Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening

Abstract

Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species.

Figure 1.

Generation and Preliminary Characterization of Transgenic Lines Expressing Antisense Constructs. (A) Antisense constructs for tomato mitochondrial fumarase (i) and mitochondrial MDH (ii) under the control of a fruit-specific B33 patatin promoter. White arrows represent the direction of transcription of the native genes. ocs, octopine synthase terminator. (B) Total fumarase activity in fumarase lines (F4, F6, F7, and F26) relative to the wild type (WT). FW, fresh weight. (C) Total MDH activity in MDH lines (M14, M21, M23, and M45) relative to the wild type. (D) and (E) Malate content in fumarase (D) and MDH (E) lines (F) and (G) Fumarate content in fumarase (F) and MDH (G) lines. The measurements for (B) to (G) were performed in pericarp of green fruits with 35 DAF. (H) and (I) Total fruit yield of the fumarase (H) and MDH (I) lines. For all parameters, determined values are presented as the mean ± se of 10 biological replicates (total fresh weight per plant is presented as the mean ± se of six biological replicates). An asterisk indicates the values that were determined by the t test to be significantly different (P < 0.05) from the wild type.

Figure 2.

Respiration in Green Fruits of the Antisense Lines Harvested at 35 DAF. (A) Respiration in mitochondrial fumarase lines. FW, fresh weight; WT, wild type. (B) Respiration in MDH lines. Data points show the evolution of ¹⁴CO₂ from isolated pericarp discs in the light. The pericarp discs were incubated in a solution containing 10 mM MES-KOH, pH 6.5, 0.3 mM Glc, supplemented with 2.32 kBq mL^–1 of [1-¹⁴C]- and [3,4-¹⁴C]-glucose at an irradiance of 200 μmol m^–2 s^–1. The ¹⁴CO₂ liberated was captured (at hourly intervals) in a KOH trap, and the amount of radiolabel released was subsequently quantified by liquid scintillation counting. Values are presented as the mean ± se of three biological replicates.

Figure 3.

Starch Metabolism in Pericarp Discs from Green Fruits Harvested at 35 DAF in the Antisense Fumarase and MDH Lines. (A) Regression analysis of malate and starch content in all genotypes studied: R_F = 0.608, R_M = 0.744, R_F+M = 0.841. Crosses, wild-type (WT/Wt) lines; closed symbols, fumarase lines; open symbols, MDH lines; FW, fresh weight. (B) and (C) Analysis of the dimerization of AGPase in fumarase (B) and MDH (C) lines. (D) Evaluation of the dimerization of AGPase following incubation of wild-type pericarp discs in 50 mM malate. Values in (B) to (D) are presented as mean ± se of determinations on 10 individual plants per line. Asterisks indicate values that were determined by the t test to be significantly different (P < 0.05) from the wild type.

Figure 4.

Gene Expression of AGPase Small Subunit and AGPase Large Subunit 1 and 2 in Antisense Lines. (A) and (B) AGPase small subunit expression in fumarase (A) and MDH (B) lines. WT, wild type. (C) and (D) AGPase large subunit 1 expression in fumarase (C) and MDH (D) lines. (E) and (F) AGPase large subunit 2 expression in fumarase (E) and MDH (F) lines. The values represent the means ± se of six individual plants. An asterisk indicates values determined by t test to be significantly from the wild-type control (P < 0.05). Analyses were determined in different ripening stages. B-3, breaker 3 d postanthesis (DPA); B-1, breaker –1 DPA; B, breaker; B+3, breaker +3 DPA; B+1, breaker + 1 DPA. Individual lines are identified in the key on the graphs.

Figure 5.

Postharvest Transpirational Water Loss of Fruits in the Antisense Fumarase and MDH Lines. (A) and (B) Percentage of weight loss from detached fruits of MDH (A) and fumarase (B) lines over a 20-d period after reaching the red-ripe stage. WT, wild type. (C) and (D) Representative photos of MDH (C) and fumarase (D) fruits 15 and 22 d after detaching from the vine. Values are presented as mean ± se of determinations on six to nine fruits per line. Asterisks indicate values that were determined by the t test to be significantly different (P < 0.05) from the wild type. Individual lines are identified in the key on the graphs. Bar = 1 cm.

Figure 6.

Microbial Infection of Wild-Type and Transgenic Fruits. Percentage of fruits infected by B. cinerea in the antisense mitochondrial fumarase (Fum) and MDH lines after harvest and storage at 100% humidity at room temperature, 3 d after inoculation. Values are presented as mean ± se of determinations on 60 individual fruits per line. Asterisks indicate values that were determined by the t test to be significantly different (P < 0.05) from the wild type.

Figure 7.

Model of the Influence of Mitochondrially Derived Malate on Tomato Fruit Starch, Soluble Sugar Content, Postharvest Shelf Life, and Bacterial Infection. Data presented from analyses of the transgenic lines characterized in this article are schematically summarized in relation to the wild type (WT). Mitochondrial MDH lines ([A]; increased malate); fumarase lines ([B]; decreased malate). The same ripening and postharvest sequence is presented for both transgenic sets. (1) Alterations in mitochondrial redox status are transmitted, either within the same cell type or from adjacent tissues, to the plastid via the malate valve as described by Scheibe (2004). (2) Altered plastidial redox status results in a reduced (MDH) or enhanced (fumarase) activation state of the reaction catalyzed by AGPase (as well as similar changes in the activation state of the plastidial MDH); whether this is mediated by the thioredoxin or the NTR-C pathway is currently unknown. (3) This leads to redox-mediated alterations in pigment biosynthesis during ripening. (4) Starch is rapidly broken down, leading to a reduced soluble solid content in red-ripe fruit in the MDH lines and an increased soluble solid content in the fumarase lines. (5) Potentially as a result of differences in cellular osmolarity, the transgenic sets oppositely display an increased water loss and wrinkling (MDH) or a decreased water loss and wrinkling (fumarase) that appears to be cell wall independent. (6) These changes in water loss and wrinkling correlate positively to the rate of opportunistic pathogen infection in the transgenic sets, while the MDH lines are increasingly susceptible to B. cincerea infection.