Alteration of the Interconversion of Pyruvate and Malate in the Plastid or Cytosol of Ripening Tomato Fruit Invokes Diverse Consequences on Sugar But Similar Effects on Cellular Organic Acid, Metabolism, and Transitory Starch Accumulation

Sonia Osorio,Marek Szecowka,Vered Tzin,Gad Galili,Ruthie Angelovici,Alisdair R Fernie,José G Vallarino,Shai Ufaz

doi:10.1104/pp.112.211094

Sonia Osorio, Marek Szecowka + Show 6 more

Open Access

https://doi.org/10.1104/pp.112.211094

Copy DOI

Abstract

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects on fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate, which suggests that an excess of oxaloacetate is converted to aspartate and reintroduced in the TCA cycle via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-ME antisense lines were characterized by no changes in respiration rates and TCA cycle flux, which together with increases of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicate that pyruvate is supplied through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening.

Highlights

Fruit ripening and development is well established to be under tight genetic control (Matas et al, 2009; Karlova et al, 2011; Klee and Giovannoni, 2011)
Eight lines for RNA interference (RNAi)-phosphoenolpyruvate carboxykinase (PEPCK) and nine lines for RNAi-malic enzyme (ME) were selected on the basis of PEPCK and NADP-ME1 gene expression by quantitative real-time-PCR (Fig. 1, A and B)
During the ripening of tomato, there is a large decrease in the organic acid content of the pericarp (Knee and Finger, 1992; Carrari et al, 2006). This decrease in organic acids could be brought about either by a restriction of their synthesis or an enhanced degradation potentially initially catalyzed by NAD-ME, NADPME, or PEPCK

Summary

Introduction

Fruit ripening and development is well established to be under tight genetic control (Matas et al, 2009; Karlova et al, 2011; Klee and Giovannoni, 2011). Certain speciesspecific differences exist in the dynamics of other metabolite pools across ripening, with, for example, grape (Vitis vinifera), strawberry (Fragaria 3 ananassa), prune (Prunus domestica), and pepper (Capsicum annuum) displaying slightly different metabolic programs from tomato (Solanum lycopersicum; Carrari et al, 2006; Zamboni et al, 2010; Lombardo et al, 2011; Osorio et al, 2011, 2012; Zhang et al, 2011) This fact notwithstanding, tomato has become the primary experimental model in which to study the development and ripening of fleshy fruits (Giovannoni, 2004; Fernandez et al, 2009). The results are discussed with respect to current models of energy metabolism within the fruit and with respect to the roles of carboxylic acids during fruit ripening and development

Objectives

Methods

Results

Conclusion