Diversity and distribution of thioredoxin family proteins in photosynthetic organisms

Thioredoxin (Trx) is a small protein that is mainly responsible for the thiol/disulfide-based redox regulation. Trx contains the highly conserved amino acid sequence WCGPC at an active site. By using two Cys residues in this sequence, Trx catalyzes a dithiol-disulfide exchange reaction with its target enzymes, enabling modulation of their functions. Therefore, Trx plays a pivotal role in transmitting reducing power to redox-sensitive enzymes and adjusting cellular functions in response to changes in redox environments.

Although the Trx-based redox regulation system is widely shared in all organisms including prokaryotes and eukaryotes, several key features are found only in photosynthetic organisms. First, the redox regulation system in photosynthetic organisms is tightly associated with the drive of electron transport reactions in the thylakoid membrane. Trx receives reducing power from photosynthetically-reduced ferredoxin (Fd) via Fd-Trx reductase (FTR) and transfers it to specific sets of redox-sensitive enzymes (e.g. some Calvin-Benson cycle enzymes). This redox pathway, so-called the Fd/Trx pathway, supports light-responsive coordination of photosynthetic metabolism at the post-translational level, which is currently accepted as the textbook knowledge (Buchanan 2016). The genetic impairment of the Fd/Trx pathway in Arabidopsis thaliana leads to severe phenotypes in growth and photosynthesis, underpinning the critical impacts of this system on photosynthetic biomass production (Yoshida et al. 2022). Second, the redox regulation system in photosynthetic organisms encompasses a large number of Trx and Trx-related proteins. For example, green plants have seven distinct subtypes even in conventional Trx (generally, f-, m-, x-, y-, z-types in plastids, o-type in mitochondria, and h-type in cytosol). Biochemical and structural studies have indicated that different Trx subtypes have different midpoint redox potentials and protein surface charges, possibly conferring their different target recognition mechanisms (e.g. Yoshida et al. 2015). In addition, some Trx-related proteins are specifically found in photosynthetic organisms. One well-known example is the NADPH-Trx reductase C (NTRC); this protein contains both an NADPH-Trx reductase (NTR) domain and a Trx domain in a single polypeptide, and thus directly uses NADPH as a source of reducing power in the redox regulation (Serrato et al. 2004). Furthermore, some proteins containing the atypical Trx-like CXXC motif, including Trx-like2 (TrxL2; WCRKC) and atypical Cys His-rich Trx [ACHT; WC(G/A)SC], have been recently identified as the factor for oxidizing and thereby downregulating photosynthetic enzymes (Yoshida et al. 2018, Yokochi et al. 2021). Our understanding of functional specificity and redundancy of these multiple Trx and Trx-related proteins (hereafter we refer the Trx family proteins) is still incomplete, which warrants further studies (Yoshida and Hisabori 2023).

A recent genome study in Arabidopsis indicated that Trx family proteins are potentially encoded by more than 40 genes in total (Chibani et al. 2021). Given this gene multiplicity, it is important to grasp the evolutionary history of Trx family proteins, particularly focusing on their diversity and distribution. Previous phylogenetic analyses have suggested that Trx family proteins in plastids have chimeric origins; they were partly inherited from cyanobacteria by endosymbiotic gene transfer (EGT) and the rest were co-opted from ancestral non-photosynthetic eukaryotic components, usually with the acquisition of plastid-targeting signal sequences (e.g. Chibani et al. 2009, Balsera et al. 2014). For example, most of plastidial Trx subtypes (m-, x-, y-, z-types) seem to have cyanobacteria-related evolutionary origins. NTRC is also thought to be inherited from cyanobacteria. By contrast, TrxL2 and ACHT are not found in cyanobacteria. These reports are helpful to gain insights into evolutionary history of Trx family proteins; however, previous phylogenetic analyses contained several limitations to reach the convincing consensus, as exemplified by following issues: (i) the saturation in amino acid substitution of the short alignment of Trx primary structure (i.e. conventional Trx consists of only approx. 110 amino acids), (ii) the difference in evolutionary speed among Trx subtypes (i.e. conventional Trx is relatively conservative, leading to the branch length-related errors in phylogenetic estimation), and (iii) the sparse taxon sampling in the analyses (i.e. only limited photosynthetic organisms have been included in the analyses). Consequently, how the redox regulation system has been constructed during the evolution of photosynthetic organisms remains to be fully elucidated. Considering the increasing availability of genome databases in the last decade, a trial of comprehensive update in this issue is currently valuable.

Figure 1 shows the maximum-likelihood phylogenetic tree of Trx family proteins. In addition to newly available data on photosynthetic organisms (One Thousand Plant Transcriptomes Initiative 2019), those on several non-photosynthetic organisms (Richter et al. 2022) were also included. A total of 194 photosynthetic and non-photosynthetic organisms (including prokaryotes and eukaryotes) were eventually subjected to the analysis (see Supplementary Text 1). The phylogenetic tree attached with each taxon label and the corresponding alignment file are available as Supplementary Fig. S1 and Supplementary Data 1, respectively. Based on these, we estimated the gene numbers of several monophyletic Trx family proteins (Supplementary Table S1). Overall, these are highly useful as a reference dataset for diversity and distribution of Trx family proteins. Here we highlight the following possibilities: (i) Eukaryote-specific plastid-localized Trx family proteins (including Trx-f, ACHT, TrxL2, and Lilium6), which form a monophyletic group in the tree, probably originated from a single acquisition event of the plastid-targeting signal sequence by an ancestral Trx-h homologue. (ii) Although Trx-z was proposed to be prokaryotic origin, its cyanobacterial counterpart has not been identified. It seems likely that Trx-z was acquired by gene duplication of Trx-y/TrxQ. (iii) In secondary plants (e.g. Haptista and Cryptista), not only plastidial Trx subtypes but also cytosolic Trx-h might be inherited via EGT events and then maintained. (iv) Cryptophyta and Glaucophyta have maintained THIOM (Thioredoxin, mitochondrial; human TXN2)-type Trx, but the majority of Rhodophyta and green plants have lost it. In green plants, THIOM seems to have been replaced by Trx-o as a mitochondrial Trx. (v) Several Trx family proteins are absent in specific phyla (e.g. PpTrx-f2 in Angiosperms, TrxL2 in Chlorophyta, and Trx-m in most of Rhodophyta), which also indicates the diversity of the redox system in photosynthetic eukaryotes different from traditional Angiosperm models.

Figure 1.

Overall phylogenetic tree of Trx family proteins. The detailed method for the construction is described in the supplementary text. Small numbers at the branches of tree are bootstrap consensus estimates (%). Bar: 1 amino acid substitution per site. Please refer to the article PDF for a high-resolution version of this figure.

In summary, we report a comprehensive dataset for diversity and distribution of Trx family proteins in photosynthetic organisms. It seems reasonable to consider that divergence or convergence of genes for Trx family proteins is the key driving force for the evolutionary development of the redox regulation system and, finally, the adaptation to local environments in photosynthetic organisms. The present in silico investigation provides insights into these issues and represents a milestone for future redox research using a variety of photosynthetic organisms.

Author Contributions

T.S. and K.Y. conceived the study. T.S. performed the phylogenetic analyses. T.S. and K.Y. interpreted the data. K.Y. wrote the original manuscript. T.S. and K.Y. reviewed the manuscript.

Conflict of Interest

None declared.

Funding

Japan Society for the Promotion of Science (JSPS) KAKENHI (22 K19130 to K.Y.; 23H02498 to K.Y.; 23H04961 to K.Y.).

Data Availability

The data underlying this article will be shared upon reasonable request to the corresponding author.