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. 2022 Oct 4;11(10):1979.
doi: 10.3390/antiox11101979.

New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts

María Ancín  1   2 , Joaquin Fernandez-Irigoyen  3   4 , Enrique Santamaria  3   4 , Luis Larraya  1 , Alicia Fernández-San Millán  1 , Jon Veramendi  1 , Inmaculada Farran  1
Affiliations

New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts

María Ancín et al. Antioxidants (Basel). .

Abstract

Post-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new in vivo approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into Nicotiana benthamiana plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The in vivo approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.

Keywords: NTRC; Nicotiana; chloroplast; proteomics; redox regulation; target proteins; thioredoxin.

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Conflict of interest statement

The authors declare no conflict of interest. The funding organisations had no role in the design of the study, the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to publish the results.

Figures

Figure 1
Figure 1
Procedure for isolation of in vivo Trx interactors. His-tagged single-cysteine mutants of Trx f, Trx m and NTRC were separately agroinfiltrated into N. benthamiana plants (1 and 2). After 5 days, leaf protein extraction was performed and the Trxs, along with their interactors, were captured in a column packed with Ni-NTA agarose (3). Finally, redox interactors were eluted with DTT (5). Trx mut: thioredoxin mutant; DTT: dithiothreitol.
Figure 2
Figure 2
Proteomic analysis of the Trx-interacting proteins. Using quantitative mass spectrometry, eluted Trxs proteomes were compared to the control (empty-vector proteome). The candidate proteins are separately shown for Trx f (green), Trx m (yellow) and NTRC (blue).
Figure 3
Figure 3
Functional classification of proteins identified as putative Trx f, Trx m or NTRC partners in chloroplasts. The plot represents the percentage of total targets for each Trx in each functional category.
Figure 4
Figure 4
Comparison of Trx specificity. Venn diagram representation showing the overlap between candidate target proteins for Trx f, Trx m and NTRC.
See this image and copyright information in PMC

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