Plastidial glyceraldehyde-3-phosphate dehydrogenase deficiency leads to altered root development and affects the sugar and amino acid balance in Arabidopsis

Abstract

Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD(+) to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism.

Figure 1.

Schematic representation of glycolysis in a plant cell. Emphasis is given to the plastidic and cytosolic glycolytic reactions catalyzed by GAPDH (GAPC, cytosolic isoform; GAPCp, plastidial isoform) and phosphoglycerate kinase (PGK, cytosolic isoform; PGKp, plastidial isoform). 1-3BisPGAP, 1,3-Bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; ENO, enolase; FAS, fatty acid synthesis; 2-PGA, 2-phosphoglycerate; PGM, phosphoglycerate mutase. Broken lines indicate several enzymatic reactions. Adapted from Schwender et al. (2003).

Figure 2.

Phylogenetic tree of the Arabidopsis phosphorylating GAPDH proteins, and gene expression levels. A, The phylogenetic tree was constructed from an alignment of deduced amino acid sequences as described in “Materials and Methods.” The bootstrap value from 100,000 replicates is given at each node. The scale shows the length of the maximum possible bootstrap value (100). Branch length is given under each segment according to the algorithm specified in “Materials and Methods.” B, Gene expression levels of the Arabidopsis GAPDH isoforms based on microarray experiments performed on 15-d-old seedlings.

Figure 3.

Subcellular localization of GAPDH isoforms by stable expression of GFP-GAPDH fusion proteins in Arabidopsis. A and B, Localization of chloroplastic and cytosolic isoforms GAPA1 and GAPC1. C and D, Chloroplastic/plastidic localization of GAPCp1 in leaves (C) and roots (D). E and F, Chloroplastic/plastidic localization of GAPCp2 in leaves (E) and roots (F). GFP fluorescence, chlorophyll fluorescence, light image, and merged image are presented from left to right. Bars = 50 μm.

Figure 4.

Expression analysis of GAPCp1 and GAPCp2 genes. A, RT-PCR analysis of GAPCp1 and GAPCp2 in the aerial part and roots of 17-d-old seedlings. B, Expression of GUS under the control of GAPCp1 and GAPCp2 promoters in seedlings (B1 and B2), cotyledons (B3 and B4), shoots (B5 and B6), and roots (B7 and B8) of 6- to 10-d-old plants. C, Relative gene expression of GAPCp1 and GAPCp2 in adult plants. Values are means ± sd (n = 3). D, Expression of GUS under the control of GAPCp1 and GAPCp2 promoters in flowers (D1 and D2), anthers (D3 and D4), pistils (D5), roots (D6), and leaves (D7–D10) of adult plants. Bars = 5 mm (B1 and B2), 1 mm (B3–B8, D1, D2, and D7–D10), and 0.25 mm (D3–D6).

Figure 5.

Characterization of gapcp1 and gapcp2 mutants. A, Genomic organization of gapcp T-DNA mutant lines. Open boxes represent exons, and solid lines represent introns. The T-DNA insertion point in each gapcp mutant is shown along with the position of primers used for genotyping. B, Detection of the GAPCp transcript in the gapcp1 and gapcp2 mutants by RT-PCR analysis. Total RNA was extracted from wild-type (WT) or mutant roots, and RT-PCR was performed with the specific primers for each T-DNA insertion located at both sides of the T-DNA insertion. Forty PCR cycles were done. For simplicity, g stands for gapcp.

Figure 6.

Phenotypic characterization of gapcp mutants and complemented lines. A, Phenotypic changes in the gapcp mutant. Plants were grown for 18 d on one-fifth-strength MS plates. Double mutants (g1.1g1.1 g2.1g2.1, g1.1g1.1 g2.2g2.2, and g1.2g1.2 g2.1g2.1) showed arrested root development. Single mutants (g1.1g1.1 G2G2), heterozygous lines (g1.1g1.1 G2g2.1), and gapcp double mutant complemented with a genomic GAPCp1 construct (g1.1g1.1 g2.1g2.1 PG1:G1) had normal root development as compared with control plants (WT). At bottom are data of the final root length, root growth rate, and aerial part fresh weight (FW) of the wild type, double mutant, and complemented line. Measurements (mean ± sd; n ≥ 30) were made in plants grown with or without 1% (w/v) Suc. B and C, Size of the root epidermal cells is reduced in the gapcp double mutant. Electron microscopy of wild-type (B) and gapcp double mutant (C) roots is shown. Measurements are means ± sd (n = 3). D and E, Bright-field inverted microscopy images showing that the size of the epidermal cells in leaves is not reduced in the gapcp double mutant (E) as compared with the wild type (D). Measurements are means ± sd (n = 3). F, gapcp double mutants (g1.1g1.1 g2.1g2.1, g1.1g1.1 g2.2g2.2, and g1.1g1.1 g2.3g2.3) are dwarf in the adult stage. Heterozygous plants (g1.1g1.1 G2g2.1) are not different from wild-type plants. G, Complementation of gapcp double mutants with a genomic GAPCp1 construct (g1.1g1.1 g2.1g2.1 PG1:G1) rescues the wild-type phenotype. For simplicity, g stands for gapcp and G stands for GAPCp. Bars = 1 cm (A), 50 μm (B–E), and 10 cm (F and G).

Figure 7.

Carbohydrate profiling of gapcp double mutants and GAPCp-overexpressing plants. Starch and total soluble sugars in the aerial part and roots of 18- to 21-d-old wild-type (WT), gapcp double mutant (g1.1g1.1 g2.1g2.1), and 35S-GAPCp-overexpressing (Oex GAPCp) plants. Seeds were sown on plates with one-fifth-strength MS medium for 8 to 10 d. After phenotypic selection of gapcp double mutants as described in “Materials and Methods,” seedlings were transferred to one-fifth-strength MS medium with or without 0.1 mm Ser for an additional 10 d. Values (mg g⁻¹ fresh weight) were normalized to the mean response of the wild type (starch: 1.75 ± 0.33 in wild-type aerial part, 0.19 ± 0.05 in wild-type roots, 2.03 ± 0.4 in wild-type aerial part with Ser, 0.17 ± 0.05 in wild-type roots with Ser; soluble sugars: 0.31 ± 0.10 in wild-type aerial part, 0.87 ± 0.15 in wild-type roots, 0.41 ± 0.06 in wild-type aerial part with Ser, 1.10 ± 0.15 in wild-type roots with Ser). For simplicity, g stands for gapcp. * Significant at P < 0.05.

Figure 8.

Ser rescues the arrested root development in gapcp double mutants. A to C, Seeds from wild-type (WT) and gapcp double mutant (g1.1g1.1 g2.1g2.1) plants were germinated in one-fifth-strength MS medium for 8 to 10 d and then transplanted for an additional 10 d to a medium with or without 0.1 mm Ser (A). The same experiment described in A was conducted with different concentrations of Ser (B) and Gly or Cys (C). Data are means ± sd (n ≥ 30). For simplicity, g stands for gapcp.

Figure 9.

Functional categorization of the genes differentially expressed in the gapcp double mutant. Down-regulated (A) and up-regulated (B) transcripts (2-fold increase) in gapcp double mutant as compared with the wild-type (WT) control were sorted by their putative functional categories and compared with the whole genome according to the FatiGO tool. For simplicity, g stands for gapcp. * Significantly enriched function at adjusted P < 0.05.

Figure 10.

Quantification of the changes in transcript levels using real-time PCR. A, A selection of up-regulated genes in gapcp double mutants. B, A selection of down-regulated genes in gapcp double mutants. At right, the putative functions of the selected genes are presented. WT, Wild type.