Distinct plastid fructose bisphosphate aldolases function in photosynthetic and non-photosynthetic metabolism in Arabidopsis

Abstract

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin-Benson cycle. Three Arabidopsis genes, AtFBA1-AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.

Keywords: Arabidopsis thaliana; 6-bisphosphate aldolase; Amino acid metabolism; Calvin–Benson cycle; fructose-1; glycolysis; phloem transport; photosynthesis; plastid metabolism.

Fig. 1.

Phylogenetic analysis of plastidial FBAs. (A) Maximum likelihood phylogenetic tree of the plastidial FBA proteins from mosses and higher plant species, with Bootstrap values. The Arabidopsis proteins (NP_565508.1, NP_568049.1, NP_178224.1) are shown in red. (B) Duplicated chromosomal regions of the Arabidopsis genome contain the FBA1 and FBA2 genes according to the Plant Genome Duplication Database. Arrows represent genes on distinct, 100 kbp regions of chromosomes 2 and 4. Lines connect duplicated genes including FBA1 and FBA2 (in red).

Fig. 2.

Expression patterns of FBA1, FBA2, and FBA3 genes in Arabidopsis. Staining of plants transformed with FBA1_pro::GUS (A, D, E), FBA2_pro::GUS (B, C, F, G), and FBA3_pro::GUS (H–L) constructs in developing leaves (A, B, C, H), sections through leaf margins (D, F), leaf vascular bundles (E, G, L), the whole seedling (I), cotyledon (E), and root cross-section (K). Representative images of expression patterns observed in at least two independent transformants are shown. In (E, F, L), the position of the xylem is indicated (x). In (L), some sieve tube cells are indicated (black arrows).

Fig. 3.

Homozygous knockout mutants of the three plastidial FBA isoforms. (A) Expression of the plastidial FBA genes in the shoot of wild-type Col-0 plants measured with RT-qPCR at midday. Values are normalized to the housekeeping genes AtYLS8, ACTIN2, and GAPC2, and are means of three biological replicates (±SE). (B) Structure of the plastidial FBA genes: black and white rectangles represent the exons and the UTR regions respectively, with introns represented as lines. The locations of the T-DNA insertions in the different mutant alleles are indicated by red triangles. Annealing sites of primers used for RT-qPCR are indicated. (C) Phenotype of the mutants 30 d after germination. Scale bar: 1 cm. (D) Expression of the FBA1, FBA2, and FBA3 genes in the rosettes of the single knock-out mutants. Values are normalized to AtYLS8, ACTIN2, and GAPC2, and are means of three biological replicates (±SE), and are expressed relative to the mean value for the wild type. (E) Total FBA enzyme activity measured in the shoots of the fba1, fba2, and fba3 mutants. The enzyme activity is normalized to the respective wild type. Values are means of three biological replicates (±SE). **P≤0.05, ***P≤0.01: significant differences from the corresponding wild type. The mean activity (±SE) of Col-0 wild type from three experimental replicates was 4.41 ± 1.00 μmol min−1 g−1 fresh weight.

Fig. 4.

Phenotypes of mutants deficient in both FBA1 and FBA2. (A) Appearance of wild-type, fba1, and fba2 plants and of representative genotypes isolated from the segregating F₂ population of a cross between fba1 and fba2. All plants were 25 d old and imaged from the growth analysis shown in (B), except the fba1fba2 double mutant, which was the sole individual identified during segregation analysis and is ×4 magnified (see Supplementary Table S6). Scale bar: 1 cm. (B) Growth rate of the different genotypes in (A) (except for the fba1fba2 double mutant) from 13–25 d after germination. Values are the means of 6–11 biological replicates (±SE). (C) Starch content of fba1, fba2, fba1^(+/−)fba2^(−/−), and of the wild type. Values are the means of four biological replicates (±SE). EoD, end of day; EoN, end of night. *P≤0.1, **P≤0.05, ***P≤0.01: significant differences from the corresponding wild type.

Fig. 5.

Photosynthesis, respiration, and diel changes in carbohydrate levels in fba1, fba2, and fba3 mutants. (A) Daytime photosynthetic carbon assimilation and night-time respiration of the aerial parts of 28-day-old plants (50-day-old for fba3), measured by infrared gas analysis (illumination at 150 μmol m⁻² s⁻¹, 20 °C, 70% relative humidity, 380 ppm CO₂). Note the difference in scale of the y-axis between positive and negative values. Mean values from four biological replicates (±SE) are given. A rate of 20 nmol g⁻¹ FW s⁻¹ for the wild type equates to approximately 5 μmol m⁻² projected leaf area s⁻¹. (B) Starch and the soluble sugars glucose, fructose, and sucrose were measured in the aerial parts of 25-day-old plants over a 12 h day (white background)–12 h night (grey background) cycle. Note the differences in the y-axis scale depending on the measured data in the different mutants. The same data for the wild type (in black) are compared against each mutant. Mean values from four biological replicates (±SE) are given.

Fig. 6.

Metabolite levels in the shoots of mutants lacking plastidial fba isoforms. Schematic representation of central carbohydrate and amino acid metabolism in photosynthetic cells. Mean metabolite values (n=3 biological replicates), normalized to the wild type, are visualized as a fold-change (FC) heatmap. (A) fba1-1 (left-hand heatmap boxes) and fba2-1 (right-hand heatmap boxes) mutants, and (B) fba3-1 mutants. *P≤0.1, **P≤0.05, ***P≤0.01: significant changes in the metabolite level compared with the respective wild type, two-tailed t-test. See also Supplementary Table S7. 1,3BPG, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; Ery, erythrose; FBA, fructose-1,6-bisphosphate aldolase; Fru, fructose; GAP, glycerylaldehyde 3-phosphate; Glc, glucose; PEP, phosphoenolpyruvate; 3PGA, 3-phosphoglycerate; Pyr, pyruvate; Rib, ribose; Ru, ribulose; Sed, sedoheptulose; Suc, sucrose; Xyl, xylulose.

Fig. 7.

Metabolite levels in the roots of the fba3 mutant. Schematic representation of central carbohydrate and amino acid metabolism in heterotrophic cells. Mean metabolite values (n=4 biological replicates), normalized to the wild type, are visualized as a fold-change (FC) heatmap. *P≤0.1, **P≤0.05, ***P≤0.01: significant changes in the metabolite level in fba3 roots compared with the wild type, two-tailed t-test. See also Supplementary Table S8. AcCoA, acetyl-CoA; DHAP, dihydroxyacetone phosphate; Ery, erythrose; FBA, fructose-1,6-bisphosphate aldolase; Fru, fructose; GAP, glycerylaldehyde 3-phosphate; Glc, glucose; PEP, phosphoenolpyruvate; 3PGA, 3-phosphoglycerate; Pyr, pyruvate; Rib, ribose; Ru, ribulose; Sed, sedoheptulose; Suc, sucrose; Xyl, xylulose.

Fig. 8.

Root growth phenotype of the fba3 mutant and its rescue by micrografting. (A) Representative seedling phenotypes of the fba3-1 mutant and its Ler wild type grown on agar plates without or with exogenous sucrose (0.1, 0.5, 1, and 1.5% (w/v)). (B) Mean root length (±SE; n=12 biological replicates) for plants grown as in (A). (C) Phenotype of chimeric plants where fba3 shoots are grafted onto wild-type roots and vice versa. Self-grafted fba3 and wild-type plants serve as controls. The genotype of the roots was determined by PCR on extracted genomic DNA to re-confirm that they were not lateral roots produced at or above the graft point. Scale bar: 1 cm.