Roles of the different components of magnesium chelatase in abscisic acid signal transduction
The H subunit of Mg-chelatase (CHLH) was shown to regulate abscisic acid (ABA) signaling and the I subunit (CHLI) was also reported to modulate ABA signaling in guard cells. However, it remains essentially unknown whether and how the Mg-chelatase-catalyzed Mg-protoporphyrin IX-production differs from ABA signaling. Using a newly-developed surface plasmon resonance system, we showed that ABA binds to CHLH, but not to the other Mg-chelatase components/subunits CHLI, CHLD (D subunit) and GUN4. A new rtl1 mutant allele of the CHLH gene in Arabidopsis thaliana showed ABA-insensitive phenotypes in both stomatal movement and seed germination. Upregulation of CHLI1 resulted in ABA hypersensitivity in seed germination, while downregulation of CHLI conferred ABA insensitivity in stomatal response in Arabidopsis. We showed that CHLH and CHLI, but not CHLD, regulate stomatal sensitivity to ABA in tobacco (Nicotiana benthamiana). The overexpression lines of the CHLD gene showed wild-type ABA sensitivity in Arabidopsis. Both the GUN4-RNA interference and overexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses. These findings provide clear evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.
KeywordsH subunit of Mg-chelatase I subunit of Mg-chelatase D subunit of Mg-chelatase GUN4 ABA binding ABA signal transduction
Abscisic acid (ABA) is an essential hormone to regulate plant growth and development and to control plant adaptation to environmental challenges (reviewed in Finkelstein and Rock 2002; Adie et al. 2007). As one of the highly complex plant cell signaling systems, ABA signaling begins with signal perception, which triggers downstream signaling cascades to induce the final physiological responses. It has been believed that the ABA signal is sensed by cells with multiple receptors including plasma membrane and intracellular receptors (Assmann 1994; Finkelstein et al. 2002; Verslues and Zhu 2007). In the past decades, ABA signal transduction has been extensively studied, and numerous signaling components, including ABA receptors, have been identified. These ABA signaling regulators involve diverse proteins, which localize to different cellular compartments including plasma membrane, cytosolic space and nucleus. Functioning on cell surface, two candidate plasma membrane ABA receptors—an unconventional G-protein-coupled receptor (GPCR) GCR2 and a novel class of GPCR-type G proteins GTG1 and GTG2—have been reported (Liu et al. 2007a, b; Johnston et al. 2007; Pandey et al. 2009), though it is controversial whether GCR2 regulates ABA-mediated inhibition of seed germination and post-germination growth (Gao et al. 2007; Guo et al. 2008). GTGs are positive regulators of ABA signaling and interacts with the sole Arabidopsis G protein α subunit GPA1 (Pandey and Assmann 2004), which may negatively regulate ABA signaling by inhibiting the activity of GTG-ABA binding (Pandey et al. 2009). Many other membrane-associated proteins, such as phospholipases C/D (Fan et al. 1997; Sanchez and Chua 2001; Zhang et al. 2004, 2009), other GPCR members (such as GCR1) and G proteins (Wang et al. 2001; Pandey and Assmann 2004; Pandey et al. 2006), and receptor-like kinases (Osakabe et al. 2005), have been reported to be involved in ABA signaling. However, whether these plasma membrane-localized proteins cooperates with plasma membrane ABA receptors to regulate early events of ABA signaling processes on the cell surface remains an interesting and open question.
Intracellular ABA signaling regulators involve numerous proteins of diverse identities such as various protein kinases, type-2C/A protein phosphatases (PP2C/A), ubiquitin E3 ligases involved in degradation of ABA signaling proteins, and various classes of transcription factors (for reviews, see Shinozaki et al. 2003; Fan et al. 2004; Seki et al. 2007; Cutler et al. 2010). Most recently, PYR/PYL/RCAR proteins, the members of a START domain superfamily, were reported to function as cytosolic ABA receptors by inhibiting directly type 2C protein phosphatases (Ma et al. 2009; Park et al. 2009; Santiago et al. 2009). A PYL/PYR/RCAR-mediated ABA signaling pathway from ABA perception to downstream gene expression has been reconstituted in vitro (Fujii et al. 2009; Cutler et al. 2010). In this PYR/PYL/RCAR-mediated ABA signaling pathway, PP2Cs relay ABA signal directly from the PYR/PYL/RCAR ABA receptors to their downstream regulators SNF1-related protein kinase 2s (SnRK2s), which activate an ABF/AREB/ABI5 clade of bZIP-domain transcription factors via a protein phosphorylation process, and finally induce physiological ABA responses (Fujii et al. 2009; Cutler et al. 2010). However, it is widely believed that the networks of ABA signaling pathways are highly complex, and connections of other numerous ABA signaling components with the PYR/PYL/RCAR ABA receptors remain to be explored.
We previously reported that the magnesium-protoporphyrin IX (Mg-ProtoIX) chelatase large subunit (Mg-chelatase H subunit CHLH/putative ABA receptor ABAR), a chloroplast/plastid protein, binds ABA and functions in ABA signaling, thus meeting the essential criteria of a candidate receptor for ABA in Arabidopsis thaliana (Shen et al. 2006; Wu et al. 2009). We further identified a CHLH-mediated ABA signaling pathway in which CHLH antagonizes a WRKY-domain transcription repressor to relieve ABA-responsive genes of inhibition (Shang et al. 2010). Although the identity of CHLH as an ABA receptor is controversial (Müller and Hansson 2009; Tsuzuki et al. 2011), we provide multiple lines of evidence to show that CHLH binds ABA (Shen et al. 2006; Wu et al. 2009; Wang et al. 2011) on the one hand, and on the other, consistent with our observations (Shen et al. 2006; Wu et al. 2009; Shang et al. 2010), evidence from independent groups reveals that CHLH mediates ABA signaling in guard cells of both Arabidopsis (Legnaioli et al. 2009; Tsuzuki et al. 2011) and peach (Prunus persica) leaves (Jia et al. 2011a). Also, it has been demonstrated that CHLH is a key component connecting the circadian clock with ABA-mediated plant drought responses in Arabidopsis (Legnaioli et al. 2009) and mediates ABA signaling in fruit ripening of both peach (Jia et al. 2011a) and strawberry (Fragaria ananassa, Jia et al. 2011b). These data consistently demonstrate that CHLH is an essential ABA signaling regulator in plant cells.
CHLH has multiple functions in plant cells. One of its functions is to chelate magnesium to protoporphyrin IX, which provides Mg-ProtoIX in the chlorophyll biosynthesis pathway (Gibson et al. 1996; Willows et al. 1996; Walker and Willows 1997; Guo et al. 1998; Papenbrock et al. 2000). The second role of CHLH is to mediate plastid-to-nucleus retrograde signaling, known as Genomes Uncoupled 5 (GUN5), and this function in the retrograde signaling may be connected with its role in catalyzing production of Mg-ProtoIX (Mochizuki et al. 2001; Nott et al. 2006). It has been well established that Mg-chelatase functions in catalyzing Mg-ProtoIX production as a hetero-tetramer, which is composed of Mg-chelatase subunits H, I (CHLI), D (CHLD) (Gibson et al. 1996; Willows et al. 1996; Walker and Willows 1997; Guo et al. 1998; Papenbrock et al. 2000) and a supplementary and essential component GUN4 (Genomes Uncoupled 4) that binds CHLH and activates Mg-chelatase (Larkin et al. 2003; Peter and Grimm 2009; Adhikari et al. 2011). A recent report showed that, besides CHLH, CHLI also mediates guard cell signaling in response to ABA (Tsuzuki et al. 2011). However, it remains essentially unknown whether Mg-chelatase heterotetramer complex or only two subunits CHLH and CHLI function in ABA signaling, and why the Mg-ProtoIX production process may differ from the CHLH-mediated ABA signaling. To explore this mechanism is of importance to understanding complex ABA signaling pathways. Here we report that, using a newly-developed surface plasmon resonance (SPR) technique, CHLH, but not CHLI, CHLD or GUN4, was shown to interact with ABA. Further findings demonstrate that CHLH and CHLI, but not CHLD nor GUN4, are ABA signaling regulators in the major ABA responses, and that the functions of CHLH and CHLI are not limited to ABA signaling in guard cells. The data provide clear and direct evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.
Interactions of CHLH/ABAR with CHLI and CHLD
CHLH, but not CHLI, CHLD or GUN4, binds ABA
In contrast to CHLH, CHLI (Fig. 1c. d), CHLD (Fig. 2e, f) or GUN4 (Fig. 2g, h) did not show substantial ABA-binding abilities. These three proteins showed only a low and nonspecific ABA-binding background (Fig. 2d, f, h), which may serve as a negative control.
New observations of a mutant allele of CHLH gene, rtl1
Upregulation of CHLI1 confers ABA hypersensitivity in seed germination, while downregulation of CHLI results in ABA insensitivity in stomatal response
We compared the mutant cs and the CHLI-RNAi and CHLI-overexpression lines with cch, rtl1 and two well characterized mutants abi4-1 and abi5-1 in their ABA sensitivity in seed germination. While confirming ABA hypersensitivity of the CHLI-overexpression line and wild-type ABA response of the cs mutant and the CHLI-RNAi line, we observed that cch mutant displayed substantially the same intensity of ABA-insensitivity as abi4-1 mutant at low ABA concentrations (<1 μM), but weaker ABA-insensitivity than abi4-1 mutant at high ABA concentrations (1 to 3 μM) (Fig. 3e). The cch mutant showed higher degree of ABA-insensitivity than abi-5 mutant, and rtl1 mutant displayed substantially the same intensity of ABA-insensitivity as abi5-1 mutant and weaker ABA-insensitivity than abi4-1 mutant (Fig. 3e).
Downregulation of CHLH or CHLI, but not CHLD, reduces stomatal sensitivity to ABA in tobacco, and upregulation of CHLD has no impact on ABA sensitivity in Arabidopsis
Down- or up-regulation of GUN4 gene does not alter ABA response
CHLH and CHLI are positive regulators of ABA signaling: new observations in Arabidopsis and tobacco
We previously showed that the cch mutation of CHLH gene reduced ABA sensitivity in all the three major ABA responses including ABA-induced inhibition of seed germination, post-germination growth arrest, and promotion of stomatal closure and inhibition of stomatal opening (Shen et al. 2006; Wu et al. 2009). The data in the present experiment (Figs. 3, 4c) are consistent with these previous observations. However, a new mutant of CHLH gene rtl1, allelic to cch, was reported to have the substantial same ABA-related phenotypes as cch, which reduced only stomatal sensitivity to ABA but did not affect other two major ABA response during seed germination and post-germination growth (Tsuzuki et al. 2011). In our present experiment, however, we showed that the rtl1 mutation reduces significantly ABA sensitivity both in seed germination and in stomatal response, though the ABA response in post-germination growth was not affected (Fig. 4), supporting the idea that the functions of CHLH are not limited to mediating stomatal signaling in response to ABA.
We observed that this inconsistency in the phenotypes in seed germination and seedling growth of the cch (and also rtl1) mutant is mainly due to variations in growth conditions. The cch mutant was identified as a light- and drought-sensitive mutant (Mochizuki et al. 2001; Shen et al. 2006). We previously emphasized that the growth conditions with a sufficient irrigation, high relative air humidity (60–70 %), moderate light intensity (about 120 μmol photons m−2 s−1) to ensure the good growth status of the cch mutant parental plants are of critical importance to their progeny to display the ABA-related phenotypes (Wu et al. 2009). Otherwise, the stressed mutant parental plants had progeny with shrunken seeds, whose insensitive phenotypes to ABA became weaker in germination and post-germination growth, but the strong ABA-insensitive phenotype in stomatal movement was not changed (Wu et al. 2009).
We reproduced well the significant ABA insensitive phenotypes of the cch mutant in all the three major ABA responses with plump seeds of quality harvested from the mutant plants grown in the above-mentioned favourable conditions (Shen et al. 2006; Wu et al. 2009; Shang et al. 2010; and this experiment). This is also true for the rtl1 mutant. The phenomenon of the conditional ABA insensitivity in seed germination and post-germination growth may be likely due to a stress-induced upregulation of ABA-responsive mechanisms independent of CHLH-mediated signaling in the cch (or rtl1) mutant (Wu et al. 2009), which needs to be clarified in the future.
The similar phenomenon of the conditional ABA-insensitive phenotypes in seed germination and post-germination growth was also observed in the CHLH-RNAi plants. We previously observed that the ABA insensitivity in stomatal response has a significant negative correlation with the CHLH levels in the RNAi lines (Shen et al. 2006). However, whereas a globally negative correlation of the ABA insensitivity with the CHLH levels was found in germination and post-germination growth, the phenotypes become weaker and dependent on good growth conditions when the CHLH levels are low to a certain extent especially when the RNAi plants show yellow leaves (Shen et al. 2006, see Supplementary Fig. 3 in this reference). We observed that the CHLH-RNAi plants showed strong ABA insensitive phenotypes in all the three ABA responses when the CHLH expression was reduced to an extent at which the residual CHLH protein levels were higher than 30 %, which could ensure that the RNAi plants remained green (Shen et al. 2006, see Fig. 2 and Supplementary Fig. 3 in this reference). These findings are essentially consistent with the above-mentioned observations in the cch or rtl1 mutants both with yellow leaves.
All the missense mutants of CHLH gene, including abar-2 (Wu et al. 2009), abar-3 (Wu et al. 2009), cch (Shen et al. 2006; Wu et al. 2009) and rtl1 (Tsuzuki et al. 2011) but except for gun5 (Mochizuki et al. 2001; Shen et al. 2006), show altered ABA sensitivity in the major ABA responses (Supplementary Fig. 1). Additionally and noteworthily, several independent groups provided evidence that CHLH mediates stomatal signaling in response to ABA in Arabidopsis (Legnaioli et al. 2009; Tsuzuki et al. 2011) as well as in peach (P. persica) (Jia et al. 2011a). In the present experiment, we showed that CHLH also regulates stomatal response to ABA in tobacco (Fig. 5). Interestingly, CHLH functions in the regulation ABA signaling in fleshy fruit ripening such as peach (Jia et al. 2011a) and strawberry (F. ananassa, Jia et al., 2011b). All these findings support the idea that CHLH is a conserved ABA signaling regulator in plant cells.
It is interesting that, previously, CHLI was shown to be involved in stomatal response to ABA in Arabidopsis (Tsuzuki et al. 2011). In the present experiment, we confirmed the role of the CHLI in stomatal response to ABA in Arabidopsis as well as in tobacco (Figs. 4, 5), and observed that down regulation of CHLI did not affect ABA responses in seed germination and seedling growth, while upregulation of CHLI1 enhanced ABA sensitivity in ABA-induced inhibition of seed germination (Fig. 4b), suggesting that CHLI likely regulates ABA signaling in both stomatal movement and seed germination. This is possible because the CHLI gene has two copies in Arabidopsis genome and double knockout mutants of two CHLI genes are lethal (Huang and Li 2009), while the leaky mutants (including the RNAi lines) may not show ABA-related phenotypes in seed germination and seedling growth.
Taken together, all the data demonstrate that CHLH and CHLI are two positive regulators of ABA signaling, which may be likely to cooperate to function in plant cell response to ABA.
CHLH, but not CHLI, CHLD or GUN4, is an ABA-binding protein
CHLH was originally isolated as an ABA-binding protein from total proteins of broad bean leaves by an ABA-affinity column that specifically binds CHLH via ABA, and was shown potentially to function in broad bean stomatal signaling in response to ABA (Zhang et al. 2002), though the ABA-affinity technique was questioned because ABA was immobilized on the affinity resin at its carboxyl group that was shown to be required for ABA’s function (Cutler et al. 2010). However, the ABA binding ability of CHLH was confirmed by both the same ABA-affinity system (Wu et al. 2009) and a 3H-labeled ABA binding assay (Shen et al. 2006; Wu et al. 2009). Further, we showed that the C-terminal half of CHLH is essential both for ABA binding and for ABA signaling that was evidenced by expression of this C-terminal protein in wild-type and cch mutant plants (Wu et al. 2009).
In contrast to the PYR/PYL/RCAR receptor for ABA, a cytosolic protein with a low molecular mass (about 20 kDa) and a highly hydrophilic nature (Ma et al. 2009; Park et al. 2009; Santiago et al. 2009), the CHLH protein is a chloroplast-membrane protein (Shang et al. 2010), and has a high molecular mass (about 150 kDa) and a slightly-hydrophobic nature. We observed that the CHLH protein aggregates and becomes rapidly unstable in solution (data not shown), which makes ABA binding assay difficult (Wang et al. 2011) and may likely be a reason why ABA binding of CHLH was not detected by other groups with the 3H-labeled ABA binding assay (Müller and Hansson 2009; Tsuzuki et al. 2011). Therefore, we adopted a new approach to test ABA binding of CHLH protein so that the data may be easily reproducible, which includes a new system to produce highly active CHLH protein with an insect cell line, and a surface plasmon resonance (SPR) system (Biacore T200, GE). The results showed that CHLH binds ABA with a saturation curve typical for receptor-ligand binding, while other Mg-chelatase components/subunits CHLI, CHLD and GUN4 do not bind ABA (Fig. 2). These data, easily reproduced with the insect-cell-produced protein and the SPR equipment, confirmed qualitatively the ABA-binding nature of the CHLH protein, and verified our previous observations (Shen et al. 2006; Wu et al. 2009).
However, the SPR system has technical limitations. In this system, CHLH protein should be linked to a sensor chip with carboxyl groups on its surface to which the sample protein is covalently immobilized via -NH2 bond of the protein, which may induce conformational change of the sample protein and reduce its activity. A structurally complex protein may partly lose its activity in some in vitro system, which was also observed in the ABA binding assay of the plasma membrane GTG1/2 receptors for ABA (Pandey et al. 2009). In addition, there is also a limitation of sensitivity to the weak signal for the signal-detecting system of the SPR equipment to test the interaction of this huge, hydrophobic CHLH protein with a small ligand ABA. The detected low ABA-affinity of CHLH protein is likely to be attributed to these technical limitations, though we previously detected high ABA affinity of this protein in a 3H-labeled ABA binding assay (Shen et al. 2006; Wu et al. 2009).
The CHLI subunit functions in ABA signaling in stomatal movement (Figs. 4, 5; and Tsuzuki et al. 2011) and also likely in seed germination (Fig. 4), while it does not directly interact with ABA (Fig. 2), suggesting that CHLI may function through interaction with CHLH. We showed that CHLI interacts directly with CHLH with stronger interaction intensity than that between CHLH and CHLD, which seems to support that CHLH interacts with CHLI to form a hetero-dimer to function in ABA signaling. We were not able to test whether CHLI affects the ABA binding activity of CHLH to be involved in ABA signaling because of the technical difficulties with the SPR system, but we could propose another possibility that CHLI may possibly modify the interactions between CHLH and its downstream regulators such as the WRKY40 transcription factor (Shang et al. 2010) to regulate ABA signaling, which needs studies in the future.
CHLH and CHLI-mediated ABA signaling is distinct from Mg-protoporphyrin IX production
Previous experiments suggested that the CHLH-mediated signaling may be distinct from Mg-ProtoIX production and chloroplast retrograde signaling. No correlation between the Mg-ProtoIX levels and stomatal response to ABA was found (Shen et al. 2006), and both the gun5 and cch mutations of CHLH/ABAR/GUN5 gene reduced Mg-ProtoIX production and affected chloroplast retrograde signaling (Mochizuki et al. 2001), while the cch mutant alone, but not gun5 mutant, showed ABA insensitive phenotypes (Shen et al. 2006; Wu et al. 2009). In the present experiment, we showed that, in contrast to CHLH and CHLI that are two positive regulators in ABA signaling, other two Mg-chelatase components CHLD and GUN4 are not involved in ABA signaling, as evidenced by CHLD-VIGS lines in tobacco (Fig. 5), CHLD-overexpression lines in Arabidopsis (Fig. 6), and GUN4-RNAi and overexpression lines in Arabidopsis (Fig. 7). Given that, as two components/subunits of Mg-chelatase, both CHLD and GUN4 are essential to the Mg-chelatase activity and Mg-ProtoIX production (Gibson et al. 1996; Larkin et al. 2003; Peter and Grimm 2009; Adhikari et al. 2011), the present data provide clear and direct evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is independent of the CHLH and CHLI-mediated ABA signaling, and give information to understand the mechanism by which the two cellular processes differs at the molecular level. Additionally, previous reports showed that CHLD (Strand et al. 2003), but not CHLI (Mochizuki et al. 2001), functions in chloroplast retrograde signaling, which contrasts with our above-mentioned findings that CHLI functions, but CHLD is not involved, in ABA signaling (Figs. 5, 6, 7), supporting the notion that the ProtoIX production and chloroplast retrograde signaling are independent of the CHLH and CHLI-mediated ABA signaling.
Materials and methods
Plant materials, generation of transgenic lines and growth conditions
Arabidopsis thaliana ecotype Columbia (Col-0) was used in the generation of transgenic plants. The mutated abi5 gene in the abi5-1 mutant (ABRC stock number CS8105; named abi5 in this report) was transferred from its background Wassilewskija (Ws) ecotype into Col-0 ecotype by backcrossing. To generate RNAi lines with down-regulated expression of CHLI1 gene (Arabidopsis genomic locus At4g18480), CHLD (At1g08520) and GUN4 (At3g59400), we chose a gene-specific fragment from each of their cDNAs. A 268-bp fragment corresponding to the region of nt 35–302 of the CHLI1 cDNA, a 183-bp fragment corresponding to the region of nt 1334 to1516 of the CHLD cDNA, and a 337-bp fragment corresponding to the region of nt 429–765 of the GUN4 cDNA, were amplified by PCR, respectively, with forward primer 5′-GCGTCGACAACTTCATCTCATCTTGCCCTAC-3′ and reverse primer 5′-CCATCGATTCTGGGCTTCTCCTTCACTCTC-3′ for CHLI1 gene, forward primer 5′-CCCAAGCTTAAATGAGCAGCAACAGGAC-3′ and reverse primer 5′-CCATCGATTTGGAAGCATTGGCTTTAT-3′ for CHLD gene, and forward primer 5′-CCATCGATTGGTAGATTCGGATACAGCGTG-3′ and reverse primer 5′-GCGTCGACGTCTGCTCCTACTCCTGCCTG-3′ for GUN4 gene. These fragments were inserted in sense orientation, respectively, into the pSK-int vector with the ClaI/SalI sites for CHLI1 gene, HindIII/ClaI sites for CHLD gene, and ClaI/SalI sites for GUN4 gene. The same fragments as above mentioned for each of three genes were amplified, respectively, with forward primer 5′-CGGAATTCTCTGGGCTTCTCCTTCACTCTC-3′ and reverse primer 5′-GGACTAGTAACTTCATCTCATCTTGCCCTAC-3′ for CHLI1 gene, forward primer 5′-CGGAATTCAAATGAGCAGCAACAGGAC-3′ and reverse primer 5′-GGACTAGTTTGGAAGCATTGGCTTTAT-3′ for CHLD gene, and forward primer 5′-TCCCCCGGGTGGTAGATTCGGATACAGCGTG-3′ and reverse primer 5′-GGACTAGTGTCTGCTCCTACTCCTGCCTG-3′ for GUN4 gene. These fragments were subsequently placed in antisense orientation, respectively, into the pSK-int vector already carrying the corresponding sense fragment with the EcoRI/SpeI sites for CHLI1 gene, EcoRI/SpeI sites for CHLD gene, and SmaI/SpeI sites for GUN4 gene. The entire RNAi cassette comprising the sense and antisense fragments interspersed by the Actin II intron was excised from the pSK-int using the flanking SacI/ApaI sites and inserted into the SacI/ApaI site of pSUPER1300(+) vector, yielding the CHLI1 RNAi, CHLD RNAi and GUN4 RNAi construct, respectively. The pSUPER1300(+) Super Promoter is a hybrid promoter combining a triple repeat of the Agrobacterium tumefaciens octopine synthase (ocs) activator sequences along with the mannopine synthase (mas) activator elements fused to the mas promoter, termed (Aocs)3AmasPmas (Ni et al. 1995). It is noteworthy that the CHLI1 RNAi construct can target to both CHLI1 and CHLI2 (At5g45930) gene transcripts. The RNAi construct for each of the three genes was introduced into Agrobacterium tumefaciens GV3101 and transformed into Col-0 by floral dip method (Clough and Bent 1998). Transgenic plants were grown on Murashige–Skoog (MS) agar plates containing hygromycin (40 μg/ml) in order to screen the positive seedlings.
To create transgenic plant lines over-expressing CHLI1, CHLD and GUN4 genes, the open reading frames (ORF) for these genes flanked by SmaI and SalI sites were isolated by PCR, using the following primers: forward primer 5′-CCCCCGGGATGGCGTCTCTTCTTGGAACATC-3′ and reverse primer 5′-GCGTCGACTCAGCTGAAAATCTCGGCGAA C-3′ for CHLI1; forward primer 5′-CCCCCGGGATGGCGATGACTCCGGTCGC -3′ and reverse primer 5′-ACTCAAGAATTCTTCAGATCAGATAG -3′ for CHLD; forward primer 5′-CCCCCGGGATGGCGACCACAAACTCTC-3′ and reverse primer 5′-GCGTCGACTCAGAAGCTGTAATTTGTTT-3′ for GUN4. These ORFs cloned into pCAMBIA-1300-221 vector harboring a 35S promoter. Transgenic manipulation was done as previously described (Wu et al. 2009). The homologous T3 generation seeds or plants were used for analysis. At least ten transgenic lines were obtained for each of the constructs.
Plants were grown in a growth chamber at 20–21 °C on MS medium at about 80 μmol photons m−2 s−1, or in compost soil at about 120 μmol photons m−2 s−1 over a 16-h photoperiod. The cs (cs1-1) and cch mutants were generous gifts from Dr. J. Chory (The Salk Institute, La Jolla, CA). The rtl1 mutant was a gift from Dr. T. Kinoshita (Nagoya University, Japan). The seed of ch1-3 mutant (CS3362) was obtained from the Arabidopsis Biological Resource Center.
Antibody production and immunoblotting
For the production of the antibody against CHLI and CHLD, the fragments corresponding to the cDNA of these genes were amplified and inserted into the EcoRI and XholI sites of pGEX4T-1 vector (Novagen). A 715-bp fragment of the CHLI cDNA was isolated using forward primer 5′-CCGGAATTC CCGGTTTATCCATTTGCAGCT-3′ and reverse primer 5′-CCGCTCGAGACTATCGAAACGAGCTCTCT-3′, which corresponds a common piece of amino-acid sequence of both CHLI1 and CHLI2. A 654-bp fragment of the CHLD cDNA was isolated using forward primer 5′-CCGGAATTCTTCTCAGAAGATAGAGGACGC-3′ and reverse primer 5′-CCGCTCGAGCTTCAGATCAGATAGTGCATC-3′. The GST-tagged fusion proteins were expressed in Escherichia coli BL21 (DE3). The affinity-purified fusion protein was used for standard immunization protocols in rabbit. The antisera were produced and tested for specificity as described previously (Wu et al. 2009). The extraction of Arabidopsis total protein, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were done essentially according to the previously described procedures (Shen et al. 2006; Wu et al. 2009).
Quantitative real-time PCR
Real-time PCR for expression of various genes was performed as previously described (Wu et al. 2009) essentially according to the instructions provided for the BioRad Real-Time System CFX96TM C1000 Thermal Cycler (Singapore). The used primers were: forward primer 5′-GGTAACATTGTGCTCAGTGGTGG-3′ and reverse primer 5′-AACGACCTTAATCTTCATGCTGC-3′ for Actin; forward primer 5′-CGATGTTCCTTACCTTGTGGCAG-3′ and reverse primer 5′-CACGACCAGCGAAAACGATTG-3′ for CHLH; forward primer 5′-GACGGTTAGAGATGCTGATTTAC-3′ and reverse primer 5′-TCACTATGTCTCCTCTCAACCC-3′ for CHLI plus CHLI2; forward primer 5′-AAGTGGCAGTATGGCATTGAA-3′ and reverse primer 5′-AACCACCACCACAAGGAAGTC-3′ for CHLD; forward primer 5′-GGCGACCACAAACTCTCTCCACC-3′ and reverse primer 5′-GTTTCGGCAGTTGTGGCGGAG-3′ for GUN4.
Expression and purification of CHLH, CHLI, CHLD and GUN4 proteins in the sf9 insect cell line
To construct ABAR/CHLH, CHLI, CHLD and GUN4 expression vectors, the ORFs of these genes flanked by SalI and KpnI sites were cloned into pFastBac™ HFT-B (Invitrogen, CA), a kind of baculo-virus transfer vector. The sf9 cells (Invitrogen, about 1 × 109) were infected with viruses expressing the Flag-tagged fusion proteins, respectively. The infected cells were seeded in flasks and cultured at 28 °C for 3 days. Cells were harvested and washed with a TBS buffer (50 mM Tris–HCl, pH 7.5, and 150 mM NaCl). Cell pellets were then lysed with sonication in the lysis buffer consisting of 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM 2-mercaptoethanol, 0.2 mg/ml trypsin inhibitor and 10 μg/ml leupeptin. After centrifugation at 17,000 g for 30 min, the supernatant was incubated with anti-FLAG M2 affinity resin (Sigma) at 4 °C for 2 h. The resin suspension was then washed with a wash buffer (10 mM, pH 7.5, 150 mM NaCl, 2 μg/ml leupeptin, and 5 mM 2-mercaptoethanol). Proteins bounding to anti-FLAG agarose, were eluted with 0.1 mM FLAG peptide (Asp Tyr Lys Asp Asp AspAsp Lys) in the wash buffer, purified by gel filtration and concentrated to 0.5–1 mg/ml by ultrafiltration.
Surface plasmon resonance (SPR) measurements were performed using a Biacore T200 (GE Healthcare) equipped with a certified CM5 sensor chip with carboxyl groups on its surface. The sample proteins (>90 % pure based on Size Exclusion Chromatography) were covalently immobilized to saturate the surface of sensor chip via -NH2 bond using amino-coupling kit from Biacore. The surface of flow cell 2 was activated for 7 min with a 1:1 mixture of 0.1 M N-Hydroxysuccinimide (NHS) and 0.1 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) at a flow rate of 10 μl/min. The sample protein was immobilized to a density that saturates the surface at a concentration of 50 μg/ml in 10 mM sodium acetate (for CHLH and GUN4 at pH 4.5, for CHLI and CHLD at pH 4.0); flow cell 1 was left blank to serve as a reference surface. The surface was then blocked with a 7 min injection of 1 M ethanolamine, pH 8.0. To collect kinetic and affinity binding data, the analyte (+)-ABA in the HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 30 mM ethylene diamine tetraacetic acid (EDTA), and 0.005 % [v/v] surfactant P20, pH 7.4) was injected over flow cell 1 and flow cell 2 at concentrations of 6 to 100 μM at a flow rate of 30 ul/min and at 25 °C. The complex was allowed to associate and dissociate for 60 s, respectively. Data were collected and globally fitted to steady-state model available within Biacore Evaluation software v1.01.
Analysis of protein interaction by yeast two-hybrid system and co-immunoprecipitation (CoIP) in yeast and in planta
Interaction between two proteins was assayed by a yeast Gal4-based two-hybrid system as described by the manufacturer (Clontech). The primers used for cloning the related cDNAs were as follows: for ABAR692–1381 (encoding C-terminal amino acid residues 692–1381 or ABARc): forward primer 5′-GGAATTCGGGAACATTCCCAATG-3′ and reverse primer 5′-ACGCGTCGACTTATCGATCGATCCCTTCGATC-3′; for ABAR1–691 (encoding N-terminal amino acid residues 1–691 or ABARn): forward primer 5′-CCGGAATTCATGGCTTCGCTTGTGTATTCTCC-3′ and reverse primer 5′-ACGCGTCGACGATAAGACTGTCGGGAAAAC-3′; for ABAR347–1038 (encoding median amino acid residues 347–1038 or ABARm): forward primer 5′-CCGGAATTCGCTTGAGGCTAGAGGTGCTA-3′ and reverse primer 5′-ACGCGTCGACGATGTTGTCAGTTCCCCAAA-3′; for the full length CHLI1: forward primer 5′-CGGAATTCATGGCGTCTCTTCTTGGAACATC-3′ and reverse primer 5′-ACCTCGAGCTCAGCTGAAAATCTCGGCGAA-3′; and for the full length CHLD: forward primer 5′-ACTGGATCCATATGGCGATGACTC-3′ and reverse primer 5′-ACGCTCGAGCTCAAGAATTCTTCAGATCAGATAG-3′. The cDNAs encoding the truncated ABARs were inserted into the pGBKT7 plasmid by the EcoRI (5′ end) and SalI (3′ end) sites to generate bait plasmids, and the cDNAs encoding CHLI1 and CHLD were cloned into EcoRI (5′ end)/XhoI (3′ end) sites and BamHI (5′ end)/XhoI (3′ end) sites of pGADT7 plasmid to generate prey plasmids, respectively. The liquid β-galactosidase assays, including measurement of β-galactosidase activity, were performed according to the manufacturer’s protocol (Clontech) by using ONPG (o-nitrophenyl-β-d-galactopyranoside; Sigma Cat No. N-1127) as substrate, which is hydrolyzed to o-nitrophenol and D-galactose.
CoIP assays were performed in the extracts of both yeast cells and Arabidopsis plants. Yeast strains were grown using SD medium deficient in Leu, Trp, His and Ade to OD600 1.0 at 30 °C. Total proteins were prepared from yeast cells with an extraction buffer (2 mL/g cells) containing 50 mM HEPES (pH 7.4), 10 % glycerol (v/v), 1 mM EDTA, 0.1 % Triton X-100 (v/v), 100 μM PMSF, and 1 μg/mL each of aprotinin, leupeptin, and pepstatin A. The antibodies used were: mouse antibody (Medical and Biological Laboratories CO., LTD) specific to MYC-tagged truncated ABAR protein, and mouse antibody specific to HA- (hemagglutinin peptide epitope, Medical and Biological Laboratories CO., LTD) tagged CHLI1 and CHLD protein. Immunoprecipitation experiments were performed with protein A/G Plus-agarose beads (Santa Cruz), following the manufacturer’s protocol. In brief, cell lysates were pre-cleared with the protein A/G Plus-agarose beads and incubated with the anti-HA serum and the protein A/G Plus-agarose beads at 4 °C overnight in the extraction buffer. The beads were washed twice extensively with buffer A [50 mM Tris pH 8.0, 150 mM NaCl, 0.1 % Triton X-100 (v/v)] and buffer B [50 mM Tris pH 8.0, 0.1 % Triton X-100 (v/v)], respectively, and then resuspended in SDS-PAGE sample buffer. The immuno-precipitates were separated on a 10 % SDS-PAGE, analyzed by immunoblotting with anti-MYC serum.
For immunoprecipitation in Arabidopsis extracts, the total proteins (6 mg) were resuspended in the yeast protein extraction buffer (1 mL) as described above. The immunoprecipitation was done with the same procedures as described above except that the anti-ABAR and anti-CHLI1/anti-CHLD serum was used instead of the anti-MYC and anti-HA serum, and the beads were washed with the extraction buffer instead of the buffer A and buffer B.
Test of protein–protein interaction by luciferase complementation imaging (LCI)
To further confirm the results of protein–protein interaction, we used a luciferase complementation imaging system according to previously described procedures (Shang et al. 2010) in which the firefly luciferase (Luc) enzyme is divided into the N- (NLuc) and C-terminal (CLuc) halves that do not spontaneously reassemble and function. Luc activity occurs only when the two fused proteins interact, resulting in reconstituted Luc enzyme. The primers used for cloning the related cDNAs were as follows: for ABAR-NLuc: forward primer 5′-GGGGTACCATGGCTTCGCTTGTGT-3′ and reverse primer 5′-ACGCGTCGACTCGATCGATCCCTTC-3′; for CLuc-ABAR: forward primer 5′-GGGGTACCATGGCTTCGCTTGTGT-3′ and reverse primer 5′-ACGCGTCGACTTATCGATCGATCCCTTC-3′; for CLuc-CHLI1: forward primer 5′-CGGGGTACCATGGCGTCTCTTCTTGGAACATC-3′ and reverse primer 5′-GCGTCGACTCAGCTGAAAATCTCGGCGAA-3′; for CHLI1-NLuc: forward primer 5′-CGGGGTACCATGGCGTCTCTTCTTGGAACATC-3′ and reverse primer 5′-GCGTCGACGCTGAAAATCTCGGCGAA-3′; for CLuc-CHLD: forward primer 5′-CGGGGTACCATGGCGATGACTCCGGTCGC-3′ and reverse primer 5′-GCGTCGACTCAAGAATTCTTCAGATCAG-3′; and for CHLD—Nluc: forward primer 5′-CGGGGTACCATGGCGATGACTCCGGTCGC-3′ and reverse primer 5′-GCGTCGAC AGAATTCTTCAGATCAGATA-3′.
The constructs were cloned into pCAMBIA-NLuc and pCAMBIA-CLuc at the KpnI and SalI sites. The constructs were mobilized into A. tumefaciens strain GV3101. Bacterial suspensions were infiltrated into young but fully expanded leaves of the 7-week old N. benthamiana plants using a needleless syringe. It is noteworthy that the amounts of the constructs were the same among treatments and controls for each group of assay. After infiltration, plants were grown first under dark for 12 h and then with 16 h light/d for 60 h at room temperature and the Luc activity were observed with a CCD imaging apparatus (Andor iXon, Andor, UK).
VIGS assay and tobacco stomata aperture assay
We used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system (Liu et al. 2002) to down-regulate the expression of CHLH, CHLI and CHLD in tobacco. The VIGS assay was performed essentially according to previously described procedures (Liu et al. 2002). The primers used for cloning the related cDNAs were as follows: for ABAR: forward primer 5′-CCGGAATTCGGGAACATTCCCAATG-3′ and reverse primer 5′-CCGCTCGAG TTATCGATCGATCCCTTCGATC-3′; for CHLI: forward primer 5′-CCGGAATTCCCGGTTTATCCATTTGCAGCT-3′ and reverse primer 5′-CCGCTCGAG CCAACAAACCAGGCTCAAAGG-3′; and for CHLD: forward primer 5′-CCGGAATTCCGAGAAAAAGTCACAATCGATG-3′ and reverse primer 5′-CCGCTCGAGCGCCCTGCCAGCTTTCCCC-3′.
The fragments corresponding to the cDNAs of these genes were cloned into the EcoRI and XholI sites of pTRV2 vector. The constructs were mobilized into A. tumefaciens strain GV3101. Agrobacterium containing pTRV1 and pTRV2 were mixed in 1 : 1 ratio and infiltrated into the lower leafs of 4-leaf stage Nicotiana benthamiana plants using a needleless syringe. Each silencing experiment was repeated at least 3 times and each experiment included at least five independent plants. We assessed the gene silencing efficiency by suppressing the expression of the phytoene desaturase (PDS) gene in N. benthamiana. A mixture of Agrobacterium culture containing the pTRV2-PDS and pTRV1 was infiltrated as described above. About 7 days after infiltration, the upper leaves of the plant exhibited the silencing effect. Silencing of PDS leads to the inhibition of carotenoid synthesis, causing the plants to a photo-bleached phenotype (Liu et al. 2002; Kumagai et al. 1995).
Then tobacco total proteins were extracted with an extraction buffer consisting of 50 mM Tris–HCl (pH 7.8), 50 mM NaCl, 10 % (v/v) glycerol, 0.1 % (v/v) Tween-20, 0.15 % (v/v) 2-mercaptoethanol. Gene silenced plants were tested by immunoblotting and were chose for stomatal aperture assay. Stomatal aperture was assayed with small pieces of tobacco leaves essentially as previously described for the assays in Arabidopsis (Shen et al. 2006; Wu et al. 2009).
For drought tolerance experiment, plants were grown on soil until they were 3-weeks old when plantlets reached the stage of five to six fully expanded leaves, and drought was imposed by withdrawing irrigation for one-half of the plants until the lethal effects was observed on most of these plants, whereas the other half were grown under a standard irrigation regime as a control.
Phenotypic analysis was done essentially as previously described (Shen et al. 2006; Wu et al. 2009; Shang et al. 2010). Briefly, for germination assay, approximately 100 seeds were planted on MS medium (Sigma, St. Louis, MO, USA; product#, M5524; full-strength MS) that contained 3 % sucrose and 0.8 % agar (pH 5.9) and was supplemented with or without (±)-ABA. The seeds were incubated at 4 °C for 3 days, and then placed at 20 °C under light conditions, and germination (emergence of radicals) was scored at the indicated times. Seedling growth was assessed by directly planting the seeds in the ABA-containing MS-medium to investigate the response of seedling growth to ABA after germination. For stomatal aperture assays, 3-week old leaves for Arabidopsis, and 5-week old leaves for tobacco were used. To observe ABA-induced stomatal closure, leaves were floated in the buffer containing 50 mM KCl and 10 mM Mes-Tris (pH 6.15) under a halogen cold-light source (Colo-Parmer) at 200 μmol m−2 s−1 for 2.5 h followed by addition of different concentrations of (±)-ABA. Apertures were recorded on epidermal strips after 2.5 h of further incubation to estimate ABA-induced closure. To study ABA-inhibited stomatal opening, leaves were floated on the same buffer in the dark for 2.5 h before they were transferred to the cold-light for 2.5 h in the presence of ABA, and then apertures were determined.
We thank Dr. T. Kinoshita (Nagoya University, Japan) for a gift of the rtl1 mutant, and Dr. Yule Liu (Tsinghua University, China) for the generous gifts of the VIGS system and TRV-PDS control. We thank also Dr. Xiangdong Li (Institute of Zoology, Chinese Academy of Sciences) for help on materials. The seeds of abi4-1 (CS8104), abi5-1 (CS8105) and ch1-3 mutants (CS3121) was obtained from the Arabidopsis Biological Resource Center. This research was supported by the National Key Basic Research ‘973’ Program of China (2012CB114300-002), National Natural Science Foundation of China (grant nos. 90817104 and 31170268), and Foundation for the Author of National Excellent Doctoral Dissertation of China (grant no. 201065).
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