-RNAi 8.3 seeds germinated (Supplemental Figure9B). After 10 d, all sdir1-1 plants had small green cotyledons,while 85.2% of wild-type, 70.1% of sdir1-1/35S:SDIRIP1-RNAi9.10, and 30.3% of sdir1-1/35S:SDIRIP1-RNAi 8.3 plants had smallgreen cotyledons (Supplemental Figure 9C). ABA and salt sen-sitivity, which were reduced in the sdir1-1 mutant, were rescuedby 35S:SDIRIP1-RNAi transformation, indicating that SDIRIP1acts downstream of SDIR1. Together, the biochemical and geneticevidence above further confirmed that SDIRIP1 is a substrateof SDIR1.

SDIRIP1 Selectively Regulates Different bZIP TranscriptionFactors Involved in ABA Signaling

Because two Col-0/35S:SDIRIP1 lines behaved similarly undersalt stress and ABA treatment, as did the Col-0/35S:SDIRIP1-RNAi lines, we selected Col-0/35S:SDIRIP1 12.3 and Col-0/35S:SDIRIP1-RNAi 4.8, respectively, for further study. We previouslyreported that SDIR1 acted upstream of ABF3, ABF4, and ABI5by positively regulating their expression under ABA treatment(Zhang et al., 2007). To investigate the function of SDIRIP1 in theABA signaling pathway, we treated Col-0, Col-0/35S:SDIRIP1,and Col-0/35S:SDIRIP1-RNAi plants with 50 mM ABA for 0, 1, 2,and 3 h and measured ABF3, ABF4, and ABI5 expression. ABI5was induced after ABA treatment in all lines. A significant dif-ference in ABI5 levels was observed after ABA treatment.Compared with the wild type, the ABI5 mRNA level was lower inCol-0/35S:SDIRIP1 but higher in the Col-0/35S:SDIRIP1-RNAitransgenic line. By contrast, the expression patterns of ABF3and ABF4 were similar among these different lines (Figure 6A).These data indicated that ABA induction of ABI5 was attenuatedby SDIRIP1 specifically and that SDIRIP1 has no significant effectson ABF3 and ABF4 expression.To investigate whether SDIRIP1 indeed acts upstream of

ABI5, we generated abi5-1/35S:SDIRIP1-RNAi transgenic plants(Supplemental Figure 10A). Wassilewskija (Ws) wild-type, abi5-1,and abi5-1/35S:SDIRIP1-RNAi seeds were germinated on half-strength MS medium containing 0, 1, or 2 mM ABA and 150 mMNaCl. The abi5-1 plants were insensitive to ABA and salt stress,whereas SDIRIP1-RNAi produced ABA- and salt-sensitive phe-notypes in wild-type plants (Figure 5A; Supplemental Figure 7B).However, compared with Ws, two abi5-1/35S:SDIRIP1-RNAi lineswere more insensitive to ABA and NaCl treatment, and no sig-nificant difference was observed between the two abi5-1/35S:SDIRIP1-RNAi lines (abi5-1/35S:SDIRIP1-RNAi 3.3 and abi5-1/35S:SDIRIP1-RNAi 8.4) and abi5-1 (Figure 6B; SupplementalFigure 10B). For example, in the presence of 2 mM ABA, after10 d, only 26% of Ws wild-type plants had green cotyledons, butall abi5 and abi5/35S:SDIRIP1-RNAi plants had small greencotyledons (Supplemental Figure 10C). ABA and salt sensitivity,which was enhanced by 35S:SDIRIP1-RNAi, was rescued by theABI5 mutation, indicating that SDIRIP1 acts upstream of ABI5.Previously, we found that SDIR1 overexpression could en-

hance the drought tolerance ability of plants by reducing therate of leaf water loss (Zhang et al., 2007). Therefore, wemeasured the rate of water loss in detached rosette leavesfrom Col-0/35S:SDIRIP1, Col-0/35S:SDIRIP1-RNAi, and wild-type plants. However, no obvious difference was observed(Supplemental Figure 11A). To confirm this observation, wechecked whether SDIRIP1 could influence the closure ofstoma under ABA treatment. Again, no obvious difference wasobserved (Supplemental Figure 11B). These data suggest thatSDIRIP1 might not be involved in the regulation of the sto-matal response to ABA. This result is correlated to the dif-ferent effects of SDIRIP1 on different bZIP transcriptionfactors. SDIRIP1 has little influence on the expression of bothABF3 and ABF4, which are known to play major roles in theplant drought response. However, the detailed mechanism isunclear.

Figure 5. SDIRIP1 Functions with SDIR1 in the ABA and Salt ResponsePathways.

(A) Seeds were grown on half-strength MS medium or half-strength MSmedium supplemented with 100 mM NaCl or 2 mM ABA. Images werecaptured 14 d after stratification. Bars = 0.5 cm.(B) Seeds were sown on half-strength MS medium and half-strength MSmedium containing 125 mM NaCl or 1 mM ABA. Images were captured14 d after stratification. Bars = 0.5 cm.

SDIR1 Destabilizes SDIRIP1 in ABA Signaling 221

DISCUSSION

Previously, we demonstrated that SDIR1 is an intracellularmembrane-localized ubiquitin ligase (E3) that acts upstream ofmany transcription factors, including ABF3, ABF4, and ABI5, inthe ABA pathway and is involved in plant responses to salt anddrought stress (Zhang et al., 2007). In this work, we identifiedthe chloroplast/nuclear protein SDIRIP1, which acts with SDIR1 inthe ABA signaling pathway.

SDIRIP1 Is Localized to the Chloroplast, Cell Periphery,and Nucleus

A previous report showed that SDIRIP1 is an Arabidopsis ho-molog of the bifunctional protein PCD/DCoH (DIMERIZATIONCOFACTOR OF HEPATOCYTE NUCLEAR FACTOR1), with dualsubcellular location (Naponelli et al., 2008). The sole copy ofPCD/DCoH has PCD activity in the mitochondria and also actsas DCoH in the nucleus in mammals (Suck and Ficner, 1996).Plants contain two copies of PCD/DCoH homologs (Naponelli

et al., 2008). Type 1 has been demonstrated to have PCD ac-tivity and is localized in the mitochondria. However, biochemicalanalysis showed that SDIRIP1 encodes a type 2 plant-specificPCD/DCoH without PCD activity (Naponelli et al., 2008). Thissuggests that the two copies of PCD/DCoH homologs in plantsmight possess different functions. One conserved copy mightshare the function of mammalian PCD/DCoH in the mitochon-dria. The other copy might act as a dimerization cofactor witha protein in the nucleus. The localization of the type 2 plant-specific PCD/DCoH, which was reported to be exclusive to thechloroplast (Naponelli et al., 2008), casts doubt on this premise.We were able to demonstrate that SDIRIP1 is not only localizedin the chloroplast but also in the cell periphery and nucleus,supporting the hypothesis of the other conserved function ofPCD/DCoH between mammals and plants. Whether SDIRIP1acts as a dimerization cofactor in the nucleus is a question forfuture studies.Several other Arabidopsis proteins have been shown to have

chloroplast and nucleus dual localization (Silva-Filho, 2003;

Figure 6. SDIRIP1 Selectively Regulates Different bZIP Transcription Factors in the ABA Signaling Pathway.

(A) Expression levels of ABI5, ABF3, and ABF4. Fourteen-day-old seedlings were treated with 50 mM ABA for different time ranges. Actin7 was used asthe internal control. Error bars represent SD. Asterisks indicate P < 0.01.(B) Seeds of abi5-1, abi5-1/35S:SDIRIP1-RNAi, and the Ws wild type were sown on half-strength MS medium and half-strength MS medium containing150 mM NaCl or 2 mM ABA. Images were obtained 4 d after stratification. Bars = 0.5 cm.

222 The Plant Cell

Caplan et al., 2008; Chen et al., 2010; Shang et al., 2010; Sunet al., 2011; Krause et al., 2012). The dual localization may beessential for signal communication at the intracellular level.However, our knowledge of the dual localization mechanism isquite limited. Via a transplastic method, it has been proven thatWHIRLY1 is translocated from the chloroplast to the nucleus,where it affects the expression of PATHOGENESIS-RELATEDPROTEIN 1 (Isemer et al., 2012). PTM is another chloroplastenvelope-bound transcription factor that possesses trans-membrane domains. In response to retrograde signals, PTM iscleaved and the N terminus of PTM is transported to the nucleusto activate ABI4 transcription (Sun et al., 2011). Protein shuttlesbetween other organelles and the nucleus have also been ob-served in signal transduction. A similar cleavage mechanismwas found in the ER-to-nucleus translocation of ETHYLENEINSENSITIVE2 (EIN2) (Ju et al., 2012; Qiao et al., 2012; Wenet al., 2012) to activate ethylene signaling. It is also possible thata chloroplast and nucleus dual localized protein is directlytargeted to the nucleus rather than to the nucleus after trans-lation. The mechanism of the multiple subcellular localization ofSDIRIP1 remains elusive and will be the focus of a future study.

SDIRIP1-SDIR1 Interaction in Vivo

SDIR1 is located on the ER membrane, while SDIRIP1 is achloroplast-, cell periphery-, and nucleus-localized protein.Where does their interaction take place? No interaction could beobserved between intact SDIR1 and SDIRIP1 in the BiFC as-says. This result was consistent with that of the coexpressionassay, in which SDIRIP1 was not detected when coexpressedwith SDIR1. This might have been due to the interaction be-tween SDIR1 and SDIRIP1 quickly driving SDIRIP1 degradationvia the 26S proteasome. Similarly, Ning et al. (2011) describedthat the colocalization of the rice (Oryza sativa) RING finger E3ligase OsDIS1 (DROUGHT-INDUCED SEVEN IN ABSENTIAPROTEIN1) with its substrate Os-Nek6 (NEVER-IN-MITOSIS/ASPERGILLUS-RELATED KINASE6) was almost undetectablein vivo, but Os-DIS1H71Y, losing E3 activity due to a point mu-tation in the RING motif, was well colocalized with Os-Nek6.SDIR1H234Y, which destroyed the E3 activity of SDIR1 but re-mained in the same cellular localization as SDIR1, could stillinteract with SDIRIP1 in yeast two-hybrid assays. Hence, weperformed a BiFC assay using SDIR1H234Y and SDIRIP1.Meanwhile, after treatment with MG132, we also detected theinteraction between SDIR1 and SDIRIP1 by BiFC. Confocalobservation showed that the fluorescence of BiFC can colocalizewith RFP-HDEL. It seems that SDIRIP1 is recognized by SDIR1 onthe ER membrane.

Most chloroplastic and nuclear proteins are translated in thecytoplasm in an ER-independent manner and are transported tothe chloroplast and the nucleus by the transit peptide and thenuclear localization signal (Inaba and Schnell, 2008; Zybailovet al., 2008). Moreover, some proteins can be translocated fromthe chloroplast to the nucleus (Isemer et al., 2012), possiblyvia the cytoplasm. Yeast two-hybrid assays indicated that theC terminus of SDIR1 interacts with SDIRIP1, and the FPP assaysdemonstrated that the C terminus of SDIR1 faces the cyto-plasm. Therefore, it is possible that the C terminus of SDIR1

recruits SDIRIP1 in the cytoplasm and then SDIRIP1 is degradedby the 26S proteasome. Because SDIR1 is localized on theER membrane, the fluorescence from BiFC can colocalize withRFP-HDEL.

SDIRIP1 Is a Substrate of SDIR1

We identified SDIRIP1 as an SDIR1-interacting protein. MG132retarded the degradation of SDIRIP1. In addition, we found thatSDIR1 could ubiquitinate SDIRIP1 in vitro and promote its deg-radation in vivo, indicating that SDIRIP1 is a substrate of SDIR1.Meanwhile, phenotypic and genetic analyses showed that RNAiknockdown of the SDIRIP1 RNA level could rescue the hypo-sensitive phenotype of the sdir1mutant under salt stress and ABAtreatment.However, SDIRIP1 can be degraded in other pathways.

SDIRIP1 was more stable in the sdir1-1 mutant than in the wild-type plants; however, SDIRIP1 in the sdir1-1 mutant could stillbe degraded under salt stress. This suggests that perhaps otherE3 ligase complexes, rather than SDIR1 only, participate inSDIRIP1 degradation or that SDIRIP1 is degraded by anotherprotein turnover mechanism in addition to the 26S proteasomepathway, as MG132 could not inhibit the degradation of SDIR-IP1 completely.It has been reported that chloroplast protein could be de-

graded by autophagy (Wada et al., 2009; Dong and Chen, 2013).E64d, an inhibitor of autophagy, could dramatically delay thedegradation of SDIRIP1, indicating that SDIRIP1 might also bedegraded partially by autophagy. However, the detailed mech-anism remains unknown.We also found that ABA could stabilize SDIRIP1. The mech-

anism remains unclear. It is possible that there is a negativefeedback loop regulated by ABA. ABA might be able to de-stabilize SDIR1 or inhibit SDIR1’s activity by another mecha-nism. It is also possible that ABA could inhibit other degradationpathways, such as autophagy.

SDIRIP1 Acts Upstream of ABI5 in ABA Signaling

In our previous work (Zhang et al., 2007), we found that SDIR1-regulated ABA affected seed germination, salt sensitivity, as wellas ABA-dependent stomata movement. In this work, we foundthat the substrate SDIRIP1 only affected the expression ofABI5 but not ABF3 or ABF4, both of which function in ABA-dependent drought responses. ABI5 is a positive regulator inthe ABA signaling pathway. The abi5 mutant exhibits severehyposensitivity to ABA and salt stress (Carles et al., 2002),and overexpression of ABI5 increases drought toleranceability by reducing the rate of transpiration (Lopez-Molinaet al., 2001). Phenotypic analysis showed that, comparedwith wild-type plants, Col-0/35S:SDIRIP1 plants were moreresistant to salt stress and ABA, whereas Col-0/35S:SDIRIP1-RNAiplants were more sensitive to these treatments. To our sur-prise, we observed no significant difference in the rate oftranspiration between these transgenic and wild-type plants.One possibility is that SDIRIP1 overexpression did not in-crease the expression of ABI5 in plants enough to affecttranspiration.

SDIR1 Destabilizes SDIRIP1 in ABA Signaling 223

SDIR1 acts upstream of ABI5, ABF3, and ABF4 and partic-ipates in ABA, salt, and drought stress pathways (Zhang et al.,2007). However, SDIRIP1 selectively regulates the expressionof ABI5 and only participates in the ABA and salt stress path-ways. Meanwhile, several other SDIR1-interacting proteins wereidentified in yeast two-hybrid assays. These results stronglyindicated that there might be other substrates of SDIR1 thatregulate other types of bZIP transcription factors, such as ABF3and ABF4, that function in the drought response. Indeed, dif-ferent substrates have been found for one particular RING fingerE3 ligase. For example, the RING E3 ligase COP1 has a numberof different substrates involved in different biological functions(Lau and Deng, 2012). In mammals, one RING finger E3 ligase,named XIAP (Galbán and Duckett, 2010), has more than 50different substrates in different cellular compartments with dif-ferent biological functions. The identification of additional sub-strates may help shed light on this process in the future.

METHODS

Plant Materials and Growth Conditions

The Arabidopsis thaliana ecotypes Col-0 and Ws were used in this study.Three mutant lines were used in this study: sdir1-1 (Salk_052702) andabi5-1 (CS8105). Arabidopsis growth and treatment were performedfollowing the protocol described previously (Zhang et al., 2007). Wild-typeNicotiana benthamiana plants were also used as host plants for Agro-bacterium tumefaciens-mediated transient expression. Plants were grownin a growth chamber at 22°C and 70% relative humidity under a 16-h-light/8-h-dark photoperiod for ;1 to 1.5 months before infiltration.

Yeast Two-Hybrid Assays

Yeast two-hybrid assays were performed following the protocol de-scribed previously (Xie et al., 1999). SDIR1H234YΔTM was fused to theGAL4 DNA binding domain in pGBKT7 as the bait construct. Prey proteinswere fused to the GAL4 activation domain in pGADT7 (Clontech). Initialscreens were performed with an Arabidopsis cDNA library constructedusing the pGADT7 vector (Zhang et al., 2011). Bait and prey constructswere cotransformed into yeast HF7C cells. Positive clones were identifiedby the ability to grow on SD medium minus histidine/leucine/tryptophanand containing 2 mM 3-aminotriazole.

Agrobacterium-Mediated Transient Expression in N. benthamiana

Agrobacterium-mediated transient expression in N. benthamiana wasperformed following the protocol described previously (Liu et al., 2010).

Pull-Down and Immunoprecipitation Assays

[35S]Methionine-labeled SDIRIP1 was generated by in vitro transcriptionand translation with the T7/SP6-coupled TNT kit (Promega). The openreading frame of SDIR1was cloned into the pMal-C2 vector (New EnglandBiolabs) and expressed in Escherichia coli. Expression of MBP fusionproteins and in vitro binding experiments were performed following theprotocol described previously (Xie et al., 1999).

For immunoprecipitation and Co-IP assays, total proteins were ex-tracted from leaf samples with native extraction buffer (50 mM Tris-MES,pH 8.0, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 5 mM DTT, 1 mMphenylmethylsulfonyl fluoride, and protease inhibitor cocktail CompleteMinitablets [Roche]). Immunoprecipitation and Co-IP were performed at4°C following the protocol described previously (Liu et al., 2010).

Chloroplast and Nucleus Isolation

Chloroplasts and nuclei were isolated from N. benthamiana leaves ex-pressing SDIRIP1-GFP. For chloroplast isolation, 2 g of leaves was ho-mogenized in 4mL of grinding buffer (50mMHEPES-KOH, pH 7.3, 0.33Msorbitol, 0.1% BSA, 1 mM MnCl2, and 2 mM EDTA). Ground tissues werefiltered through two layers of Miracloth. Chloroplasts were pelleted bycentrifugation at 2600g for 5 min. The crude chloroplasts were re-suspended in 0.4 mL of grinding buffer with 40% and 80% Percoll. A totalof 3.2 mL of 40% (v/v) Percoll was gently layered on top of 1 mL of 80%(v/v) Percoll. Then, the resuspended chloroplast pellet was gently layeredon top of the Percoll gradient and centrifuged at 1500g for 10 min. Weharvested the band between the two phages, which contained intactchloroplasts.

Nucleus isolation was performed using a plant nucleus isolation kit(CelLytic kit; Sigma-Aldrich) following the protocol for highly pure prep-aration of nuclei in the technical bulletin.

BiFC Assays

SDIR1, SDIR1H234Y, and SDIRIP1 were fused to the N and C termini ofVenus (Waadt et al., 2008) to produce VN(R)-SDIR1, VN(R)-SDIR1H234Y,and SDIRIP1-VC, respectively. VC and VN(R) were used as negativecontrols. VN(R)-SDIR1/VC, VN(R)/SDIRIP1-VC, VN(R)-SDIR1/SDIRIP1-VC, VN(R)-SDIR1H234Y/VC, and VN(R)-SDIR1H234Y/SDIRIP1-VC were tran-siently expressed inN. benthamiana. Leaveswere collected 3 d after infiltration.The fluorescencewasdetectedusing aLeica TCSSP5confocal laser scanningmicroscope.

FPP Assays

Arabidopsis protoplasts were isolated from rosette leaves using the Tape-Arabidopsis Sandwich method (Wu et al., 2009). Protoplast transformationwas performed following the protocol described previously (Yoo et al.,2007).

FPP assays were performed using a modified procedure. After in-cubation overnight in W5 solution (2 mM MES, pH 5.7, 154 mM NaCl,125mMCaCl2, and 5mMKCl), protoplasts were washedwithW5 solutionthree times and then resuspended in WI solution (4 mM HEPES, pH 7.5,and 20 mM KCl). To increase the permeability of the plasma membrane,digitonin was added to the medium to a final concentration of 50 mM andincubated at room temperature for 5 min. After that, the same volume ofWI solution containing 2% low-melting-point agarose was added to theprotoplast solution. The protoplasts were spread onto a glass slide.Photographs were taken before trypsin treatment. Then, WI solutioncontaining 4 mM trypsin was added onto the slide. After 1 min of in-cubation, photographs were taken.

In Vitro Degradation Assays

Total protein was extracted from leaf samples with native extraction buffer(50 mM Tris-MES, pH 8.0, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA,5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitorcocktail Complete Minitablets [Roche]). Samples were incubated at roomtemperature (for SDIRIP1) for various time periods.

Protein Gel Blot Analysis and Image Quantification

Protein gel blot analysis was performed following the protocol describedpreviously (Liu et al., 2010) with primary anti-MYC antibody (9E10; SantaCruz Biotechnology) (1:1500 diluted), anti-GFP antibody (JL-8; Clontech)(1:1500 diluted), anti-RFP antibody (AbM59004-25-PU; Beijing ProteinInnovation) (1:1500 diluted), anti-ubiquitin antibody (Liu et al., 2010)(1:3000 diluted), anti-PAG1 antibody (raised in our laboratory) (1:10,000

224 The Plant Cell

diluted), anti-Arabidopsis PsbA antibody (AS01 016; Agrisera) (1:6000diluted), and anti-Arabidopsis histone H3 antibody (AS10 710; Agrisera)(1:6000 diluted) followed by secondary goat anti-mouse antibody or goatanti-rabbit antibody conjugated to horseradish peroxidase and visualizedusing ECL solution (Amersham Pharmacia). Protein gel blot pictures werescanned, and the intensity of the images was quantified by ImageJ(National Institutes of Health).

In Vitro Ubiquitination Assay

For the in vitro ubiquitination assay, crude extract containing recombinantwheat (Triticum aestivum) E1 (GI: 136632), At-UBC10 (40 ng), purifiedSDIR1 (1 mg) fused with the MBP tag, purified Arabidopsis ubiquitin(UBQ14; At4g02890; 2 mg) fused with the His tag, and purified GST-MYC-SDIRIP1 (2 mg) fused with both the GST and MYC tags were used for theassay. MBP was used as the negative control of MBP-SDIR1. Reactionswere performed following the protocol described previously (Xie et al.,2002). The reaction was stopped by the addition of 43 protein loadingsample buffer (258 mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.4%Coomassie Brilliant Blue, and 0.4 M b-mercaptoethanol) and boiling for10 min.

RT-PCR and Quantitative RT-PCR Assays

DNase I-treated total RNA was denatured and subjected to reversetranscription reaction using SuperScript II (200 units per reaction; Invi-trogen) at 37°C for 1 h followed by heat inactivation of the reversetranscriptase at 70°C for 15 min. For coexpression assays, PCR wasperformed using the primers shown in Supplemental Table 1 for 25 cycles.Quantitative RT-PCRwas performed using a SsoFast EvaGreen Supermixkit (Bio-Rad) and the primers listed in Supplemental Table 1.

Measurement of the Transpiration Rate

Plants were grown on half-strength MS medium for 7 d and thentransplanted to soil to grow for 21 d. To minimize the experimentalvariation, whole aerial parts were detached from plants and placed onPetri dishes with the abaxial side of every leaf up. The leaves did notoverlap. Samples were kept at room temperature and weighed at differenttimes.

Measurement of Stomatal Aperture

In this assay, we used the same plants that were used to measuretranspiration rates. Fresh leaves at similar developmental stages wereharvested. These leaves were then incubated in a buffer (5 mM KCl,50 mM CaCl2, and 10 mM MES-Tris, pH 6.1) at room temperature underhigh light for 3 h to ensure the opening of all stomata. After that, ABA wasadded to the buffer to a final concentration of 5mM. After 5 h of incubation,photographs were taken, and stomatal apertures were measured byImageJ.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative database under the following accession numbers: SDIR1,At3g55530; SDIRIP1, At5g51110; HY5, At5g11260; ABI5, At2g36270;ABF3, At4g34000;ABF4, At3g19290;Actin7, At5g09810;PAG, At2g27020;PsbA, AtCg00020; Histone H3, At1g01370.

Supplemental Data

Supplemental Figure 1. Detection of SDIRIP1-GFP in the NuclearFraction by Protein Gel Blot.

Supplemental Figure 2. Schematic Illustration of the Principle behindthe FPP Assay to Reveal the C-Terminal Orientation of SDIR1.

Supplemental Figure 3. Controls for BiFC Assays.

Supplemental Figure 4. SDIRIP1 Is Unstable and Degraded by the26S Proteasome Pathway.

Supplemental Figure 5. The Stability of SDIRIP1.

Supplemental Figure 6. Ubiquitination of SDIRIP1 by SDIR1 in Vitro.

Supplemental Figure 7. Statistical Analysis of Rates of Germinationand Cotyledon Greening for Col-0 Wild-Type, Col-0/35S:SDIRIP1, andCol-0/35S:SDIRIP1-RNAi Plants.

Supplemental Figure 8. Salt Treatment of Col-0 Wild-Type, Col-0/35S:SDIRIP1, and Col-0/35S:SDIRIP1-RNAi Plants in Soil.

Supplemental Figure 9. Statistical Analysis of Rates of Germinationand Cotyledon Greening for Col-0 Wild-Type, sdir1-1, and sdir1-1/35S:SDIRIP1-RNAi Plants.

Supplemental Figure 10. Statistical Analysis of Rates of Germinationand Cotyledon Greening for Ws Wild-Type, abi5-1, and abi5-1/35S:SDIRIP1-RNAi Plants.

Supplemental Figure 11. SDIRIP1 Has No Obvious Influence onTranspiration and Effects of ABA on Stomatal Aperture.

Supplemental Table 1. Primers Used in This Work.

ACKNOWLEDGMENTS

We thank Gang Zeng for technical support. This research was supportedby 973 Program Grant 2011CB915402 from the National Basic ResearchProgram of China and Grant NSFC 31030047/90717006 from the NationalScience Foundation of China. Y.-R.W. was supported by 973 ProgramGrant 2012CB114300.

AUTHOR CONTRIBUTIONS

H.-W.Z. and Q.X. designed the research. H.-W.Z., F.C., L. Liu, M.-M.T.,Y.-S.N., L. Lou, K.S., Y.-R.W., and S.-Y.T. performed research. H.-W.Z.and Q.X. analyzed data. H.-W.Z. and Q.X. wrote the article.

Received November 12, 2014; revised December 17, 2014; acceptedJanuary 6, 2015; published January 23, 2015.

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SDIR1 Destabilizes SDIRIP1 in ABA Signaling 227

DOI 10.1105/tpc.114.134163; originally published online January 23, 2015; 2015;27;214-227Plant Cell

Sanyuan Tang and Qi XieHuawei Zhang, Feng Cui, Yaorong Wu, Lijuan Lou, Lijing Liu, Miaomiao Tian, Yuese Ning, Kai Shu,

ArabidopsisDegradation to Modulate the Salt Stress Response and ABA Signaling in The RING Finger Ubiquitin E3 Ligase SDIR1 Targets SDIR1-INTERACTING PROTEIN1 for

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