JXB Advance Access published online on March 28, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern026
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Integrated metabolite and gene expression profiling revealing phytochrome A regulation of polyamine biosynthesis of Arabidopsis thaliana
1Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 Japan
2Department of Applied Environmental Biology, Graduate School of Pharmaceutical Science, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 Japan
* To whom correspondence should be addressed. E-mail: okazawa{at}bio.eng.osaka-u.ac.jp
Received 19 October 2007; Revised 17 January 2008 Accepted 18 January 2008
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Abstract |
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In this study, metabolite profiling was demonstrated as a useful tool to plot a specific metabolic pathway, which is regulated by phytochrome A (phyA). Etiolated Arabidopsis wild-type (WT) and phyA mutant seedlings were irradiated with either far-red light (FR) or white light (W). Primary metabolites of the irradiated seedlings were profiled by gas chromatography time-of-flight mass spectrometry (GC/TOF-MS) to obtain new insights on phyA-regulated metabolic pathways. Comparison of metabolite profiles in phyA and WT seedlings grown under FR revealed a number of metabolites that contribute to the differences between phyA and the WT. Several metabolites, including some amino acids, organic acids, and major sugars, as well as putrescine, were found in smaller amounts in WT compared with the content in phyA seedlings grown under FR. There were also significant differences between metabolite profiles of WT and phyA seedlings during de-etiolation under W. The polyamine biosynthetic pathway was investigated further, because putrescine, one of the polyamines existing in a wide variety of living organisms, was found to be present in lower amounts in WT than in phyA under both light conditions. The expression levels of polyamine biosynthesis-related genes were investigated by quantitative real-time RT-PCR. The gene expression profiles revealed that the arginine decarboxylase 2 (ADC2) gene was transcribed less in the WT than in phyA seedlings under both light conditions. This finding suggests that ADC2 is negatively regulated by phyA during photomorphogenesis. In addition, S-adenosylmethionine decarboxylase 2 and 4 (SAMDC2 and SAMDC4) were found to be regulated by phyA but in a different manner from the regulation of ADC2.
Key words: Arabidopsis thaliana, gene expression profiling, metabolite profiling, phytochrome A, polyamine biosynthesis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Photosynthetic organisms, from bacteria to higher plants, perceive and utilize light by various wavelength-specific photoreceptors (reviewed by Fankhauser and Chory, 1997; Sullivan and Deng, 2003) to obtain energy and gain information for themselves. One of the plant photoreceptors is a phytochrome, which is important in the regulation of a wide range of developmental responses, for example seed germination, seedling de-etiolation, development of functional photosynthetic apparatus, and flowering (reviewed by Quail et al., 1995; Nagy and Schäfer, 2002). In Arabidopsis, there are five phytochromes, termed phyA–phyE (Clack et al., 1994). Phytochrome A (phyA), the only type I photolabile phytochrome, is a unique photoreceptor responsible for the far-red light high irradiance response (FR-HIR) of etiolated seedlings, including inhibition of hypocotyl elongation and cotyledon expansion (Whitelam et al., 1993).
These phyA-mediated photomorphogenic responses involve global changes in gene expression. A microarray-based analysis of gene expression profiles in wild-type (WT) and phyA-null mutants of Arabidopsis seedlings revealed that a minor proportion (8%) of the total of 812 phyA-regulated genes exhibited altered expression within 1 h after the onset of FR irradiation. These genes were defined as ‘early-response’ genes (Tepperman et al., 2001). On the other hand, the majority of phyA-regulated genes showed changes in their expression after several hours under FR, including photosynthesis- and chloroplast-related genes. Therefore, many downstream components are regulated after a longer duration of light treatment.
In the field of plant biology, research concerning metabolite profiling has been carried out extensively by using gas chromatography–mass spectrometry (GC/MS) (Fiehn et al., 2000; Roessner et al., 2000; Roessner-Tunali et al., 2003; Urbanczyk-Wochniak and Fernie, 2005). Such a comprehensive approach provides metabolic snapshots and has been considered as a powerful tool to investigate the regulatory mechanisms of metabolic pathways in plants. Recently, several studies have been carried out through the combination of transcriptomic, proteomic, and metabolomic approaches (Hirai et al., 2004; Weckwerth et al., 2004; Carrari et al., 2006). Transcriptional and metabolite profiling data have been integrated, in order to examine the regulation of various biochemical pathways in Arabidopsis during photomorphogenesis (Ghassemian et al., 2006). In addition, a combination of metabolite profiling with selective mRNA and physiological profiling has been demonstrated in germinating Arabidopsis seeds (Fait et al., 2006). These studies have emphasized that metabolite profiling is one of the practical tools for the study of relationship(s) between signal transduction and metabolic pathways.
In this study, the aim was to find novel information regarding phyA signalling by primary metabolite profiling. The differences in metabolite profiles between WT Arabidopsis and its phyA-null mutant seedlings under distinct light conditions were investigated by GC/time-of-flight (TOF)-MS. In combination with quantitative real-time RT-PCR analysis of a selected pathway, possible phyA-regulated genes have been identified. This study emphasizes the utility of metabolite profiling for the study of plant biology.
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Materials and methods |
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Plant material and light treatments
Arabidopsis thaliana ecotype Columbia was used in this experiment. Seeds of the WT and phyA-211 mutant (phyA) were sterilized and sown on moistened GF/A glass microfibre filters (Whatman, Middlesex, UK) in sealed square Petri dishes before irradiation with FR (10 µmol m–2 s–1) for 25 min. FR was supplied by FR-emitting diodes (MIL-F18; Sanyo Electric, Osaka, Japan) with filters (Deraglass A-900; Asahikasei, Tokyo, Japan) to avoid contamination by red light (<700 nm). After stratification for 2 d at 4 °C in darkness, germination was induced by irradiation with white fluorescent light (W) for 2 h. After 4 d at 21 °C in darkness, the etiolated seedlings were irradiated with W (10 µmol m–2 s–1) or FR (10 µmol m–2 s–1), or retained in darkness as control samples. The samples were harvested at 0, 6, and 24 h after the onset of irradiation. The tissues (50±0.1 mg) were harvested under green light, frozen immediately in liquid nitrogen, and stored at –80 °C. Two independent experiments were performed. Each experiment consisted of two replications for metabolite profiling and one for quantitative real-time RT-PCR.
Extraction, derivatization, and analysis of Arabidopsis seedling metabolites using GC/TOF-MS
Extraction, separation, and derivatization were performed according to the procedures previously reported by Fukusaki et al., (2006) with some modifications. Seedlings were homogenized and disrupted with MM 301 mixer mills (Retsch GmbH, Haan, Germany) and then extracted with 1 ml of methanol/water/chloroform (2.5:1:1, v/v/v). A ribitol solution (40 µl, 0.2 mg ml–1) was added as an internal standard (IS). The extraction process was performed with MM 301 mixer mills at 20 Hz for 5 min. The solutions were then centrifuged at 16 000 g for 10 min. The hydrophilic phase (800 µl) was transferred to a new Eppendorf tube and mixed with 400 µl of Milli-Q water. The hydrophilic fraction was separated again by centrifugation and a portion (900 µl) was transferred to a new Eppendorf tube for evaporation under vacuum for drying. Derivatizations were carried out at 30 °C for 90 min with methoxyamine hydrochloride in pyridine (100 µl, 20 mg ml–1), and at 37 °C for 30 min with 100 µl of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (GL Science, Tokyo, Japan). The derivatized sample (1 µl) was injected via an Agilent 7683 autosampler into an Agilent 6890N gas chromatograph (Agilent, Palo Alto, CA, USA) equipped with a 30 mx0.25 mm i.d. fused-silica capillary column coated with 0.25 µm CP-SIL 8 CB low bleed (Variance, Palo Alto, CA, USA) and coupled to a Pegasus II mass spectrometer (LECO, St Joseph, MI, USA). The injector temperature was 230 °C and the helium gas flow rate through the column was 1 ml min–1. The column temperature was held at 80 °C for 2 min, then raised by 15 °C min–1 to 325 °C, and held there for 9 min. The transfer line and the ion source temperatures were set at 250 °C and 200 °C, respectively. The ions were generated by a 70 eV electron beam; two scans per second were recorded in the mass range of 50–600 m/z. The acceleration voltage was turned on after a solvent delay of 230 s.
Data analysis for metabolite profiles
Peak deconvolution, identification, and quantification were performed using the Pegasus software package ChromaTOFTM Ver 2.32 (LECO, St Joseph, MI, USA). Automatic peak identification was carried out by comparing mass spectra with those in the NIST98 Mass Spectral Database Ver 1.0.0.1
[EC]
2. In addition, commercially available standard compounds were derivatized and analysed in parallel with the samples. The obtained mass spectra and retention time were used to identify several metabolites. Unique fragment ions were assigned to an individual metabolite, and used for a peak area calculation. The specific ion used for quantifying each metabolite is given in Supplementary Table S1 available at JXB online. The peak areas were exported to Microsoft Excel 2003 for normalization to the IS, followed by weight correction and statistical analyses. The difference between two observations was determined to be statistically significant (P <0.05) by Student's t-test.
RNA isolation, cDNA synthesis, and gene expression analysis
For each sample, 30–50 mg of seedlings were collected and ground. Total RNA was extracted from the whole seedlings using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocol. Genomic DNA was removed using an RNase-Free DNase Set (Qiagen). Double-stranded cDNAs were synthesized from 1.5 µg of total RNA, in the presence of 5.5 mM MgCl2, 0.5 mM of each dNTP, 2.5 µM oligo(dT)16, 0.4 U µl–1 of RNase inhibitor, and 1.25 U µl–1 of MultiScribe reverse transcriptase from TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). The mixture was incubated at 25 °C for 10 min and at 48 °C for 60 min. Finally, the mixture was inactivated at 95 °C for 5 min.
Quantitative real-time RT-PCR was performed on a GeneAmp 5700 Sequence Detection System, using the SYBR Green PCR Master Mix Kit (Applied Biosystems). The sequences of the gene-specific primers used in this study are shown in Table 1: arginine decarboxylase (ADC1, ADC2), agmatin iminohydrolase (AIH), N-carbamoylputrescine amidohydrolase (NCPAH), spermidine synthase (SpdSyn1 and SpdSyn2), spermine synthase (SpmSyn and ACL5), and S-adenosylmethionine decarboxylase (SAMDC1, SAMDC2, SAMDC3, and SAMDC4). All primers were designed with the software PRIMER EXPRESS 2.0 (Applied Biosystems). For a 20 µl PCR solution, 2.5 µl of cDNA templates were mixed with 200 nmol l–1 each of forward and reverse primers and 2x SYBR Green PCR Master Mix. The reaction was first incubated at 50 °C for 2 min, then at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Quantitative real-time RT-PCR was performed in three replications for each sample. Gene expression levels were computed relative to the expression of the actin 2 gene (Actin2) using the 2–
CT method (Livak and Schmittge, 2001). The expression levels of light-irradiated samples were calculated relative to those of dark control samples at the same light irradiation period intervals. For each time point, genes of the WT that deviated in expression by 
2-fold from that of the WT in darkness and phyA were classified as phyA-regulated genes.
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Results |
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Response of amino acids to FR and W irradiation
WT Arabidopsis and phyA mutants were grown under darkness on moistened microfibre filters. Etiolated WT and phyA seedlings were subjected to W or FR, or kept in darkness (as control samples). Seedlings under each condition were harvested at 0, 6, and 24 h after the onset of irradiation. Under FR, no greening was observed in either WT or phyA seedlings. The expansion of cotyledons was observed in the WT after 24 h under FR, but not in phyA. Unlike the WT, phyA could not undergo photomorphogenesis under FR. The de-etiolation process occurred in both WT and phyA seedlings as early as after 6 h of W irradiation. The greening and expansion of cotyledons of seedlings were reported as well-known characteristics of photomorphogenesis in previous studies (reviewed by McNellis and Deng, 1995).
Dark-grown seedlings of both WT Arabidopsis and phyA mutants harvested at 0, 6, or 24 h showed no morphological differences. Although the levels of most metabolites were unaltered upon incubation in darkness, some differences in metabolite levels were observed (see Supplementary Table S2 at JXB online). Therefore, the normalization of FR- or W-treated samples with their dark control samples at the same time points was necessary to compensate for these differences.
The effects of FR and W irradiation on amino acid levels in etiolated seedlings were examined by GC/TOF-MS analysis. The relative levels of amino acids were profiled and are illustrated in Fig. 1 (raw data can be found in Supplementary Table S3 at JXB online). The amino acid levels in WT seedlings irradiated with FR (WT/FR versus WT/D) showed no significant changes during 6 h of irradiation. However, many amino acids decreased significantly as the irradiation time increased from 6 h to 24 h (Fig. 1A). These compounds were alanine, β-alanine, cysteine, glutamate, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. In contrast, amino acids in phyA under FR (phyA/FR versus phyA/D) showed constant levels over the FR irradiation period. The amino acid levels of both the W-irradiated WT (WT/W versus WT/D) and the phyA seedlings (phyA/W versus phyA/D) decreased slightly or remained the same over the light irradiation period (Fig. 1B). In WT seedlings, aspartate and glutamate transiently accumulated during 6 h after light irradiation and decreased thereafter. While alanine, isoleucine, leucine, phenylalanine, and valine decreased in the WT seedlings, glycine, leucine, and phenylalanine decreased in the phyA mutant from 6 h to 24 h under W irradiation.
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-aminobutyric acid; 4Hy-proline, 4-hydroxyproline.Table 2 summarizes the metabolites showing significant differences in their levels between the WT and phyA. To specify metabolic changes mediated by phyA, fold changes (phyA/WT) were calculated as the ratio of relative metabolite levels (metabolite level that was normalized with its controls as presented in Figs 1–3) between phyA mutant and WT seedlings. Comparison of amino acid contents between WT and phyA seedlings under W indicated that there was no significant difference between WT and phyA (phyA/W versus phyA/D and WT/W versus WT/D), except for methionine and phenylalanine. These two amino acids were found in greater quantities in phyA than in WT seedlings after 24 h of W irradiation. On the other hand, under FR, phyA and WT seedlings exhibited significant differences (phyA/FR versus phyA/D and WT/FR versus WT/D) in the levels of many amino acids. After 6 h FR irradiation, the WT contained a higher amount of aspartate, cysteine, and methionine than phyA. However, following 24 h FR irradiation, methionine, threonine, isoleucine, proline, valine, leucine, serine, cysteine, glycine, phenylalanine, and tyrosine were decreased in the WT.
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Response of organic acids, sugars, and other metabolites to FR and W irradiation
Along with amino acids, other primary metabolites such as organic acids, sugars, sugar alcohols, and nitrogen compounds were readily detected in a single analysis by GC/TOF-MS. The relative levels of organic acids and other metabolites were profiled and are illustrated in Fig. 2. The levels of organic acids and nitrogen compounds in both WT/FR versus WT/D and phyA/FR versus phyA/D seedlings showed no significant changes (P <0.05), except for phosphate and putrescine (Fig. 2A). Phosphate and putrescine levels in WT/FR seedlings declined from 6 h to 24 h of irradiation. A similar tendency was found in the seedlings under W (Fig. 2B). Glycolate, lactate, malate, n-butylamine, and succinate levels in W-treated WT seedlings decreased significantly from 6 h to 24 h of irradiation. Seedlings of phyA accumulated fumarate under W, whereas the threonate level declined.
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Major sugars and sugar alcohols were identified and profiled as illustrated in Fig. 3A and B for FR and W irradiation, respectively. These results clearly demonstrated that the sugar levels of WT seedlings were significantly decreased during 6–24 h FR irradiation, while those of phyA remained the same or decreased slightly. These significant metabolites included fructose, galactose, glucose, inositol, ribose, sucrose, and xylose.
With the statistical analysis, the differences in organic acid, sugar, sugar alcohol, and nitrogen compound levels in phyA and in the WT under distinct light irradiation regimes were tested, and significant metabolites are also summarized in Table 2. Although the same phenotypic characteristics were observed for WT and phyA under W, significant differences in metabolite levels were found as early as 6 h after the onset of irradiation. Putrescine was more abundant in phyA than in the WT. In contrast, the malate level in phyA was lower than in the WT. There was no significant difference in other sugar contents between WT and phyA during W irradiation.
Expression profiles of genes involved in polyamine biosynthesis
Several biochemical pathways have been previously reported as being controlled via the phyA signalling network (Ma et al., 2001; Ghassemian et al., 2005). Nonetheless, phyA-mediated regulation of polyamine biosynthesis has not been examined in detail. According to profiling data described above, the expression of genes coding for enzymes involved in polyamine biosynthesis was studied further as the level of putrescine was found to be significantly different between WT and phyA under both FR and W. The expression levels of 12 genes after FR and W irradiation were investigated by quantitative real-time RT-PCR, and the results are shown in Figs 4 and 5. Genes were classified as phyA regulated if two criteria were met. First, the expression level in WT/FR seedlings was >2- or <0.5-fold different from that of WT/D seedlings. Secondly, the expression level in WT/FR (relative to WT/D) was statistically >2- or <0.5-fold different from that of phyA/FR seedlings at the same time point. Three of the 12 genes ADC2, SAMDC2, and SAMDC4, met these criteria (Fig. 4). The SAMDC2 expression of WT seedlings markedly increased after 6 h of FR irradiation, while that of phyA showed no change. After 24 h, the SAMDC2 expression in the WT was reduced to a level lower than that in phyA. The fold differences between phyA and WT were 0.33 for SAMDC2 after 6 h of FR irradiation. In contrast, the expression level of SAMDC4 in the WT substantially decreased under FR from 6 h of FR irradiation. The fold differences for SAMDC4 between phyA and the WT were 2.24 after 6 h and 2.18 after 24 h of FR irradiation. It was found that the expression of SAMDC1 and SAMDC3 in the WT was slightly decreased from 6 h to 24 h FR irradiation. The ADC2 gene obviously showed a negative deviation of its expression level in WT/FR from the dark control seedlings, and from the phyA/FR. The repression of ADC2 gene expression was observed in the WT at 6 h after the onset of FR irradiation. Compared with phyA, ADC2 expression in the WT was decreased 3.38 and 9.08 times, respectively, following 6 h and 24 h FR irradiation.
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The transcription of the genes involved in polyamine biosynthesis pathways was increased for both WT/W and phyA/W, with the exception of ADC2 (Fig. 5). Comparison of gene expression revealed two genes, ADC2 and SAMDC2, showing significant differences in their expression levels (
2-fold) between WT and phyA. The differences were observed at 6 h after the onset of W irradiation, but not at 24 h. The expression of ADC2 in the WT was reduced immediately after W irradiation, similar to the observation under FR. In contrast, phyA under W showed distinct accumulation of ADC2 compared with the WT after 6 h W irradiation. The ADC2 levels in phyA gradually decreased to the same level as in the WT under W, while no reduction was observed under FR. The SAMDC2 expression in the WT markedly increased compared with phyA after 6 h W irradiation. However, a significant difference (2-fold) in SAMDC2 expression was not observed between the WT and phyA after 24 h W irradiation. |
Discussion |
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In addition to biochemical, molecular, and genetic approaches, metabolomics recently has been considered as an important approach for understanding metabolic responses in plants. Furthermore, metabolic profiles can be utilized as biochemical phenotypes associated with loss or gain of gene functions. In this study, metabolite profiling by GC/TOF-MS was selected as a tool for clarification of the biochemical phenotype associated with loss of phyA during seedling de-etiolation. GC/TOF-MS as an analytical tool has high sensitivity, reliability, good reproducibility, and wide coverage of metabolite species (Fiehn et al., 2000). Under continuous FR, phyA is only the active phytochrome inhibiting hypocotyl elongation and stimulating cotyledon expansion through the FR-HIR (Quail et al., 1995; von Arnim and Deng, 1996). The absorption of light by phyA is coupled with the modulation of gene expression leading to various morphological changes. Plants possess complicated networks for maintenance of metabolic balance, especially primary metabolism. In some cases, loss of gene function is accompanied by changes in mRNA levels, protein accumulation, enzyme activities, and finally metabolite levels. In this study, metabolome analysis showed that the accumulation pattern of amino acids, sugars, and polyamines changed according to the loss of phyA function in Arabidopsis seedlings during FR irradiation. As evident from Fig. 6, illustrating global changes in metabolite levels, FR light caused significant changes in metabolism during irradiation in both the WT and phyA.
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According to the profiles of primary metabolites, the reduction of some amino acid levels in the WT under FR was observed, while there was no significant change in phyA. Amino acid levels in cells should be determined by the balance between biosynthesis of amino acids and the amino acid-consuming translation activity. The present observations can be explained in one of two possible ways: (i) the down-regulation of amino acid biosynthesis; or (ii) the up-regulation of protein synthesis. The latter seems to be more plausible because many genes related to several biosynthetic pathways are known to be up-regulated under FR (Ma et al., 2001; Tepperman et al., 2001). The biosynthesis of S-adenosylmethionine (SAM) from methionine is catalysed by S-adenosylmethionine synthetase (SAM-S), which is reported as a gene induced late by FR (Tepperman et al., 2001). SAM is involved in the production of polyamines and numerous transmethylation reactions, including the biosynthesis of lignin (Shen et al., 2002). It is well known that phenylalanine ammonia-lyase (PAL) is induced by FR (Attridge et al., 1974; Tepperman et al., 2001). PAL is one of the key enzymes in the phenylpropanoid pathway (Bate et al., 1994). The significant reduction of phenylalanine only in the WT after 24 h FR irradiation would be caused by the activation of PAL, leading to the accumulation of anthocyanins in WT seedlings (Neff and Chory, 1998). Additionally, the phenylalanine level in phyA was higher than in the WT after 24 h W irradiation, suggesting a specific role for phyA in the activation of the phenylpropanoid pathway even under W. The amino acids involved in protein biosynthesis such as the branched-chain amino acids (valine, leucine, and isoleucine), serine, and glycine significantly decreased in WT seedlings upon FR irradiation. The reduction in phenylalanine, valine, leucine, and isoleucine observed in both WT and phyA under 24 h of W irradiation possibly implies the incorporation of these amino acids into proteins. Other amino acids remained unchanged upon W irradiation compared with dark controls. It may be due to the ongoing release from storage proteins or the amino acid biosynthesis balancing the incorporation into proteins. As a result, the net amino acid level remains constant.
Most of the sugars are consumed after 24 h, except for in phyA under FR. This corresponds well to the morphological difference as only phyA under FR exhibits skotomorphogenesis. Consumption of the sugars might be required for the light responses, such as the conversion of sugar into fatty acids during photomorphogenesis (Ghassemian et al., 2006). The metabolite profiles suggested that sugar metabolites were down-regulated by phyA in WT seedlings during the late response to FR. The down-regulation of these metabolites is an important clue to extend the knowledge of the phyA-regulated biochemical pathways. Ma et al. (2001) revealed that genes involved in photosynthesis, the TCA cycle, starch synthesis, photorespiration, amino acid biosynthesis, and chlorophyll biosynthesis were activated and up-regulated during light irradiation. The increase in gene expression should result in the higher accumulation of metabolites in WT seedlings. However, the present results conflict with their transcriptome results. A possible explanation for the low accumulation of metabolites is that the biosynthesis rate is slower than the consumption rate. This conflict clearly shows the importance of metabolomics to understand cell responses fully under a specific condition.
Despite the remarkable decrease in sugars and amino acids in WT/FR, the levels of organic acids in both WT/FR and phyA/FR showed some significant changes during the irradiation. This indicates the relatively steady states of organic acid pools in the metabolic pathways compared with those of amino acids and sugars. One of the exceptions is the decrease of glycolate in WT/W seedlings. It had been indicated that transcription of the glycolate oxidase (GLO) gene, one of the genes involved in photorespiration, was enhanced by light (Barak et al., 2001). Barak et al. (2001) had also reported that some signals from the developing plastids were important for the enhancement of the transcription of GLO. Their observation fits well with the present results as the glycolate level decreased only in WT/W that possesses an immature plastid, but not in WT/FR. Interestingly, the glycolate level in phyA did not change by W, indicating the involvement of phyA in the regulation of glycolate metabolism. Previously, it has been reported that the enhancement of fumarate hydratase activity is mediated by phytochrome (Bajracharya et al., 1976). Therefore, the accumulation of another organic acid, fumarate, in phyA after 24 h of W irradiation might be explained by the absence of fumarate hydratase activity.
PhyA is uniquely responsible for the FR-HIR. Upon FR irradiation, etiolated WT seedlings undergo photomorphogenesis, while phyA mutant seedlings still exhibit etiolation. During de-etiolation in the FR late response, accumulation of anthocyanins, expansion of the cotyledons, and inhibition of hypocotyls were observed in WT seedlings with the associated reduction of several amino acids and sugar compounds (Fig. 6). This implies that primary metabolites might be consumed to support the enhancement of biosynthetic processes needed for seedling de-etiolation. Therefore, it is possible to say that light regulation of these metabolite levels is mediated directly or indirectly by phyA.
Besides those compounds mentioned above, putrescine, one of the metabolites in metabolism of polyamines, is involved in the discrimination between WT and phyA in the FR late response. Since the putrescine level was reduced in WT/FR but not in phyA/FR, the FR signal mediated by phyA finally repressed the biosynthesis of putrescine. The Arabidopsis polyamine biosynthesis pathway involves the synthesis of putrescine, spermidine, and spermine. However, spermidine and spermine contents in the samples used in this study did not reach detectable amounts using the profiling method. It is possible that spermidine and spermine may occur in bound forms rather than in the free forms. The light regulation of putrescine via phyA signalling has not been examined in detail, compared with the metabolism of other biosynthesis pathways such as chlorophyll and carotenoid. Hence, further attention was focused on the regulation by phyA of genes involved in polyamine pathways.
In plants, putrescine is synthesized through two biosynthetic pathways; one is from arginine and the other is from ornithine. In Arabidopsis, putrescine is formed only from arginine by ADC, AIH, and NCPAH involving the intermediates, agmatine and N-carbamoylputrescine. The ornithine decarboxylase (ODC) gene is absent in Arabidopsis (Hanfrey et al., 2001). The activity of ADC has been shown to be rate limiting in the pathway (Hanfrey et al., 2001). There are two ADC paralogues, ADC1 and ADC2, in the Arabidopsis genome (Watson et al., 1997). Both of them are required for production of polyamines that are essential for normal seed development (Urano et al., 2005). During seedling development of Arabidopsis, ADC paralogues were found to show different promoter activities (Hummel et al., 2004). The high ADC2 promoter activity was detected in cotyledons, petioles, and limbs of first leaves, as well as in roots. No ADC1 promoter activity was observed in petioles and roots of seedlings under the same conditions. It was also reported that the promoter activity of ADC2, not ADC1, was regulated by light under temperate conditions (Hummel et al., 2004). Spermidine is further synthesized from putrescine by SpdSyn and the addition of an aminoproply group acquired from decarboxylated S-adenosylmethionine (dcSAM). The conversion of spermidine to spermine is catalysed by SpmSyn and ACL5 with the additional aminopropyl group.
Tepperman et al. (2001) reported that genes present in the Affymetrix Arabidopsis 8K microarray were induced or repressed in the WT in response to FR irradiation, but not in the phyA null mutant. The time course of the ADC1 gene expression profile under FR indicated the induction of this gene in the WT at the maximum level at 6 h, which is consistent with the present real-time RT-PCR results (Fig. 4). However, ADC2 was not previously reported as an induced or a repressed gene. In the present study, the ADC2 expression level of the WT was significantly reduced in comparison with phyA under FR. The reduction in ADC2 expression in the WT but not the phyA mutant implicates the involvement of phyA in putrescine biosynthesis. Generally, there is a positive relationship between an increase in ADC mRNA levels, activity, and changes in putrescine levels (Masgrau et al., 1997; Watson et al., 1998). It is important to note that there was the delayed response of putrescine level compared with the response of ADC2 expression under FR irradiation. However, the putrescine level and ADC2 expression were markedly reduced after 24 h irradiation. The intracellular level of free putrescine depends on the rate of its synthesis, degradation, transport, and conjugation. In addition, post-transcriptional and post-translational regulation may play a role in determining the level of putrescine (Martin-Tanguy, 2001; Hanfrey et al., 2002). Therefore, it is not surprising that ADC2 expression and putrescine levels did not correlate exactly. Compared with the ADC1 gene, ADC2 has been reported to be more responsive to environmental signals, such as hyperosmotic stress (Soyka and Heyer, 1999), exogenous hormones (Perez-Amador et al., 2002), salt stress (Urano et al., 2004), and water deficiency (Alcázar et al., 2006). ADC2 is also considered as the key gene for the production of putrescine. The overexpression of the ADC2 gene in Arabidopsis leads to a very high level of accumulation of putrescine (Alcázar et al., 2005). Since there was no change observed in the ADC1 expression level in FR-irradiated WT, the ADC1 gene may not be regulated by phyA. In contrast to ADC1, ADC2 was considered to be negatively regulated by phyA. The reduction of putrescine in WT seedlings irradiated with FR for 24 h was considered to be due mainly to the decrease of ADC2 expression. Further studies are required to solve the physiological meaning of the down-regulation of ADC2 by phyA.
It has been reported previously that four other SAMDC genes have been detected in the Arabidopsis genome sequence in addition to the SAMDC1 and SAMDC2 genes (Franceschetti et al., 2001). Because there were no expressed sequence data, it was assumed that the SAMDC3–SAMDC6 genes were not expressed or were expressed only at low levels. Recently, sequence analysis suggested that SAMDC5 and SAMDC6 are enzymatically inactive. For this reason, it was decided to observe the expression of SAMDC3 and SAMDC4 rather than of SAMDC5 and SAMDC6, in addition to SAMDC1 and SAMDC2. The SAMDC2 expression in WT, in contrast to ADC2 expression, markedly increased after 6 h, followed by a sudden decrease, while there was no change in phyA seedlings. In contrast, the expression level of SAMDC4 is similar to that of ADC2, substantially decreased by FR irradiation. No significant difference was observed in SAMDC1 and SAMDC3 expression between the WT and phyA under FR. It has been reported that both phytochrome- and blue light photoreceptor-mediated pathways were involved in the light regulation of SAMDC genes in Pharbitis nil (Yoshida et al., 1999). Nonetheless, a specific member of the SAMDC gene family, which is under phytochrome regulation, has not been characterized yet. The results of the present study suggest that phyA may play a role in the regulation of SAMDC2 and SAMDC4, but not of other SAMDC genes, in the later response of Arabidopsis to FR.
The expression of genes involved in the polyamine biosynthesis pathway in both WT and phyA had been induced under W, except ADC2 whose transcription decreased immediately. The increase in gene expression did not result in the accumulation of putrescine. The significant difference in gene expression levels between WT and phyA was observed for ADC2 and SAMDC2 genes at 6 h of W irradiation. No difference was found even when the seedlings were exposed to W for a longer period of time (24 h). The reduction in ADC2 expression and the increase in SAMDC2 expression resulted in the decrease of the putrescine level of WT/W for 6 h. As mentioned before, the significant decrease of the putrescine level was also observed in WT/FR after 24 h irradiation. ADC2, SAMDC2, and SAMDC4 expression also decreased in the WT under FR (Fig. 4). The observation that putrescine decreased even when SAMDC2 and SAMDC4 expression decreased under FR in the WT may be due to the different localization of the two enzymes. While ADC has been suggested to be localized in the chloroplast (Borrell et al., 1995), SAMDC is localized in the cytoplasm (Slocum et al., 1991). Also, it has been proposed that plant polyamine biosynthesis may be located in the chloroplast. Therefore, it is proposed here that the reduction of putrescine in FR-treated WT seedlings might be derived mainly from the decrease in ADC2 expression.
In conclusion, the metabolomics approach has revealed changes in several significant metabolites that are mediated by phyA during Arabidopsis seedling de-etiolation. These findings strongly confirmed that metabolite profiling is a practical tool to plot specific biochemical pathways and that it can complement genome-wide transcriptional investigation, such as microarray analysis. The differences in the phenotypes of WT and phyA seedlings under FR irradiation were accompanied by the decrease in the level of several amino acids, sugars, and putrescine in the WT. The examination of the expression of polyamine biosynthesis-related genes revealed that Arabidopsis ADC2 is important in putrescine production and is negatively regulated by phyA during photomorphogenesis. Furthermore, SAMDC2 and SAMDC4 expression was also considered to be regulated by phyA.
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Supplementary material |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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The following supplementary material is available at JXB online.
Table S1. Specific ion used for quantifying each metabolite.
Table S2. Relative metabolite contents of dark control samples.
Table S3. Relative metabolite content of FR- or W-irradiated seedlings normalized to dark control seedlings in the metabolite profiles presented in Figs 1, 2, and 3.
Table S4. Significant metabolites increasing or decreasing after FR or W irradiation.
Table S5. Fold changes in metabolite contents between the phyA mutant and WT at 6 h and 24 h after FR or W irradiation.
Table S6. Gene expression data as presented in Figs 4 and 5.
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Acknowledgements |
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We would like to thank Professor Takayuki Kohchi (Kyoto University) for useful discussions. We also thank Kazuteru Takagi and Ryo Yoshida for excellent technical assistance, and Benesh Joseph for suggestions in improving the manuscript. Useful comments from the anonymous reviewers who reviewed an earlier version of the manuscript are also acknowledged. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO to EF) and a Grant-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT to AO), Japan.
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