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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.
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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.
Figure 9. The proposed working model of AMP1 in abiotic stress. AMP1 acting as an ER-located carboxypeptidase is important in regulating the balance of carbon (C)/nitrogen (N) metabolism. In amp1 mutants, imbalanced energy metabolism disturbs the cellular hormone homeostasis via affecting abscisic acid (ABA) biosynthesis and signaling. The stability of AMP1 protein is negatively regulated by ABA. In addition, activation of reactive oxygen species (ROS) signaling also occurs due to the disturbance of ROS scavenging system in amp1 mutants. Hence, AMP1 serves as a negative regulator to modulate freezing and drought stress responses. CK, cytokinin; GA, gibberellin; ET, ethylene.
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