Characterization and Functional Analysis of Pyrabactin Resistance-Like Abscisic Acid Receptor Family in Rice
- Xiaojie Tian†1, 2,
- Zhenyu Wang†1,
- Xiufeng Li1, 2,
- Tianxiao Lv1, 2,
- Huazhao Liu1, 3,
- Lizhi Wang4,
- Hongbin Niu5Email author and
- Qingyun Bu1Email author
© Tian et al. 2015
Received: 13 February 2015
Accepted: 23 July 2015
Published: 11 September 2015
Abscisic acid (ABA) plays crucial roles in regulating plant growth and development, especially in responding to abiotic stress. The pyrabactin resistance-like (PYL) abscisic acid receptor family has been identified and widely characterized in Arabidopsis. However, PYL families in rice were largely unknown. In the present study, 10 out of 13 PYL orthologs in rice (OsPYL) were isolated and investigated.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that expression of OsPYL genes is tissue-specific and display differential response to ABA treatment, implying their functional diversity. The interaction between 10 OsPYL members and 5 protein phosphatase 2C in rice (OsPP2C) members was investigated in yeast two-hybrid and tobacco transient expression assays, and an overall interaction map was generated, which was suggestive of the diversity and complexity of ABA-sensing signaling in rice. To study the biological function of OsPYLs, two OsPYL genes (OsPYL3 and OsPYL9) were overexpressed in rice. Phenotypic analysis of OsPYL3 and OsPYL9 transgenic rice showed that OsPYLs positively regulated the ABA response during the seed germination. More importantly, the overexpression of OsPYL3 and OsPYL9 substantially improved drought and cold stress tolerance in rice.
Taken together, we comprehensively uncovered the properties of OsPYLs, which may be good candidates for the improvement of abiotic stress tolerance in rice.
Abscisic acid (ABA) plays pivotal roles in regulating plant growth and development, including seed dormancy, germination, and seedling growth. More importantly, ABA is the key phytohormone that functions in a plant’s response to abiotic stressors such as drought, high salinity, and extreme temperature (Cutler et al. 2010). During abiotic stress, ABA biosynthesis is activated, resulting in an increase in ABA levels in the plant. ABA binds to the pyrabactin resistant-like/regulatory components of ABA receptors, PYL/RCAR (hereafter referred to as PYLs for simplicity), an ABA receptor family that promotes the interaction between PYL with protein phosphatase 2C (PP2C), which then results in the release of SNF1-related protein kinase (SnRK)) from the repression of PP2C. Finally, the active SnRK phosphorylates and activates downstream transcriptional factors that promote the expression of ABA-regulated genes. An ABA signal is then generated, which in turn results in the acquisition of abiotic stress resistance in plants (Ma et al. 2009; Park et al. 2009; Cutler et al. 2010; Klingler et al. 2010). ABA receptor PYL proteins, which contain a conserved steroidogenic acute regulatory-related lipid transfer (START) protein domain, are the core components of this ABA sensing signaling pathway (Ma et al. 2009; Park et al. 2009). To date, PYL proteins have been identified from distinct plant species, including Arabidopsis, rice, tomato, and soybean, which present highly conserved PYL-mediated ABA-sensing signaling pathways (Ma et al. 2009; Park et al. 2009; Kim et al. 2012; Bai et al. 2013; Gonzalez-Guzman et al. 2014; He et al. 2014). The biochemical property and structure of PYLs have been extensively studied in the dicot plant model Arabidopsis (Ma et al. 2009; Melcher et al. 2009; Klingler et al. 2010; Joshi-Saha et al. 2011). Some AtPYLs are monomers that facilitate interactions with PP2C in the absence of ABA; some AtPYLs are in a dimeric state and require ABA to form a complex with PP2C (Hao et al. 2011). Recent studies have investigated the biological functions of various AtPYLs. The overexpression of AtPYL5 leads to ABA hypersensitivity during early seedling development, as well as enhanced drought stress tolerance (Klingler et al. 2010). AtPYL8 is involved in root growth and development, which is in line with its root-specific expression pattern (Antoni et al. 2013). Although AtPYL13 is not an ABA receptor, it can positively regulate the ABA signaling pathway by interacting with and inhibiting both the PYL receptors and the PP2C co-receptors (Li et al. 2013; Zhao et al. 2013). AtPYL4A194T forms stable complexes with PP2CA in the absence of ABA, and the overexpression of AtPYL4A194T increases a plant’s sensitivity to ABA-mediated inhibition of germination and seedling establishment, as well as enhances drought resistance (Pizzio et al. 2013). In addition, AtPYLs are largely functionally conserved, and the analysis of higher order mutants have indicated that AtPYLs regulate stomatal conductance (Gonzalez-Guzman et al. 2012).
Unlike AtPYLs, information on PYL homologs in rice is limited. Studies have predicted that rice has 13 OsPYL members that share high sequence similarity with AtPYLs (Kim et al. 2012; He et al. 2014). Core components in ABA signaling, including OsPYLs, OsPP2C, OsSAPK2, and OsOREB1, have been identified in rice, and the ABA signaling transduction pathway has also been reconstituted in a protoplast system (Kim et al. 2012). The biochemical properties and structure of OsPYLs have also been recently reported (He et al. 2014). However, to date, only OsPYL/RCAR5 has been identified as a positive regulator in seed germination, early seedling growth, and drought and salt stress tolerance (Kim et al. 2012; Kim et al. 2014). Other features of OsPYLs such as expression pattern, subcellular localization, interaction specificity with OsPP2C, and biological function, have not been examined.
The present study examined the tissue-specific expression pattern and distinct response of OsPYL members to ABA treatment, which was suggestive of its differential biological function. An overall interaction map between 10 OsPYLs and 5 OsPP2C members indicated that OsPYLs were selective of their interaction partner, thus indicating the complexity and specificity of the rice ABA-sensing signaling pathway. Furthermore, using OsPYL3 and OsPYL9, we determined that OsPYLs play pivotal roles in ABA-mediated inhibition of seed germination, as well as drought and cold stress tolerance, which thereby could serve as potential targets for the improvement of abiotic stress tolerance in rice.
Expression Pattern of OsPYL Members
OsPYLs are also differentially expressed after ABA treatment (Fig. 1b). Some OsPYLs were downregulated such as OsPYL1, OsPYL2/9, and OsPYL3 (Fig. 1b), whereas OsPYL4 was upregulated (Fig. 1b). The expression of OsPYL5, OsPYL7/8, and OsPYL10 was not affected by ABA treatment (Fig. 1b). These findings suggest that OsPYL members play diverse roles in sensing the ABA signal.
OsPYLs are Localized in the Cytosol and Nucleus
OsPYL Members Selectively Interact with OsPP2C Members
OsPP2C Determines the Subcellular Localization of the OsPYLs-OsPP2C Complex in an ABA-independent Manner
The results of the BiFC assay indicated that the subcellular localization of the fused green fluorescent proteins varied among different OsPYLs and OsPP2C members, and most of the OsPYL-OsPP2C complexes were localized in the nucleus (Fig. 4), which is not consistent with the localization of OsPYLs (Fig. 2a). To explore the underlying mechanism of this activity, the subcellular localization of five OsPP2C members was investigated. OsPP2C53 was localized to both the nucleus and cytosol, whereas other OsPP2C members were detected only in the nucleus (Fig. 2b). Comparative analysis showed a similarity between the subcellular localization of OsPP2C members and that of OsPYLs-OsPP2C complexes (Figs. 2b and 4). In the BiFC assay, ABA was also injected to test whether ABA affected the interaction between OsPYLs and OsPP2Cs. The results showed that ABA treatment only enhanced interaction strength, and did not affect the subcellular localization of the OsPYL-OsPP2C complexes (Fig. 4). Consequently, the findings indicated that OsPP2C members can determine the subcellular localization of OsPYL-OsPP2C complexes in an ABA-independent manner.
Overexpression of OsPYL3 and OsPYL9 Confers ABA Hypersensitivity during Seed Germination
Overexpression of OsPYL3 and OsPYL9 Enhances Drought Stress Tolerance
Plants lose water mainly through stomata transpiration. To determine whether stomatal closure is involved in the enhanced drought tolerance of OsPYL3 and OsPYL9 overexpression lines, a water-loss assay was performed using detached leaves. The water loss rate of the OsPYL3 and OsPYL9 overexpression lines was significantly slower than that observed in the controls (Fig. 6d and e). Relative water content (RWC) more accurately reflects the physiological consequence of cellular water deficit. Fig. 6e and g shows that with drought stress, the OsPYL overexpression lines lost water at a slower rate and have a lower minimum relative water content that helped the plants survive during severe drought conditions. These results indicated that the OsPYL3 and OsPYL9 overexpression lines have a faster rate of stomatal closure and were hypersensitive to drought treatment compared to the controls, thereby contributing to a high level of drought stress tolerance.
Overexpression of OsPYL3 and OsPYL9 Increases Cold Stress Tolerance
Expression of ABA-Regulated Genes is Enhanced in OsPYL3 and OsPYL9 Overexpression lines
Members of the PYL protein family such as the ABA receptors and the core component in ABA-sensing signaling have been investigated in various plant species, including Arabidopsis, soybean, rice, and cucumber (Ma et al. 2009; Wang et al. 2012; Bai et al. 2013; He et al. 2014; Kim et al. 2014). Compared to the extensive studies on AtPYLs, the property, function, and mechanism of OsPYLs are largely unknown. In the present study, based on amino acid sequence analysis of AtPYLs and predicted OsPYLs in the rice genome annotation project, 10 out of the 13 OsPYLs were cloned and studied. OsPYLs were differentially expressed in the leaves, stems, roots, panicles, embryo, and endosperm, and some OsPYL members showed pronounced tissue-specific expression patterns (Fig. 1a). OsPYLs also displayed a differential response to ABA treatment (Fig. 1b). The diverse expression patterns of OsPYLs were indicative of their functional diversity. All OsPYLs were localized to the cytoplasm and nucleus (Fig. 2a), which was consistent with the results of previous reports on the subcellular localization of AtPYLs and GmPYLs (Ma et al. 2009; Bai et al. 2013).
The interaction between the OsPYLs and OsPP2Cs was investigated in both yeast and tobacco, and an overall interaction map was generated between 10 OsPYLs and 5 OsPP2C members in the absence or presence of ABA treatment (Figs. 3 and 4). OsPYLs selectively interacted with OsPP2Cs in ABA-dependent or ABA-independent manner. Combined with a recent report that OsPYLs inhibit OsPP2C activity (He et al. 2014), these data suggest that OsPYLs are functional rice ABA receptors.
In addition to the discovery of PYLs as ABA receptors, the present study has shown that these proteins also regulate ABA signaling and improve tolerance to abiotic stress. Overexpression of AtPYL4 and AtPYL5 leads to enhanced ABA hypersensitivity during seed germination and improved drought tolerance (Santiago et al. 2009; Pizzio et al. 2013). In addition, the root-specific expression of AtPYL8 regulates ABA-mediated inhibition of root growth (Antoni et al. 2013). However, information on the biological function of OsPYLs in rice, which is considered a monocot model, is limited. OsPYL5 has been shown to positively regulate seed germination and drought stress response (Kim et al. 2012; Kim et al. 2014). In the present study, transgenic rice overexpressing OsPYL3 and OsPYL9 were produced and analyzed in detail (Fig. 5a and b). First, overexpression of OsPYL3 and OsPYL9 conferred ABA hypersensitivity during seed germination (Figs. 5c-i). In addition, overexpression of OsPYL3 and OsPYL9 in the absence of ABA significantly delayed seed germination, and OsPYL9 played more important roles, which was consistent with the upregulation of OsPYL9 in the embryo compared to that of OsPYL9 (Fig. 5c-i). Second, overexpression of OsPYL3 and OsPYL9 enhanced drought stress tolerance, which can be partly explained by a slower water loss rate through the stomata in transgenic lines (Fig. 6). Third, OsPYL3 and OsPYL9 overexpression lines show increased membrane stability during cold stress and enhanced cold stress tolerance (Fig. 7). Last, expression of ABA-regulated genes in OsPYL3 and OsPYL9 overexpression lines was significantly higher than that in the controls (Fig. 8), which accounted for the increased stress tolerance of transgenic plants. Interestingly, compared to the controls, OsPYL3 and OsPYL9 overexpression lines did not show any negative effect on growth, grain yield, and other observable phenotypes, whereas these were observed in OsPYL5 (Kim et al. 2014). One possible explanation for this finding is that OsPYL5 was directed by the maize ubiquitin promoter, which is a very strong promoter in monocots, and OsPYL3 and OsPYL9 was directed by the 35S promoter, whose efficiency was less than that of ubiquitin in rice. Taken together, these results indicate that OsPYL3 and OsPYL9 can be used as a target gene for the improvement of abiotic stress tolerance in rice.
In the present study, we study the expression pattern of PYL orthologs in rice (OsPYL), generate an overall interaction map between 10 OsPYL members and 5 protein phosphatase 2C in rice (OsPP2C) members and show the biological function ofOsPYL3 and OsPYL9. Taken together, we comprehensively uncovered the properties of OsPYLs, which may be good candidates for the improvement of abiotic stress tolerance in rice.
Plant materials, and rice transformation
For cloning of OsPYLs and OsPP2Cs genes, Oryza sativa L. ssp. Japonica cv. Nipponbare was used. The coding sequence of OsPYLs and OsPP2Cs were cloned from the cDNA using standard PCR-based protocol. The primers used are listed at Additional file 1: Table S1. Full-length sequences of OsPYLs and OsPP2Cs were cloned into pENTR/D-Topo (Invitrogen), resultant constructs were confirmed by sequencing and saved for later use. To over express the OsPYLs in rice, OsPYLs was transferred to pH7WG2 vector via LR recombination reaction of the Gateway system. The resultant 35S:OsPYLs constructs, in which OsPYLs was drived by the cauliflower mosaic virus (CaMV) 35S promoter, were transferred into Agrobacterium tumefaciens EHA105. Finally, OsPYLs were transformed into Oryza sativa L. ssp. Japonica cv. Longjing 11 by the Agrobacterium-mediated co-cultivation method.
Yeast two-hybrid assay
Full-length sequences of OsPYLs and OsPP2Cs were cloned into pDEST32 or pDEST22 vector via an LR recombination reaction and used as baits or preys respectively. The resultant constructs were transformed into the yeast strain Y2H gold. Presence of the transgenes was confirmed by growth on a SD/-Leu/-Trp plate. To assess protein interactions, the transformed yeast cells were suspended in liquid SD/-Leu/-Trp to OD600 = 1.0. The suspended cells were spread on plates containing SD/-His/-Leu/-Trp medium supplied with indicated concentration of 3-AT (3-amino-1, 2, 4-triazole) and ABA. The interactions were observed after 4 days of incubation at 30 °C. The experiments were repeated three times with similar results.
Subcellular localization and Bimolecular fluorescence complementation in tobacco
For subcellular localization of OsPYLs and OsPP2Cs, full-length sequences of OsPYLs and OsPP2Cs were cloned into the pH7WGF vector via an LR recombination reaction, in which the OsPYLs and OsPP2Cs fused with green fluorescent protein (GFP) was drived by 35S promoter. For Bimolecular fluorescence complementation (BiFC) assays, OsPYLs were fused with the C-terminal portion of the yellow fluorescent protein (YFP) via an LR recombination reaction; OsPP2Cs were fused with the N-terminal portion of the yellow fluorescent protein (YFP) via an LR recombination reaction. For transient expression, the fusion constructs were transferred into Agrobacterium tumefaciens GV3101. Agrobacterium tumefaciens strain harboring each construct along with the p19 strain were infiltrated into 4-week-old N. benthamiana leaves. For staining of the nuclei, 10 mg/ml 4, 6-diamidino-2-phenylindole (DAPI) was infiltrated into N. benthamiana leaves 3 h before observation. For microscopic analyses, leaf discs were cut 3 d after infiltration. The fluorescence signal was observed using confocal microscopy.
Analysis of genes expression
Total RNA was extracted using TRIzol (Invitrogen) and treated with DNaseI. cDNA was synthesized from 2 µg of total RNA using SuperscriptII Reverse Transcriptase. Real-time PCR was performed with SYBR Green PCR master mix (TransStart). Data were collected using Bio-Rad chromo 4 real-time PCR detector. All expressions were normalized against the Ubiquitin gene. The primers used are listed at Additional file 1: Table S1.
Germination and abiotic stress tolerance assay
Dehulled seeds were surface-sterilized and planted on half-strength MS medium supplemented with indicated concentration of ABA (A1049, Sigma-Aldrich). Seed germination was defined as the coleoptiles emerged from the seed and scored every 12 h for 5 days. Three independent T3 homologous transgenic lines and the control Longjing 11 were used for stress tolerance experiments. For the dehydration treatment, rice plants grown for 3 weeks were withhold water for 10 days and then rehydrated and grown under normal conditions for 7 days. For cold treatment, 14-day-old seedlings were transferred to 10 °C for 4 days and then returned to normal growth conditions for 7 days. The survival rates were recorded. Approximately 50 seedlings of each line were used for each experiment, and three replicates of each experiment were performed. Tests for statistical analysis between transgenic lines and the controls were performed using Microsoft excel 2007.
Water loss assay and measurement of relative electrolyte leakage
For water loss assay, leaves of control and OsPYLs transgenic plants grown under normal conditions were detached from 3-week-old seedlings and weighed immediately on a piece of weighing paper, and then placed on a laboratory table and weighed at indicated time intervals. Three replicates were performed for each line.
For relative water content measurement (RWC), Three-week-old seedlings of control and three independent OsPYLs transgenic lines were withhold water. The protocol was as described by (Barrs and Weatherley 1962). RWC was measured until the leaf cannot expand during dipped in the water.
Two-week-old seedlings of control and three independent OsPYLs transgenic lines were transferred into a 10 °C chamber. At 0 d, 2 d and 3 d, 0.5 g of leaves were harvested from each of ten plants. Leaf fragments were immersed in 6 mL deionized water and shaken at 100 rpm at 25 °C for 2 h, and electrical conductivity was determined (C1). The samples were then boiled for 20 min, and the total conductivity was determined again (C2) after cooling to room temperature. Relative ion leakage (%) was calculated as C1/C2 × 100.
This work was supported by Hundred–Talent-Program of Chinese Academy of Sciences, National Natural Science Foundation of China (No. 31070255), Excellent Academic Leaders of Harbin (RC2014XK002003), National Key Project of Transgenic Crops of China (2014ZX0800205B-003) and the 863 High Tech Program of Ministry of Science and Technology of China (2012AA101105).
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