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Peptidases are ubiquitous proteins found in all living organisms. They have fundamental roles in intracellular protein turnover which involve selective and bulk removal of proteins in many cellular processes. For instance, several peptidases play a role in the degradation of specific regulatory gene products, the maintenance of free amino acids and the elimination of malfunctioning proteins and nutrient recycling (Smalle & Vierstra, 2004). Thus, peptidases are involved in almost all aspects throughout the life cycle of the cell.
The recent availability of numerous complete genome sequences revealed that the peptidases represent a large group of proteins. They are classified into six distinct groups based on the mechanism of catalysis. Of these, aminopeptidases (APs), which are exopeptidases that liberate amino acid from the N-terminal end of proteins/peptides, have attracted great interest. Accumulating evidence indicates the biological significance of mammalian APs belonging to the M1 and M17 families. Extensive studies have revealed the importance of M1 and M17 APs in generating antigenic peptides, in processing of bioactive peptide hormones, and the implications in vesicle trafficking to the plasma membrane (Albiston et al., 2004; Tsujimoto & Hattori, 2005). In prokaryotes, physiological functions of APs likely display greater redundancy than eukaryotes. APs fundamentally serve for proteolytic process, function as potential virulence factors in some pathogenic bacteria (Jobin & Grenier, 2003) and are required for replication (Devroede et al., 2006).
In plants, APs are believed to be involved in a wide range of physiological processes, including stress response and osmoregulation (Chao et al., 1999). So far, the most extensive studies of APs have been on the tomato leucine aminopeptidases (LAPs) belonging to the M17 family (Chao et al., 2000; Gu & Walling, 2000; Pautot et al., 2001; Tu et al., 2003; Walling, 2006; Fowler et al., 2009). Elucidation of tomato LAP-A in regulating the defense and wound signaling pathways has been described (Fowler et al., 2009). By contrast, information of APs from other plant species is very scarce, and thus implications of the plant APs are still largely unknown. In Arabidopsis thaliana, the classification used in the MEROPS database revealed over 800 sequences annotated as putative peptidases (http://merops.sanger.ac.uk). Of these, at least 28 genes encode proteins belonging to APs (Walling, 2006). The physiological importance of two Arabidopsis APs belonging to the M1 family, namely MPA1 (meiotic prophase aminopeptidase) and APM1 (aminopeptidase M1), has been reported. It has been shown that MPA1 is important for meiotic recombination and that a loss of function resulted in reduction in the fertility (Sánchez-Morán et al., 2004). On the other hand, APM1 is required for normal cell division throughout embryogenesis, and has a function for regulation of auxin transport (Peer et al., 2009). In addition to the M1 family, a possible role of the Met AP (MAP), which belongs to the M24A family, has been described. It has been suggested that a minimum amount of cytoplasmic MAP is required for normal development (Ross et al., 2005).
In this study, we performed functional and expression analyses of one putative AP, At4g30920 (LAP2), which is a member of the M17 family. Impacts of a loss of function of LAP2 to a variety of growth processes and stress sensitivity have been demonstrated. In vitro analysis using synthetic substrates revealed that LAP2 hydrolyzed efficiently Met- and Leu-4-methylcoumaryl-7-amides (aminoacyl-MCAs) and moderately the Phe-4-MCA. Integration of global gene expression and metabolite analyses suggest that LAP2 regulates intracellular amino acid turnover. Further, our data strongly suggest that metabolic flux of leucine in the mutant was compensated by up-regulating the biosynthetic pathway of leucine, which consequently influenced the amount of glutamate. Considering these findings, results presented here provide valuable insights into the molecular mechanism and physiological importance of LAP2. Our results also contribute to further understanding of the APs having several implications in higher plant cellular processes and biology.
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- Materials and Methods
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In this study, we demonstrated that Arabidopsis LAP2 regulates a variety of cellular functions by controlling intracellular amino acid turnover. In fact, AP has long been considered to have a primary role in protein degradation and intracellular amino acid turnover (Smalle & Vierstra, 2004; Turk, 2006). Moreover, the role of AP in protein maturation has been described (Matos et al., 1998). There is evidence that proteolysis by 20S proteosome releases free amino acids and the smallest products (two to six amino acids) are directly degraded by APs, mainly LAPs (Polge et al., 2009). Thus, the loss of function of LAP possibly affects the changes in intracellular amino acids in lap2 mutants. The expression studies by promoter-GUS showed that LAP2 perhaps functions in amino acid turnover in the quiescent center, apices, and vascular tissues. It would be interesting to study the function of LAP2 in these sites in the future. So far, no direct suggestion as to the role of LAP in these tissues has been provided. LAPs have been extensively studied in tomato. Analysis of the expression of a LAPA1:GUS gene in transgenic tomato indicated that the LapA1 promoter was active during floral and fruit development, and was used during vegetative growth only in response to wounding (Gu & Walling, 2000).
Biochemical analysis of the recombinant LAP2 clearly showed an AP hydrolytic activity towards Leu-, Met- and Phe-MCAs (Fig. 2c). This result implied that the LAP2 is an AP that liberates N-terminal leucine, methionine and phenylalanine from proteins/peptides. The AP activity of LAP2 was modulated by divalent cations (i.e. Mn2+, Ni2+ and Co2+), which is a striking feature of the M17 AP family. The Arabidopsis LAP2 was remarkably activated by 2 mM Mn2+, which is in line with the other plant (Arabidopsis PM25 and tomato LAP-A) or animal (bovine lens LAP) M17 APs that are also stimulated by 0.5–5 mM Mn2+ (Bartling & Weiler, 1992; Gu & Walling, 2002). Given that the manganese concentration in tobacco (Hirschi et al., 2000) is roughly estimated to be c. 0.15 mM, it is tempting to speculate that Arabidopsis LAP2 functions as an AP in vivo, like its homologous or orthologous APs. In particular, Mn2+ is accumulated mostly in vacuole and chloroplast (McCain & Markley, 1989) and it is crucial for photosynthesis as part of the oxygen-evolving complex in the PSII. Therefore, a strong activation of the Arabidopsis LAP2 by Mn2+ might reflect the physiological importance of LAP2 in a specific tissue or compartment. Our genetic and molecular analyses also revealed that LAP2 influenced PSII, as we could identify that several components of PSII, genes involved in chlorophyll biosynthesis and regulation, were repressed in lap2-1 (Table S4). This evidence suggests that the physiological function of the Arabidopsis LAP2 might be directly or indirectly associated with PSII.
The LAP2 is an AP that liberates N-terminal leucine, methionine, and phenylalanine from proteins/peptides. We initially anticipated that the mutation of LAP2 might alter or modulate composition of these amino acids in vivo. However, there were no differences in the amounts of these amino acids between wild-type and lap2-1 in all conditions tested, but significant changes in nitrogen-rich amino acids, such as glutamate and glutamine, were observed (Table 2). It was shown that depletion of glutamate in the Arabidospsis glt1-T mutant displayed a reduction in growth. Glutamate concentration was significantly reduced in the glt1-T mutant (c. 70%) compared with the wild-type (Lancien et al., 2002). In the present study, glutamate concentration of the lap2 mutant was reduced to c. 80% compared with the glt1-T mutant (Table 2). It should be noted that in both mutants, the amounts of other nitrogen-rich amino acids were also reduced (Table 2) (Lancien et al., 2002). These results suggest the significance of nitrogen-rich amino acids for plant growth and development.
Biosynthesis of leucine shares the initial steps with valine, and it branches off at the final intermediate, 2-ketoisovalerate. Four enzymes, isopropylmalate synthase (IPS), isopropylmalate isomerase (IPM), IMD, and BCAT4, further catalyze to generate leucine. The final step catalyzed by BCAT4 is an interconversion of 4-methyl-2-oxopentanoate and l-glutamate to form l-leucine and 2-oxoglutarate, respectively. Functional analysis of BCAT4 showed activities toward leucine, methionine, and glucosinolate metabolites with different affinities (Schuster et al., 2006). These data clearly suggest that the biosynthetic pathways of leucine and glucosinolate are interdependent. In higher plants, branched-chain amino acids are important compounds in many aspects. Besides their function as building block of proteins, they play a pivotal role in the synthesis of a number of secondary metabolites in plants (Diebold et al., 2002). In Arabidopsis, glucosinolates are a group of unique secondary metabolites. These organic compounds are known to be produced only in the order Brassicales. In our microarray data and RT-PCR analysis, many genes for glucosinolate biosynthesis were up-regulated (Table S3). Extensive study of MYB76 and MYB29 (positive regulators for glucosinolate biosynthesis) revealed that the plants overexpressing lines increased glucosinolate concentrations with an unchanged growth phenotype or caused growth retardation (Gigolashvili et al., 2008). We speculate that a variety of glucosinolates would be modulated in lap2. Analysis of glucosinolate in loss and gain of function of LAP2 would be worth investigating in future studies.
The reduced expression of LAP2 does not lead to changes in the concentration of leucine. Thus, the cells seem to regulate leucine concentration by decreased catabolism or increased expression of several biosynthetic genes. Metabolomics analysis revealed a difference of amino acid pool (Table 2) and GABA content (Table 3) between the wild-type and lap2 mutant. The suppression of GAD expression in the lap 2-1 mutant together with the up-regulation of BCAT4 in lap2-1 might lead to reduced glutamate concentrations, which might finally stimulate a decrease in GABA by the reduction of substrate availability. Moreover, glutamate is used as a donor of amino groups in the biosynthesis of some amino acids. Therefore, the decrease in glutamate may result in decreases in several amino acids, such as glutamine, alanine, glycine, and aspartate.
Loss of function of LAP2 rendered plants more sensitive to various stresses (Fig. 5). We suggest that this sensitivity might be the result of the suppression and/or alteration of metabolic pathways, including metabolites that play a protective role under stress conditions. For example, a significant decrease in GABA concentrations was observed in all conditions tested (Table 3). GABA is a nonprotein amino acid that is present in all living organisms. It is well known as a neurotransmitter in mammalian cells. In plants, it has been proposed that GABA contributes to C : N balance, regulation of cytosolic pH, and functions as an osmoregulator (Bouché & Fromm, 2004). Genes involved in GABA biosynthesis and the GABA shunt pathway, including GABA transport, were functionally analyzed in Arabidopsis (Bouché & Fromm, 2004; Meyer et al., 2006; Miyashita & Good, 2008). The contribution of GABA in the response to abiotic stresses has been reported (Miyashita & Good, 2008; Sawaki et al., 2009; Urano et al., 2009). The decrement of GABA in lap2 would support previous studies because lap2 displayed sensitive phenotype under various stresses.
Early leaf senescence was observed in lap2. To date, a number of mutants with altered leaf senescence have been isolated. It was shown that some of the mutated genes encode for enzymes involved in proteolysis (Doelling et al., 2002; Golldack et al., 2002). It is believed that the decrease in protein turnover was accompanied by an increase in the damaged proteins during senescence (Woo et al., 2001), and therefore APs would play crucial roles in the elimination of malfunctioning protein(s) and amino acid recycling to provide nitrogen compounds to plants. A decrease in nitrogen-rich compounds in lap2 would have an effect on nutrient recycling in plants as well. The difference in amino acid composition (Table 2) would possibly affect and participate in the early-senescent phenotype of the lap2 mutant. We determined the amino acid concentrations of plants (42-d-old) and found that nitrogen-rich amino acids were decreased (data not shown). It should be noted that in recombinant inbred lines of Arabidopsis showing leaf-senescent phenotypes, amino acids, namely glutamate, glutamine, aspartic acid and asparagine, were found to be modulated (Diaz et al., 2005).
In summary, we performed functional and expression analyses of the Arabidopsis LAP2. Biochemical and physiological data strongly suggest that LAP2 is indeed an enzymatically active AP involved in the regulation of plant development and stress response. Regulated proteolysis is an important mechanism in all stages of the plant life cycle. LAP2 together with other plant APs might be related to the process of plant peptide hormone. Detailed analysis of the interaction of LAP2 with plant peptide hormones and a further exploration of natural substrates are topics of great interest for future studies.