Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis

Abstract

In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1, TRX m2, and TRX m4 (TRX ms), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis (Arabidopsis thaliana). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m-silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC , we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway.

Redox‐control of chlorophyll biosynthesis mainly depends on thioredoxins

“…This conclusion is supported by the interaction of CHLM with m-type TRX [11] and the analysis of TRXf1,2 mutant as well as TRXm (1,2,4) triple-silencing plants, which do show ntrc-like alteration of the redox state and de-stabilization of CHLM [11,14]. In addition, the redox-dependent de-stabilization of GLUTR observed in ntrc is not visible in TRX f-and TRX m-deficient plants [11,14] suggesting that either NTRC or one of the other TRXs are more important to maintain GLUTR's redox-state. Although the demand for Chl is reduced compared to young developing tissues, the increased Chl content in older ntrc leaves could be also explained by an increased activity of the FTR system and a prevailing impact of TRX on the redox-regulation of TBS enzymes at a later stage of development.…”

“…The new data correspond with a main TRX function for redox control of plastid-localized enzymes in Chl synthesis rather than NTRC. This conclusion is supported by the interaction of CHLM with m-type TRX [11] and the analysis of TRXf1,2 mutant as well as TRXm (1,2,4) triple-silencing plants, which do show ntrc-like alteration of the redox state and de-stabilization of CHLM [11,14]. Additionally, a recovery of Chl content was also observed in older leaves of the ntrc mutant [4].…”

“…Thus, TRXs compensate the lack of NTRC for 2CP reduction on the cost of reducing equivalents needed for the reduction of other TRXs targets [12]. This conclusion is supported by the interaction of CHLM with m-type TRX [11] and the analysis of TRXf1,2 mutant as well as TRXm (1,2,4) triple-silencing plants, which do show ntrc-like alteration of the redox state and de-stabilization of CHLM [11,14]. Despite the published evidence for the interaction and impact of NTRC on the redox status of GLUTR, CHLM, and other TBS enzymes [9,10,13], we do not exclude to reconsider the function of NTRC on plastid-localized proteins.…”

“…As NTRC interacts with GLUTR and CHLM, respectively, and redox regulation of CHLM was shown to be crucial for its activity [13] and stability [9,11], we analyzed the amount of TBS enzymes in the mutants (Fig. 2).…”

“…The NTRC knockout mutant (ntrc) shows a pale green phenotype, which was attributed to altered activities and stability of glutamyl tRNA-reductase (GLUTR) and Mg-protoporphyrin IX methyltransferase (CHLM), two important enzymes for the synthesis of heme and chlorophyll (Chl) [9]. However, the redox status of Chl synthesis enzymes, such as CHLM, was cumulatively affected in plants with deficiency of NTRC and three TRX m isoforms [11].…”