Proteome Approach to Characterize the Methylmalonate

In this study, semi-dwarf mutant Tan-ginbozu,26 a GA biosynthesis mutant, and its wild-type Ginbozu was used. After treatment with or without 5 μM GA...
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Proteome Approach to Characterize the Methylmalonate-Semialdehyde Dehydrogenase that Is Regulated by Gibberellin Naoki Tanaka,† Hideyuki Takahashi,‡ Hidemi Kitano,§ Makoto Matsuoka,§ Shoichiro Akao,| Hirofumi Uchimiya,‡ and Setsuko Komatsu*,† Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan, Bioscience Center, Nagoya University, Nagoya 464-0814, Japan, and Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan Received April 23, 2005

Proteins regulated by gibberellin (GA) in rice were determined by proteome analysis. Proteins extracted from suspension culture cells of slr1, a constitutive GA response mutant of rice, were separated by two-dimensional polyacrylamide gel electrophoresis, and three proteins were greatly accumulated in the mutant. The most up-regulated protein was methylmalonate-semialdehyde dehydrogenase (MMSDH), and the amount of protein was 7-fold that of wild type. In this study, the function of MMSDH in rice was analyzed. MMSDH gene expression in suspension culture cells, roots, and leaf sheaths of slr1 was higher than that in its wild-type. MMSDH expression in wild-type roots was increased by exogenous GA3. Analyzed by in situ hybridization, MMSDH mRNA was expressed in root primordia of slr1, where cells are undergoing growth. MMSDH gene expression in the root zone of tissue differentiation was higher than in the elongation zone or meristem. Transgenic rice expressing antisense MMSDH showed that its seminal roots were thinner than that of control, and that the leaf sheath elongation was slightly inhibited compared to control. Concentrations of TCA cycle metabolites were decreased in the antisense plants as compared with the control plants, suggesting that acetyl-CoA was reduced in the antisense plants. These results suggest that one of the regulations by GA signal transduction including SLR1 is the expression of MMSDH, and that MMSDH may play a role in root development and leaf sheath elongation in rice. Keywords: gibberellin • metabolome • methylmalonate-semialdehyde dehydrogenase • proteome • rice • slr1

The plant hormone gibberellin (GA) plays an essential role in many aspects of plant growth and development. GA is also required for seed germination and for complete anther, seed, flower, and fruit development.1 The biosynthesis and catabolism pathways of GA have been revealed to use GA-deficient mutants, which have a reduced ability to carry out step(s) in the GA biosynthetic pathway. Recently, the genes encoding most of the enzymes involved in GA biosynthesis and the initial steps in catabolism were isolated and characterized.2 Examination of the expression patterns of these genes is revealing the sites of GA metabolism during development and the complex regulatory mechanisms by which endogenous developmental cues the concentrations of bioactive GAs. GA signaling operates as a de-repressible system that is moderated by DELLA-domain proteins, which are transcrip-

tional regulators that repress GA responses. DELLA proteins are highly conserved among different species, including Arabidopsis, barley, Brassica, grape, maize, rice, and wheat.3 In Arabidopsis, rive DELLA protein genes (GA-Insensitive [GAI], Repressor of ga1-3 [RGA], RGA-like1 [RGL1], RGL2, and RGL3) have been identified.4 The DELLA proteins were localized in nuclei and were rapidly degraded in response to GA signaling.5 F-box proteins SLEEPY1 (SLY1) that mediate this degradation (as part of the E3 ubiquitin ligase SCF complex) have been identified in Arabidopsis.6 The GA signal negative regulator SPINDLY (SPY), an O-linked N-acetylglucosamine transferase, has been identified.3 In addition, the chromatin-remodeling factor PICKLE (PKL) is thought to function as a positive regulator of GA response.7 Short internodes (SHI) act as a negative regulator of GA responses through transcriptional control.8

* To whom correspondence should be addressed. Tel: +81-298-38-7446. Fax: +81-298-38-7464. E-mail: [email protected]. † National Institute of Agrobiological Sciences. ‡ University of Tokyo. § Nagoya University. | Miyazaki University.

Recently, the role of GAs in the germination of Arabidopsis seeds was examined by proteomic or DNA microarray approaches.9,10 Proteomic analysis showed that R-2,4 tubulin, the cytoskeleton component, appears to depend on the action of GAs during germination. GA also controlled the abundance of

Introduction

10.1021/pr050114f CCC: $30.25

 2005 American Chemical Society

Journal of Proteome Research 2005, 4, 1575-1582

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research articles a β-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.9 In the case of microarray analysis, a subset of GAresponsive genes involved in cell elongation and cell division, GA biosynthesis, transport, and signaling of other hormones were identified.10 The technique of proteome analysis, using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), mass spectrometry (MS), and Edman sequencing, has in fact been used for resolving hormone responses concerning rice. GA-responsive proteins in the leaf sheath of rice have been analyzed, and calreticulin showed different isoelectric points and expression levels in response to GA treatment.11 In rice suspension culture cells, methylmalonate semi-aldehyde dehydrogenase (MMSDH) protein was accumulated when treated with auxin or GA.12 The MMSDH accumulation was also observed in the suspension culture cells of the slr1 mutant.12 The slr1 mutant showed a constitutive GA-response and a slender phenotype with elongated stem, leaf sheath, and blade similar to that of rice plants treated exogenously with GA3.13 SLR1 was an ortholog of Rht in wheat, d8 in maize, and GAI, RGA, and RGL in Arabidopsis, and was a negative regulator for the GA signal transduction pathway.5,13 These results suggest that the MMSDH expression is involved in GA signal transduction. MMSDH is a mitochondrial enzyme involved in the distal part of the valine and pyrimidine catabolic pathways, where it catalyzes the oxidative decarboxylation of malonate and methylmalonate-semialdehydes to acetyl- and propionyl-CoA, respectively. MMSDH also have a function that mediated regulation of the long chain fatty acylation.14 These findings were identified in bovine, rat, or human MMSDHs,14,15 and, there are no reports concerned about plant’s MMSDH functions. In this study, to understand the function of MMSDH, which is a candidate protein regulated in the downstream of GA transduction in rice, the expressions of gene and protein in different tissues were analyzed.

Experimental section Plant Materials. The following two wild types and two mutants of rice plants (Oryza sativa L.) were used in this study: Nipponbare (japonica); slr1, a constitutive gibberellin response phenotype mutant derived from Nipponbare;13 Ginbozu (japonica); and Tan-ginbozu, a semi-dwarf mutant derived from Ginbozu. The rice plants were grown under fluorescent white light (600 µmol m-2 s-1, 12 h light period per day) at 25 οC and 70% relatively humidity in a growth chamber. Rice suspension culture cells were cultured in N6 liquid medium16 supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid, under conditions shaking in an incubator at 22 οC. The cultures were subcultured using the same medium every 2 weeks. Protein Extraction. The basal region of seedlings were homogenized with a lysis buffer17 containing 8 M urea, 2% nonidet P-40 (NP-40), 0.8% Ampholine (pH 3.5-10 and pH 5-8), 5% 2-mercapthoethanol and 5% poly(vinylpyrrolidone)40, using a grass mortar and pestle on ice. The homogenates were centrifuged twice at 15 000 rpm in a RA-50 JS rotor (Kubota, Tokyo, Japan) for 5 min each. The supernatants were subjected to 2D-PAGE. Two-Dimensional Polyacrylamide Gel Electrophoresis. Prepared samples were separated by 2D-PAGE in the first dimension by isoelectric focusing (IEF) or immobilized pH gradient (IPG) tube gels (Daiichi Kagaku, Tokyo, Japan) and in the second dimension by SDS-PAGE. An IEF tube gel measuring 1576

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11 cm length and 3 mm diameter was prepared. The IEF gel solution consisted of 8 M urea, 3.5% acrylamide, 2% NP-40, 2% Ampholine (pH 3.5-10 and pH 5-8). Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 600 V for 1 h. For IPG electrophoresis, samples were applied to the acidic side of gels. Electrophoresis using IPG tube gels (pH 6.0-10.0) of 11 cm length and 3 mm diameter was carried out at 400 V for 1 h, followed by 1000 V for 16 h and 2000 V for 1 h. After IEF or IPG, SDS-PAGE in the second dimension was performed using 15% polyacrylamide gels with 5% stacking gels. The gels were stained with Coomassie brilliant blue (CBB), and image analysis was performed. Gel Image Analysis. 2D-PAGE images were synthesized and position of individual proteins on gels was evaluated automatically with Image Master 2D Elite software (version 2.0; Amersham Bioscience, Piscataway, NJ). The pI and molecular mass of each protein was determined using 2D-PAGE marker (BioRad, Richmond, CA). The amount of a protein spot was estimated using the Image Master 2D Elite software. The amount of a protein spot was expressed as the volume of that spot which was defined as the sum of the intensities of all the pixels that make up the spot. To correct the variability due to CBB-staining and to reflect the quantitative variations in intensity of protein spots, the spot volumes were normalized as a percentage of the total volume in all of the spots present in the gel. Cleveland Peptide Mapping. Following separation by 2DPAGE, gel pieces containing protein spots were removed and the protein was electroeluted from the gel pieces using an electrophoretic concentrator (Nippon-Eido, Tokyo, Japan) at 2 W constant power for 2 h. After electroelution, the protein solution was dialyzed against deionized water for 2 days and lyophilized. The protein was dissolved in 20 µL of SDS sample buffer containing 0.5 M Tris-HCl (pH 6.8), 10% glycerol, 2.5% SDS and 5% 2-mercaptoethanol, and applied to a sample well in an SDS-PAGE gel. The sample solution was overlaid with 20 µL of a solution containing 10 µL of Staphylococcus aureus V8 protease (0.1 µg µL-1; Pierce, Rockford, IL) and 10 µL of the SDS sample buffer. Electrophoresis was performed until the sample and protease were stacked in the stacking gel, interrupted for 30 min to digest the protein.18 Electrophoresis was then continued, and the gels were stained with CBB. N-terminal and Internal-Amino Acid Sequence Analysis. To analyze an N-terminal and internal-amino acid sequences following separation by 2D-PAGE or Cleveland peptide mapping, the proteins were electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane (Pall, Port Washington, NY) and detected by CBB staining. The stained protein spots were excised from the PVDF membrane and directly subjected to Edman degradation on a gas-phase protein sequencer (Procise 494, Applied Biosystems, Foster City, CA). RNA Isolation and RNA Gel Blot Analysis. Total RNA was isolated from leaf blades, leaf sheaths, roots and cultured suspension cells by the method described by Chomczynski and Sacchi.19 Twenty micrograms was electrophoresed on a 1.2% agarose-formaldehyde gel and then transferred to a Hybond N+ membrane (Amersham Bioscience). Hybridization was performed at 42 °C in UltraHyb buffer (Ambion, Austin, TX). The MMSDH gene-specific probe was amplified from an EST clone of rice MMSDH and labeled with [R-32P]dCTP (Amersham Bioscience). Filters were washed twice with 2 × SSC and 0.1% SDS at 42 °C for 5 min and once with 0.1 × SSC and 0.1% SDS at 42 °C for 20 min. The hybridization signals were detected

Methylmalonate-Semialdehyde Dehydrogenase

and analyzed with Typhoon 8600 k variable imager (Amersham Bioscience). The relative amount of MMSDH mRNA was estimated using ImageQuant software (Amersham Bioscience). In Situ Hybridization. Plant materials were fixed in 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, overnight at 4 °C, dehydrated through a graded ethanol series followed by a tert-butyl alcohol series, and finally embedded in Paraplast. Microtome sections (10 µm thick) were mounted on grass slides. Digoxygenin (Roche Diagnostics, Indianapolis, IN)-labeled RNA probe was prepared from a MMSDH EST pBluescript SK+ plasmid. Hybridization and immunological detection of the hybridized RNA probe that was labeled with digoxygenin was performed according to the method of Kouchi and Hata.20 Construction of Antisense MMSDH Transgenic Rice. For construction of antisense transgenic plants, MMSDH cDNA sequence in the pBluescript SK+ (Stratagene, La Jolla, CA) vector was amplified by PCR using primer pairs of 5′-ACGTGAGCTCGCAGACCTTGGTCCAGTGAT-3′ (5′ side, SacI site italicized as a linker) and 5′-ACGTGCGGCCGCTTTCGCATTGGTGCATGTGT-3′ (3′ side, NotI site italicized as a linker). The resulting PCR product was ligated between the 35S promoter and nopaline synthase terminator in the binary vector pIG121Hm.21 The pIG121-Hm/Anti-MMSDH construct was confirmed by restriction mapping and sequencing. The pIG121-Hm/AntiMMSDH plasmid and the vector control, which was pIG121Hm only, were then transformed into Agrobacterium tumefaciens EHA10522 and transformed into rice as described.23 Transgenic plants were selected on medium containing hygromycin. The hygromycin-resistant plants were transplanted to soil and grown to maturity at 30 °C in 16 h light/8 h dark cycle in an isolation greenhouse. Metabolites Analysis. One-hundred and fifty mg of the frozen sample was powdered in liquid nitrogen and added 100 µL of methanol. Fifty µL of 400 µM PIPES and Milli-Q water were added into the sample solutions and centrifuged at 15 000 × g for 5 min. Then the supernatant was transferred into Millipore 5-kDa-cutoff filter (Millipore, Billerica, MA) for removal of protein, phospholipids, and chlorophyll. After centrifugation at 15 000 × g for 30 min, the resulting solution was used. Metabolites were determined by capillary electrophoresis mass spectrometry (CE/MS) according to Soga et al.24 The samples were separated on a poly(ethylene glycol) coating capillary (DB-WAX, 100 cm × 50 µm I. D., Agilent Technologies, Palo Alto, CA) and separation voltage was -20 kV. Electrolyte was 20 mM ammonium acetate (pH 6.8) and sheath liquid was 2 mM ammonium acetate in 50% methanol. Condition of mass spectrometry was selective ion monitoring and negative ion mode. Compound identification was performed by comparing m/z value and migration time of standard solution containing selected compounds.

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Results

Figure 1. 2D-PAGE pattern of proteins in suspension culture cells from wild-type rice and the GA response mutant, slr1. Proteins were extracted from wild type (A) and slr1 (B) suspension culture cells, separated by 2D-PAGE and detected by CBB staining. In the first dimension, IEF (low pI range) and IPG (high pI range) were used and overlapped. The pI and relative molecular weight of each protein were determined using 2D-PAGE Marker. Arrows indicated the proteins that were much accumulated in slr1, CS307, CS326, and CS697. (C) Protein spots of CS307, CS326 and CS697 in suspension culture cells were quantified densitometrically relative to the wild type.

Identification of Proteins Regulated Downstream of GA Signal Transduction. To identify proteins that were regulated by GA signal transduction, proteins from suspension culture cells of slr1 mutant was analyzed by 2D-PAGE. Because the GA signal was not regulated negatively in the mutant, GA regulated proteins could be identified. Compared with the pattern of 2D-PAGE of the wild type, three proteins in slr1 were up-regulated more than 3-fold higher than that in its wild type (designated as CS307, CS326, and CS697 in Figure 1A,B). The three proteins were identified as β-tubulin (CS307), MMSDH

(CS326), and osmotin (CS697) (Table 1). The relative amount of β-tubulin, MMSDH and osmotin in slr1 was approximately 3-, 7-, and 5-fold, respectively, compared with the wild type (Figure 1C). MMSDH was focused as the most accumulated protein in slr1. In our previous study, MMSDH protein accumulation was accelerated by GA3 treatment in the suspension culture cells and MMSDH accumulation in slr1 was higher than that in the wild type.12 Taken together, MMSDH accumulation was regulated under the GA signal transduction cascade. In this Journal of Proteome Research • Vol. 4, No. 5, 2005 1577

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Table 1. Identification of Proteins Accumulated in Suspension Culture Cells of slr1 spot no. kDa

CS307 CS326

CS697 a

pI

sequencea

54.8 4.8 N-MREILHIQGG 53.7 5.5 I-STAAASXLS I-HACGMGTLQM I-LVKRASSLVV I-MECYKEEIFG 33.0 7.8 N-DYAPMTLTIV

homologous protein

accession no.

β-tubulin S52008 Methylmalonate- AF045770 semialdehyde dehydrogenase Osmotin

BAB67891

N-, N-terminal amino acid sequence; I-, internal amino acid sequence.

Figure 2. MMSDH gene expression in different tissues of Nipponbare and slr1 and in response to gibberellin in Nipponbare. (A) Total RNAs were extracted from suspension culture cells (CS), and from roots (RO), leaf sheaths (LS) and leaf blades (LB) of 2-week-old seedlings of Nipponbare or slr1. The level of MMSDH mRNA in Nipponbare tissues was set as 100. (B) Total RNAs were extracted from Nipponbare suspension culture cells (CS), and roots (RO), leaf sheaths (LS) and leaf blades (LB) of 2-week-old seedlings were treated with (+) or without (-)10 µM GA3 for 24 h. The value of without GA3 treated tissues was set as 100. Total RNAs (10 µg per lane) were separated by agaroseformaldehyde gel, and blotted onto nylon membrane. The blot was hybridized with the MMSDH probe. The values are averages of three independent experiments.

study, intact plant was used to analyze MMSDH of rice in detail whether MMSDH was regulated by GA in different tissues. Expression of MMSDH in slr1 Mutant and Response to GA Application. To examine the expression pattern of MMSDH mRNA in various tissues of slr1 and its wild-type Nipponbare, Northern blot analysis was performed (Figure 2A). In slr1 and Nipponbare, the MMSDH gene expression in suspension culture cells and roots were stronger than that in leaf sheaths and leaf blades. The gene expression in suspension culture cells, roots, and leaf sheaths of slr1 were higher than those of Nipponbare (Figure 2A). The high level expressions of MMSDH gene in slr1 indicated that the gene expression was regulated under the downstream of SLR1. To determine the MMSDH gene expression is regulated under the downstream of GA signal, the gene expression upon exogenous GA application was analyzed. In suspension culture cells, roots, leaf sheaths, and leaf blades of Nipponbare, 1578

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Figure 3. MMSDH gene expression in roots. (A) In situ hybridization of the MMSDH mRNA in longitudinal-sections from leaf sheaths of slr1. Longitudinal-sections of leaf sheath from 2-weekold slr1 seedling were hybridized to DIG-labeled antisense RNA prepared from MMSDH EST (right). DIG-labeled sense RNA was used as a negative control (left). Arrow shows root primodia. Bars ) 400 µm. (B) Expression of MMSDH gene in different root zone. Diagram of crown and root of rice seedling (left). Basal region includes leaf sheath basal region that contained crown and shoot primodia. R1 includes lower root region that had no lateral roots and hairs. R2 includes middle root region that contained lateral roots and hairs. R3 includes upper root region that contained lateral roots and primary roots. Northern blot analysis (right). Total RNAs (10 µg per lane) were isolated from roots of 2-weekold Nipponbare seedlings and separated by agarose-formaldehyde gel, and blotted onto nylon membrane. The blot was hybridized with the MMSDH probe. The level of MMSDH mRNA in R1 was set as 100.

MMSDH gene expression in roots was dramatically increased and in leaf sheaths was slightly increased after GA3 treatment (Figure 2B). On the other hand, the gene expressions in suspension culture cells and leaf blades did not change after GA3 treatment (Figure 2B). MMSDH Gene and Protein Expression in Roots. As described above, MMSDH gene expression in roots and leaf sheaths of slr1 was slightly higher than that of Nipponbare (Figure 2B). For more precise analysis of the site of the MMSDH expression in crown of young seedlings where included the primordia of leaf sheath and root, in situ hybridization on the tissue was performed using the tissue. Higher-level MMSDH mRNA was expressed mainly in the root primordia (arrow in Figure 3A right), where the cells are undergoing growth. This result was revealed that MMSDH expression was involved in root growth. Since the MMSDH gene expression in roots was higher than that in the other tissues, the expression in roots was analyzed in detail. The roots from 2-week-old seedlings were divided into three parts (Figure 3B left): lower root region that had no lateral

Methylmalonate-Semialdehyde Dehydrogenase

roots and hairs, middle root region that contained lateral roots and hairs, and upper root region which contained lateral roots and primary roots. MMSDH gene expression in the middle and upper regions were higher than that of the lower region and leaf sheath basal region that contained crown and shoot primodia (Figure 3B right). The middle and upper root regions are differentiation zone where hairs actively elongate.25 To characterize MMSDH in more detail, a time-course of changes in level of MMSDH was examined, comparing with untreated rice plant daily for 6 days (data not shown). Upon addition of GA3, the level of MMSDH was elevated, and remained high with the passage of time. As found in these results, a constant level of MMSDH was maintained. MMSDH Antisense Transgenic Plant. To assess the effects of the loss of function of the MMSDH on rice growth and development, the cDNA of MMSDH was introduced into the rice cell in antisense orientation under the control of the CaMV 35S promoter in a pIG121-Hm binary vector by means of Agrobacterium-mediated transformation (Figure 4A). In total, five antisense transgenic plants were isolated. Of these transgenic rice plants, root tissue of an antisense transgenic rice plant had different morphologies compared with that of the control plant. In the antisense transgenic plant, the seminal roots were thinner than that of the vector control, and the number of root hairs was increased significantly compared with the control (Figure 4B). The expression level of MMSDH in the transgenic roots was dramatically decreased (Figure 4C). The aboveground phenotype of the antisense transgenic plants exhibited various degrees of repressed growth and were about 80% shorter than the vector control or did not change the height when thy reached maturity (Figure 5A). RNAs were isolated from three antisense and one control lines and assessed for the expression levels of MMSDH sense transcripts (Figure 5B). Two lines had slightly reduced levels of sense MMSDH mRNA when compared to vector control, their growth were also slightly repressed whose root phenotype was altered described above, while MMSDH mRNA of line 39 was not efficiently inhibited, its growth was no different from that of control plant (Figure 5B). MMSDH Expression in Leaf Sheath Elongation. MMSDH protein was regulated downstream of GA signal transduction from analysis of slr1 suspension culture cells. Whether the MMSDH gene and protein expression was increased by GA3 treatment or not, the gene and protein expression pattern in elongating leaf sheaths of a rice GA biosynthesis mutant was analyzed. In this study, semi-dwarf mutant Tan-ginbozu,26 a GA biosynthesis mutant, and its wild-type Ginbozu was used. After treatment with or without 5 µM GA3 for 24 or 48 h, RNA or protein from leaf sheaths of Tan-ginbozu and Ginbozu were extracted. MMSDH gene and protein expression in Ginbozu without GA3 treatment was higher than that in Tan-ginbozu (Figure 6A). While the gene and protein expression was increased by GA3 treatment in Tan-ginbozu (Figure 6A). In addition, MMSDH expression in a GA signal transduction mutant was performed. In slr1, GA signal was not regulated negatively.12 Analyzing a rice GA-insensitive dwarf mutant, gid2, it is revealed that the SLR1 was high level accumulation and the SLR1 degradation was caused by SCF complexes.27 The expression level of MMSDH mRNA and protein in the constitutive GA response phenotype mutant was higher than in Nipponbare (Figure 6B). Metabolome Analysis of Antisense Transgenic Plant. According to the function of MMSDH, acetyl-CoA and propionyl-

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Figure 4. Construction of transgenic rice plants constitutively overexpressing the antisense MMSDH and phenotype of roots. (A) Binary vector pIG121-Hm harboring MMSDH in the antisense direction under the control of the CaMV 35S promoter. (B) Phenotype of roots of transgenic rice plant and the vector control plant. Transgenic rice plants were grown for 3 months in the soil in the isolation greenhouse. (C) Expression levels of MMSDH mRNA in roots of transgenic plants. Total RNAs were extracted from roots of the antisense transgenic rice (line 2) and the vector control, grown for 3 months. Total RNAs (10 µg per lane) were separated by agarose-formaldehyde gel and blotted onto nylon membrane, and then hybridized with the MMSDH probe. (D) Fresh weight of seminal roots and number of root hair per seedling. The fresh weight of seminal roots was measured and number of root hair per seedling of vector control, transgenic line 2, 14, and 56 was counted. Data equal average of 5 seedlings ( SD.

CoA were produced from malonate- and methylmalonatesemialdehydes, respectively. Since the antisense MMSDH transgenic rice plant was suppressed the MMSDH gene expression, amount of acetyl-CoA in the transgenic rice cell might be decreased and metabolisms included acetyl-CoA might be changed. Acetyl-CoA is important substrate in plants; such as tricarboxylic acid (TCA) cycle, fatty acids synthesis or isoprenoids synthesis and so forth. In this study, metabolism was focused on TCA cycle, and the amounts of metabolites in the cycle (pyruvate, citrate, cis-aconitate, 2-oxoglutarate, suucinate, fumalate, and malate) were analyzed by metabolomics approach (Table 2). These metabolites were analyzed capillary electrophoresis mass spectrometry (CE/MS) from shoots of one-week-old seedlings from the antisense transgenic rice and Journal of Proteome Research • Vol. 4, No. 5, 2005 1579

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Figure 6. Expression of MMSDH gene and protein in response in genetic mutants exhibiting abnormal heights. (A) Two-weekold Tan-ginbozu and Ginbozu seedlings were treated with (+) or without (-) 5 µM GA3. (B) Two-week-old Slr1 and Nipponbare seedlings were treated with (+) or without (-) 5 µM GA3. Total RNAs (10 µg per lane) extracted from leaf sheath after GA3 treatment for 24 h were blotted onto nylon membrane, and then hybridized with the MMSDH probe. rRNA stained with ethidium bromide was used as loading control. The level of MMSDH mRNA in Ginbozu without GA3 treatment (A) and Nipponbare without GA3 treatment (B) were arbitrarily set as 100. Proteins were extracted from leaf sheaths after GA3 treatment for 48 h, separated by 2D-PAGE and detected by CBB staining. 2D-PAGE was performed as described in Figure 1. Arrow indicates MMSDH. Figure 5. Phenotype of the aboveground of the transgenic rice plants constitutively overexpressing the antisense MMSDH. (A) Phenotype of aboveground of the antisense transgenic rice plants and the vector control plant. The transgenic rice plants were same ones as shown in Figure 4. (B) Expression levels of MMSDH mRNA and protein in the leaf sheath of transgenic lines. Total RNAs and proteins were extracted from leaf sheath of the antisense transgenic rice and the vector control, grown for 3 months. Total RNAs (10 µg per lane) were hybridized with the MMSDH probe. The level of MMSDH mRNA in control was set as 100. Crude proteins were extracted and separated by 2DPAGE. Growth curves of the MMSDH antisense and a vector control transgenic lines show.

the vector control. The decrease of MMSDH expression resulted in alteration in the metabolite composition of TCA cycle. In shoots from the transgenic plant and control, the concentration of pyruvate, precursor metabolite for TCA cycle, were almost same, however, the concentration of metabolites involved in TCA cycle were slightly different between them (Table 2).

Discussion In the present study, to identify the GA-regulated proteins, proteins from the GA response mutant slr1 were analyzed using differential display of protein with 2D-PAGE. Then, MMSDH was identified as the most accumulated protein in slr1 (Figure 1C). In our previous study, it was reported that the MMSDH was accumulated by GA3 or auxin treatments for rice suspension cultured cells and that MMSDH were increased in the suspension cultured cells of slr1.12 Thus, in this study, the expression of rice MMSDH and function were further analyzed. MMSDH has been reported to be localized in the matrix space of bovine, rat, and human mitochondria where it 1580

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Table 2. Metabolite Concentrations of TCA Cycle in Shoot from the Antisense Transgenic Rice and the Vector Control Ricea concentration (nmol g-1 FWb)

b

metabolite

control

antisense

Pyruvate Acetyl-CoA Citrate cis-Aconitate Isocitrate 2-oxoglutarate Succinyl-CoA Succinate Fumalate Malate Oxaloacetate

206.4 ( 16.5 NDc 6064.7 ( 684.1 139.1 ( 9.2 ND 478.7 ( 23.6 ND 902.1 ( 49.6 172.0 ( 14.1 9796.7 ( 446.7 ND

201.2 ( 23.3 ND 5447.2 ( 153.7 83.6 ( 3.4 ND 288.9 ( 12.7 ND 887.2 ( 13.4 121.8 ( 8.0 10600.6 ( 384.6 ND

a Values presented are the mean ( SE of three independent determinants. FW, fresh weigh. c ND, not determined.

catalyzes the oxidative decarboxylation of malonate- and methylmalonate-semialdehydes to acetyl- and propionyl-CoA, respectively.14,15 Acetyl-CoA is a central metabolic intermediate in the TCA cycle and a substrate of fatty acyl-CoA in fatty acid synthesis. Propionyl-CoA is also fed into the TCA cycle after conversion to succinyl-CoA. In contrast to mammalian systems, the function of MMSDH in plants is still unclear. Mitochondrial proteome analyses have revealed that MMSDH is localized in the mitochondria of Arabidopsis28,29 and rice.30 Plant and vertebrate MMSDH show significant homologies on the amino acid level (data not shown), suggesting that they might have similar functions. MMSDH were accumulated in slr1 described above, suggesting that MMSDH was involved in slr1 growth. In fact, the

Methylmalonate-Semialdehyde Dehydrogenase

slr1 mutant show increased shoot elongation caused by enhanced cell expansion; on the other hand, root number and length are reduced,5 indicating that GA evokes different effects in different organs. In this report presented that MMSDH expression level in roots was higher than that in leaf sheaths or in leaf blades in slr1; gene expression levels in slr1 roots were slightly higher than that in wild-type Nipponbare (Figure 2A). Furthermore, the MMSDH expression in roots of Nipponbare was enhanced by GA3 treatment (Figure 2B). Thus, the MMSDH gene is expressed mainly in roots and is controlled by the GA signaling pathway, including SLR1. In leaf sheaths, the MMSDH level also was higher in slr1 than in Nipponbare (Figure 2A). Taken together, these results suggest that GA stimulates cell elongation in the leaf sheath, while it suppresses cell elongation in roots. In situ hybridization of MMSDH mRNA in slr1 clearly showed expression in root primordia (Figure 3A right), suggesting an involvement of MMSDH in root growth. In addition, the MMSDH expression in the differentiated root zones that carried crown roots, lateral roots, or root hairs was higher than in the elongation and meristematic zones (Figure 3B),25 suggesting that MMSDH is involved in root tissue differentiation or thickening growth rather than root cell division or elongation. The effect on the appearance of the root primodia cells may be stimulation de novo initiation of adventitious root formation.31 In the slr1 mutant plant, in which MMSDH was increased, the seminal roots were not thinner than that of its wild type, and the number of root hairs was decreased compared with its wild type. Antisense MMSDH overexpressing transgenic rice roots were thinner than the vector control roots (Figure 4B), supporting that MMSDH plays an important role in root development. Concerning root development, many phytohormone synthesis or signaling mutants exhibit abnormal root growth or development.32 The Arabidopsis rooty (rty) mutant showed high endogenous auxin concentrations and an extreme proliferation of roots.33 The maize slr1 and slr2 (short lateral roots1 and 2) mutants displayed short lateral roots as a result of impaired root cell elongation, and no major influence on the aboveground performance of the affected plants.34 Recently, a regulation of the GA signaling pathway by auxin has been reported in pea or Arabidopsis. In pea shoots, auxin (indole3-acetic acid) promotes the biosynthesis of the active GA by controlling the GA synthesis genes PsGA3ox1, PsGA2ox1, and PsGA2ox2.35 In Arabidopsis, auxin is necessary for GA-mediated control of root growth, and auxin transport or signaling delays the GA-induced disappearance of RGA, a repressor protein of GA signaling, from roots cell nuclei.36 Our previous study revealed that the MMSDH expression was increased when auxin stimulated root development.12 On the other hand, it has been reported that increases in fructose-bisphosphate aldolase activity stimulate the glycolytic pathway and may play an important role in the GA-induced growth of roots.37 And, the activation of glycolytic pathway function accelerates root growth and GA3-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associated with vacuolar H+-ATPase in roots and may regulate the vacuolar H+ATPase mediated control of cell elongation that determines root length.38 These findings suggest that MMSDH expression changed by GA and auxin may be important as one of the regulation for root cell growth and development in rice seedlings. In addition to the effects on root growth, antisense MMSDH transgenic rice plants showed slightly reduced height

research articles (Figure 5), suggesting that MMSDH also functions in stem elongation. Because the gene expression level in roots and leaf sheaths were quite different (Figure 2A), the phenotypic effects caused by gene suppression were differed. Furthermore, MMSDH protein accumulation in the rice semi-dwarf mutant Tanginbozu was low level and increased during shoot elongation stimulated by GA3 treatment (Figure 6). The slr1 mutant showed a constitutive GA-response and a slender phenotype with elongated stem, leaf sheath, and blade similar to that of rice plants treated exogenously with GA3. These results suggest that MMSDH is regulated under GA signal transduction and that MMSDH is involved in rice leaf sheathes elongation. The regulation of shoot elongation by GA has been discussed mostly with a focused on cell wall extensibility. In pea, seedling root elongation was regulated by GA3 which induced changes in the galactose content of the cell wall and enhanced cell wall extensibility.39,40 Similarly, stem elongation was also enhanced by GA through increased cell wall extensibility.41 In rice internodes, two (OsXET1 and OsXET3) of the four genes encoding proteins related to the cell wall-loosening enzyme XET (OsXET1, OsXET2, OsXET3, and OsXET4) were up-regulated by GA.42 Because the loosening of the cell wall is an important process in cell elongation, GA-stimulated plant growth might depend on a wall-loosing mechanism. Concentrations of TCA cycle metabolites in the leaf blade and leaf sheath from antisense MMSDH rice and control plants were analyzed. Because MMSDH was controlled by GA signal and MMSDH was involved in acetyl-CoA generation, GA was involved in energy production by MMSDH induction and acetyl-CoA generation. In the transgenic rice, the concentrations were relatively low compared to the vector control, while the levels of the precursor metabolite of acetyl-CoA, pyruvate, were similar (Table 2). However, the concentration of malate was higher in transgenic leaf sheaths than in control plants. Malate is located in mitochondria as an intermediate of the TCA cycle, and also in the cytosol, chloroplasts, peroxysomes, and glyoxysomes.43 Malate transport systems in mitochondria have been analyzed. The malate-2-oxoglutarate translocator and the malate-oxaloacetate translocator exchange 2-oxoglutarate and oxaloacetate, respectively, for malate.43 In the leaf blade of transgenic rice, 2-oxoglutarate levels were reduced (Table 2). These altered metabolite concentrations might reflect a disturbation of the malate level in cell by the suppression of MMSDH in the antisense plants. Our results suggest that the concentration of acetyl-CoA, the precursor of TCA cycle, is reduced in transgenic plants and that therefore the concentration of metabolites of the TCA cycle is altered. Recently, metabolic profiling has been shown to be a versatile tool in plant functional genomics.44-46 In the present study, metabolic analysis has focused on the TCA cycle, and its metabolites in antisense MMSDH rice was found to be altered as compared to the control. The metabolic differences between the antisense and control plants are quite complex, and additional metabolic pathways that involve acetyl-CoA such as fatty acid synthesis need to be analyzed. To clarify the metabolic pathway between the reduction of MMSDH and the change of intermediates in TCA cycle, more experiments in details will be needed. Nonstandard Abbreviations: MMSDH, methylmalonatesemialdehyde dehydrogenase; GA, gibberellin; 2D-PAGE, twodimensional polyacrylamide gel electrophoresis; IEF, isoelectric focusing; IPG, immobilized pH gradient; CBB, Coomassie brilliant Blue; PVDF, poly(vinylidene difluoride); TCA, tricarboxylic acid. Journal of Proteome Research • Vol. 4, No. 5, 2005 1581

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