Research Articles

Arabidopsis C3HC4-RING finger E3 ubiquitin ligase AtAIRP4 positively regulates stress-responsive abscisic acid signaling

Liang Yang1, 2, Qiaohong Liu1, Zhibin Liu1, Hao Yang1, Jianmei Wang1, Xufeng Li1 and Yi Yang1*

1

Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of

Life Sciences, Sichuan University, Chengdu 610064, China, 2The Crop Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China.

*Correspondence: [email protected]

Edited by: Robert E Sharp, University of Missouri, USA Running Title: AtAIRP4 involved in ABA signaling

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1111/jipb.12364] This article is protected by copyright. All rights reserved.

Received: January 31, 2015; Accepted: April 23, 2015

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Abstract

Degradation of proteins via the ubiquitin system is an important step in many stress

signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA) - insensitive RING protein 4 (AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4-RING finger domain in its C-terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T-DNA insertion mutant atairp4 effectively recovered the ABA-associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild-type and atairp4 mutant plants. In addition, the expression levels of ABA- and drought-induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild-type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA-mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis.

Key words: AtAIRP4; E3 ligase; abscisic acid; drought stress; salt stress

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INTRODUCTION Abiotic stresses such as drought, high salinity, and extreme temperatures can adversely affect plant growth, development, and productivity. These unfavorable conditions cause plants to rectify their cellular physiological and biochemical processes via signal transduction events, which lead to changes in expression of stress-related genes. Although numerous genes induced by drought and salt stress have been identified and studied (Xiong et al. 2002; Zhu 2002), the biological functions of many of these genes are essentially unknown in higher plants. Therefore, it is desirable to study the functions of genes regulated by abiotic stresses to improve crop adaptation to drought and salt stresses. Abscisic acid (ABA) is a crucial phytohormone that functions during the adaptive response to environmental stresses. Seed dormancy and germination, plant growth, and regulation of stomatal aperture are particularly regulated by ABA. For example, under drought stress, ABA induces stomatal closure to prevent water loss through transpiration and launches a series of protection mechanisms by regulating gene expression (Xiong et al. 2002; Yamaguchi-Shinozaki and Shinozaki 2006). Ubiquitination is a type of post-translational modification that mediates several important processes in plants, such as biotic and abiotic stress responses, cell cycle progression, and subcellular localization (Callis and Vierstra 2000; Hellmann and Estelle 2002; Serino and Xie 2013; Zhang et al. 2013). Attaching ubiquitin to target proteins needs a succession of catalyzed reactions by three enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) (Glickman and Adir 2004; Smalle and Vierstra 2004). Genome sequencing has revealed that over 6% of Arabidopsis thaliana protein-coding genes are directly or indirectly dedicated to the ubiquitin 26S proteasome system (UPS). For example, there are more than 1400 genes encoding E3 ligases, which is much more than E1s (2 coding genes) and E2s (37 coding genes). Such a large number of E3 ligases demonstrate that they play a crucial role in determining the specificity of target substrates. In Arabidopsis, there are at least 477 RING domain-containing E3 ligases genes (Stone et al. 2005). These RING-containing E3 ligases participate not only in growth and developmental processes, but also in regulated mechanisms in response to abiotic stresses (Stone and Callis 2007; Guo et al. 2013). For example, 3      1     

 

RING-H2 E3 ligase XERICO confers drought and salt tolerance by increasing ABA synthesis (Ko et al. 2006). Recent studies have revealed that RING domain-containing E3 ligases SDIR1, AtAIRP1, AtAIRP2, AtAIRP3, RHA2a and RHA2b are positively involved in regulating ABA signal transduction. Meanwhile, overexpression of these RING E3 ligase genes in plants enhances drought resistance via increasing ABA-induced stomatal closure (Zhang et al. 2007; Bu et al. 2009; Ryu et al. 2010; Cho et al. 2011; Li et al. 2011; Kim and Kim 2013). The atairp1, atairp2 and atairp3 loss-of-function mutants exhibit ABA-insensitive phenotypes (Ryu et al. 2010; Cho et al. 2011; Kim and Kim 2013). Expression levels of these AtAIRP genes are induced by ABA, drought, NaCl and cold. Moreover, ABA-induced expression of AtAIRP2, which plays combinatory roles with AtAIRP1, is not observed in the snrk2.2/2.3/2.6 triple mutant, suggesting that transcription of AtAIRP2 in response to stress is regulated via these ABA-activated protein kinases (Ryu et al. 2010; Cho et al. 2011). Another C3HC4-type RING E3 ligase, AtAIRP3, plays dual functions in ABA-mediated drought stress responses and in an amino acid export pathway in Arabidopsis (Kim and Kim 2013). Here, we identified and characterized a C3HC4-type RING E3 ligase, AtAIRP4, which has strong sequence similarity to AtAIRP2. The expression level of AtAIRP4 can be induced by ABA and other abiotic stresses, including osmotic, oxidative and cold stresses. The 35S::AtAIRP4 transgenic lines and the atairp4 mutant showed contrary phenotypes in terms of ABA-responsive root elongation and stomatal function.AtAIRP4 also functions as a negative regulator of NaCl signaling. Furthermore, overexpression of AtAIRP4 could confer drought avoidance in Arabidopsis. These results indicate that AtAIRP4 is a C3HC4-type RING E3 ubiquitin ligase, which may be involved in positive regulation of ABA-dependent drought avoidance and negative regulation of salt tolerance.

RESULTS Expression pattern of AtAIRP4 To identify E3 ligase genes that are responsive to abiotic stresses in Arabidopsis thaliana, the expression pattern of some single subunit RING-finger E3 ligase genes was analyzed in the publicly available Arabidopsis microarray databases (Kosarev et al. 2002; Stone et al. 2005; Winter et al. 2007; Hruz et al. 2008). AtAIRP4 (At5g58787), a gene with a RING domain in its C-terminal side based on deduced peptide sequence particularly piqued our interest. Microarray 4      1     

 

analysis showed that its transcription level could be induced by heat (37℃), osmotic (300 mM mannitol) and oxidative stresses. Further investigation indicated that AtAIRP4 was also induced by ABA (Figure 1A). These results suggested that AtAIRP4 may be involved in abiotic stress response. To validate the in silico results, 10-d-old Arabidopsis seedlings were treated with ABA or several abiotic stresses. The analysis using quantitative reverse transcript-PCR (qRT-PCR) demonstrated that the transcript abundance of AtAIRP4 was up-regulated by ABA, mannitol, H2O2 and cold in a time-dependent manner, but not by NaCl (Figure 1A). In particular, the expression level of AtAIRP4 reached a peak of 4.2-fold and 4-fold after being treated with ABA (1.5 h) and H2O2 (6 h), respectively. Subsequently, the mRNA of AtAIRP4 was detected in different tissues of Arabidopsis, including roots, stems, leaves, old leaves, flowers and siliques (Figure 1B). Our results showed that AtAIRP4 was expressed in all organs; roots showed the highest expression, whereas the lowest was in the stem. To further confirm these results, the upstream region (~1.5 kb) of the AtAIRP4 ATG start codon fused with the β-glucuronidase (GUS) gene was constructed and transformed into the wild-type plants. Histochemical staining showed that GUS activity could be monitored at all developmental stages, indicating that AtAIRP4 is expressed throughout the entire life cycle of Arabidopsis (Figure 1C). The GUS staining was first detected in the emerging radicle of germinated seeds after 24 h from planting (Figure 1C, a) and became more obvious in 2- and 3-d-old seedlings (Figure 1C, b–d). With the seedling growth, GUS activity was found mainly in the vascular tissues of shoots and roots, shoot apical meristems and root tips (Figure 1C, e). At the late developmental stage, GUS expression was observed in the leaf tips and generative tissues including stigmas, anthers, the vascular tissues of sepals and petals, and siliques of mature plants (Figure 1C, f–l). Treatment with ABA or abiotic stresses significantly induced GUS expression throughout the 10-d-old seedlings compared with the mock (Figure S2). These results confirmed that AtAIRP4 is a stress-responsive gene.

AtAIRP4 is a RING E3 ubiquitin ligase and localized in the cytoplasm The coding sequence (CDS) of AtAIRP4 is made up of 729 base pairs, which encodes a polypeptide of 242 amino acid residues (molecular weight of 28.1 kDa). Sequence analysis showed that AtAIRP4 contains a C3HC4- RING domain in its C-terminal side (Figure S1). In 5      1     

 

Arabidopsis, there are two orthologs of AtAIRP4 including At3g47160 (78% similarity) and AtAIRP2 (63% similarity) (Figure 2A). Moreover, AtAIRP4 is 58% to 71% identical to the deduced unknown protein in rice (Oryza sativa), cacao (Theobroma cacao), maize (Zea mays), poplar (Populus trichocarpa), ricinus (Ricinus communis), grape (Vitis vinifera) and sorghum (Sorghum bicolor) (Figure S1). Since AtAIRP4 has no homologs in mammals or prokaryotes, it can be speculated that AtAIRP4 and its homologs may be specific to plants. In general, proteins with a C3HC4-RING domain exhibit E3 ubiquitin ligase activity (Wenzel et al. 2011; Cheng et al. 2012; Kim and Kim 2013). To demonstrate whether AtAIRP4 is also a functional E3 ligase, it was fused with GST protein tag, expressed in Escherichia coli and purified by GSH affinity chromatography to carry out in vitro E3 ligase assay. Ubiquitination activity was observed by anti-Ub antibody in the presence of ATP, human E1 (UBA1) and E2 (UBCh5b) (Figure 2C). Poly-ubiquitin chains could not be formed in the absence of any of the reaction components, suggesting that AtAIRP4 possesses E3 ligase activity in vitro. The

cellular

localization

of

AtAIRP4

was

investigated

by

transforming

the

35S::AtAIRP4-GFP and 35S::GFP plasmids into Arabidopsis leaf protoplasts using the polyethylene glycol (PEG)-mediated method. The fluorescence signal of AtAIRP4-GFP fusion was predominantly found in the cytoplasm similar to the localization of free GFP control, indicating that AtAIRP4 is a cytosolic protein (Figure 2D).

AtAIRP4 is a positive regulator in ABA-mediated root growth and stomatal closure A reverse genetics approach was used to investigate the biological functions of AtAIRP4 in Arabidopsis. A T-DNA insertion mutant line, atairp4 (Salk_115341), was obtained from the Arabidopsis Biological Resources Center (ABRC). The T-DNA insertion in the atairp4 mutant was located in the second exon of AtAIRP4 (Figure 3A). No full-length AtAIRP4 transcript was detected in the atairp4 mutant by reverse transcription (RT)-PCR analysis, suggesting that atairp4 is a loss-of-function mutant (Figure 3B). Subsequently, a construct containing AtAIRP4 under the control of the cauliflower mosaic virus 35S promoter was transformed into the atairp4 homozygous lines via the Agrobacterium tumefaciens-mediated floral dip method (Clough and Bent 1998). Three independent 35S::AtAIRP4 transgenic plants (lines 2, 6 and 12) were obtained using hygromycin as a selection marker. qRT-PCR result showed that the transcript levels of AtAIRP4 in these transgenic lines were overexpressed at least 200-fold higher than in the 6      1     

 

wild-type plants (Figure 3C). Furthermore, immunoblotting with HA-tag antibody demonstrated that the AtAIRP4 overexpression plants could effectively express AtAIRP4 protein (Figure 3D). Seed germination was initially tested in the presence of ABA (0, 0.2, 0.4 and 0.8 μM). No differences were observed among wild-type, atairp4 mutant, and 35S::AtAIRP4 plants grown on plates containing various concentration of ABA. Thereafter, the analysis of ABA sensitivity of AtAIRP4 was extended to post-germination root growth. Wild-type, atairp4 mutant and 35S::AtAIRP4 seeds were imbibed on half-strength MS medium for three days under normal germination conditions. Germinated evenly-grown seedlings were then transferred to half-strength MS plates supplemented with or without ABA. Three 35S::AtAIRP4 transgenic lines showed shorter primary roots compared with the atairp4 mutant under ABA treatment (Figure 4A, B). When the seedlings were grown for 9 days on half-strength MS medium with 10 μM ABA, the root length of 35S::AtAIRP4 plants was 55.2–82.5% shorter than that of the mutant. At 20 μM ABA, the atairp4 mutant root was 29.8–48.7% longer than that of the 35S::AtAIRP4 plants. Wild-type seedlings exhibited intermediate phenotypes between the atairp4 mutant and 35S::AtAIRP4 transgenic lines under the different ABA concentrations. Stomatal closure is an important ABA-regulated process that helps to restrict water loss under drought stress (Leung and Giraudat 1998). To investigate the function of AtAIRP4 in ABA-mediated stomatal closure, we measured the stomatal apertures in the wild-type, atairp4 mutant and 35S::AtAIRP4 plants under ABA treatment. The 4-week-old rosette leaves of Arabidopsis were first pretreated with stomatal opening solution and then treated with 10 μM ABA for 2 h. As observed by light microscopy, there were no significant differences in stomatal apertures among the leaves of different genotypes without the treatment of ABA (Figure 4C, D). However, the stomatal apertures of the AtAIRP4 overexpressing lines were reduced to 0.24 (line 2) or 0.2 (line 12) in the treatment with 10 μM ABA, which were approximately 2-fold lower than that of the atarip4 mutant. In the wild-type plants, the stomatal aperture was between that of the atairp4 mutant and 35S::AtAIRP4 plants. Stomatal movement regulates the transpiration rate of plants by controlling water evaporation and gas exchange (Farquhar and Sharkey 1982). In this study, water loss rates of detached rosette leaves were evaluated. The rosette leaves of 4-week-old plants were cut and placed under normal growth conditions. As shown in Figure 4E, the 35S::AtAIRP4 plants showed a lower water loss rate than wild-type plants, while the atairp4 mutant plants exhibited the highest rate among all 7      1     

 

samples. According to the above results, overexpressing AtAIRP4 in the atairp4 mutant enhanced the sensitivity of root growth and stomatal closure to ABA. Therefore, AtAIRP4 may function as a positive regulator in ABA responses during post-germination growth.

AtAIRP4 enhances drought avoidance in Arabidopsis Because AtAIRP4 is induced by ABA and positively involved in ABA responses during post-germination growth (Figure 4), it was speculated that there might be altered responses to drought stress in the atairp4 mutant and 35S::AtAIRP4 plants. To verify our hypothesis, we examined the drought stress responses of soil-grown atairp4 mutant and 35S::AtAIRP4 plants (Figure 5). Wild-type, atairp4, and 35S::AtAIRP4 plants (lines 2 and 12) were grown for 2 weeks in soil under normal growth conditions and then subjected to water deficit. After withholding water for 24 days, the wild-type and especially the atairp4 mutant plants exhibited a severely withered phenotype, whereas the 35S::AtAIRP4 plants retained a more vigorous appearance. Consistent with the differential leaf water loss rates shown in Figure 4E, the more extreme withering in the atairp4 mutant plants was associated with the greatest decrease in relative soil water content among the different lines (Figure S4). Conversely, the 35S::AtAIRP4 plants exhibited the least decrease in soil water content. After rewatering, only 23% of the mutant plants survived compared with over 95% of the 35S::AtAIRP4 plants (Figure 5B). To further demonstrate that the findings in Figure 5A were due to differential soil drying between the lines, we repeated the water deficit treatment with wild-type, atairp4, and 35S::AtAIRP4 plants (lines 2 and 12) planted in one container. In this case, relatively small phenotypic differences among the different lines were observed during a soil drying and rewatering cycle (Figure 5C). Taken together, the results in Figures 4–5 and Supplemental Figure S4 indicate that overexpression of AtAIRP4 could confer drought avoidance via decreased rates of stomatal water loss. With the consideration that AtAIRP4 was positively involved in ABA responses, we speculated that AtAIRP4 may act as a positive regulator in ABA-dependent drought stress response.

AtAIRP4 results in a hypersensitive phenotype to salinity during germination and early seedling development, but to osmotic stress only during germination Treatment with exogenous ABA can trigger salt and drought responses, which activate a series of ABA-dependent responses in plants (Zhu 2002). Nearly all ABA-deficient and ABA-insensitive 8      1     

 

mutants are insensitive to salt during seed germination (Kang et al. 2002).To ascertain the effect of AtAIRP4 on salinity tolerance of plants, seed germination was assayed at gradually increased NaCl concentrations (0, 75, 100, 125 and 150 mM). The analysis showed no obvious differences when seeds were plated on half-strength MS agar medium (Figure 6A). However, in the presence of 100 or 150 mM NaCl, the 35S::AtAIRP4 plants showed much lower germination than the wild-type and atairp4 mutant plants (Figure 6B). After 2 days on 100 mM NaCl, more than 80% of the wild-type and atairp4 mutant seeds germinated, while only 30% of 35S::AtAIRP4 seeds did (Figure 6D, left panel). When grown on the half-strength MS medium containing 150 mM NaCl for 2 days, only 18% seeds germinated in 35S::AtAIRP4 plants compared to 78% in atairp4 mutant and 62% in the wild-type plants (Figure 6D, left panel). To further study the effects of high salinity on post-germination growth, root lengths of plants were analyzed under the treatment with different NaCl concentrations. The atairp4 mutant and 35S::AtAIRP4 seedlings showed opposite phenotypes under salt treatment. In the presence of NaCl, the seedlings of atairp4 mutant were more vigorous than those of 35S::AtAIRP4 plants. The primary roots of atairp4 mutant were 18-29% and 26-34% longer than those of 35S::AtAIRP4 plants at 100 mM and 125 mM NaCl, respectively (Figure 7). Salt injury causes a variety of stresses to plants, mainly including hyper-ionic and hyper-osmotic stresses (Verslues et al. 2006). To identify whether AtAIRP4 functions in salt-specific or osmotic response during the germination stage, the seeds of different genotypes were grown on half-strength MS medium with or without mannitol. Our studies demonstrated that osmotic stress inhibited seed germination of 35S::AtAIRP4 plants much more than in the atairp4 mutant and wild-type plants (Figure 6C, D). Subsequently, 3-d-old seedlings were grown for another 6 days on different concentrations of mannitol and there were no significant differences observed in primary root length (Figure S5A). As an alternative to mannitol, high-molecular-weight polyethylene glycol (PEG) was used to impose osmotic stress treatment to the seedlings of different genotypes. 5-d-old seedlings were transferred to PEG-infused plates (– 0.5 MPa) or to a plate without PEG (– 0.25 MPa). Similarly to the findings with mannitol, the seedlings did not exhibit obvious differences in root length on the PEG-infused plates (Figure S5B). These results suggested that the overexpression of AtAIRP4 can enhance sensitive responses to salt and osmotic stresses at seed germination, but not for post-germination root growth. 9      1     

 

Altered expression of AtAIRP4 leads to altered expression of ABA-induced drought stress responsive genes Our results have shown that AtAIRP4 positively regulated the drought stress response in an ABA-dependent manner (Figure 4, 5). Many genes responding to ABA and drought stress responses have been used as markers for examining stress response pathways. To further investigate whether the transcript level of ABA-responsive and drought-responsive genes might be affected by AtAIRP4 overexpression, we compared the mRNA levels of MYB2, RD20, RD29A and RD29B in the wild-type, atairp4 mutant and 35S::AtAIRP4 plants. According to the analysis by qRT-PCR, the expression levels of these ABA signaling marker genes in 35S::AtAIRP4 plants were nearly 2-fold higher than those in atairp4 mutant even under normal growth conditions (Figure 8). After treatment with 10 μM ABA for 2 h, higher transcript abundances of those ABA-mediated genes were detected in 35S::AtAIRP4 plants in comparison with those in wild-type plants, while the lowest transcript abundances of those genes were detected in atairp4 mutant plants (Figure 8). In addition, we also investigated the effect of altered expression of AtAIRP4 on ABI2, ABF3 and ABI5. The analysis demonstrated that there were no differences in the transcript abundances of ABI2, ABF3 and ABI5 among wild-type, atairp4 mutant and 35S::AtAIRP4 plants under either normal growth conditions or ABA treatment (Figure 8). Hence, our results suggest that AtAIRP4 may function as a positive regulator in ABA-mediated drought avoidance by up-regulating some ABA- and drought-responsive genes.

DISCUSSION ABA is an essential mediator in triggering plant responses to adverse environmental stimuli (Leung and Giraudat 1998). Drought and salt stresses specifically trigger the production of ABA, which in turn causes stomatal closure to reduce water loss and induces the expression of stress-related genes (Shinozaki and Yamaguchi-Shinozaki 2007). Recent studies revealed that plant RING-type E3 ligase isoforms play crucial roles in abiotic stress signal transduction. For example, AtAIRP1 and AtAIRP2 function coordinately in ABA mediated drought stress responses in Arabidopsis (Ryu et al. 2010; Cho et al. 2011), and AtAIRP3 is a positive regulator in the ABA-mediated drought and salt stress tolerance mechanisms (Kim and Kim 2013). These studies show the significant role of RING-type E3 ligase in ABA-mediated stress responses. In 10      1     

 

this study, we showed that AtAIRP4 may act as a positive regulator of ABA-mediated stress responses. Publicly available Arabidopsis microarray database analysis and qRT-PCR results indicated that AtAIRP4 was up-regulated by ABA and other abiotic stresses (Figure 1A). Further analysis of its promoter by using the PLACE program (http://www.dna.affrc.go.jp/PLACE/) detected some abiotic stress-related cis-regulatory elements (ABRE, MYB and MYC) in the 2-kb region upstream from the initiation codon of AtAIRP4, suggesting that this gene may be regulated by ABA or other abiotic stresses (Prestridge 1991; Higo et al. 1999). ProAtAIRP4::GUS transgenic plants under different abiotic stresses showed stronger GUS activities than those of the control plants (Figure S2), consistent with the microarray databases and qRT-PCR analysis. By analyzing the protein motifs in AtAIRP4, a C3HC4-RING domain was found at its C-terminal side. AtAIRP4 was shown to have in vitro E3 ligase activity and was localized in the cytoplasm (Figure 2B - D), consistent with other RING domain proteins (Ko et al. 2006; Zhang et al. 2007; Ryu et al. 2010; Cho et al. 2011; Kang et al. 2011; Kim and Kim 2013). ABA-associated phenotypic analysis indicated that 35S::AtAIRP4 transgenic lines and atairp4 mutant plants had opposite phenotypes in terms of root elongation and stomatal aperture: the overexpressors and the mutant were hypersensitive and less-sensitive to exogenous ABA, respectively (Figure 4). However, there were no significant differences between the atairp4 mutant and AtAIRP4-overexpressing plants during seed germination in the absence or presence of ABA. In fact, signal transduction pathways occurring during germination are distinct from those affecting post-germination pathways (Price et al. 2003). Some regulators involved in ABA signaling function only during the germination stage or the post-germination stage. For example, ARCK1, which belongs to the RLK sub-family and encodes a cytosolic protein kinase, plays a negative role in ABA signaling only during the post-germination stage (Tanaka et al. 2012). In contrast, as negative regulators in ABA signaling transduction, DWA1 and DWA2 function during the seed germination stage, but have no effects at the post-germination stage (Lee et al. 2010).These results suggest that AtAIRP4 may play a positive role in ABA signaling during the post-germination growth stage, but not during the germination stage. Stomatal closure is an important ABA-controlled event in dealing with water deficit conditions. Some ABA-insensitive mutants (i.e., abi1 and abi2) are hypersensitive to water deficit because of impaired stomatal aperture regulation (Hetherington 2001). Our results suggest 11      1     

 

that stomatal movement is regulated by AtAIRP4. The 35S::AtAIRP4 plants displayed reduced water loss rate and enhanced drought avoidance compared with the wild-type and atairp4 mutant plants (Figure 4E, 5). Meanwhile, stomatal closure in the epidermis of AtAIRP4-overexpressing plants was more sensitive than in the wild-type and atairp4 plants after ABA treatment (Figure 4C). These results further indicate that AtAIRP4 may be a positive regulator in the drought avoidance by regulating stomatal movement in an ABA-mediated signaling pathway. Both drought and salt stress signal transduction pathways involve osmotic homeostasis, and ABA plays an important role in some of these processes. For example, many studies have observed that mutants with altered ABA sensitivity are affected in germination on salt- and/or mannitol- containing media (Kang et al. 2002; Guo et al. 2009). The AtAIRP4-overexpressing plants also revealed an increased sensitivity to salt and osmotic stresses during seed germination (Figure 6). Therefore, AtAIRP4 may be involved in regulating early growth of seedlings by suppressing seed germination under adverse growth conditions, similar to other E3 ligases that function in stress-responsive regulation during seed germination, such as its ortholog AtAIRP2 (Cho et al. 2011) and the U-box E3 ligase PUB44/SAUL1 (Salt et al. 2011). Meanwhile, the root elongation assay indicated that 35S::AtAIRP4 plants displayed sensitivity to salt stress during post-germination

(Figure

7).

However,

whether

the

seedlings

were

planted

on

mannitol-containing or PEG-infused agar plates, 35S::AtAIRP4 plants did not exhibit an obvious increased sensitivity in terms of root growth (Figure S5). Thus, it appears that AtAIRP4 may be involved in ABA-regulated rather than osmotic response in the salt signaling pathway, which is similar to SDIR1 (Zhang et al. 2007), AtAIRP3 (Kim and Kim 2013) and AtRDUF1 (Li et al. 2013). Similarly, the phenotypes of seedlings under osmotic stress (Figure S5B) and water deficit treatment (Figure 5C) indicated that the drought avoidance of 35S::AtAIRP4 plants was not dependent on osmotic signaling, but may be related to ABA-mediated stomatal movement regulation. Increased ABA levels in response to drought and high salinity induce the expression of many genes that appear to play multifaceted roles in dehydration response in both vegetative tissues and seeds (Fujita et al. 2011). By analyzing the expression patterns of several ABA-induced drought stress-related genes, we found that the mRNA levels of MYB2, RD20, RD29A and RD29B were up-regulated in 35S::AtAIRP4 plants compared with the atairp4 mutant, even if they were under normal conditions (Figure 8). MYB2 encodes a R2R3 MYB domain-containing 12      1     

 

transcription factor that regulates several ABA-dependent salt and drought stress responsive genes (Abe et al. 2003; Yanhui et al. 2006). RD20 encodes a member of the caleosin protein family and can be highly and rapidly induced by water deficit and ABA treatment through the AREB1 transcription factor acting as a molecular switch (Fujita et al. 2005). The RD29A promoter contains several DREs and one ABRE and the RD29B promoter contains several ABREs and one DRE (Uno et al. 2000; Sakuma et al. 2006a; Sakuma et al. 2006b). However, both RD29A and RD29B can be significantly induced by exogenous application of ABA (Zimmermann et al. 2005; Nemhauser et al. 2006). In view of these results, we suggest that AtAIRP4 may enhance ABA signal transduction by up-regulating some ABA-induced downstream marker genes, which lead to drought stress acclimation. However, overexpressing AtAIRP4 in atairp4 mutant had no effect on the expression levels of ABI2, ABF3, and ABI5 (Figure 8). It is well documented that ABI2, ABF3, and ABI5 are the key factors in ABA signaling during seed germination (Holdsworth et al. 2008; Penfield and King 2009; Mittal et al. 2014). As an ABA-responsive protein phosphatase 2C, ABI2 negatively regulates ABA signaling during seed dormancy and germination (Leung et al. 1997; Rodriguez et al. 1998). In contrast, the ABA-induced basic Leu zipper transcription factor ABF3 is functionally redundant to ABI5 in positively regulating ABA signaling during seed development and germination (Finkelstein and Lynch 2000; Finkelstein et al. 2005; Yoshida et al. 2010). In particular, ABI5 is required for the germination and post-germination developmental arrest checkpoint (Lopez-Molina et al. 2001). With this evidence, we speculate that the same germination phenotypes of the 35S::AtAIRP4 and atairp4 mutant plants under ABA treatment may be due to the same expression levels of ABI2, ABF3, and ABI5, which were not influenced by the expression of AtAIRP4. Since AtAIRP4 and AtAIPR2 share a high level of similarity in their sequence (Figure S1), it is likely that they have partial functional redundancy. However, there are still some differences in their expression patterns and their overexpression phenotypes. For example, in terms of the expression pattern, AtAIRP2 is mainly expressed in leaf hydathodes and shoot apical meristems, but not in roots; whereas AtAIRP4 was expressed in all organs, with the highest expression in roots (Figure 1B, C). 35S::AtAIPR2 transgenic plants were hypersensitive to exogenous ABA at both germination and post-germination stages(Cho et al. 2011), whereas the 35S::AtAIRP4 plants were hypersensitive to ABA only at the post-germination stage (Figure 4). After ABA treatment, 13      1     

 

the mRNA levels of ABI2 and ABF3 were markedly higher in the 35S::AtAIPR2 plants than in the wild-type plants; however, there were no differences between the 35S::AtAIRP4 and the wild-type plants in the expressions of ABI2 and ABF3 (Figure 8). These differences can be explained by the fact that AtAIRP4 and AtAIPR2 may have different target(s). Thus, AtAIRP4 may play a combinatory role with AtAIPR2 in ABA and drought stress responses. However, we cannot exclude the possibility that AtAIRP4 may affect ABA signal transduction indirectly. To further confirm its role in ABA-mediated stress- responsive signaling, it is necessary to identify the targets of AtAIRP4, and this will be the next work in the future. In conclusion, our study demonstrated that AtAIRP4 is a C3HC4 RING E3 ligase, which may play a positive role in ABA-mediated drought avoidance and a negative role in salt tolerance in Arabidopsis.

MATERIALS AND METHODS Plant materials and growth conditions The background of Arabidopsis thaliana used in this study was Columbia (Col-0). The atairp4 (Salk_115341) T-DNA insertion mutant was obtained from the ABRC (Alonso et al. 2003). Seeds were surface-sterilized with 20% (v/v) bleach solution for 15 min and washed three times with sterile distilled water, then kept in the dark at 4°C for 48 h to break dormancy and grown on soil or half-strength MS medium (with 1% sucrose and 0.8% agar, pH 5.8). Plants were grown in a temperature controlled chamber at 22°C and ~60% relative humidity with a 16-h photoperiod at a light intensity of ~110 μmol photons m-2 s-1. The half-strength MS medium was supplemented with ABA, NaCl, mannitol, or H2O2 as indicated. For germination assay, seeds were collected at the same time and dried in an incubator at 30℃ for two weeks. For root length assay, 3- or 5-d-old seedlings growing in normal half-strength MS medium were transferred to culture dishes containing different concentrations of ABA, NaCl, or PEG as needed. PEG-infused agar plates were prepared using PEG-8000 according to Verslues et al. (2006)

Sequence alignment and phylogenetic analysis To obtain the homologs of AtAIRP4, BLASTP program (http://www.ncbi.nlm.nih.gov/BLAST/) was performed. Phylogenetic analysis was conducted in MEGA software version 4 using the Neighbor-Joining method (Tamura et al. 2007; Ryu et al. 2010). Alignment of AtAIRP4 and its homologous amino acid sequences was carried out by DNAMAN software version 8. 14      1     

 

Transformation vectors and construction of transgenic plants For AtAIRP4 stable expression in Arabidopsis, the full-length cDNA sequence (729 bp) of AtAIRP4 was amplified and cloned into the BamH I-Sac I restriction enzyme sites of the pZh01 vector, in which the expression of AtAIRP4 was controlled by the CaMV 35S promoter. For the AtAIRP4 promoter-β-glucuronidase (proAtAIRP4:GUS) construct, a ~1.5-kb fragment upstream of the AtAIRP4 transcription start site was amplified from Arabidopsis genomic DNA and cloned into the Xho I-BamH I restriction enzyme sites of the pBI121 vector (Clontech, California, USA) to replace the 35S promoter. Primers used for PCR amplifying were listed in Table S1. Transformation of Arabidopsis was performed by the floral dip method using Agrobacterium tumefaciens strain EHA105 (Clough and Bent 1998). T0 seeds were germinated on half-strength MS medium containing 25 mg/mL hygromycin (Roche, Indianapolis, USA) for the pZh01 vector or 50mg/mL kanamycin for the pBI121 vector. The resistant plants were then transferred into soil until obtained homozygous T3 seeds. For the phenotypic and AtAIRP4 expression pattern analysis, independent lines of homozygous T3 plants were used for detailed analysis.

Subcellular localization The full-length AtAIRP4 coding region was amplified using primers listed in Table S1 and cloned into the BamH I-Sal I restriction enzyme sites of the pBI221-GFP transient expression vector. The constructed fusion genes and empty GFP vector were transformed into the wild-type Arabidopsis protoplasts by means of PEG treatment (Yoo et al. 2007). The expressions of AtAIRP4-GFP and GFP were monitored after transforming for 16 h. Transformed protoplasts were placed on the slide glass and observed using a confocal fluorescence microscope (Leica TCS SP5 II system, Leica, Wetzlar, Germany) to capture the photos, then analyzed by Leica Application Suite (LAS) software version 4.1 (Leica, Wetzlar, Germany).

Protein extraction and western blot Total proteins were extracted from detached rosette leaves (0.1 g) of 4-week-old plants by grinding in liquid nitrogen, then suspended in protein extraction solution (0.1 M Tris-HCl, pH 8.0, 30% w/v sucrose, 10 mM EDTA, 1 mM PMSF, 1 mM DTT, 4% w/v SDS, 2% v/v βMercaptoethanol). The extractions were incubated on ice for 30 min, then centrifuged at 12, 000 15      1     

 

rpm 4 ℃ for 30 min. The supernatant as protein extraction was collected and added with 1 × SDS loading buffer. After boiling for 10 min, the protein extraction was analyzed by SDS-PAGE and western blot with anti-HA antibody (Santa Cruz, Texas, USA).

In vitro assay of E3 ubiquitin ligase activity The full-length AtAIRP4 coding region was amplified using primers listed in Table S1 and cloned into the BamH I-Sma I restriction enzyme sites of the pGEX-6p-1 vector (GE Healthcare, Fairfield, CT, USA). GST-AtAIRP4 fusion protein was expressed in Escherichia coli strain Rosetta and purified using glutathione sepharose beads (GE Healthcare, Fairfield, CT, USA). In vitro E3 ligase assay of AtAIRP4 was performed as described (Liu et al. 2014). Human E1 (UBA1, GI: 23510338), yeast ubiquitin (GI: 209599), and human E2 (UBCh5b, GI: 4507773) were purchased from Sigma Company (Sigma‐Aldrich, St Louis, MO, USA).

Drought treatment and water-loss analysis For drought avoidance in Figure 5A, seeds were plated in separate containers (height×top diameter×bottom diameter = 110×120×90 mm; 16 plants each) with the same weight soil (soil was a mixture of 50% peat and 50% vermiculite) for 2 weeks. The total weight (dry weight) of soil and container was 170g±3.7g. Containers were irrigated with water to saturation and weighed at the start of water deficit stress treatment (initial weight) and then periodically throughout the treatment period. Relative soil water content was calculated as (final fresh weight – dry weight)/(initial weight – dry weight). After 24 d, plants were rewatered and survival rate was assessed 3 days after rewatering. For the drought stress experiment in Figure 5C, seeds were planted in one container for 3 weeks under normal growth conditions and then subjected to water deficit for 19 d. Photographs were taken after rewatering for 3 d. For water-loss rate analysis, six detached leaves of 4-week-old plants were weighed at the indicated times. Water-loss rate was expressed as the ratio between water loss and plant initial fresh weight. Each experiment was carried out at least three times.

Stomatal aperture measurement Mature leaves of the 4-week-old wild-type, atairp4, and 35S::AtAIRP4 plants were used for the observation of stomatal aperture. Detached leaves were incubated for 2 h in stomatal opening 16      1     

 

solution (10 mM KCl, 100 mM CaCl2, 10 mM MES, pH 6.1) and then transferred to stomatal opening solution supplemented with 10 μM ABA for 2 h. Subsequently, the adaxial surface of each leaf was applied to clear tape to peel off the epidermal layer, then mounted on glass slides and observed with Leica DM1000 microscope (Leica, Wetzlar, Germany). Images were captured with a cool CCD camera and imported into LAS software version 4.1 (Leica, Wetzlar, Germany). The ratio of width to length of the stomata was measured. Over 60 guard cells from each sample were used to measure stomatal aperture.

RT-PCR and real-time qRT-PCR analysis Total RNA was extracted using RNAprep pure Plant kit (Tiangen, Beijing, China) from 10-d-old seedlings that had been subjected to ABA and other abiotic stresses. Each cDNA was synthesized using 1 μg of total RNA with the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan). Real time-PCR was performed on a CFX Connect Real-Time PCR Detection System using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). PCR program was as followed: 95℃ for 3 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 40 s. Data were obtained using the CFX Manager software version 3.1 (Bio-Rad, Hercules, CA, USA), then normalized to the Actin2 (At3g18780) or Tubulin2 (At5g62690) mRNA levels as indicated. All qRT-PCR experiments were performed three technical replicates and three independent biological repetitions. Primers used in RT-PCR and qRT-PCR analysis are listed in Supplemental Table S1.

Histochemical GUS staining For the histochemical GUS assay, seedlings and tissues were gently immersed in 90% acetone on ice for 30 min, then transferred into a GUS staining solution containing 2 mM X-Gluc, 0.5 mM K3Fe (CN)6, 0.5 mM K4Fe (CN)6, 0.1% v/v Triton X-100 (Sigma‐Aldrich, St Louis, MO, USA) in 50 mM sodium phosphate buffer (pH 7.0), vacuum infiltrated for 10 min, and incubated for 12 h at 37℃. After GUS staining, seedlings or tissues were washed with 70% ethanol to remove chlorophyll.

ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (31171586, 31271758, and 31300996) and National 973 Project (2013CB733903). 17      1     

 

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SUPPORTING INFORMATION Figure S1. Sequence alignment of AtAIRP4 amino acids with its homologs AtAIRP4 was aligned with NP_001176229 in O. sativa, XP_002313384 in P. trichocarpa, XP_002525634 in R. communis, XP_002447334 in S. bicolor, EOY33736 in T. cacao, XP_002277921 in V. vinifera, and NP_001147625 in Z. mays. Identical amino acid residues were shaded in black. Highly conserved amino acid residues (at least 4 identical residues) were shaded in gray. C3HC4- RING finger domain was labeled by black line. Figure S2. Enhanced GUS staining in AtAIRP4 promoter-GUS transgenic plants after treated with different stresses 10-d-old GUS transgenic seedlings were treated with indicated concentration of ABA, NaCl, mannitol, H2O2, or cold for the indicated times. Figure S3. GST-AtAIRP4 fusion protein was analyzed by SDS-PAGE M: protein marker; 1: purified GST-AtAIRP4 eluted from glutathione resin with reduced glutathione. The molecular weight of GST-AtAIRP4 was about 54 kDa. Figure S4. Relative soil water content of different genotypes in Figure 5A Data represent means ± SD (n=3). Asterisks indicate significant differences (*P