On the role of the plant mitochondrial thioredoxin system during abiotic stress

ABSTRACT

Thiol-disulfide redox exchanges are widely distributed modifications of great importance for metabolic regulation in living cells. In general, the formation of disulfide bonds is controlled by thioredoxins (TRXs), ubiquitous proteins with two redox-active cysteine residues separated by a pair of amino acids. While the function of plastidial TRXs has been extensively studied, the role of the mitochondrial TRX system is much less well understood. Recent studies have demonstrated that the mitochondrial TRXs are required for the proper functioning of the major metabolic pathways, including stomatal function and antioxidant metabolism under sub-optimal conditions including drought and salinity. Furthermore, inactivation of mitochondrial TRX system leads to metabolite adjustments of both primary and secondary metabolism following drought episodes in arabidopsis, and makes the plants more resistant to salt stress. Here we discuss the implications of these findings, which clearly open up several research avenues to achieve a full understanding of the redox control of metabolism under environmental constraining conditions.

Involvement of mitochondrial TRX in abiotic stress responses

Redox reactions substantially influence the activity of several proteins and participate in the regulation of crucial cellular processes.¹ Accordingly, a growing body of evidence has highlighted the importance of the thioredoxins (TRXs) for the redox control of plant metabolism.^2,3 TRXs are small proteins containing a redox active disulfide group within its catalytic domain. Being widely distributed throughout most living cells,⁴ TRXs are involved in a variety of cellular redox reactions. Numerous isoforms of TRXs are present in plants, differing in both amino acid sequence and subcellular localization. Over 20 isoforms have been identified in the genome of arabidopsis and grouped into seven subfamilies (f-, m-, h-, o-, x- y-, and z-types).^5,6 The vast majority of TRXs isoforms are located in the chloroplast, but TRXs are also present in mitochondria (TRXs o and h) and cytosol (TRX h), where the presence of highly similar isoforms of NADPH-dependent TRX reductase (NTR), A and B, complete a functional TRX system.^7,8

Non-plastid TRX system also includes proteins located at the nucleus (TRXs h-type in wheat seeds,^9,10 NTRA in arabidopsis¹¹ and pea¹² and TRX o1 in pea leaves¹³), endoplasmic reticulum¹⁴ (TRXs h2, h7, and h8) and attached to the plasma membrane¹⁵ (TRX h9). The redox regulation of chloroplast function has received considerable attention over the last decades; however, the functional role of redox processes in other cell compartments remains unclear. For instance, the functional role of the extraplastidial NTR/TRX system remains poorly understood, most likely due to the extensive functional redundancies between TRXs and glutaredoxins (GRXs) and also between NTRs found in both mitochondria and cytosol.^5,16 The introduction of proteomics-based approaches to TRX studies have greatly contributed to the identification of putative TRX targets in plants, especially in the mitochondria.^7,17–20 Since then, hundreds of putative targets involved in a broad spectrum of mitochondrial processes have been identified, including photorespiration, tricarboxylic acid (TCA) cycle and associated reactions, ATP synthesis, hormone synthesis, and stress-related reactions.^17,18 However, it is important to emphasize that such proteomic approaches provide substantial false-positive interactions and also lack the information whether the TRX-target interaction identified in vitro also occurs in vivo. Thus, further experiments with mutants and/or recombinant proteins are needed to fully elucidate the functionality of the TRX-target proteins interaction. In this vein, experimental validation successfully demonstrated that alternative oxidase (AOX)²¹ and some of the enzymes of the TCA cycle including isocitrate dehydrogenase,²² citrate synthase,^23,24 succinate dehydrogenase, and fumarase are regulated by the mitochondrial TRX system in vitro and/or in vivo.²⁵ Furthermore, cytosolic malate dehydrogenase has also been demonstrated to be regulated by TRXs.²⁶

The mechanisms that coordinate mitochondrial TRX regulation and its implications for the dynamic of plant metabolism under different stresses conditions are poorly understood. However, the significance of mitochondrial redox metabolism in cellular signaling processes coupled with studies of different loss-of-function mutants have recently enabled us to grasp the importance of mitochondrial TRXs under stress conditions.^25,27–29 In order to gain further insights into the physiological and metabolic function of the mitochondrial TRX/NTR system, we have recently investigated the significance of the mitochondrial TRX system under consecutive drought episodes. Using the arabidopsis ntra ntrb double mutant and two independent mitochondrial trxo1 mutants, our study demonstrated that the lack of a functional mitochondrial NTR/TRX system enhances drought tolerance following single and multiple events of drought.²⁷Extensive metabolic and physiological analyses of these mutants revealed multiple and complex responses following both single and repetitive drought episodes.²⁷ Notably, TRX o1 transcripts were more highly expressed under drought, an effect that was even stronger during repetitive drought/recovery events.²⁷ Compelling evidence from several plant species indicates that AOX transcript and protein increase during drought (reviewed in³⁰). Given that TRX o1 may function in the reductive activation of AOX,¹² further investigation to elucidate whether the proposed protective role for TRX o1 under drought²⁷ and salinity^28,29 is possibly also associated to the redox modulation of AOX. Notwithstanding, we further showed that the levels of a large number of secondary metabolites (glucosinolates, anthocyanins, flavonol glycosides, and hydroxycinnamates) increased in at least one of the TRX mutants following two cycles of drought.²⁷ This result reinforces the idea that secondary metabolism is redox-regulated by TRX system.^25,31 What remains unclear and thus deserve further investigation is which enzymes of the secondary metabolism are the target of TRXs. Additionally, our study demonstrated that TRX-mediated redox regulation seems to be crucial for stomatal function following the plant recovery from a second drought event.²⁷

Similar to the situation observed under water limitation, compensatory readjustments were also observed in the responses of plants lacking the mitochondrial TRX o1 following salt stress. These responses included alterations in the glutathione redox state and/or up-regulation of AOX³² and antioxidant enzymes^28,29 (Figure 1). Moreover, Attrxo1 seeds germinated faster and accumulated higher H₂O₂ content under salinity, suggesting that TRX o1 could act as a sensor under salinity and/or an inducer of H₂O₂ accumulation.²⁸ The expression of the antioxidant enzymes AtPRXIIF and AtSRX was not altered during germination in either water or NaCl.²⁸ Conversely, in pea plants the expression of TRX o1 and PRXIIF increased in response to short-term salt stress^32,33 highlighting to the heterogeneity of the antioxidant system depending on the stress condition, which may in turn be related to specific TRX o1 targets following seed germination, a point that clearly deserves further investigation.²⁸ Collectively, these observations are in good agreement with our proposal that perturbation of mitochondrial TRX affects cellular redox metabolism in general. It seems likely that in the absence of a functional mitochondrial TRX system the main mechanisms used to maintain redox homeostasis under drought/salt include an increased concentration of secondary metabolites and higher activities of enzymes of redox metabolism such as superoxide dismutase and catalase (Figure 1). Moreover, stomatal behavior – allowing higher stomatal closure during salt stress²⁹ and better recovery of stomatal conductance following rehydration²⁷ (Figure 2) – was shown to be a key factor for the maintenance of plant growth.

Figure 1.

Schematic model showing the main responses of TRX o1 mutant plants to salt and/or drought/drought recovery. Two independent studies have recently demonstrated that several physiological processes related to metabolism, stomatal function and antioxidant metabolism are affected in Arabidopsis thaliana plants with knockout of the mitochondrial TRX o1.^27,29 The inactivation of TRX o1 leads to increases and decreases in both primary and secondary metabolism during drought and recovery, respectively²⁷ (further details are found in the main text). Moreover, stomatal function is affected in the mutants under salinity and drought recovery: lower water loss and higher stomatal closure under salinity but higher stomatal conductance (g_s) following drought recovery were observed. Although higher levels of H₂O₂ and lipid peroxidation were found in these plants, it seems that increased activities of antioxidative enzymes such as Mn-SOD, Fe-SOD, Cu/Zn-SOD, GR and catalase are likely able to counteract TRX o1 deficiency in arabidopsis mutant plants under salinity.

Abbreviations: Mn-SOD, manganese superoxide dismutase; Fe-SOD, iron superoxide dismutase; Cu/Zn-SOD, copper-zinc, GR, glutathione reductase

Figure 2.

Relationship between the ratio of electron transport rate (ETR) and net photosynthesis (A_N) versus stomatal conductance (g_s) in TRX arabidopsis knockout mutants. The genotypes used here were: ntra ntrb, trxo1-1 and trxo1-2, and wild-type plants (WT) during dehydration and following rehydration. Data was calculated from da Fonseca-Pereira et al. 2019.²⁷ Regression coeficiente and P value are shown.**Means the significant difference of the relationship between A_N/ETR x g_s. in a two-tailed Fisher’s exact test (P< 0.001).

To further elucidate the physiological performance of the TRX mutant lines after drought, here we compared the relationship between the ratio of electron transport rate (ETR) and net photosynthesis (A_N) versus stomatal conductance (g_s) (extracted from²⁷). Both parameters can be employed as physiological status indicators.^34,35 ETR/A_N ratio reflects the energy transfer to the photosynthesis. Under optimum conditions, A_N is the main sink, however, under stress A_N is more sensitive and reduces faster than ETR, promoting the ratio increase and indicating an energy impairment that can be associated to ROS production.³⁴ Accordingly, following drought recovery after drought (irrigation for 3 days) an inverse relationship between both parameters was observed, where WT plants displayed higher ETR/A_N (indicating higher physiological stress) accompanied by lower g_s; meanwhile TRX mutant lines showed comparatively lower ratios with higher g_s for the same recovery period (Figure 2).

Perspectives in understanding mitochondrial redox regulation

While the concept that perturbations in mitochondrial homeostasis exert a major influence on different cellular processes,³⁶ many uncertainties remain concerning the interacting pathways that underpin each process. Moreover, many of the mechanisms and components involved in the redox control of plant responses to stress situations, in general, remain to be fully identified.

Given the multiplicity of targets that TRXs are able to reduce and the yet unknown processes in which they are likely involved,³⁷ it will be interesting, in the coming years, to focus on determining the specific targets of the mitochondrial TRX enzymes in vivo. Furthermore, the characterization of mutants lacking multiple TRXs or compensatory TRX proteins such as GRXs, peroxiredoxins, and sulfiredoxins will be important to unravel the redundant and the complementarity of the different components of the redox metabolism, especially under stress conditions. The combination of proteomics with metabolomics seems to be an excellent strategy to improve our understanding regarding redox regulation of the metabolism. Such approaches will likely be of paramount importance in providing useful information for improved metabolic engineering to develop stress-tolerant plants.

Funding Statement

This work was supported by funding from the Max Planck Society, the CNPq (National Council for Scientific and Technological Development, Brazil) [grant No. 402511/2016-6, and research fellowships to A.N.N., D.M.D., and W.L.A.]; the FAPEMIG (Foundation for Research Assistance of the Minas Gerais State, Brazil) [grant Nos. APQ-01078-15, APQ-01357-14 and RED-00053-16]. CAPES (Coordination for the Improvement of Higher Education Personnel, Brazil) [scholarship to P.F.P.]; and the Spanish Government [the research program ‘Juan de la Cierva’ post-doc grant to J.G.].

Acknowledgments

We acknowledge the helpful comments made by the anonymous referees on previous versions of our manuscript.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.