Abstract

Reduction-oxidation (redox) regulation in metabolic pathways is governed by oxidoreductases. In autotrophic tissues, there are two major systems namely ferredoxin-thioredoxin (Fdx-TRX) system and NADPH-thioredoxin reductase-thioredoxin (NTR-TRX) system. Many reports have indicated the significance of both systems in regulating broad metabolic processes in chloroplast, while the redox regulation in non-photosynthetic plant metabolism remains unclear. In the present study, the impact of these thiol-redox systems on non-photosynthetic metabolism was investigated by analyzing (i) the role of mitochondrial TRX (TRXh2 and TRXo1) in Arabidopsis plants, and (ii) the role of plastidial NADPH-thioredoxin reductase C (NTRC) in heterotrophic tomato fruits. To analyze the role of mitochondrial TRXs, two T-DNA insertion lines, trxh2.1 and trxo1.1, and a cross double mutant, trxh2.1trxo1.1, were used for further assays. In the single and double mutants, the expression of corresponding genes decreased by over 95% compared to wild-type level. The mutant lines showed comparable growth phenotype to the wild type, even though there was a small decrease in leaf size depending on the light conditions. Nevertheless, the trxh2.1trxo1.1 line tended to accumulate more soluble sugars and sugar phosphates at the end of day, suggestive of enhancement in photosynthetic processes, while the levels of several phosphate intermediates, such as phosphoenopyruvate (PEP) and adenine nucleotides (ATP and ADP), decreased in the trxh2.1trxo1.1 line. This further implied that jointly decreasing the expression of TRXh2 and TRXo1 might affect energy metabolism. Metabolite profiling of mutants lines harvested at the end of night revealed an overall decrease in the metabolites of Gly decarboxylation and tricarboxylic acid cycle, while this change diminished in the samples from the end of day. This indicated that down regulating both mitochondrial TRXs affected metabolic processes in mitochondrion as well as peroxisome. Furthermore, the trxh2.1trxo1.1 line showed a better photosynthetic performance compared to the wild type either in the condition of high CO2 concentration or fluctuating light situation. This implied the potential of TRXh2 and TRXo1 in affecting extra-mitochondrial processes, such as photosynthesis. In addition to its role in chloroplasts, NTRC is also present in non-photosynthetic plastids. To investigate the role of NTRC, a NTRC RNA interference (NTRC-RNAi) construct controlled by a fruit-specific promoter was introduced into tomato plants, which eventually led to a 60%-80% decrease in NTRC expression level in transgenic tomato fruits during their development. The NTRC-RNAi lines tended to generate smaller and lighter fruits with less accumulation of dry matters, compared to the wild type. At an early developmental stage, the accumulation of transient starch was greatly reduced in response to NTRC down-regulation, which subsequently resulted in a significant decrease in ripe fruits. This mainly attributed to the inhibition on the redox-activation of two key enzymes in starch biosynthesis, namely ADP-Glc pyrophosphatase (AGPase) and soluble starch synthase. Furthermore, down-regulation of NTRC perturbed the redox poise of NAD(P)(H), which led to a large increase in the reductive states of NAD(H) and NADP(H). Through performing metabolomics, it was found that NTRC down-regulation led to a general decrease in the level of sugars, while the levels of amino acids and organic acids were mainly increased, implying the potential of NTRC in regulating osmotic balance. These changes in metabolite level are anticipated to influence the taste and flavor of tomato fruits. Overall, this indicates that NTRC serves as a central component in modulating carbon metabolism and redox balance, which ultimately affects fruit growth as well as quality. In summary, these results indicate that in leaves mitochondrial TRXs are involved in interorganellar cross-talk, affecting photosynthetic processes in the chloroplast, while plastidial NTRC in important to regulate metabolism and redox-balance in heterotrophic fruits, affecting fruit size and quality.