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First published online September 26, 2008; 10.1104/pp.108.128769 Plant Physiology 148:1655-1667 (2008) © 2008 American Society of Plant Biologists Characterization of Arabidopsis Lines Deficient in GAPC-1, a Cytosolic NAD-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase1,[C]Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, CONICET/UNSAM, 7130, Chascomús, Argentina (S.P.R., D.F.G.-C.); Centro de Estudios Fotosintéticos y Bioquímicos, Universidad Nacional de Rosario, 2000, Rosario, Argentina (P.C.); and Laboratorio de Enzimología Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000, Santa Fe, Argentina (A.A.I.)
Phosphorylating glyceraldehyde-3-P dehydrogenase (GAPC-1) is a highly conserved cytosolic enzyme that catalyzes the conversion of glyceraldehyde-3-P to 1,3-bis-phosphoglycerate; besides its participation in glycolysis, it is thought to be involved in additional cellular functions. To reach an integrative view on the many roles played by this enzyme, we characterized a homozygous gapc-1 null mutant and an as-GAPC1 line of Arabidopsis (Arabidopsis thaliana). Both mutant plant lines show a delay in growth, morphological alterations in siliques, and low seed number. Embryo development was altered, showing abortions and empty embryonic sacs in basal and apical siliques, respectively. The gapc-1 line shows a decrease in ATP levels and reduced respiratory rate. Furthermore, both lines exhibit a decrease in the expression and activity of aconitase and succinate dehydrogenase and reduced levels of pyruvate and several Krebs cycle intermediates, as well as increased reactive oxygen species levels. Transcriptome analysis of the gapc-1 mutants unveils a differential accumulation of transcripts encoding for enzymes involved in carbon partitioning. According to these studies, some enzymes involved in carbon flux decreased (phosphoenolpyruvate carboxylase, NAD-malic enzyme, glucose-6-P dehydrogenase) or increased (NAD-malate dehydrogenase) their activities compared to the wild-type line. Taken together, our data indicate that a deficiency in the cytosolic GAPC activity results in modifications of carbon flux and mitochondrial dysfunction, leading to an alteration of plant and embryo development with decreased number of seeds, indicating that GAPC-1 is essential for normal fertility in Arabidopsis plants.
Glyceraldehyde-3-P dehydrogenases (GAPDHs) are enzymes conserved in all living organisms, where they play a central role in the carbon economy of the cells. Higher plants possess four distinct isoforms of GAPDHs: (1) GAPC, a cytosolic, phosphorylating, NAD-specific GAPDH catalyzing the conversion of glyceraldehyde-3-P (Ga3P) to 1,3-bisphosphoglycerate; (2) NP-GAPDH, a cytosolic non-phosphorylating NADP-dependent GAPDH that catalyzes the oxidation of Ga3P to 3-phosphoglycerate (3PGA; Valverde et al., 2005  ); (3) GAPA/B, a phosphorylating, NADP-specific GAPDH involved in photosynthetic CO2 fixation in chloroplasts (Cerff and Chambers, 1979); and (4) GAPCp, involved in glycolytic energy production in non-green plastids (Petersen et al., 2003).
Much is known concerning the gene structure, evolution, and functional properties of GAPDHs in algal systems (Koksharova et al., 1998
In recent years, detailed studies have been carried out on the structure-function relationships (Mateos and Serrano, 1992
The importance of GAPC in photosynthetic tissues has been widely reported; however, only a few reports have described its function in heterotrophic tissues (Fernie et al., 2004 In this work, we isolated and characterized two lines exhibiting a GAPC-1 deficiency: a null mutant line of Arabidopsis deficient in GAPC-1 expression (gapc-1, SALK_010839) and a transgenic line expressing the antisense version of the GAPC-1 gene (At3g04120, as-GAPC1). Both lines exhibited defects in fertility, with alterations in seed and fruit development, suggesting that GAPC-1 is essential in these organs. The molecular, biochemical, and physiological studies of these lines indicate that this enzyme plays critical and pleiotropic roles, being essential for the maintenance of cellular ATP levels and carbohydrate metabolism and required for full fertility in Arabidopsis.
Identification of a gapc-1 Mutant and Construction of the Antisense as-GAPC1 Line To evaluate the possible function(s) of the GAPC-1 gene in Arabidopsis, we selected a T-DNA insertion mutant from the Arabidopsis Biological Resource Center (The Ohio State University, Columbus, OH) seed stock (SALK_010839, gapc-1). GAPC-1 (At3g04120; 1,017 bp) is composed of nine exons and eight introns, and in gapc-1 plants the T-DNA is inserted in the ninth exon (Fig. 1A). gapc-1 homozygous plants were isolated using PCR screening, and segregation analysis and DNA gel-blot hybridization indicate that the mutant plants contained only one copy of T-DNA (data not shown).
To obtain a second GAPC-1-deficient line, we constructed a transgenic antisense line (as-GAPC1) by transformation with pCAMBIA 2200 plasmid (Hajdukiewiez et al., 1994 ; Fig. 1B). The expression of GAPC-1 in null mutants and as-GAPC1 plants verified by reverse transcription (RT)-PCR analysis shows that GAPC-1 mRNA is absent in gapc-1 plants, confirming that the T-DNA insertion impairs GAPC expression (Fig. 1C). In the as-GAPC1 line, there is a 30% decrease in GAPC-1 transcripts compared to wild-type plants (Fig. 1C). The GAPC protein levels determined by western blot using specific antibodies show a decrease of 23% ± 5% and 27% ± 8% in gapc-1 and as-GAPC1 plants, respectively (Fig. 2A).
Moreover, we determined that total GAPC activity is 50% decreased in leaves and flowers of both lines (Fig. 2B). Although we observed a complete absence of the GAPC-1 transcript in the gapc-1 line, the amount of protein was reduced in a way that the decrease in GAPC total activity was only about 50% of that determined for wild-type plants. This very likely results from the activity of: (1) other GAPC isoforms encoded by GAPC-2 (At1g13440), GAPCp-1 (At1g16300), or GAPCp-2 (At1g79530) in the Arabidopsis genome; or (2) the GAPA/B isoform, which preferentially uses NADPH but also is active in the presence of NADH (Sparla et al., 2004).
Phenotypic Characterization of gapc-1 and as-GAPC1 Plants Under normal growth conditions, gapc-1 and as-GAPC1 plants exhibited retarded growth at different developmental stages compared to wild-type plants (Fig. 3, A and B). While we did not observe differences in the morphology of roots and leaves, flowers and fruits were severely affected in their morphology, with fruits displaying alterations in their size and weight (Fig. 3C). Wild-type plants have normal amounts of seeds per silique (45 ± 5) and normal morphology (Fig. 3D), whereas gapc-1 and as-GAPC1 plants showed aberrant seed development and seed number deficiency (Fig. 3, E and F). The reduced number of seeds (about 12% and 6% for gapc-1 and as-GAPC1 plants, respectively) was frequently accompanied by empty embryo sacs (Table I). These results suggest that GAPC activity is essential for zygotic and/or early embryo development. Because GAPC-1-deficient plants have abnormal fruit development, we investigated whether or not pollen viability and/or pollen tube elongation was affected in post-anthesis flowers of these plants. Pollen grains from gapc-1 and as-GAPC1 lines showed apparently normal morphology. However, they were not fertile and showed defects in pollen tube germination in vitro. We observed 23% and 21% viable pollen grains in gapc-1 and as-GAPC1 lines, respectively, compared to more than 95% of germinated pollen in wild-type plants (Fig. 4).
When the gapc-1 plants were fertilized with wild-type pollen, the resulting F1 plants exhibited a silique morphology and seed production similar to the wild-type phenotype, with 43 ± 6 seeds per silique and the absence of empty sacs or aborted seeds observed in the mutant line (Fig. 5, A and B). This result suggests that the reduced fertility of the mutant was due to defects in male organs and the female fertility was unaffected.
Analysis of gapc-1 Plants To have a better assessment of the way by which GAPC activity changes determine the observed phenotype, we next compared the transcriptome of gapc-1 and wild-type plants. Results were analyzed to state a primary relationship between transcript levels and changes in enzyme activities and/or protein function. In addition, we also sought further evidence of changes determining levels of protein and/or activity for many of the implied enzymes. Microarray analysis revealed alterations in the expression of genes encoding for glycolytic and Krebs cycle enzymes in gapc-1 plants. As shown in Table II, we observed a down-regulation of several genes involved in these pathways: GAPC-2, NP-GAPDH, two phosphofructokinase (PFK) genes, two pyruvate kinase (PK) genes, and a triose-P isomerase (TPI) gene. Conversely, we observed an up-regulation of two malate dehydrogenase (NAD-MDH) genes. As shown in Table II, there is also an alteration in the transcript levels of several genes related to photosynthesis, such as Rubisco SSU (At1g67090), Rubisco LSU (At1g14030 and At4g20130), and RCA (for Rubisco activase; At2g39730). Furthermore, several genes involved in the TCA cycle showed decreased expression: citrate synthase (At3g60100), succinate dehydrogenase (SDH; At3g27380), and aconitase (ACO; At2g05710 and At4g26970) genes. Also, several genes encoding for enzymes related to stress responses showed altered expression, such as different peroxidase (PER) isoforms, a peroxiredoxin (PEROX; At1g65970) isoform, an alternative oxidase (AOX; At5g64210) isoform, and three isoforms of catalase (CATs). Thus, microarray results suggest that mutants have an altered metabolism, which can induce a stress situation that could account for the delayed growth observed in the GAPC-deficient plants.
As described above, GAPC and NP-GAPDH participate in a bypass step of the glycolysis in plants, catalyzing the production of 3PGA, NADH, and ATP or 3PGA and NADPH, respectively. To further characterize the regulation of this bypass, we analyzed the expression of NP-GAPDH in gapc-1 and as-GAPC1 plants. A decrease of 25% and 92% in the expression of NP-GAPDH in gapc-1 and as-GAPC1, respectively, was observed (Fig. 6), in agreement with the decrease determined by microarray analysis (Table II) and also with the decrease in NP-GAPDH-specific activity (Fig. 7A). This is in contrast to the results obtained with np-gapdh mutant plants, where the expression of GAPC-1 is induced in the absence of NP-GAPDH. In addition, we did not observe an up-regulation of genes encoding for Glc-6-P dehydrogenase (G6PDH) isoforms either by microarrays or RT-PCR experiments as was observed in the np-gapdh plants (Rius et al., 2006 ). These data suggest that differences in the regulation or in the compensatory responses of GAPC-1 and NP-GAPDH genes exist in np-gapdh and gapc-1 mutant lines. In addition to GAPC-1, other cytosolic GAPDH isoforms could contribute to the maintenance of the carbon flux through the glycolytic pathway, such as GAPC-2. Therefore, the expression of GAPC-2 was analyzed. As shown in Figure 6, we found 48% and 37% decreases of GAPC-2 mRNA levels in gapc-1 and as-GAPC1 lines, respectively.
As mentioned above, microarray data from gapc-1 plants showed an alteration in the level of transcripts encoding for glycolytic and energy metabolism enzymes (Table II). To assess the impact of alterations in gene expression observed in microarrays, we evaluated the activity of several enzymes involved in both pathways (Fig. 7A). A 25% decrease in PK activity was detected in gapc-1 plants. This correlates with the 74% and 57% decreases in the two PK genes expression determined by RT-PCR for gapc-1 and as-GAPC1 plants (Fig. 7B) and microarray experiments with gapc-1 mutants (Table II). We also observed decreases in the activity of other enzymes in the mutant plants: 49% for phosphoenolpyruvate carboxylase (PEPC), 24% for NAD-malic enzyme (NAD-ME), and 39% for G6PDH (Fig. 7A). The decrease in several glycolytic transcripts such as the two PK genes and the inhibition of PK activity (the primary point of regulation of plant glycolysis), as well as the inhibition of other glycolytic enzymes, gives strong support that GAPC-1 deficiency lead to an inhibition of carbohydrate metabolism.
Russell and Sachs (1989) As described above, some genes related to photosynthesis, for example, Rubisco SSU and RCA, were decreased in microarray analysis. We confirm the decrease in RCA transcript levels (about 50%) by RT-PCR analysis. However, a slight increase of Rubisco SSU mRNA expression was observed (Fig. 7B). Furthermore, the transcription of many genes encoding for enzymes related to stress responses such as the three catalases was repressed (Fig. 7B; see also Table II). Thus, microarray and RT-PCR results suggest that mutants have an altered photosynthetic and carbon metabolism, which can induce a stress situation that could account for delayed growth.
To evaluate the effect of the GAPC deficiency in glycolytic or TCA intermediates, we quantified the levels of pyruvate, the end product of glycolysis, and malate, the product of the alternative glycolytic pathway. Furthermore, we determined the levels of two metabolites of the TCA cycle, citrate and isocitrate. The gapc-1 and as-GAPC1 plants accumulate 31% and 22%, respectively, of the pyruvate levels compared to the wild type, whereas there is an increase of about 1.7-fold in the accumulation of malate in both lines. Indeed, gapc-1 and as-GAPC1 plants showed a reduction in TCA intermediate levels such as citrate (40% and 32%, respectively) and isocitrate (54% and 27%, respectively; Table III).
gapc-1 Plants Show Reduced Levels of Oxygen Uptake and ATP, and Reduced Expression and Activity of TCA Cycle Enzymes
It has been demonstrated that GAPC-1 and other glycolytic enzymes are associated with the mitochondrial outer membrane in Arabidopsis (Giege et al., 2003
The energetic status of the gapc-1 line was evaluated by measuring total ATP levels. We found 38% and 29.4% decreases in the ATP concentration in mutant leaves and flowers, respectively (Fig. 8B). The results suggest that GAPC-1 deficiency has an important effect on ATP levels, in correlation with the decrease in the respiration rate observed in the mutant plants (Fig. 8A). The decrease in ATP pools and the reduction of the respiration rate in mutant plants correlate with a reduced expression of genes involved in the TCA cycle such as ACO and SDH determined by RT-PCR (Fig. 8C). This is in agreement with the observed decrease in aconitase (15% and 20% in gapc-1 and as-GAPC1 plants, respectively) and SDH activity (about 20% in both lines; Fig. 8C). Taken together, our results suggest that the GAPC-1 deficiency affects not only the glycolysis but also the function of other respiratory pathways, and that this metabolic deficit could be the cause of the phenotypes observed in reproductive tissues, which depend mainly on respiration to obtain energy.
Recently, it has been suggested that GAPC plays a regulatory role in reactive oxygen species (ROS) signaling in plants (Hancock et al., 2005
Microarray data also show that transcript levels for several genes encoding proteins that participate in oxidative stress response, such as peroxidases (PER62, PER41, PER52, PER9, and PER2) and superoxide dismutase [Cu-Zn], show increased levels in gapc-1 plants. In addition, we measured by RT-PCR the mRNA levels of other genes encoding for proteins involved in stress responses, such as PEROX and AOX (Sweetlove et al., 2002 ), which showed a slight change in expression (Fig. 9C). All together, these data support the existence of increased oxidative stress in GAPC-1-deficient plants.
To investigate other possible physiological functions of GAPC-1, we characterized two GAPC-deficient lines: a T-DNA insertional mutant (SALK_010839, gapc-1) and an antisense line, as-GAPC1. We found evidence that GAPC-1 plays an important role and is required for full fertility in Arabidopsis plants. The knockout mutant gapc-1 is null in terms of GAPC-1 expression, and the as-GAPC1 line displayed a decrease in the expression of GAPC-1 transcript. Accordingly, both lines showed lower levels of GAPC protein by western-blot analysis (Figs. 1 and 2) and reduced activity in leaves and flowers (Fig. 2B). Western-blot analysis performed in gapc-1 and as-GAPC1 plants revealed the presence of a protein band that reacts with a-GAPC antibodies. This can be due to the expression of GAPC-2, a cytosolic isoform of GAPC having high structural similarity with GAPC-1, whereas GAPC-2, GAPCp, and/or GAPA/B isoforms could contribute to the NADH-dependent residual activity observed in the two lines.
Both gapc-1 and as-GAPC1 plants exhibited a delay in growth. Indeed, both lines presented an alteration in silique morphology and seed production. The defect in pollen tube germination and the restoration of viable seed production after crossing experiments using wild-type pollen indicate that GAPC-1 function is important in male organs and suggest that the decrease in GAPC-1 activity impairs the mitochondrial function required for normal pollen production (Heiser et al., 1997
In a previous work, Hajirezaei et al. (2006)
In plants, the glycolytic carbon flux can pass through the GAPC enzyme producing 3PGA, NADH, and ATP or through a metabolic bypass catalyzed by NP-GAPDH producing 3PGA and NADPH, but not ATP. This alternative pathway has energetic and metabolic consequences; it has been proposed to participate in a shuttle of triose-P/phosphate that indirectly transfers photosynthetically reduced NADP+ from chloroplast to cytoplasm during photosynthesis (Kelly and Gibbs, 1973b
The deficiency of GAPC-1 activity shows a direct effect on the production of energy, as we measured a decrease in ATP cellular levels in flowers and leaves of the gapc-1 plants. Indeed, the decrease of pyruvate levels and TCA cycle intermediates suggests a decrease in the carbon flux through the glycolytic pathway to the mitochondria. Pollen development in the anther and the growth of the pollen tube are highly energy-demanding processes. It has been reported that pollen granules contain 20 times more mitochondria per cell than normal vegetative tissues in maize (Gass et al., 2005
Two major sites of ROS production in plant cells have been reported, one in the chloroplast, where ROS is produced in the photosynthetic electron transport chain, and the other in the mitochondria (Millar et al., 2001 In conclusion, in this work, we studied the effect of the disruption of the GAPC-1 gene in Arabidopsis plants using gapc-1 null mutants and antisense lines deficient in GAPC-1 expression. Both lines showed defects in fertility, with alterations of seed and fruit development, suggesting that GAPC-1 and the presence of a full glycolytic pathway are essential in these organs and have an important role in fertility in Arabidopsis. Analysis by microarrays, RT-PCR, and enzymatic activity suggest that a deficiency in GAPC-1 results in an inhibition of glycolysis, mitochondrial dysfunction, and increase of oxidative stress.
Plant Material and Growth Conditions Arabidopsis (Arabidopsis thaliana) var Columbia was used as the wild type. The gapc-1 mutant plant contains a T-DNA insertion in the ninth exon of the GAPC-1 gene At3g04120 (SALK_010839; Fig. 1A). The gapc-1 mutant seeds were obtained from the T-DNA Express Collection at the Salk Institute (http://signal.salk.edu/cgi-bin/tdnaexpress). Seeds were germinated directly in soil and kept at 4°C for at least 72 h before light treatment. Plants were grown in greenhouse conditions at 25°C under fluorescent lamps (Grolux, Sylvania and Cool White, Philips) with an intensity of 150 µmol m–2 s–1 using a 16-h-light/8-h-dark photoperiod.
The position of the T-DNA insert was determined by PCR using the following primers: LBb1 (GCGTGGACCGCTTGCTGCAACT; http://signal.salk.edu) and GAPC-1 (Table IV).
Genomic DNA was extracted from leaves using the cetyl-trimethyl-ammonium bromide method described by Sambrook et al. (1989)
Isolation of RNA and RT-PCR Analysis Total RNA from 6-week-old fully expanded rosette leaves collected from pools of six plants was extracted using TRI Reagent (Sigma-Aldrich). First, cDNA synthesis was obtained using total RNA (3 µg) in the presence of random hexamers and moloney murine leukemia virus RT (USB) according to the manufacturers' instructions. An aliquot (1 µL) from the RT reaction was used as template in PCR reactions with the corresponding oligonucleotides (Table IV). Semiquantitative RT-PCR analysis was performed on the amplification of products after 16, 20, 24, and 28 PCR cycles using (at least) three independent samples. Appropriate number of cycles was determined for each cDNA to obtain data during the exponential phase of the PCR reaction. β-ACTIN was used as internal control. Specific primer pairs were designed based on the cDNA sequence reported in GenBank for the desired genes. PCR products were analyzed on agarose gels and visualized using ethidium bromide staining and/or transferred onto Hybond N+ membranes (Amersham Biosciences). Probe labeling and membrane hybridization were performed according to the ECL Direct Nucleic Acid Labeling and Detection System protocol (Amersham Biosciences).
Arabidopsis oligonucleotide microarrays were fabricated by the University of Arizona and contain 26,000 oligonucleotides (http://www.ag.arizona.edu/microarray/). All analyses were completed as described previously (Rius et al., 2006
The relative levels of mRNA transcripts for the different genes were determined by densitometric analysis using the Gel Pro Analyzer program (Media Cybernetics).
Oxygen consumption was measured at 25°C using an air-tight chamber fitted with a Clark type electrode (Hansatech Leaf Disc Electrode unit). Calibration was achieved by a simple 2-point calibration between air (21% O2) and the injection or removal of a known volume of air from the chamber. Zero oxygen was achieved by equilibration with N2 to displace all the O2 present in the chamber. Plants were kept in the dark for 15 min before measurement. Detached Arabidopsis leaves and bud flowers (200–300 mg) were placed in the oxygen electrode chamber. Oxygen concentration was monitored for 15 min.
Leaf extracts obtained from pools of six plants were electrophoresed on 12% SDS-polyacrylamide gels and electroblotted onto a nitrocellulose membrane. Immunoblotting was revealed using affinity-purified antibodies raised against recombinant GAPC of Triticum aestivum, and antigenic polypeptides were detected using an alkaline-phosphatase-conjugated secondary antibody (dilution 1:10,000), as described previously (Plaxton, 1989
In vitro pollen germination was determined by incubating released pollen with medium containing 17% (w/v) Suc, 2 mM CaCl2, 1.65 mM H3BO3, 0.6% (w/v) agar, and 5 mM MES, pH 5.8, as described previously (Busi et al., 2006a
Six-week-old rosette leaves and bud flowers from wild-type and gapc-1 plants frozen with liquid nitrogen were ground to a powder using a chilled mortar and pestle. The powder (20 mg) was homogenized with 60 µL of 0.1 M HCl in a 1.5-mL microtube. The homogenate was centrifuged at 20,000g for 10 min at 4°C. The supernatant was filtered through a micro-concentrator Microcon YM-3 (Amicon) at 14,000g at 4°C. After neutralization with 1 M Tris-HCl, pH 7.4, the filtrates were used for the measurement of ATP levels. Total cellular ATP content was determined with an ATP bioluminescent assay kit (Sigma-Aldrich) and an LD-400 Luminescence detector (Beckman-Coulter; Wulff and Döppen, 1985
Metabolite levels were assayed spectrophotometrically as described previously (Chen et al., 2002
The histochemical detection of ROS was performed according to Hempel et al. (1999)
Homogenates used to determine enzyme activity were prepared as described (Eastmond et al., 2000
Protein concentration was determined by the modified Bradford assay (Bollag et al., 1996
General molecular techniques such as plasmid DNA isolation, restriction digestion, modification and ligation of DNA, PCR, agarose gel electrophoresis, northern blots, transformation, and culture of Escherichia coli were carried out according to standards protocols (Sambrook et al., 1989
We are grateful to Jose Luis Burgos (Comision de Investigaciones Científicas) for excellent technical assistance and the Arabidopsis Biological Resource Center and the stock donor(s). We thank Drs. María Victoria Busi, Guillermo Santa María, and Alejandro Araya for helpful discussions and critical reading of the manuscript and Dr. Carlos Bartoli for helping with the respiration measurements. Received August 30, 2008; accepted September 22, 2008; published September 26, 2008.
1 This work was supported by CONICET (grant nos. PIP–6241 to D.F.G.-C., and PIP–6357 and CAI+D'06 to A.A.I.), by ANPCyT (grant nos. PICT'06-00614 to D.F.G.-C.; PICTO03–13241 and 05–13129, PICT'03–14733, and PAV'03–137 to A.A.I.; and PICT'03–13278 to P.C.), and by Fundación Antorchas (grant no. 4306–5 to P.C.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors is: Diego F. Gomez-Casati (diego.gomezcasati{at}intech.gov.ar).
[C] Some figures in this article are displayed in color online but in black and white in the print edition. www.plantphysiol.org/cgi/doi/10.1104/pp.108.128769 * Corresponding author; e-mail diego.gomezcasati{at}intech.gov.ar.
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