ALTERED MERISTEM PROGRAM1 (AMP1) encodes a glutamate carboxypeptidase that plays an important role in shoot apical meristem development and phytohormone homeostasis.
We isolated a new mutant allele of AMP1, amp1-20, from a screen for abscisic acid (ABA) hypersensitive mutants and characterized the function of AMP1 in plant stress responses.
amp1 mutants displayed ABA hypersensitivity, while overexpression of AMP1 caused ABA insensitivity. Moreover, endogenous ABA concentration was increased in amp1-20- and decreased in AMP1-overexpressing plants under stress conditions. Application of ABA reduced the AMP1 protein level in plants. Interestingly, amp1 mutants accumulated excess superoxide and displayed hypersensitivity to oxidative stress. The hypersensitivity of amp1 to ABA and oxidative stress was partially rescued by reactive oxygen species (ROS) scavenging agent. Furthermore, amp1 was tolerant to freezing and drought stress. The ABA hypersensitivity and freezing tolerance of amp1 was dependent on ABA signaling. Moreover, amp1 had elevated soluble sugar content and showed hypersensitivity to high concentrations of sugar. By contrast, the contents of amino acids were changed in amp1 mutant compared to the wild-type.
This study suggests that AMP1 modulates ABA, oxidative and abotic stress responses, and is involved in carbon and amino acid metabolism in Arabidopsis.
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The Arabidopsis AMP1 gene encodes an endoplasmic reticulum (ER) membrane-localized glutamate carboxypeptidase that has been implicated in the small peptide signaling process (Helliwell et al., 2001; Vidaurre et al., 2007). The orthologs of this presumptive glutamate carboxypeptidase – VP8 (Zea mays), PLA3 (Oryza sativa) and TRICOT (Lotus japonicus) – are involved in the regulation of phytohormone homeostasis (Suzuki et al., 2008; Kawakatsu et al., 2009; Suzaki et al., 2012). VP8 modulates meristem development and seed maturation by controlling the accumulation of abscisic acid (ABA) and embryonic regulators such as LEAFY COTYLEDON1 (LEC1)/B3 domain transcription factors (Suzuki et al., 2008). PLA3 regulates various developmental processes and plant hormone homeostasis. A pla3 loss-of-function mutant maintains a slightly higher concentration of cytokinin but a lower amount of ABA than the wild-type, and it displays an ABA-insensitive phenotype (Kawakatsu et al., 2009). In Arabidopsis, AMP1 regulates embryonic and postembryonic growth and development by affecting plant hormone biosynthesis and signaling (Chaudhury et al., 1993; Vidaurre et al., 2007). The amp1-1 mutant was reported to have increased zeatin content and leaf number, and an enlarged apical meristem in the shoot (Chaudhury et al., 1993; Riou-Khamlichi et al., 1999). Several alleles of AMP1 that have pleiotropic phenotypes in response to different plant hormones have been isolated. A weak missense allele, amp1-7, exhibits decreased hypocotyl elongation when exposed to ethylene and GA3 in light (Saibo et al., 2007). Another amp1 mutant was isolated as a suppressor of an monopteros/auxin response factor 5 (mp/arf5) mutant, suggesting a role for AMP1 in meristem-niche-associated auxin signaling (Vidaurre et al., 2007). One recent study reported that the amp1 mutation has different effects on dormancy and on ABA concentrations in different accessions (Griffiths et al., 2011).
ABA regulates many important aspects of physiological processes, including seed dormancy and germination, vegetative growth and plant responses to environmental stresses (Leung & Giraudat, 1998; Finkelstein et al., 2002). The biosynthesis of ABA involves five essential enzymes, encoded as ABA1, ABA2, ABA3, NCED3 and AAO3. These genes can be rapidly induced by abiotic stress and exogenous ABA is capable to rescue their hypersensitivite phenotypes to freezing and salt stress of these ABA deficient mutants (Llorente et al., 2000; Barrero et al., 2006). By screening of ABA insensitive mutants, several key components in the ABA signaling pathway, including ABI1 to ABI5 (ABA insensitive1-5), have been characterized (Koornneef et al., 1989; Finkelstein, 1994). ABI1 and ABI2 are PP2C (phosphatase type-2C) proteins with negatively regulatory roles in ABA signaling (Allen et al., 1999; Merlot et al., 2001). ABI3 (a B3 domain transcription factor) and ABI5 (a bZIP transcriptional factor) mainly function in ABA-dependent seedling growth arrest during seed germination and postgermination growth stages (Giraudat et al., 1992; Finkelstein & Lynch, 2000b). ABI4 is a member of the ERF/AP2 transcription factor family (Finkelstein et al., 1998).
Unfavorable environments such as osmotic stress and salinity induce oxidative stress and promote reactive oxygen species (ROS) overproduction in chloroplasts, mitochondria and other cellular components (Miller et al., 2008; Jaspers & Kangasjarvi, 2010; Suzuki et al., 2012). Accumulated ROS are involved in various cellular responses and ultimately lead to cell death (Noctor et al., 2007; Taylor et al., 2009; Suzuki et al., 2012). Besides its toxic effect, ROS also act as key molecules which can trigger the transcription of downstream stress response genes (Foyer & Noctor, 2005; Fujita et al., 2006; Miller et al., 2008; Jaspers & Kangasjarvi, 2010). To keep the balance of ROS and protect cellular homeostasis from membrane system injury and oxidative stress, the excessive ROS are scavenged by enzymatic and nonenzymatic antioxidants, such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, ascorbic acid and glutathione (Mittler et al., 2004; Noctor et al., 2007; Foyer & Noctor, 2009; Miller et al., 2011). However, the precise mechanism of cellular ROS production in response to abiotic stresses is still unclear. Recent evidence shows that ROS generated from plasma membrane and mitochondria are involved in ABA signaling pathway to regulate the process of root growth, stomata movement and seed germination (Kwak et al., 2003; Liu et al., 2010; He et al., 2012). Overexpression of a ROS-induced transcription factor AtWRKY15 showed tolerance to salt and osmotic stress (Vanderauwera et al., 2012). Arabidopsis lsd1 and chs2 mutants with ROS overaccumulation showed sensitivity to low temperatures (Huang et al., 2010a,b). Oxidative stress occurrence in Arabidopsis cells are reported to affect carbon metabolism by inhibiting the TCA cycle in mitochondria, leading to decreased amino acid content and disturbed balance of metabolic coordination (Baxter et al., 2007; Takahashi & Murata, 2008; Suzuki et al., 2012).
In this study we report that amp1 mutants displayed hypersensitivity to ABA and oxidative stress during germination and postgermination growth. Conversely, overexpression of AMP1 resulted in early germination and insensitivity to ABA and oxidative stress. The concentration of ABA accumulation increased in amp1- and decreased in AMP1-overexpressing plants under osmotic stress. Consistently, loss-of-function of AMP1 conferred enhanced freezing and drought tolerance. We also demonstrated that the accumulation of sugar and amino acids was affected in amp1 mutants. Thus, our results suggest that AMP1 is a novel component that is involved in ABA, oxidative and abitoc stress responses, and mediates carbon and amino acid metabolism in Arabidopsis.
Materials and Methods
Plant materials and growth conditions
The Arabidopsis thaliana (L.) Heynh ecotypes Columbia (Col-0) and Landsberg erecta (Ler) were used in this study. The mutants aba2-1 (Leon-Kloosterziel et al., 1996), abi4-1 (Finkelstein et al., 1998), abi5 (Finkelstein & Lynch, 2000a), pyr1 pyl1 pyl4, pyr1 pyl1 pyl2 pyl4 (Park et al., 2009), amp1-1 (CS8324), amp1-13 (SALK_022988), amp1-14 (SALK_087303), amp1-22 (SALK_138749) are in the Col background, while abi1-1 (Meyer et al., 1994), abi2-1 (Leung et al., 1997) and abi3-1 (Giraudat et al., 1992) are in the Ler background. The Arabidopsis plants were grown at 22°C with a photoperiod of 16-h light : 8-h dark on MS medium (Sigma-Aldrich) containing 2% sucrose and 0.8% agar, unless otherwise indicated.
For the germination assay, sterilized seeds were plated on MS medium containing 0.8% agar, supplemented with different concentrations of ABA, Methyl violgen (MV) or glucose. Plates were stratified at 4°C in darkness for 3 d and then transferred to 22°C. Plants showing open green cotyledons were scored to calculate for germination rate at day 6 after the end of stratification. Three independent experiments were carried out and each experiment contained three replications with 30 seedlings for each replicate.
Screening of amp1 and map-based cloning of AMP1
In order to isolate ABA hypersensitive mutants, T2 plant seeds from an Arabidopsis T-DNA insertion collection that was generated using the activation-tagging vector pSKI015 (Qin et al., 2003) were planted on MS medium supplemented with 0.5 μM ABA, and grown at 22°C for 6 d. Plants that were hypersensitive to ABA were rescued on MS medium and transferred to soil to set seeds, and T3 seeds were used to recheck ABA sensitivity.
The amp1-20 mutant with Col background was crossed to Ler, and the resulting F2 seeds were collected. A total of 500 amp1-20 mutant seedlings were chosen from the segregating F2 population. Genomic DNA was extracted and used for PCR-based mapping with simple sequence length polymorphism (SSLP) and cleaved amplified polymorphic sequence (CAPS) markers. Candidate genes in this region were sequenced in amp1-20 to identify the mutation.
Plasmid construction and plant transformation
For complementation of amp1-20, a 3.1-kb AMP1 genomic fragment was amplified by PCR using the primers AMP1-3F and AMP1-3R (Supporting Information Table S1) and cloned into pCAMBIA1300 (CAMBIA, Canberra, Australia) containing the 2.3-kb promoter region of AMP1. To construct pSuper:AMP1-GFP and pSuper:AMP1ΔN-GFP, the AMP1 cDNA fragment was amplified by PCR using the primers AMP1-4F and AMP1-4R and AMP1-5F and AMP1-5R, respectively, and subsequently cloned into a pSuper1300 vector (Yang et al., 2010) containing a GFP tag.
All constructs were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into plants via the floral dip transformation (Clough & Bent, 1998).
Freezing and drought tolerance assays
The freezing tolerance assay was carried out as described (Shi et al., 2012). Briefly, 2-wk-old plants were placed in a freezing chamber set to −1°C and programmed to cool by decreasing the temperature at a rate of −1°C h−1. The plates were removed at −6°C, −7°C or −8°C, and incubated at 4°C for 12 h before transferred to 22°C. Survival seedlings were scored after a 2-d recovery periopd. For drought-tolerant assay, plants were grown in soil for 4 wk without watering. Then the plants were irrigated, and the survival rates were scored 4 d later.
Ion leakage and sugar content were measured as described (Lee et al., 2002). Total soluble sugar (sucrose, glucose and fructose) content was measured as described (Stitt et al., 1989; Strand et al., 1999). Proline content was measured as described (Bates et al., 1972). For the ABA content measurement, 2-wk-old seedlings grown at 22°C were treated with or without 40% PEG8000 for 6 h. ABA contents were determined as described previously (Kojima et al., 2009). Total chlorophyll content was determined as described (Huang et al., 2009).
RNA extraction and real-time PCR
Total RNA extraction from 2-wk-old plants and real-time PCR were performed as described (Huang et al., 2010a). Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green PCR Master Mix kit (Takara, Tokyo, Japan). Three RT-PCR reactions were repeated independently using Actin2/8 gene as an internal control. The relative expression levels were calculated as described (Huang et al., 2010a). The primers used are listed in Table S1.
Five-day-old AMP1-OX and pSuper:GFP seedlings were treated with 50 μM ABA for 6 h. GFP fluorescence in roots was imaged using a confocal laser scanning microscope (LSM510; Carl Zeiss, Oberkochen, Germany).
Protein preparation and immunoblot assay
Soluble and membrane proteins were isolated as described (Hua et al., 2001). HSP90 monoclonal antibody was used as a soluble marker after fractionation.
For immunoblots, 10-d-old seedlings were treated with 500 μM CHX (cycloheximide; Berberich & Kusano, 1997) for 2 h, or with 50 μM ABA or 50 μM ABA plus 50 μM MG132 for 0–6 h. AMP1-GFP fusion proteins were detected by immunoblot using an anti-GFP antibody (1 : 1000 dilution; Sigma-Aldrich). Rubisco stained by Ponceau S (GenView, CA, USA) was used as a loading control.
Amino acid measurement
Amino acids were prepared from fresh seedlings as described previously (Wu et al., 1996). Briefly, c. 0.05 g seedlings were collected and extracted by adding 500 μl of HClO4 extraction buffer (Perchloric acid), followed by centrifugation at 10 000 g for 10 min. Subsequently, 250 μl 2 m K2CO3 was added to the supernatant and centrifuged at 10 000 g for 10 min. The supernatant was used for subsequent fluorometric HPLC methods described (Wu et al., 1997).
Measurement of ROS in plants
Nitroblue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB) staining with 2-wk-old seedlings was performed to detect superoxide as described previously (Shi et al., 2007). H2O2 was measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich; Munemasa et al., 2007). All experiments were repeated more than three times.
Affymetrix GeneChip ATH1 arrays representing 24 000 Arabidopsis genes were used for the analysis of whole genome gene expression profile in wild-type Col, amp1-20 and AMP1-OX2 overexpressing plants. Total RNA was extracted from 2-wk-old seedlings grown on MS medium containing 2% sucrose. The raw microarray data were analyzed using Gene Ontology Annotations (http://www.arabidopsis.org/tools/bulk/go/index.jsp) and visualized with MapMan software (http://mapman.gabipd.org; Ariel et al., 2012).
Disruption of AMP1 causes hypersensitivity to ABA in Arabidopsis
In order to identify novel components involved in the plant response to ABA, mutants that were hypersensitive to high concentrations of ABA were isolated by screening individual lines derived from an Arabidopsis T-DNA insertion collection with Col-0 background (Qin et al., 2003). One mutant, amp1-20, was selected for further study (Fig. 1a). Genetic analysis showed that no T-DNA insertions in amp1-20 were linked to the mutant phenotype. Consequently, positional cloning was used to map the mutation locus to a 200-kb region on the bottom arm of chromosome 3 between the markers F24B22 and F28P10 (Fig. S1a). The sequencing of candidate genes in this region revealed a 37-bp deletion at the C-terminus of the AMP1 gene (Chaudhury et al., 1993; Helliwell et al., 2001). This deletion causes a frameshift and generates a premature stop codon (Fig. S1b). RT-PCR showed that this deletion has no obvious effects on the transcription of the truncated AMP1 fragment (Fig. S1c).
In order to determine whether the hypersensitivity of amp1-20 could be attributed to the loss of AMP1 function, three T-DNA insertion lines, amp1-13, amp1-14 (Griffiths et al., 2011) and amp1-22 (SALK_138749), were isolated (TAIR; http://www.arabidopsis.org). There was no detectable full-length AMP1 transcript in any of these three mutants (Fig. S1c). All three mutants exhibited significant hypersensitivity to exogenous ABA in seed germination compared to the wild-type (Fig. 1c,d). Similar to amp1-1 (Chaudhury et al., 1993), amp1-20 showed severe morphological defects, including more leaves, and shorter petioles and primary roots than the wild-type (Fig. S1d).
We next transformed amp1-20 with a wild-type genomic fragment harboring the 5.4-kb AMP1 genomic region. All of the transgenic lines that were obtained complemented the amp1-20 phenotypes in terms of ABA sensitivity and morphology, as listed above (Fig. 1a), further confirming amp1-20 as a new mutant allele of AMP1.
ABA response during germination in amp1-20 and AMP1 overexpressing plants
In order to further explore the role of AMP1 in ABA response, we generated transgenic plants overexpressing AMP1 fused with GFP (AMP1-OX) under the control of a Super promoter derived from octopine and mannopine synthase genes (Ni et al., 1995) in the wild-type Col and amp1-20 mutant. The AMP1-OX lines in amp1-20 fully rescued the amp1-20 morphological phenotypes (Fig. S1d), indicating the AMP1-GFP is functional. We chose two independent lines (AMP1-OX2 and AMP1-OX7) in the Col background – in which AMP1 protein was overexpressed (Fig. 2a) – for further analysis. These transgenic plants were indistinguishable morphologically from wild-type plants.
In contrast with amp1, the AMP1-OX lines displayed significantly early greening on ABA-free medium (Fig. 2b,c). Furthermore, the AMP1-OX lines showed increased ABA insensitivity in assays of ABA-conferred inhibition of cotyledon greening (Fig. 2d,e). Taken together, these results indicate that amp1 mutants are hypersensitive to ABA, while AMP1 overexpression reduces ABA sensitivity; thus implicating AMP1 plays a role in ABA response during the germination stage.
ABA response in seedling growth in amp1-20 and AMP1 overexpressing plants
Next, we investigated the function of AMP1 in the response of postgermination growth to ABA. Seedlings were grown on MS medium for 4 d and subsequently transferred to MS medium containing 30 μM or 50 μM ABA for 10 d, wild-type seedlings showed an increasing inhibition of leaf emergence and primary root growth with increased amounts of ABA. Compared with wild-type plants, the primary root growth of amp1-20 was significantly arrested by ABA. By contrast, the growth retardation by ABA was obviously suppressed in the AMP1-OX2 line (Fig. 2f,g). These results indicate that AMP1 is required for the ABA response at the seedling growth.
Global gene expression analysis in amp1-20 and AMP1-overexpressing plants
Previous study indicated that the homologous genes of AMP1 in several other species affect ABA metabolism (Suzuki et al., 2008; Kawakatsu et al., 2009). To further dissect the function of AMP1 in Arabidopsis, we performed Affymetrix ATH1 Genome Array with amp1-20, AMP1-OX2 and wild-type Col-0 plants. Then we analyzed microarray data to search for genes that are possibly regulated by AMP1. A total of 684 genes were upregulated in the amp1-20 and downregulated in the AMP1-OX2 line (Table S2), and 253 genes were downregulated in the amp1-20 and upregulated in the AMP1-OX2 line compared with the wild-type (more than two-fold changes, P <0.05; Table S3). The AMP1 gene was 26-fold upregulated in the AMP1-OX2 line compared with wild-type, indicating that our microarray experiment is efficient. In total, there were 937 genes whose expression was changed oppositely in the amp1-20 and AMP1-OX2 lines. Amongst these genes, we identified 30 cytochrome P450 genes and six amylase related genes, which are consistent with the previous study of Helliwell et al. (2012). We classified these genes with microarray data analysis tool Mapman (Ariel et al., 2012; Fig. S2). The most significantly changed expressing genes in phytohormone categories belong to the IAA, ABA and ethylene pathways (Fig. S2a). A group of endoplasmic reticulum (ER) stress-associated genes such as heat shock protein (Hsp) genes dramatically decreased; while cold and drought stress-related gene as well as dismutase/catalase genes increased in amp1-20 mutant (Fig. S2b,c). Three enzymatic families were also changed obviously, which include UDP glucosyl and glucuronyl transferases, cytochrome P450 families and glutathione-S-transferases (Fig. S2d). Based on the transcriptomic analysis, we suggest AMP1 affects many metabolic processes including phytohormones, oxidative stress, UDP glucosyl and glucuronyl transferases, and amylase metabolisms.
ROS accumulation in the amp1-20 mutant
It has been reported that osmotic stress stimulates ROS production to regulate seed germination and stress response via activating the ABA signaling pathway (Mustilli et al., 2002; Kwak et al., 2003). We therefore monitored the accumulation of ROS in the amp1-20 mutant. Two-week-old mutant and wild-type seedlings were treated with NBT or DAB, which (respectively) stain two major ROS, superoxide () and hydrogen peroxide (H2O2). NBT staining revealed a higher concentration of accumulated in amp1-20 seedlings (Fig. 3a). By contrast, DAB staining showed that H2O2 accumulation in mutant and wild-type was at similar, very low concentrations (Fig. S3a). We then detected H2O2 by fluorescence using DCFH-DA staining. To our surprise, there was less extensive fluorescent spotting observed in the amp1-20 mutant than in the wild-type leaves (Fig. S3b). In plants, molecules are catalyzed by the main antioxidant enzymes superoxide dismutases (SODs) to form H2O2. Next we examined the SOD activities of amp1, AMP1-OX and wild-type seedlings. SOD activities were decreased in amp1 mutants but increased in AMP1-OX lines compared with the wild-type (Fig. 3b). This result suggests that AMP1 might affect the activity of SOD enzymes, which results in the over accumulation of in amp1 mutants.
Glutathione S-transferases (GSTs) are thought to play major roles in oxidative stress. One of the Arabidopsis GSTs named GUST17 has been recently reported to affect the accumulation of GSH and ABA, mutations of GUST17 exhibit ABA hypersensitivity during seed germination (Chen et al., 2012). Using qRT-PCR, we found that expression of a set of GST genes significantly decreased in the amp1-20 mutant compared with the wild-type (Fig. 3c), which is consistent with our microarray data analysis (Fig. S2d). Moreover, we examined total glutathione ((GSH) + (GSSG)) content and reduced glutathione ratio ((GSH) : ((GSH) + (GSSG))) in amp1-20 and AMP1-OX2 seedlings. Less total glutathione accumulated in amp1-20, while more total glutathione accumulated in the AMP1-OX2 seedlings than in the wild-type (Fig. S3c). However, compared with the wild-type, no significant difference of the (GSH) : ((GSH) + (GSSG)) ratio was observed in amp1-20 and AMP1-OX2 seedlings (Fig. S3c).
Methyl viologen (MV) is an electron donor that induces oxidative stress to damage plant photosynthesis. In a seed germination assay, we found that the cotyledon greening rates of amp1-20 were significantly decreased in the presence of 0.6 μM MV (Fig. 3d,e). These results indicate that amp1 mutants are hypersensitive to oxidative stress.
Next we asked whether the increased ROS concentrations in amp1 mutants were correlated with their MV and ABA hypersensitive phenotypes. In the presence of the ROS scavenging agent dithiothreitol (DTT), the MV-conferred cotyledon greening inhibition of amp1-20 was almost completely rescued (Fig. 3f,h). The ABA-conferred cotyledon greening inhibition of both amp1-20 and the wild-type was partially compromised by DTT (Fig. 3g,i). Moreover, the relative rescue degree of amp1-20 by DTT was higher than that of wild-type (Fig. 3g,i). These results suggest that AMP1 mediates ABA and MV responses at least partially dependent on ROS.
The freezing tolerance of the amp1-20 mutant
Based on the results that the endogenous homeostasis of ABA and ROS has been disrupted in amp1-20, we wondered whether its responses to abiotic stress are affected. Therefore, we examined the freezing tolerance of amp1. Intriguingly, amp1-20 exhibited constitutively freezing-tolerant phenotypes. Nonacclimated amp1-20 mutants were more tolerant to freezing at −6°C than wild-type seedlings (Fig. 4a,b). To determine whether the amp1 mutation also affects cold acclimation, we tested the freezing tolerance of seedlings pre-grown at 4°C for 4 d and then treated them at −8°C for 1 h. The amp1 mutants showed higher survival rates at −8°C than the wild-type seedlings (Fig. 4a,b). These results demonstrate that amp1 mutations affect both the basal and acquired freezing tolerances of plants. Besides amp1-20 mutant, several other amp1 mutants exhibited the enhanced freezing tolerance (Fig. 4c).
The best-characterized cold signaling pathway in plants is the CBF/DREB1 transcriptional regulatory cascade (Thomashow, 1999). We next examined whether the cold-regulated genes in the CBF pathway are involved in the freezing tolerance of amp1-20. qRT-PCR analysis showed that the levels of CBF1-CBF3 in amp1-20 were similar to those in wild-type plants before or after cold treatment (Fig. S4a). However, the expression of RD29A in amp1-20 was consistently higher than in the wild-type with or without cold treatment (Fig. S4a,b). COR47 was also upregulated in amp1-20 under cold stress (Fig. S4a). Our microarray analysis also showed that several COR genes, including COR15a, COR15b, RD29B, and COR78 were upregulated in amp1-20 (Table S2). Therefore, constitutive expression of COR genes could be attributable to the enhanced freezing tolerance of amp1-20.
The drought tolerance of amp1-20 and AMP1-OX plants
We also explored whether amp1 and AMP-OX plants have an altered response to drought stress. Wild-type, amp1 and AMP-OX plants were grown for 2 wk in soil and then subjected to dehydration treatment for an additional 2 wk. After rewatering, the survival rate of the wild-type was 44% and nearly all amp1-20 plants survived; however, the survival rates for AMP1-OX2 and AMP1-OX7 plants were only 3% and 8%, respectively (Fig. 4d,e). These results demonstrate that amp1 mutants enhance drought tolerance, while overexpression of AMP1 confers reduced drought tolerance.
The expression of ABA biosynthesis and signaling genes in amp1-20 and AMP-OX plants
Based on the above observations, we hypothesized that the altered ABA sensitivity of amp1 and AMP1-OX2 seedlings might be attributed to the regulation of the expression of ABA biosynthesis or signaling genes by AMP1. To test this hypothesis, we evaluated the transcript levels of ABA biosynthesis genes by qRT-PCR in amp1 and AMP1-OX2 plants. In the absence of ABA, the transcript levels of ABA1, ABA3 and NCED3 were clearly higher in amp1 than in wild-type plants (Fig. 4f). Moreover, the ABA-induction of ABA1, ABA3 and NCED3 was more pronounced in amp1 than in the wild-type.
The expression of ABA signaling (ABI1 and ABI5) and ABA responsive (ABF3) genes was also examined in amp1 and AMP-OX2 seedlings. The ABA-induced expression of ABI1 and ABI5 was increased in amp1, and decreased in AMP-OX2 seedlings compared to the wild-type (Fig. 5a). Consistently, the transcript levels of ABF3 induced by ABA were consistently greater in amp1 compared to the wild-type (Fig. 4f). Therefore, AMP1 negatively regulates the expression of genes in ABA biosynthesis and signaling pathway.
ABA concentrations in amp1-20 and AMP1-OX plants under stress
In order to further examine whether AMP1 plays a role in the regulation of ABA biosynthesis, we measured the endogenous ABA contents of amp1-20 and AMP1-OX2 seedlings. Under normal conditions, low concentrations of ABA were found in both amp1-20 and AMP-OX2 seedlings, similar to the concentration observed in wild-type plants. The endogenous ABA content was dramatically elevated after 40% PEG treatment for 6 h. However, the ABA content in amp1-20 was higher than that in the wild-type under the same conditions. By contrast, the ABA content in AMP1-OX2 seedlings was lower than that in the wild-type (Fig. 4g). These results indicate that AMP1 negatively regulates stress-induced ABA concentrations in Arabidopsis.
The effect of ABA on AMP1 at the mRNA and protein levels
Because the amp1 mutants were hypersensitive to ABA, we asked whether the AMP1 transcript level was regulated by ABA. qRT-PCR showed that AMP1 expression in wild-type plants was not affected by exogenous ABA (Fig. 5a). We next analyzed the expression of AMP1 in mutants deficient in ABA biosynthesis and signaling, including aba2-1, abi4-1, abi5, abi1-1, abi2-1, pyr1 pyl1 pyl4 and pyr1 pyl1 pyl2 pyl4 (Park et al., 2009). AMP1 expression was consistently unchanged in the genotypes tested compared to the wild-type (Fig. 5b), indicating that the transcript level of AMP1 is not regulated by endogenous ABA biosynthesis and signaling.
We then investigated whether ABA influences the AMP1 protein concentration. AMP1-OX2 seedlings were treated with 50 μM ABA and concentrations of AMP1-GFP were detected by immunoblot analysis. The AMP1 protein concentration decreased after 3 h of ABA treatment, but remained unchanged in the absence of ABA treatment (Fig. 5c). Consistently, the AMP1-GFP signal was strongly reduced in the root after 6 h of ABA treatment in AMP1-OX2 plants (Fig. 5d). Thus, ABA may affect AMP1 accumulation at the post-transcriptional concentration.
In order to determine whether the AMP1 protein degradation occurs via the 26S ubiquitin-proteasome pathway, we treated AMP1-OX2 seedlings with the proteasome inhibitor MG132 (Smalle & Vierstra, 2004). MG132 treatment did not inhibit the ABA-induced AMP1 degradation (Fig. 5e). These results suggest that the ABA-induced AMP1 protein degradation is not mediated by the 26S proteasome pathway.
ABA hypersensitivity and freezing tolerance of amp1 abi double mutants
In order to further dissect the genetic interactions between amp1 and the ABA signaling pathway, we generated double mutants of amp1-20 with five ABA signaling mutants: abi1-1 (Meyer et al., 1994), abi2-1 (Leung et al., 1997), abi3-1 (Giraudat et al., 1992), abi4-1 (Finkelstein et al., 1998) and abi5 (Finkelstein & Lynch, 2000a). As abi1-1, abi2-1 and abi3-1 are in the Ler background; the double mutant combinations of these abi mutants with amp1-20 are in the mixed background. The abi mutants partially to fully rescued the ABA hypersensitive phenotype of amp1-20 during germination (Fig. 6a,b). This result indicates that the ABA hypersensitivity of amp1-20 was at least partially suppressed by these abi mutants.
In order to decipher the correlation between the ABA signaling and freezing tolerance of amp1-20, the phenotypes of the amp1 abi double mutant were analyzed under freezing conditions. Among these double mutants, the amp1 abi1 and amp1 abi3 seedlings showed survival rates intermediate between the amp1-20 mutant and the abi single mutants (Fig. 6c,d). Accordingly, the ion leakages of amp1 abi1 and amp1 abi3 double mutants were higher than that of the amp1 seedlings, but lower than that of the abi mutants after freezing treatment (Fig. 6e), indicating that abi1 and abi3 partially compromised the freezing tolerance of amp1-20 seedlings. These results suggest that the freezing tolerance of amp1-20 seedlings is partially dependent on ABA signaling.
Deletion of membrane domain affects AMP1 function
AMP1 protein is reported to localize to the ER membrane (Vidaurre et al., 2007). To determine whether the membrane localization is important for AMP1 function, we generated a truncated form of AMP1 fused to GFP under the control of a Super promoter (AMP1ΔN-GFP), in which the N-terminal membrane-localization signal peptide was deleted, and transformed this construct into amp1-20 to generate AMP1ΔN-GFP/amp1-20 plants (Fig. 7a). The AMP1ΔN-GFP protein was not localized at the membrane any more (Fig. 7b). All the AMP1ΔN-GFP/amp1-20 transgenic plants showed growth defects and ABA hypersensitive phenotypes which are similar to amp1-20 (Fig. 7c,d), indicating that the membrane localization of AMP1 is required for its functions.
In plants, increasing evidence indicates that abiotic stress-associated ROS production induces ER stress in plant cell (Chu et al., 2010; Jaspers & Kangasjarvi, 2010). To determine whether AMP1 can affect plant ER-stress response, we tested the phenotype of the amp1-20 and AMP-OX2 lines in the presence of 0.01 nM ER stress-inducer tunicamycin (TM). Wild-type and AMP1-OX2 seedlings exhibited the same growth inhibition after germination for 2 wk; however, the growth of the amp1-20 mutant was totally arrested under the same conditions (Fig. S5). Together with the effect of amp1 mutations on ROS accumulation, these results suggest that a lack of AMP1 contributes to ER stress via disruption of ROS homeostasis.
Changes in AMP1 expression affect sugar accumulation and amino acid metabolism
Sugar plays an important role during plant growth and development, and moderates abiotic stress by regulating carbohydrate metabolism. High sugar accumulation is reported to be increased under freezing stress to protect enzymes and cell membrane from dehydration (Price et al., 2004). The concentration of total soluble sugar was two-fold higher in amp1 mutants than in the wild-type at 22°C (Fig. 8a).
In our microarray data, a set of amylase related genes were upregulated in the amp1-20 mutant (Table S2). The enzyme β-amylase is essential for the breakdown of starch to provide an immediate source of soluble sugars during cold stress (Kaplan & Guy, 2004; Kaplan et al., 2007). We analyzed the expression of β-amylase genes, including BAM3 and BAM9, in amp1 seedlings grown at 22°C. Expression of BAM3 and BAM9 was higher in amp1 mutants than that in the wild-type; however, BAM3 expression in AMP1-OX2 plants was lower than that in the wild-type (Fig. 8b). This finding is consistent with a previous study showing that β-amylase genes are upregulated in plants bearing amp1/pt alleles (Helliwell et al., 2001). Moreover, the expression of the galactinol and raffinose synthase genes AtGolS1, AtGolS2 and AtGolS3 were also upregulated in amp1 mutants, while AtGolS2 and AtGolS3 were downregulated in AMP1-OX2 plants (Fig. 8b).
We further investigated whether amp1 and AMP1-OX lines exhibited an altered sugar response. With 5% and 7% glucose, the relative primary root lengths were much shorter in the amp1 mutants and higher in the AMP1-OX lines compared to the wild-type (Fig. 8c,d). These results indicate that AMP1 negatively regulates sugar accumulation and sugar sensitivity. Furthermore, the amp1 abi3, amp1 abi4, and amp1 abi5 double mutants showed dramatically decreased sensitivity to glucose during greening (Fig. S6), demonstrating that abi3, abi4 and abi5 could at least partially rescue the sugar sensitivity of amp1-20.
Proline (Pro) is an osmolyte that accumulates under abiotic stress to protect plants. We tested whether amp1-20 accumulates more Pro than the wild-type. Intriguingly, the accumulation of Pro dramatically decreased before and after 4°C treatment (Fig. S4c). This observation prompts us to propose that AMP1 as a small peptidase may also modulate amino acid metabolism. To test this hypothesis, total amino acids were extracted from 2-wk-old Arabidopsis seedlings, and were measured by HPLC. Twelve out of 23 amino acids measured, including Arg, Cit, Gly, His, Leu, Lys, Orn, Phe, Ser, Trp, Tyr and Val, significantly decreased in amp1 mutants compared to the wild-type. By contrast, Asn, Glu and Try increased in the AMP1-OX lines compared to the wild-type (Fig. 8e). Taken together, our results suggest that AMP1 is associated with sugar and amino acid metabolism.
Glutamate carboxypeptidases are ubiquitous in various species of eukaryotes (Rawlings & Barrett, 1997; Barinka et al., 2008), but the functions of AMP1 are not yet well characterized in plants. Two homologs of AMP1, VP8 in maize and PLA3 in rice, have been shown to regulate the homeostasis of ABA, and loss-of-function in these two genes results in an ABA-deficient phenotype in seeds, such as viviparity and insensitivity to ABA stress (Suzuki et al., 2008; Kawakatsu et al., 2009). One recent study showed that the ABA levels of three amp1 alleles in three different accessions are different, but do not correlate with the level of seed dormancy (Griffiths et al., 2011). In this study, we isolated a new allele of AMP1 and made a series of new observations of loss-of-function amp1 mutants especially in response to abiotic stress. Also we identified some antagonistic phenotypes of AMP1-overexpressing lines. These findings shed more light on understanding the functional mechanism of AMP1 gene in Arabidopsis.
At present, the diverse biological functions of AMP1 and homologs in different plant species are still elusive due to their unknown substrates. Arabidopsis amp1 mutants were hypersensitive to ABA during seed germination and postgermination growth. These phenotypes of amp1 in Arabidopsis are opposite with the phenotypes in monocots (Suzuki et al., 2008; Kawakatsu et al., 2009). Conversely, the overexpression of AMP1-induced early germination and ABA insensitivity during germination and postgermination growth. Moreover, elevated endogenous ABA was detected in amp1 under osmotic stress, which is consistent with the upregulation of genes involved in ABA biosynthesis, signaling and ABA-associated abiotic stress. Intriguingly, AMP1 protein might be influenced by stress-induced ABA accumulation because the exogenous ABA treatment negatively regulated AMP1 protein concentration. As expected, the abi mutants, including two dominant negative mutations in ABI1 and ABI2, and the mutations in ABI3, ABI4 and ABI5, obviously rescued the ABA hypersensitivity and freezing tolerance of amp1-20, which further indicates that the phenotype of amp1-20 is dependent on ABA signaling. Thus, we conclude AMP1 is a negative regulator of the ABA biosynthesis pathway under stress conditions in Arabidopsis.
ABA stimulates the accumulation of ROS, especially H2O2 and superoxide. It has been shown that atrbohD/atrbohF double mutants display ABA insensitive phenotype during seed germination and root growth due to their impaired stress-induced ROS production and ABA signaling pathway (Kwak et al., 2003). In our study, we interestingly found that superoxide was overproduced in amp1 mutants, but less H2O2 was detected in amp1 mutants. This is probably due to the reduced activity of SOD in amp1 mutants. Therefore, AMP1 probably mediates cellular ROS detoxification in Arabidopsis. This hypothesis is also supported by the hypersensitive response of amp1 mutants to MV-induced oxidative stress. Application of exogenous ROS scavenging agent DTT alleviates growth inhibition of amp1 and wild-type by ABA, with more alleviation trends in amp1. Thus, the mutation of AMP1 probably stimulates the production of ROS, thereby impairing the plant response to ABA. However, ROS imbalance in amp1 is not the only determinant for its ABA-hypersensitive phenotype, because application of DTT could not totally restore amp1-20 to the wild-type phenotype in response to ABA.
Plants exhibiting enhanced freezing and drought tolerance often accumulate more osmolytes, such as soluble sugar and proline. Consistent with its freezing and drought tolerance, an increased concentration of soluble sugar was detected in amp1 mutants. The inhibition of growth by high sugar concentration was also observed in amp1 mutants. Previous studies indicate that sugar has a tight connection with ABA during seed germination. Screening for sugar-insensitive mutants identified several ABA signaling or biosynthesis deficient mutants, including aba2, abi4 and abi5 (Arenas-Huertero et al., 2000; Laby et al., 2000). Indeed, the primary root length inhibition of amp1 by high concentration of glucose could be compromised by five abi mutations. These results suggest that the high concentrations of sugar in amp1 could account for the hypersensitivity of ABA.
Proline is proven to be an important osmolyte (Nanjo et al., 1999). Despite its enhanced freezing tolerance, amp1 contained much less proline than the wild-type. Moreover, proline is not the only amino acid with a lower content in amp1. Of 23 amino acids measured, most decreased significantly in amp1, except for Cys, Met and a further nine amino acids. Thus, amp1 seems to be amino acid starving and disturbs the balance of carbon and nitrogen (C/N) metabolism. Carbon assimilation is required for amino acids biosynthesis, and C/N metabolism is subject to ABA regulation and ROS generation (Foyer et al., 2003; Taylor et al., 2004). Previously, Arabidopsis glutamate receptors (AtGLR1.1) have been shown to coordinate C/N metabolism and ABA synthesis; the loss-of-function of AtGLR1.1 results in sensitivity to elevated exogenous C : N ratio and ABA (Kang & Turano, 2003; Kang et al., 2004). It has also been reported that altered carbon metabolism in both chloroplasts and mitochondria could generate excessive ROS (Suzuki et al., 2012). Enzymes involved in TCA cycle are sensitive to oxidative stress, which leads to the decrease in amino acids (Baxter et al., 2007). Thus, the imbalance of C/N metabolism might induce ROS accumulation in amp1 and, in turn, causes its sensitivity to oxidative stress. Considering the potential peptidase activity of AMP1, it is tempting to speculate that AMP1 and/or its natural substrates might be responsible for the activity of N-metabolic enzymes.
In summary, we proposed a working model depicting the action of AMP1 in the regulation of abiotic stress responses (Fig. 9). AMP1 protein is negatively regulated by abiotic stress, possibly through ABA. The amp1 mutations disturb the balance of C/N metabolism, which may promote ABA biosynthesis and impair ROS scavenging system. On the one hand, ABA biosynthesis and signaling activate transcription of stress responsive genes and enhance abiotic stress tolerance of amp1; on the other, imbalance of ROS scavenging system fails to protect cells from oxidative stress, which may induce cellular ROS burst and ER-stress. This finding raises the possibility that AMP1 catalyzes its natural substrates to regulate C/N metabolism, thereby modulating ABA and abiotic stress.
We thank Drs Sean R. Cutler and Zhizhong Gong for kindly providing seeds. This work was supported by National Key Basic Research Program of China (2011CB915401 and 2009CB119100), the China National Funds for Distinguished Young Scientists (31225003), and the National Natural Science Foundation of China (31121002).