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Keywords:

  • amino acid;
  • aminopeptidase;
  • Arabidopsis;
  • senescence;
  • stress response

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Peptidases are known to play key roles in multiple biological processes in all living organisms. In higher plants, the vast majority of putative aminopeptidases remain uncharacterized.
  • In this study, we performed functional and expression analyses of the Arabidopsis LAP2 through cDNA cloning, isolation of T-DNA insertional mutants, characterization of the enzymatic activity, characterization of gene expression and transcriptomics and metabolomics analyses of the mutants.
  • Loss of function of LAP2, one of the 28 aminopeptidases in Arabidopsis, reduced vegetative growth, accelerated leaf senescence and rendered plants more sensitive to various stresses. LAP2 is highly expressed in the leaf vascular tissue and the quiescent center region. Integration of global gene expression and metabolite analyses suggest that LAP2 controlled intracellular amino acid turnover. The mutant maintained free leucine by up-regulating key genes for leucine biosynthesis. However, this influenced the flux of glutamate strikingly. As a result, γ-aminobutyric acid, a metabolite that is derived from glutamate, was diminished in the mutant. Decrements in these nitrogen-rich compounds are associated with morphological alterations and stress sensitivity of the mutant.
  • The results indicate that LAP2 is indeed an enzymatically active aminopeptidase and plays key roles in senescence, stress response and amino acid turnover.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and growth conditions

Arabidopsis thaliana (L.) Heynh. seeds (ecotype Columbia) were used in this study. Thirty T-DNA insertion mutants were provided by the ABRC Arabidopsis stock center. These mutants represent 18 putative metallopeptidases in Arabidopsis (Supporting Information, Table S1). All plants were grown in soil or on Murashige and Skoog (MS) standard medium, pH 5.7, containing 0.8 g l−1 phytoagar at c. 100–120 μmol m−2 s−1 for a 16 h photoperiod at 22°C, unless otherwise stated.

Identification of T-DNA insertion mutants for the LAP2 gene

The lap2-1 and lap2-2 T-DNA insertion lines were isolated from the SALK T-DNA collection (SALK_053879 and SALK_150009). T-DNA insertions for lap2-1 and lap2-2 were identified using the following primers: T-DNA-LBa1, LAP2-ex6 and LAP2-ex10. The homozygous lap2-1 and lap2-2 mutants were identified by PCR. Transcripts of the homozygous mutants were confirmed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) using the primer pairs mLAP2-F and mLAP2-R. Ubiquitin primer pairs UBQ-F and UBQ-R were used as a positive control. The list and sequence of primers used in this study are shown in Table S2.

Transgenic constructs and plant transformation

For complementation analysis of the lap2 mutant, the LAP2 cDNA was amplified by PCR using the primer pairs LAP2-1 and LAP2-2, and cloned into a Gateway donor vector pCR8/GW/TOPO (Invitrogen). A full-length cDNA clone was transferred into the binary vector, pGWB2 (Nakagawa et al., 2007). The resulting plasmids were electroporated into Agrobacterium tumefaciens strain GV3101, which was used to transform the lap2 mutants by the floral dipping method (Clough & Bent, 1998). To select transgenic plants, seeds were surface-sterilized and plated on to agar-solidified MS medium containing 1% sucrose and supplemented with 25 mg l−1 hygromycin and 50 mg l−1 kanamycin. Independent transgenic lines that segregated 3 : 1 for the transgene antibiotic-resistance marker in the heterozygous T1 generation were carried through T2 and T3 generations to isolate progeny that are homozygous for the transgene. The T3 homozygous plants of the single copy transgene were used for further and detailed analysis.

For GUS reporter gene analysis, a DNA fragment containing c.1.2 kb of the upstream region of the LAP2 was amplified by PCR using the primer pairs LAP2-GUS1 and LAP2-GUS2, and cloned into a Gateway donor vector pCR8/GW/TOPO. The cloned cDNA was transferred into the binary vector pGWB3 (Nakagawa et al., 2007). Transformation and selection were carried out as described in the complementation analysis. Histochemical localization of GUS activities in the transgenic plants was observed by incubation in 5-bromo-4-chloro-3-indolyl- glucuronide (X-gluc) buffer (50 mM sodium phosphate buffer, pH 7.0 : 0.1% Triton X-100 : 0.5 mM potassium ferrocyanide : 0.5 mM potassium ferricyanide/1 mg ml−1 X-gluc) at 37°C for 16 h.

Stress treatments

Seeds of the wild-type and the lap2 mutants were sown on MS medium (pH 5.7), and grown under standard growth conditions at c. 100–120 μmol m−2 s−1 for a 16 h photoperiod at 22°C. Seventeen-day-old seedlings were used for stress treatments. For nitrogen starvation, plants were transferred to nitrogen-free MS medium (depleted ammonium nitrate) and were grown for another 3 d. Treated plants were harvested and immediately immersed in liquid nitrogen and stored at −80°C. For the drought stress experiment, plants were transferred to a filter paper to absorb excess water, and then placed on a clear plastic tray. The trays were transferred in a clear plastic container with a cover to avoid rapid dehydration. After 24 h, the dehydrated plants were rapidly frozen in liquid nitrogen and stored at −80°C. Frozen plant tissues were also used for the amino acid analyses, including measuring the γ-aminobutyric acid (GABA) contents. Stress treatments on soil-grown plants were also performed. Two-week-old seedlings of plants on MS medium were transferred to soil and subsequently subjected to stresses. For low pH, plants were watered with 1/10 MS basic salts (pH 3.5) for 16 d. For salt stress, plants were watered with 1/10 MS basic salts (pH 5.7) supplemented with the indicated NaCl concentrations (one tray containing eight pots, and watered with 150 ml per tray). Salinity in solution was measured by a refractometer. Salt stress was applied stepwise by adjusting NaCl concentrations to 100, 150, 200, and 250 mM in the watered solution, each with a 4 d interval. For drought stress, plants were watered for 6 d, following which the water was withheld for another 14 d.

Expression of recombinant LAP2

The LAP2 cDNA was amplified by the primer pairs LAP2-Nde and LAP2-Eco, and cloned into pGEM-T Easy vector (Promega). The full-length LAP2 fragment was prepared by double digestion with NdeI and EcoRI and ligated into the corresponding sites of pCold I vector (Takara, Tokyo, Japan). A nonsynonymous mutation (Arg440 to Ala) was introduced at codon 440 of the LAP2 gene using a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The sequences of designed primers were as follows: sense primer, 5′-ACCGATGCTGAAGGTGCACTAACACTTGCAGAT-3′; and antisense primer, 5′-ACTTGCAAGTGTTAGTGCACCTTCAGCATCGGT-3′. The plasmids of LAP2 were introduced into Escherichia coli strain Rosetta-gami B (DE3) pLysS, and recombinant LAP2s were induced with 0.2 mM isopropyl β-d-thiogalactopyranoside at 15°C. After 16 h, cells were harvested by centrifugation at 3100 g for 15 min, and the bacterial pellets were resuspended in buffer A (50 mM Tris, pH 7.5, 100 mM NaCl). After sonication, the supernatant was supplemented with 1% Triton X-100 and 50 mM imidazole, and then incubated for 1 h on ice. The clarified supernatant was loaded on to a HisTrap HP column (GE Healthcare, Piscataway, NJ, USA). The column was washed extensively with buffer A containing 50 mM imidazole, and then recombinant LAP2s were eluted with buffer A containing 300 mM imidazole.

The AP activity of recombinant Arabidopsis LAP2 was examined with various fluorogenic substrates. Aminoacyl-4-methylcoumaryl-7-amides (aminoacyl-MCAs) are the synthetic substrates of APs and are conventionally used for detection of their activities (Peptide Institute, Osaka, Japan). The reaction mixture containing 100 μM of aminoacyl-MCA and the enzyme (0.75 μg in 0.1 ml of 50 mM Tris/HCl buffer (pH 8.5) with or without 2.0 mM Mn2+ ion) was incubated at 37°C for 30 min. The amount of 7-amino-4-methylcoumarin released was measured by spectrofluorophotometry (F-2000; Hitachi, Tokyo, Japan) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. All measurements were performed in triplicate. To examine the effect of divalent cations on the AP activity of LAP2s, Leu-MCA was employed as a substrate.

Total RNA extraction, DNA microarray analysis and data mining

Above-ground tissues of 3-wk-old seedlings of Arabidopsis plants grown on MS medium were harvested and used for DNA microarray analysis. The deep-frozen plants (20 plants pooled × two replications) were transferred to a prechilled (in liquid N2) mortar and pestle and ground to a very fine powder. Total RNA was extracted from c. 60 mg sample powder using the Qiagen RNeasy Mini Kit (Qiagen). To verify the quality of this RNA, the yield and purity were determined spectrophotometrically (NanoDrop, Wilmington, DE, USA) and visually confirmed using formaldehyde-agarose gel electrophoresis.

A whole Arabidopsis (V4) genome 4 × 44K oligo microarray kit (Design ID: 21169, G2519F; Agilent Technologies, Palo Alto, CA, USA) was used for RNA analysis. Total RNA (800 ng) was labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit. Fluorescently labeled targets of control, as well as the lap2-1 mutant samples, were hybridized to the same microarray slide with 60 mer probes. A flip labeling (dye-swap or reverse labeling with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA (Cho et al., 2008). Hybridization and wash processes were performed according to the manufacturer’s instructions, and hybridized microarrays were scanned using an Agilent Microarray scanner G2565BA. For the detection of significantly differentially expressed genes between control and lap2 mutant samples, each slide image was processed using Agilent Feature Extraction software (version 9.5.3.1). This program measures Cy3 and Cy5 signal intensities of whole probes. Dye bias tends to be signal intensity-dependent, and therefore the software selected probes using a set by rank consistency filter for dye normalization. Said normalization was performed by locally weighted linear regression (LOWESS) which calculates the log ratio of dye-normalized Cy3 and Cy5 signals, as well as the final error of log ratio. The significance (P) value was based on the propagate error and universal error models. In this analysis, the threshold of significant differentially expressed genes was < 0.01 (for the confidence that the feature was not differentially expressed). In addition, erroneous data generated as a result of artifacts were eliminated before data analysis using the software. The differentially expressed gene lists (up- and down-regulated genes) were generated and annotated using the GeneSpring version GX 10 (Agilent). The raw data have been deposited in a MIAME (Minimum Information About a Microarray Experiment)-compliant database with accession number GSE22000.

RT-PCR analysis

Five micrograms of the total RNA was reverse-transcribed using the Superscript II RT kit (Invitrogen) according to the manufacturer’s instructions. The PCR amplification was performed with oligonucleotides specific for targeted genes (Table S2). The PCR-amplified samples were electrophoresed on 1.2% (w/v) agarose gels, and were detected with 0.1 μg ml−1 ethidium bromide staining. All RT-PCR experiments were repeated at least three times.

Other methods

The maximum quantum efficiency of photosystem II (PSII) was measured with a pulse amplitude-modulated fluorometer (MINI-PAM; H. Walz, Effeltrich, Germany). Pigments were extracted from rosette leaves and total chlorophyll was measured (Porra et al., 1989). Free amino acids and GABA were extracted from Arabidopsis seedlings as previously described (Waditee et al., 2005). Briefly, plant tissues were homogenized in absolute methanol. The supernatant was collected and the pellet was re-extracted with 90% methanol. The combined methanol extract was dried in a vacuum rotary evaporator at 45°C. The dry residues were re-extracted with a mixture of water and chloroform (1 : 1 v/v). The upper aqueous phase was filtered through a 0.22 μm membrane filter. The filtrate was dried in a vacuum and stored at −20°C until use. Amino acid and GABA contents were analyzed using a Hitachi L-8500A amino acid analyzer (Hitachi, Tokyo, Japan).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Morphological phenotypes of lap2 alleles

Thirty mutant lines of the Arabidopsis APs were screened to isolate a mutant altered in growth/development. We found the mutants for At4g30920 (LAP2) exhibiting considerably smaller sizes than that of the wild-type, that is, in the rosette leaf diameter. Two Arabidopsis mutant lines, SALK_053879 (lap2-1) and SALK_150009 (lap2-2), harbored T-DNA insertions within intron 6 and exon 7 of At4g30920, respectively (Fig. 1a). Knockout mutant plants lacked LAP2 transcripts as demonstrated by RT-PCR analysis (Fig. 1b).

image

Figure 1. Genotypes and phenotypes of Arabidopsis lap2 mutants. (a) Schematic representation of the LAP2 gene structure (At4g30920) with exons (black boxes) and introns. The T-DNA locations for two lap2 knockouts are indicated. (b) Reverse transcription polymerase chain reaction (RT-PCR) of the LAP2 transcript. Ubiquitin (UBQ) was used as a positive control. (c) Phenotype of lap2 mutants (3-wk-old seedlings). Bar, 10 mm. LB, Left Border; RB, Right Border; ATG, start codon; TAA, stop codon).

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The lap2 mutants were backcrossed twice with the wild-type plants, and genetic segregation of F1 generation was performed. We obtained both lap2-1 and lap2-2 mutants with only a single T-DNA insertion. In the case of backcrossing between lap2-1 with the wild-type, of 134 F2 seedlings analyzed, 31, 69, and 34 seedlings revealed the wild-type, heterozygous, and homozygous mutant genotypes, respectively. When lap2-2 plants were backcrossed with the wild-type, of 158 F2 seedlings analyzed, 47, 72, and 39 seedlings revealed the wild-type, heterozygous, and homozygous mutant genotypes, respectively. These results conformed to a theoretical segregation ratio of 3 : 1, representing a T-DNA insertion at a single locus of the genome. The two homozygous mutant lines displayed phenotypic changes throughout the plant life cycle. Vegetative growth of the lap2 mutants was reduced to c. 70% as determined by rosette leaf diameter and FW measurements. The lap2 mutants exhibited early flowering and a slight reduction in plant height (Table 1). We further generated transgenic plants for complementation of the lap2 mutants. Ten independent transgenic lines for 35S::LAP2 were used for phenotypic analyses. We found that over 80% of transgenic 35S::LAP2 lines appeared as similar phenotypes. The phenotype of a representative complementation line (35S::LAP2 line A8) was compared with wild-type and lap2 mutants. As shown in Fig. 1(a,c) complemented lap2 mutant carrying 35S::LAP2 rescued the phenotype of lap2 mutant plants.

Table 1.   Phenotypic characteristic of wild-type Arabidopsis, lap2-1 and lap2-2
 Wild-typelap2-1lap2-2
  1. aRosette diameter was obtained from 21-d-old plants by measuring the longest rosette leaf of each plant.

  2. bFW was measured from above-ground tissues of 20 plants from (a).

  3. cThe flowering time was scored when the first flower appeared.

  4. dSix-week-old plants.

  5. eSilique length is the average lengths of the siliques in the position 5–10.

  6. fNumber of seeds per silique counted from the siliques in the position 5–10 from (d).

  7. The measurements are means ± SD from 20 soil-grown plants per line from three biologically independent replications. ND, not determined.

  8. *Significant differences (P < 0.05) from wild-type plants (Student’s t-test).

Rosette diameter (mm)a22.1 ± 1.817.1 ± 1.7*18.1 ± 1.1*
FW (mg)b199.4 ± 10.8144.8 ± 7.2*ND
Flowering time (d)c29 ± 1.823 ± 2.3*24 ± 2.2*
Plant height (cm)d30 ± 225 ± 2*27 ± 3*
Silique length (mm)e14.1 ± 0.813.8 ± 1.213.6 ± 0.7
No. of seeds per siliquef59.3 ± 4.460.3 ± 2.161.3 ± 1.9

Enzymatic properties of LAP2

To characterize enzymatic properties of the Arabidopsis LAP2, we expressed recombinant LAP2 with a 6xHis tag at the N-terminus in E. coli. Recombinant wild-type LAP2 was purified to homogeneity in a single step from crude E. coli lysate using Ni2+-chelating sepharose chromatography.

Purified recombinant wild-type LAP2 had a molecular mass of c. 60 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was expected from total amino acid number of the enzyme (Fig. 2a). Although in the absence of Mn2+ no hydrolytic activity was observed, apparent Leu-MCA hydrolytic activity was detected in wild-type LAP2 under the 2 mM Mn2+ condition (Fig. 2b). To confirm that this activity was from recombinant LAP2, a catalytically inactive mutant of LAP2 was generated. It was suggested that Arg431 of tomato LAP-A plays an essential role in catalysis, and the substitution of the residue with Ala caused a marked decline in AP activity (Gu & Walling, 2002). We thus introduced Ala substitution at Arg440 (R440A), which corresponds to Arg431 of tomato LAP-A. The expression and purification efficiencies between wild-type and R440A LAP2s were similar; Leu-MCA hydrolytic activity in parallel with optical density corresponding to LAP2 was detected only in the wild-type eluates (Fig. 2b). This result clearly revealed that recombinant wild-type LAP2 had an AP activity and also indicated that carryover of bacterial LAP during the purification step was barely detectable in our recombinant LAP2s.

image

Figure 2. Purification of recombinant LAP2, specificity and effect of metal ions. (a) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of recombinant Arabidopsis wild-type and R440A LAP2s purified by Ni2+ sepharose high-performance column chromatography. Purified LAP2s were visualized by Coomassie brilliant blue staining. (b) Aminopeptidase (AP) activity of wild-type and R440A LAP2s toward Leu-MCA. AP activity was examined with Leu-MCA in the presence of 2.0 mM Mn2+ ion. (c) Specificity of wild-type LAP2 toward fluorogenic substrates with (black bars)/without (gray bars) 2.0 mM Mn2+ ion. (d) Effect of metal ions on LAP2 activity. Data are presented as means ± SD of three independent replications.

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We next examined AP activity of recombinant wild-type LAP2 toward various synthetic substrates. Among 10 substrates tested, LAP2 hydrolyzed Met- and Leu-MCA efficiently and Phe-MCA moderately under the 2 mM Mn2+ condition, whereas, in the absence of Mn2+, hydrolytic activities toward all substrates were hardly measurable (Fig. 2c). Because modulation of the AP activity by divalent cations is one of a distinctive feature of the M17 family of APs (Gu & Walling, 2002), the effects of divalent cations on the activity of recombinant wild-type LAP2 were examined. As shown in Fig. 2(d), the Leu-MCA hydrolytic activity of recombinant wild-type LAP2 was markedly enhanced by addition of Mn2+. The Ni2+ and Co2+ also moderately enhanced the LAP2 activity. On the other hand, Mg2+ and Ca2+ did not promote AP activity of LAP2.

LAP2 is highly expressed in the leaf vascular tissue and the quiescent center region

The spatial expression of the LAP2 gene was monitored by analyzing 12 independent transgenic Arabidopsis lines containing the GUS reporter gene under the control of the LAP2 promoter. The expression was strongly observed in the leaf vascular tissue (Fig. 3a) and in the quiescent center cells in the root meristem (Fig. 3b). A strong GUS staining was also seen in the shoot apical meristem (Fig. 3a, shown by the yellow arrowhead). A relatively weak expression could be detected in the anthers (data not shown). No expression was detected in the nongreen tissues (such as petals), stems, and siliques.

image

Figure 3. Analysis of spatial expression pattern using promoter::GUS transgenic plants. (a) Above-ground tissues of Arabidopsis plant grown in MS basal medium for 14 d (arrowhead shows a strong GUS staining in the shoot apical meristem). (b) The main root of plant grown in Murashige and Skoog (MS) basal medium for 14 d. GUS activity was seen after 16 h in 1.0 mM 5-bromo-4-chloro-3-indolyl- glucuronide (X-gluc). GUS staining was observed from 12 independent lines. Bars: 5 mm (a); 0.1 mm (b).

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Loss of function of LAP2 accelerates leaf senescence

As shown in Fig. 1(c) and Table 1, loss of function of LAP2 influenced vegetative growth and flowering time. When plants grew further, the first symptoms of senescence, characterized by a decrease in leaf chlorophyll, were observed earlier in lap2 mutants (Fig. 4a). To determine whether the LAP2 mutation affects photosynthesis, the photosynthetic efficiency was measured. The quantum yields of the lap2 mutants declined progressively after 42 d of germination (Fig. 4b). Chlorophyll contents were also found to be considerably depleted in the mutants (Fig. 4c). These results indicated that loss of function of LAP2 resulted in early leaf senescence. Expression of the LAP2 gene under control of the cauliflower mosaic virus 35S promoter rescued early leaf senescent phenotype of lap2 mutant plants (Fig. 4a).

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Figure 4. The Arabidopsis lap2 mutants showed early leaf senescence. (a) Leaf phenotype at 44 d after germination (DAG; bar, 10 mm). (b) Quantum yield (Fv/Fm). (c) Leaf chlorophyll concentration. All parameters were determined from leaf 4. Data are presented as means ± SD from eight to 15 plants per line of three independent replications. WT, wild-type.

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Loss of function of LAP2 renders plants more sensitive to various stresses

We next examined phenotypes of the lap2 mutants under abnormal environmental factors. Stress sensitivity of lap2 mutants was performed in the vegetative stage (Fig. 5). When plants were subjected to low pH, lap2 mutants appeared as slightly pale green with curly-leaf phenotypes compared with the control, and chlorosis symptoms appeared after a long growing period, that is, 1 month after being subjected to pH stress. Chlorophyll contents were much lower in the mutants (data not shown). When plants were exposed to NaCl, lap2 mutants showed early bleaching and chlorosis. For drought stress exposure, water was withheld for 2 wk, and lap2 mutants showed obvious decreases in plant size and development. Expression of the LAP2 gene under control of the cauliflower mosaic virus 35S promoter rescued the stress-sensitivity phenotype of lap2 mutant plants (Fig. 5). These results indicate that loss of function of LAP2 rendered plants more sensitive to various stresses.

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Figure 5. Stress sensitivity of the lap2 mutants. Two-week-old Arabidopsis seedlings of plants on Murashige and Skoog (MS) medium were transferred to soil and subsequently subjected to stresses as described in the Materials and Methods section. Plant phenotypes were observed from at least 30 soil-grown plants per line from three independent replications.

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Transcriptional profiles reveal functional compensation of leucine and detoxification process in lap2

To provide clues and gain insight into molecular mechanisms of LAP2, a genome-wide expression analysis was performed. A cDNA microarray chip containing 43803 Arabidopsis gene probes was used to compare expression patterns between lap2 and wild-type plants grown in MS media for 3 wk (for details, see the Materials and Methods section). Gene expression profiles led to the identification of 647 differentially regulated genes. Of these, 226 genes were up-regulated and 421 genes were down-regulated. Among the up-regulated genes, we found high abundances of transcripts of four distinct groups involved in amino acid metabolism, glucosinolate biosynthesis, detoxification and transcription-related (Table S3). In the first group, four genes that are responsible for leucine biosynthesis, 3-isopropylmalate dehydrogenase (IMD), aconitase C-terminal domain-containing proteins (ACO) and branched-chain aminotransferase 4 (BCAT4), were identified. Another two genes in this group, isocitrate lyase and aspartate kinase, are important as metabolic intermediates of tricarboxylic acid cycle (TCA) and in the biosynthesis of aspartate family amino acids. The second group comprises genes encoding proteins involved in the biosynthesis of glucosinolate. The third group comprises genes encoding proteins known to function in detoxification. In the last group, transcription factors that play a crucial role in regulating flowering time were identified.

The induced expressions of leucine and glucosinolate biosynthetic pathways would suggest an increase in the synthesis for both pathways in lap2. In fact, it has been shown that leucine and glucosinolate are synthesized by sharing the same enzyme in some steps (Schuster et al., 2006), thus their biosynthetic pathways are interdependent in Arabidopsis (Schuster et al., 2006; Knill et al., 2008).

Relative expressions of selected genes for leucine and glucosinolate biosynthetic pathways were verified by semiquantitative RT-PCR. We found a good correlation between the microarray result and RT-PCR analysis (Fig. S1).

LAP2 influences cell wall metabolism and components of cell growth, photosynthesis, and nitrogen metabolism

Gene expression profiles revealed a down-regulation in a substantial number of the transcripts involved in cell wall metabolism, structure components, photosynthesis, metabolisms, transport and transcription-related (Table S4). The down-regulated cell wall-associated genes and cell growth components included cellulose synthase-like, cell wall invertase, expansin, extensin, putative chitinases and putative cell wall protein precursor. In relation to photosynthesis and chlorophyll biosynthesis, 13 genes were identified. The expression of eight genes encoding transcription factors was influenced by LAP2. Several of these genes have been shown to be associated with photoperiod (CCA1, LHY, DDF1, and DDF2) and stress responses (DREB1A, CBF1, CBF2, CBF4/DREB1D, DDF1, and DDF2). Another two major down-regulated groups included genes involved in nitrogen metabolism, GLUTAMINE SYNTHETASE 1;5 (GLN1;5), glutamate decarboxylase 4 (GAD4), glutamate decarboxylase 5 (GAD5), ARABIDOPSIS THALIANA GLUTAMINE DUMPER 6 (ATGDU6), glutamine-hydrolyzing, l-asparaginase, and several transporters. Diminished expressions of some metabolic genes for nitrogen metabolism were verified by semiquantitative RT-PCR (Fig. S1). Results revealed that GLN1;5, GAD4 and GAD5 were significantly reduced. Thus, it is plausible that the LAP2 mutation may be involved in the change of nitrogen metabolism and remobilization of metabolites.

Alterations of free amino acids and related metabolites in lap2-1

Comparative gene expression profiles by DNA microarray and RT-PCR analysis revealed that metabolic pathways of leucine and glutamate were particularly influenced in lap2-1. We hypothesized that free amino acids and metabolites derived from amino acid biosynthesis would be modified in lap2-1. To address this question, free amino acids in wild-type and lap2-1 were determined. Levels of free amino acids were measured from rosette leaves of 3-wk-old seedlings which were the same stage as used for DNA microarray analysis. Amino acids which showed wild-type/lap2-1 ratio ≥ 1.2 or ≤ 0.8 represent increased and decreased levels, respectively. As shown in Table 2, amino acid profiles revealed that alanine, glycine, glutamate, glutamine, aspartic acid, serine, tryptophan, and tyrosine were substantially decreased, while arginine and asparagine were elevated in lap2-1. It was shown by DNA microarray and RT-PCR that several genes involved in leucine biosynthesis were up-regulated in lap2-1 while amino acid profiles showed similar concentrations of leucine and other branched-chain amino acids in wild-type and lap2-1 in all conditions tested. Thus, there was a significant difference in terms of composition of amino acid pools between wild-type and lap2-1, although leucine concentration did not change (Table 2). These results suggest that the LAP2 mutation resulted in modification of amino acid contents, particularly for nitrogen-rich amino acids.

Table 2.   Amino acid content in rosette leaves of Arabidopsis wild-type (WT) and lap2-1
Amino acidAmino acid content (nmol mg−1 FW)t-testRatio
WTlap2-1WT/lap2-1
  1. Above-ground tissues of 3-wk-old seedlings of WT and lap2-1 grown on Murashige and Skoog (MS) medium (pH 5.7) under 16 h photoperiod were used for amino acid analysis. Data are presented as means ± SD of three independent replications.

Ala0.505 ± 0.0310.311 ± 0.0220.00091.6
Arg0.914 ± 0.0591.454 ± 0.0600.00040.6
Asp2.214 ± 0.1301.889 ± 0.0900.06171.2
Asn1.196 ± 0.1001.486 ± 0.0800.01720.8
Gly0.544 ± 0.0300.247 ± 0.0100.00012.2
Glu4.079 ± 0.3113.373 ± 0.1980.02951.2
Gln19.46 ± 1.12116.41 ± 0.8090.01881.2
His0.145 ± 0.0210.143 ± 0.0120.89301.0
Ile0.271 ± 0.0180.265 ± 0.0160.68831.0
Lys0.354 ± 0.0190.310 ± 0.0210.05441.1
Leu0.205 ± 0.0110.204 ± 0.0150.93051.0
Met0.022 ± 0.0020.021 ± 0.0010.48181.0
Phe0.219 ± 0.0130.206 ± 0.0090.22751.1
Pro0.926 ± 0.0510.856 ± 0.0590.19551.1
Ser1.775 ± 0.1401.375 ± 0.1100.01751.3
Thr0.613 ± 0.0300.619 ± 0.0290.81851.0
Trp0.075 ± 0.0080.064 ± 0.0050.11361.2
Tyr0.191 ± 0.0070.143 ± 0.0070.02641.3
Val0.462 ± 0.0180.419 ± 0.0210.05801.1
Total amino acid34.17 ± 2.12029.795 ± 1.5740.04551.1

We further analyzed the metabolites involved in amino acid metabolism. Intriguingly, we found that GABA, a nonprotein amino acid, was strikingly decreased in lap2-1. Under normal growth conditions, GABA concentration in lap2-1 was c. 50% compared with the wild-type (Table 3). When plants were subjected to nitrogen starvation, 2% glucose or drought stress, GABA concentration was also diminished in lap2-1 (Table 3).

Table 3.   Comparison of γ-aminobutyric acid (GABA) in rosette leaves of Arabidopsis wild-type (WT) and lap2-1
ConditionGABA content (nmol mg−1 FW)t-testRatio
WTlap2-1 WT/lap2-1
  1. Control, plants were grown on Murashige and Skoog (MS) medium + 1% sucrose, pH 5.7, for 3 wk; nitrogen starvation, plants were grown on MS medium + 1% sucrose, pH 5.7, for 17 d and subsequently transferred to nitrogen-depleted medium for 3 d (see the Materials and Methods section); glucose, plants were grown on MS medium supplemented with 2% glucose, pH 5.7, for 3 wk; drought stress, plants were grown on MS medium (pH 5.7) for 20 d and subjected to drought stress (see the Materials and Methods section) for 24 h. Data are presented as means ± SD of three independent replications.

Control0.168 ± 0.0120.081 ± 0.0050.00032.1
Nitrogen starvation0.129 ± 0.0080.075 ± 0.0060.00071.7
2% glucose0.369 ± 0.0370.223 ± 0.0010.00241.6
Drought stress0.418 ± 0.0310.246 ± 0.0170.00111.7

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the Foreign Postdoctoral Researcher’s Program from RIKEN (to R.W.S.) and the Program for Promotion of Basic Research Activities for Innovation Bioscience (PROBRAIN) (to T.N.). We thank the Arabidopsis Biological Resource Center and the SALK Institute for providing the T-DNA-tagged lines.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 RT-PCR analysis of selected up-regulated transcripts involved in leucine and glucosinolate biosynthetic pathways and selected down-regulated transcripts involved in nitrogen metabolism.

Table S1 T-DNA insertion mutants used in this study

Table S2 Primers used in this study

Table S3 Representative expression levels of up-regulated genes in lap2-1

Table S4 Representative expression levels of down-regulated genes in lap2-1

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