Members of the Capsicum annuum CaTrxh Family Respond to High Temperature and Exhibit Dynamic Hetero/Homo Interactions

Climate change adversely affects the water and temperature conditions required for plant growth, leading to a decrease in yield. In high temperatures, oxidative stress causes cellular damage in plant cells, which is a negative factor for crop production. Thioredoxin (Trx) is a small redox protein containing a conserved WC(G/P)PC motif that catalyzes the exchange of disulfide bonds. It is known to play an important role in maintaining cellular redox homeostasis. Trx proteins are widely distributed across various subcellular locations, and they play a crucial role in responding to cellular stresses. In this study, seven CaTrxh-type genes present in pepper were identified and the CaTrxh-type family was classified into three subgroups. CaTrxh genes responded to heat stress. Moreover, subcellular locations of the CaTrxh family exhibited dynamic patterns in normal conditions, and we observed relocalizations in heat stress conditions. Each CaTrxh family protein member formed homo-/heteromeric protein complexes in BiFC assay. Unexpectedly, subgroup III CaTrxh9 and CaTrxh10 can recruit subgroup I and II CaTrxh proteins into the plasma membrane. Thus, the function of the CaTrxh-type family is expected to play a protective role in the cell in response to high-temperature stress via protein complex formations. CaTrxh may have potential applications in the development of crops with enhanced tolerance to oxidative stress.


Introduction
Crop production is decreasing due to global warming.Extreme temperature changes, droughts, and floods place great stress on plant growth [1].In response to high temperatures, plants produce a variety of reactive oxygen species (ROS) as signaling molecules, and excessive ROS accumulation causes oxidative damage to organelles, proteins, and membranes [2].To protect against oxidative damage, eukaryotic cells are equipped with a dynamic redox regulatory system [3].In general, redox reactions affect the activity of various proteins that play important roles in metabolic processes.
Thioredoxin (Trx) is evolutionarily conserved in diverse organisms and plays important roles in many cellular processes involving redox regulation [4,5].Trx is an about-12-kDa small protein with a redox-active disulfide motif (WCGPC) in a catalytic domain.Trx has a dithiol-disulfide oxidoreductase activity and functions as a reversible disulfide reductase that reduces target substrates [6].During the reducing process, two cysteine residues at the 'WCGPC' active site are oxidized and inactivated.Maintaining basal levels of intracellular ROS is an essential process for cell differentiation and proliferation.Trx can associate with antioxidant enzymes to protect against intracellular oxidative damage via excess ROS accumulation in the cell.For example, Arabidopsis thaliana Trx-h2-overexpressing Brassica napus transgenic plants have shown enhanced antioxidant activity and have exhibited strong salt stress tolerance [7].When Banana MaTrx12 was heterologously overexpressed in Trx-deficient yeast cells, the mutant yeast exhibited improved viability against oxidative stress conditions [8].Overexpression of Drohophila Trx-2 has also been shown to prolong lifespan and increase resistance to oxidative stress [9].These reports suggest that evolutionarily conserved Trx is a valuable genetic resource for the development of crops that can tolerate oxidative stress.
Plant Trx has evolved extensively, displaying a high degree of diversity compared to other organisms.The human has two TRX isoforms: TRX-1, found in the extracellular milieu, cytoplasm, and nucleus [10]; and TRX-2, which is located in the mitochondria and regulates mitochondrial metabolic activity [11].In the Arabidopsis genome, a total of 41 Trx genes exist, which are further divided into 18 typical (WCGPC active site) and 23 atypical (XCXXC active site) Trx genes [12,13].Plants higher than Arabidopsis are expected to have a more diverse Trx family.For example, 125 Trx were found in soybean (Glycine max) [14].Nearly 86 GhTrx (40 typical and 46 atypical types) were identified in the cotton genome (Gossypium hirsutum L.) [15].The Trx subfamily is also classified into seven types, namely, f, h, m, o, x, y, and z, based on sequence similarity, intracellular localization, and molecular function [13,16].Among them, the largest family of Trx is the h-type, with 10 isoforms in Arabidopsis.AtTrxh-type is again divided into subtypes I (h1, h3, and h4), II (h2, h7, and h8), and III (h9, CxxS1, CxxS2, and CxxC2) [16].In the rapeseed (Brassica napus) genome, it was confirmed that the Trxh subfamily consists of about 36 genes [7].
The Trxh-type function is still largely unknown.The AtTrxh-type proteins are expected to be present mainly in the cytoplasm, but most of the Trx proteins in crops are unknown.AtTrxh5 is associated with cytoplasmic oligomers of non-expressor PR1 (NPR1).NPR1 is translocated into the nucleus as a monomeric NPR1 and functions as a defense activator [17].It has also been found that AtTrxh5 has a selective protein denitrosylation activity in plant immunity [18].Interestingly, AtTrxh2 has been shown to exhibit an RNA chaperone-like activity under cold stress conditions and can distribute mRNA from the nucleus to the cytoplasm [19].Recently, tomato (Solanum lycopersicum) SlTrxh-overexpressing tobacco plants have shown enhanced oxidative stress tolerance under excess nitrate stress, and SITrxh protein is associated with tomato peroxiredoxin (SlPrx) protein [20].It can be expected that the functions of the subfamily belonging to the Trxh-type will be diverse.Identification of the Trxh-type in crops has not yet been [21] performed.
Pepper (Capsicum annuum), a member of the Solanaceae family, stands as one of the most crucial vegetable crops cultivated worldwide.Its versatility is showcased through its various uses as a spice, a medicinal ingredient, and as a staple in foods such as kimchi [21,22].As a thermophilic vegetable, peppers contribute to the global market [23].In this study, the CaTrxh-type subfamily identified in the hot pepper genome was analyzed.A total of seven Trxh-types were found, and we performed analysis of the CaTrxh protein sequence, gene expression, and subcellular localization.As a result, CaTrxh-type was highly conserved in the Solanaceae family of vegetables, including potato, tomato, and tobacco.The CaTrxh genes exhibited a significant response under heat stress conditions.The subcellular localizations of CaTrxh-type proteins have been observed in various intracellular locations.This diversification may imply complex biological functions of the CaTrxh-type family.In addition, CaTrxh protein members specifically formed protein complexes via homo-/heteromeric associations without the redox-active disulfide motif.It is expected that the characterization of the Trxh subfamily will enable the development of crops with strong antioxidant and chaperon-like functions that can respond to climate change.

Genome-Wide Identification and Characterization of the CaTrxh-Type Family in Pepper
While studies on Trx in various crops exist, research on the Trxh family in peppers is still lacking.Therefore, we first identified the Trxh-type family in peppers.Putative hot pepper Trxh-type members were identified using BLAST searches against the genome sequence for hot pepper C. annuum cv.CM334 and Zunla-1 (Table 1).We found seven CaTrxh-type genes through pepper genome analysis.The Trx domain structure is annotated by SMART (http://smart.embl-heidelberg.de/smart/batch.pl,accessed on 1 January 2023).The molecular weights and theoretical isoelectric points of each CaTrxh-type protein were analyzed using ExPASy (https://www.expasy.org/,accessed on 1 January 2023) (Table 2).From the identification data, we identified seven CaTrxh-type members.To confirm the phylogenetic relationship between Arabidopsis AtTrxh, which is well known for its function, and other important crop plants, a phylogenetic tree was constructed for the Trxh-type protein sequence.As shown in Figure 1A, CaTrxh family proteins were divided into three subgroups according to the classification of AtTrxh.OsTrxh4, OsTrxh5, OsTrxh7, and OsTrxh8 are considered redundant types.These results indicate that the functional and evolutionary properties of CaTrxh present in Solanaceae crops may be similar.

Gene Accession Number
Arabidopsis thaliana  Among the properties of the protein, the N-terminus is highly conserved with glycine in Trxh subgroups II and III.The glycine involved in N-myristoylation was not clearly present in subgroup I (Figure 1B).These results indicate that differences in post-translational modifications between group I and groups II-III result in functional diversity.It has been known that the attachment of myristoyl groups is essential for proteins involved in a variety of biological functions such as subcellular localization and signal transduction [24,25].Furthermore, subgroup I had putative N-α-acetylation Ala-2 residue.In subgroup III, Trxh-type proteins contained both putative Cys residues for palmitoylation and Gly residues for myristorylation (Figure 1B) [26].CaTrxh-type proteins also exhibited highly Among the properties of the protein, the N-terminus is highly conserved with glycine in Trxh subgroups II and III.The glycine involved in N-myristoylation was not clearly present in subgroup I (Figure 1B).These results indicate that differences in post-translational modifications between group I and groups II-III result in functional diversity.It has been known that the attachment of myristoyl groups is essential for proteins involved in a variety of biological functions such as subcellular localization and signal transduction [24,25].Furthermore, subgroup I had putative N-α-acetylation Ala-2 residue.In subgroup III, Trxhtype proteins contained both putative Cys residues for palmitoylation and Gly residues for myristorylation (Figure 1B) [26].CaTrxh-type proteins also exhibited highly conserved features of N-terminal modifications.This indicated that the N-terminal extension of CaTrxh-type proteins which has been discovered so far may play a more diverse role via post-translational modification.In CaTrxh-type proteins, the active site Cys residue and catalytic motif 'WCXPC' were completely conserved (Figure 1B).

Cis-Regulatory Element Analysis of CaTrxh-Type Gene Promoter
To understand the function of the CaTrxh family, we obtained a 1.5 kb promoter region of CaTrxh-type genes and analyzed the cis-acting regulatory element motifs (Table 3).It was found that motifs mainly responding to plant hormones were distributed in the promoter (Figure 2A,B).Abscisic acid (ABA)-responsive elements such as ABRE and MYC motifs were present in significant numbers of CaTrxh promoters.Basically, ABA regulates the regulation of these diverse processes, including physiological responses to cold, drought, and salinity stresses [27,28].ABRE, an ABA response element belonging to the G-box family, contains an 'ACGT' sequence known to be recognized by the plant bZIP transcription factor [29,30].Interestingly, five ABRE motifs were found in the promoter of CaTrxh9 (Figure 2A).Several MYC motifs are known to respond to cold stress and involved in regulating CBF gene by R2R3-type MYB transcription factors [31,32].Six MYC motifs were found in the promoter of CaTrxh1-2 and were abundantly included in the promoters of other CaTrxh genes (Figure 2A).Additionally, cis-acting elements related to the cold-induced expression (LTR) motif, MYB-binding motif, and drought-inducible (MBS) motif were each found to be associated with abiotic stress.Thus, we speculated that the LTR and MBS motifs might play a role in regulating the expression of CaTrxh genes in response to cold and drought stresses.
Table 3. Types and numbers of known cis-elements in the promoter regions of CaTrxh-type genes.

Cis-Element
Hormonerelated MYC cis-acting element involved in the abscisic acid responsiveness TCA-element cis-element involved in salicylic acid responsiveness

Tissue-Specific Gene Expression of CaTrxh Family
To gain further insight into the development and functional role of CaTrxh-type in each tissue, the expression pattern of CaTrxh-type was investigated via semi-quantitative RT-PCR.Gene expression of the CaTrxh-type family was confirmed in mature leaves (ML), senescent leaves (SL), flower (F), green fruit (GF), and breaker fruit (BF).All CaTrxh types was expressed in ML, SL, F, GF, and BF.CaTrxh1-1, CaTrxh1-2, CaTrxh1-3, CaTrxh2-1, and CaTrxh2-2 showed little change in expression levels; whereas subgroup III CaTrxh9 and CaTrxh10 showed reduced gene expression levels in BF (Figure 3).For CaTrxh10, gene expression is reduced in SL compared to ML.Thus, some CaTrxh genes are expected to affect developmental processes.

Tissue-Specific Gene Expression of CaTrxh Family
To gain further insight into the development and functional role of CaTrxh-type in each tissue, the expression pattern of CaTrxh-type was investigated via semi-quantitative RT-PCR.Gene expression of the CaTrxh-type family was confirmed in mature leaves (ML), senescent leaves (SL), flower (F), green fruit (GF), and breaker fruit (BF).All CaTrxh types was expressed in ML, SL, F, GF, and BF.CaTrxh1-1, CaTrxh1-2, CaTrxh1-3, CaTrxh2-1, and CaTrxh2-2 showed little change in expression levels; whereas subgroup III CaTrxh9 and CaTrxh10 showed reduced gene expression levels in BF (Figure 3).For CaTrxh10, gene expression is reduced in SL compared to ML.Thus, some CaTrxh genes are expected to affect developmental processes.

Expression Analysis of CaTrxh Genes in Response to Heat and Cold Stresses
The Trxh genes have been reported to be involved in oxidative tolerance in abiotic stress [33,34].We therefore investigated the gene expression of the CaTrxh family in response to cold and heat stresses.CaTrxh1-3, CaTrxh2-1, CaTrxh2-2, and CaTrxh9 responded to heat stress at 12 h.The expression levels of CaTrxh1-3 and CaTrxh2-1 in pepper decreased under heat stress compared to the mock control (Figure 4).The expression levels of CaTrxh9 were highly increased under heat stress at 12 h (Figure 4).However, transcript levels of CaTrxh1-1, CaTrxh1-2, and CaTrxh10 were not significantly changed under both stresses.

Expression Analysis of CaTrxh Genes in Response to Heat and Cold Stresses
The Trxh genes have been reported to be involved in oxidative tolerance in abiotic stress [33,34].We therefore investigated the gene expression of the CaTrxh family in response to cold and heat stresses.CaTrxh1-3, CaTrxh2-1, CaTrxh2-2, and CaTrxh9 responded to heat stress at 12 h.The expression levels of CaTrxh1-3 and CaTrxh2-1 in pepper decreased under heat stress compared to the mock control (Figure 4).The expression levels of CaTrxh9 were highly increased under heat stress at 12 h (Figure 4).However, transcript levels of CaTrxh1-1, CaTrxh1-2, and CaTrxh10 were not significantly changed under both stresses.

Subcellular Localization of the CaTrxh Family
Trxh proteins present in plants have evolved various isoforms involved in the redox regulation of a wide range of metabolic processes in various intracellular compartments [35].The subcellular localization of Trxh family proteins with different targets or substrates makes significant differences [36,37].The subcellular localization of Trxh-type in crops is not yet well understood.Therefore, we decided to fuse CaTrxh genes with GFP to determine their subcellular localization.As shown in Figure 5A, the GFP signals of 35S::CaTrxh1-1-GFP, 35S::CaTrxh1-2-GFP, and 35S::CaTrxh1-3-GFP, classified into subgroup I, were equally observed in the cytoplasm and nucleus.Thus, subgroup I of CaTrxh is predicted to have a similar function not only in the cytoplasm but also in the nucleus.
In subgroup II, OsTrxh4 has been identified as an interactor of rice phosphate overaccumulator 2 (OsPHO2), which is an E2 ubiquitin conjugase, to regulate phosphate homeostasis.Interestingly, subcellular localizations of OsTrxh4 were detected mainly in the cytoplasm and ER/Golgi membrane [38].To further confirm their subcellular localization, 35S::CaTrxh2-1-GFP and 35S::CaTrxh2-2-GFP were expressed together with Golgi-resident fusion protein (Man49-mCherry), respectively.GFP signals of 35S::CaTrxh2-1-GFP and 35S::CaTrxh2-2-GFP were perfectly merged with Golgi marker (Figure 5B and Figure S2).Although the effective reducing agents of chloroplasts are mainly known as Trx y, Trx x, Trx m, and Trx f [39], the CaTrxh-type family was not located in chloroplasts.For subgroup II, GFP signals were occasionally detected in the cytoplasm and the nucleus (Figure S1A).In the case of subgroup III, GFP was observed not only in the cell membrane but also weakly in the nucleus (Figure S1B).The subcellular localization of CaTrxh is believed to be highly variable.In subgroup III, GFP signals of 35S::CaTrxh9-GFP and 35S::CaTrxh10-GFP were merged with plasma membrane (PM)-localized SAUR63-CFP marker (Figure 5C).Likewise, the subcellular localization of AtTrxh9 was at the plasma membrane [40].SlTrxh9 and NtTrxh9 proteins formed a small clade in subgroup III (Figure 1A).This clade was expected to have specialized functions in the cell membrane through N-myristoylation.

Subcellular Localization of the CaTrxh Family
Trxh proteins present in plants have evolved various isoforms involved in the redox regulation of a wide range of metabolic processes in various intracellular compartments [35].The subcellular localization of Trxh family proteins with different targets or substrates makes significant differences [36,37].The subcellular localization of Trxh-type in crops is not yet well understood.Therefore, we decided to fuse CaTrxh genes with GFP to determine their subcellular localization.As shown in Figure 5A, the GFP signals of 35S::CaTrxh1-1-GFP, 35S::CaTrxh1-2-GFP, and 35S::CaTrxh1-3-GFP, classified into subgroup I, were equally observed in the cytoplasm and nucleus.Thus, subgroup I of CaTrxh is predicted to have a similar function not only in the cytoplasm but also in the nucleus.

CaTrxh Protein Changes in Subcellular Localization under Heat Stress
It has been reported that under cold stress conditions, demyristoylation of cytoplasmic AtTrxh2 allows for intracellular translocation into the nucleus and the subsequent dissociation from CBF oligomers, a transcriptional regulator [41].As shown in Figure 1, Trxh-type subgroups II and III had characteristics of N-myristoylation, which showed a conserved Gly residue in position 2.These biochemical properties of N-myristoylation are known to alter their intracellular localization [26].Furthermore, subgroup III contained conserved N-terminal Cys residue to be palmitoylated (Figure 1B).This Cys residue is also known to be an important modification site for plasma membrane localization [26].In subgroup II, OsTrxh4 has been identified as an interactor of rice phosphate overaccumulator 2 (OsPHO2), which is an E2 ubiquitin conjugase, to regulate phosphate Based on the results that the CaTrxh2-2 and CaTrxh9 genes' response to heat stress (Figure 4), we hypothesized that heat stress may cause changes in the subcellular localization of CaTrxh protein.CaTrxh1-1-GFP showed no change in its subcellular location

CaTrxh Proteins Were Able to Make Complexes via Homomeric Association
Trx protein is an important intracellular protein that regulates protein activity and is known to interact with various targets [13,42].However, since research on the interaction between thioredoxin family proteins has not yet been conducted, we used bimolecular fluorescence complementation (BiFC) analysis to determine the interaction between the CaTrxh-type protein family (Figure 7A).In CaTrxh subgroup I, combinations of CaTrxh1-1-YFP N and CaTrxh1-1-YFP C or CaTrxh-1-2-YFP N and CaTrxh1-2-YFP C or CaTrxh1-3-YFP N and CaTrxh1-3-YFP C were transiently expressed in tobacco plants.The reconstituted YFP signals were observed in the nucleus and cytoplasm (Figure 7B).The results showed that all CaTrxh subgroups I formed homodimers.Similarly, homodimerization of subgroup II members CaTrxh2-1 and CaTrxh2-2 was tested.The YFP signals detected that CaTrxh2-1-YFP N and CaTrxh2-1-YFP C or CaTrxh2-2-YFP N and CaTrxh2-2-YFP C each formed a homodimeric complex in the Golgi region (Figure 7C).CaTrxh9 and CaTrxh10, corresponding to group III, each formed a homodimeric complex in the plasma membrane (Figure 7D).Thus, CaTrxh family proteins within each group will function through homodimerization.

CaTrxh Proteins Were Able to Make Complexes via Homomeric Association
Trx protein is an important intracellular protein that regulates protein activity and is known to interact with various targets [13,42].However, since research on the interaction between thioredoxin family proteins has not yet been conducted, we used bimolecular fluorescence complementation (BiFC) analysis to determine the interaction between the CaTrxh-type protein family (Figure 7A).In CaTrxh subgroup I, combinations of CaTrxh1-1-YFP N and CaTrxh1-1-YFP C or CaTrxh-1-2-YFP N and CaTrxh1-2-YFP C or CaTrxh1-3-YFP N and CaTrxh1-3-YFP C were transiently expressed in tobacco plants.The reconstituted YFP signals were observed in the nucleus and cytoplasm (Figure 7B).The results showed that all CaTrxh subgroups I formed homodimers.Similarly, homodimerization of subgroup II members CaTrxh2-1 and CaTrxh2-2 was tested.The YFP signals detected that CaTrxh2-1-YFP N and CaTrxh2-1-YFP C or CaTrxh2-2-YFP N and CaTrxh2-2-YFP C each formed a homodimeric complex in the Golgi region (Figure 7C).CaTrxh9 and CaTrxh10, corresponding to group III, each formed a homodimeric complex in the plasma membrane (Figure 7D).Thus, CaTrxh family proteins within each group will function through homodimerization.

CaTrxh Proteins Exhibited Heteromeric Protein Complexes
We previously showed through BiFC that CaTrxh proteins form homodimerization with each other.Then, since CaTrxh exists in the same intracellular location within each group, we hypothesized that it may function through a heterodimer.The combination of CaTrxh1-1-YFP N and CaTrxh1-2-YFP C or CaTrxh1-1-YFP N and CaTrxh1-3-YFP C or CaTrxh1-We then hypothesized whether CaTrxh, which has different subcellular localizations, forms heterodimeric complexes through protein interactions.To test this, we selected CaTrxh from each group and performed BiFC.Interestingly, strong YFP signals were observed partially in the Golgi region and nucleus in the group 1 CaTrxh1-1-YFP N and group 2 CaTrxh2-1-YFP C (Figure 8D).Through heterodimerization of different groups of Catrxh proteins, the intracellular localization of heteromeric CaTrxh proteins is altered.Similarly, we confirmed that group III CaTrxh9, which mainly exists in the plasma membrane, moves group I CaTrxh1-1 or group II Catrxh2-1 to the cell membrane (Figure 8E,F).These results show that the intracellular location of CaTrxh can be regulated in various ways by forming homodimeric-/heterodimeric complexes and are expected to affect target protein regulation.

Thioredoxin Protein Activity Is Not Related with CaTrxh Protein Complex and Relocalization
Since it is already well known that the N-myristoylation site is an important factor in determining the intracellular localization of Trxh, we tested whether mutations in the redox-active disulfide motif, which is an important part of CaTrxh enzymatic activity, affect its subcellular localization and protein interactions.First, the Cys residue of the catalytic motif 'WCXPC' mutated to 'WAXPA'.CaTrxh1-1(C/A)-GFP, CaTrxh2-1(C/A)-GFP, and CaTrxh9(C/A)-GFP constructs were transiently expressed in N. benthamiana.The results showed that mutations in the catalytic motif did not affect subcellular localization (Figure S4).We tested whether mutations in the catalytic motif affected the formation of homodimers and heterodimers.As shown in Figure 9A-C, the homodimer of CaTrxh was observed normally without any intracellular changes.In the case of the heterodimer of CaTrxh, CaTrxh2-1(C/A)-YFP N corresponding to group II interacted with CaTrxh1-1(C/A)-YFP C of group I, resulting in a strong YFP signal in the Golgi and nucleus (Figure 9D).Group I CaTrxh1-1(C/A)-YFP N or group II CaTrxh2-1(C/A)-YFP N formed a strong YFP signal at the plasma membrane with CaTrxh9(C/A)-YFP C (Figure 9E,F).As a result, the cysteine catalytic motif is an important region in redox regulation but is not related to the homo-/heterocomplex or intracellular localization of CaTrxh.
We found that some CaTrxh proteins have altered intracellular localization upon hightemperature stress.And the function of CaTrxh proteins does not seem to depend on the catalytic motif.The diverse intracellular locations and relocalizations of CaTrxh through protein interactions result in the production of a variety of intracellular target proteins.Based on our results, the regulatory actions of CaTrxh are briefly presented in Figure 10.We found that some CaTrxh proteins have altered intracellular localization up high-temperature stress.And the function of CaTrxh proteins does not seem to depe on the catalytic motif.The diverse intracellular locations and relocalizations of CaTr through protein interactions result in the production of a variety of intracellular tar proteins.Based on our results, the regulatory actions of CaTrxh are briefly presented in Figure 10.
Figure 10.A model for subcellular localization and protein-protein interactions of the CaTrxh family under heat stress.The CaTrxh-type family is largely divided into three groups, and their intracellular locations are quite specific subcellular localizations.Subgroup I is located in the nucleus and cytoplasm.Subgroup II is predominantly located in the Golgi apparatus and cytosolic regions.Subgroup III is mainly located in the plasma membrane.Interestingly, all CaTrxh family proteins can form homodimers and heterodimers.In the BiFC results of subgroup III and subgroup I or subgroup II present in the plasma membrane, these CaTrxh subgroup III proteins can move subgroup I or subgroup II proteins to the cell membrane.Additionally, when exposed to heat stress, subgroup I showed no change in intracellular location, whereas subgroup II and subgroup III showed a shift toward the tonoplast.This intracellular localization is expected to play a role in regulating the redox form of various target proteins under high-temperature stress or protecting cells through chaperone function.'?' indicates the possibility that CaTrxh directly regulates the activity of a transcription factor (TF).

Discussion
Trx proteins present in plants are largely divided into six major clusters based on Arabidopsis.Among them, the Trxh-type has a variety of isotypes compared to other Trx family genes.In a representative functional study, biological and abiotic stresses on Arabidopsis AtTrxh2 and AtTrxh5 were known.These Trxh-type proteins have disulfide reductase activity as well as chaperon activity or denitrosation activity [18,19].These results lead to interest in the functions of the Trxh-type present in various crops [43].CaTrxh-type is highly conserved in Solanaceae crops such as tomato, potato, and tobacco.Some of the CaTrxh genes showed a tendency to decrease or increase gene expression under heat stress (Figure 4).Thus, CaTrxh-type family could play an essential role in abiotic stresses.Additionally, CaTrxh family proteins were localized in the cytoplasm and other subcellular organelles.There were CaTrxh-type proteins present at specific subcellular locations in the cell.This study serves as the basis for functional studies of the CaTrxh-type family, which is essential for future developments in crops with improved stress responses.

Discussion
Trx proteins present in plants are largely divided into six major clusters based on Arabidopsis.Among them, the Trxh-type has a variety of isotypes compared to other Trx family genes.In a representative functional study, biological and abiotic stresses on Arabidopsis AtTrxh2 and AtTrxh5 were known.These Trxh-type proteins have disulfide reductase activity as well as chaperon activity or denitrosation activity [18,19].These results lead to interest in the functions of the Trxh-type present in various crops [43].CaTrxh-type is highly conserved in Solanaceae crops such as tomato, potato, and tobacco.Some of the CaTrxh genes showed a tendency to decrease or increase gene expression under heat stress (Figure 4).Thus, CaTrxh-type family could play an essential role in abiotic stresses.Additionally, CaTrxh family proteins were localized in the cytoplasm and other subcellular organelles.There were CaTrxh-type proteins present at specific subcellular locations in the cell.This study serves as the basis for functional studies of the CaTrxh-type family, which is essential for future developments in crops with improved stress responses.CaTrxh-type proteins are identified as potential antioxidant proteins or chaperon-like proteins.
Although Trxh-type mainly presented in the cytoplasm, the intracellular localization of Trxh-type protein is not considered critical.Recently, it has been reported that the perennial plant (Saussurea involucrata) SikTrxh, belonging to h-type subgroup I, is completely present in chloroplasts [44].AtTrxh2 and AtTrxh9 are also present in the mitochondria and plasma membrane, respectively [40].Similarly, we also found that CaTrxh9-GFP is localized to the plasma membrane and CaTrxh2-2-GFP in located in the Golgi and cytosolic regions (Figure 5).The intracellular localization of Trxh-types can be predicted but cannot be specified, and their functions will also be more diversified.On the other hand, it can be predicted that Trx proteins of similar groups in the phylogenetic tree will have almost-similar subcellular localizations or functions.For example, loss-of-function mutant phenotypes of AtTrxh9 showed strong dwarfism and developmental defects [40].If the CaTrxh9 gene is suppressed by virus-induced gene silencing (VIGS) in hot pepper, it will be developmentally deficient, similar to the AtTrxh9 mutant.Furthermore, membranelocalized Trxh-type proteins could associate with a target protein different from that of cytosolic Trxh proteins.
There are some reports that Trxh is a useful genetic resource for overcoming various oxidative stresses.In other words, the enzymatic properties of redox regulation, which are specialized for photosynthesis, seem to form various isotypes in plants as a way to overcome additional environmental stress.Recently, AtTrxh2 have been shown to regulate CBF transcription factor activity upon cold stress.It was reported that AtTrxh2 protein and mRNA expression levels were not different under cold stress or warm conditions [41].Unlike Arabidopsis, CaTrxh-type genes present in pepper did not respond significantly to cold stress.However, we found that CaTrxh2-1, CaTrxh2-2, and CaTrxh9 genes response to high temperature.Thus, the CaTrxh-type gene would be functionally related to climate change phenomena such as temperature stress.AtTrxh3 has chaperone activity, and transgenic Arabidopsis plants overexpressing it showed improved heat stress tolerance even without disulfide reduction activity [45].Our results showed that mutations in the CaTrxh cysteine active motif did not affect the subcellular localization or protein interactions.The CaTrxh-type family can act as both chaperones and disulfide reductases and play an important role in preserving the functional integrity of key target proteins; in particular, its function will be even more important under oxidative stress conditions.
Many members of the Trxh-type gene family and their diverse subcellular locations may be closely related to their functions.The most effective approach to engineering crops to overcome climate change is to adopt methods that enhance tolerance to oxidative stress [43].In this study, we identified seven CaTrxh-types genes and confirmed that CaTrxhtype genes respond to abiotic stress.We also found that CaTrxh proteins were present at specific subcellular locations in plant cells and that their locations changed in response to heat stress (Figure 4).It is necessary to make transgenic plants using the CaTrxh gene and to test what kind of phenotype they have in response to high-temperature stress.CaTrxh-type genes may have a useful high-temperature tolerance phenotype.
Recently, the THIOREDOXIN INTERACTING RECEPTOR KINASE (TIRK) transmembrane receptor was discovered as a new target for AtTrxh5.AtTrxh5 modulates the S-nitrosylation of TIRK and participates in redox-dependent regulation of this receptor [46].In particular, it is well known that NPR1 undergoes structural changes through protein interaction with AtTrxh5 and migrates to the nucleus to activate defense [17,47].We therefore wondered if CaTrxh-family protein could interact with CaNPR1 present in pepper, and whether this is a specific and evolutionally conserved regulation mechanism.Consequently, the diversified CaTrxh-type family is thought to have various strategies for coping with oxidative stress in crops.

Plant Materials
Seeds of hot pepper (Capsicum annuum L. cv.Bugang) and Nicotiana benthamiana were sown in commercial bed soil.After the germination of pepper or tobacco seeds on a plate with 100% moisture, the plants were individually transplanted into 500 mL pots.The plants were cultivated under the following conditions: a growth room/growth chamber with a 16 h LED light (color temperature ranging from 5000 K to 6500 K) and an 8 h dark photoperiod at 22 ± 2 • C with 60-70% relative humidity.Four-week-old seedlings were utilized for the subsequent treatments.For heat and cool treatments, hot pepper plants were grown for 4 weeks, provided with enough water, and then treated with mock (22 • C), heat (42 • C), and cold (4 • C) for 0, 12, and 24 h.Three biological replicate samples were mRNA extracted via isolRNA kit (TaKaRa, Kusatsu, Japan) in accordance with the commercial protocol.Briefly, the sample tissue was rapidly frozen using liquid nitrogen (LN2) and subsequently finely ground with a mortar and pestle.Following this, 1000 µL of Trizol RNAiso solution was added, thoroughly mixed, and left at room temperature for 20 min.The mixture was then centrifuged at 13,000 rpm and 4 • C for 5 min.The upper layer was carefully transferred to a new tube, and 500 µL of isopropanol was added.After incubating at room temperature for 10 min, the solution was centrifuged at 13,000 rpm for an additional 10 min at 4 • C to obtain the RNA pellet.For cDNA synthesis, total RNA 5 µg was reacted with MMLV cDNA synthesis kit (ELPIS BIO).For qPCR experiments, SYBR Green kit (Biorad and KAPAbio) was used.Data normalization was performed with a reference gene, CaActin.All primers used for semi-quantitative RT-PCR and qPCR are presented in Supplementary Table S1.

Identification, Protein Sequence Alignment, and Phylogenetic Tree of CaTrxh-Type Subfamily
CaTrxh-type genes were manually searched and selected via the Solanaceae Genomics Network (https://solgenomics.net/, accessed on 12 December 2022) and Pepper genome (http://peppergenome.snu.ac.kr/blast.php,accessed on 20 December 2022).Molecular weight and isoelectric point of pepper CaTrxh-type proteins were calculated by a protein isoelectric point calculator.CaTrxh-type protein sequences were aligned with identified Arabidopsis, tobacco, and tomato Trxh protein sequences via the neighbor joining method.Phylogenetic analysis was performed in MEGA-X [48].

BiFC Analysis and Subcellular Localization Analysis
For the BiFC assay, the CaTrxh gene was cloned into pSPYNE and pSPYCE vectors, each containing a split YFP at the C-terminus, and subsequently transformed into Agrobacterium tumefaciens GV3101.Agro-cells containing the 35S::CaTrxh-YFP N and 35S::CaTrxh-YFP C constructs were co-delivered to tobacco leaves.The reconstructed YFP signal was observed via confocal microscopy.Additionally, to determine the subcellular localization of the CaTrxh protein, all genes of the CaTrxh-type family were fused with GFP at the N-terminus.

Figure 1 .
Figure 1.Phylogenetic analysis and multiple sequence alignment of hot pepper CaTrxh-type proteins.(A) Phylogenetic analysis of the CaTrxh-type family in five plant species.Phylogenetic analysis was performed in MEGA-X, and trees were generated using the neighbor joining method with a bootstrap of 1000 cycles.Trxh proteins are divided into three subgroups as indicated.(B) Protein sequence alignment analysis of hot pepper CaTrxh-type with other Trxh-types.Predicted N-α-acetylation Ala-2 in subgroup I is indicated in blue boxes.In subgroups II and III, Gly-2, which undergoes N-myristoylation, is indicated in red boxes.N-terminal Cys residue in subgroup III, predicted to be palmitoylated, is indicated in purple boxes.The active-site Cys residues and the catalytic motif are indicated by 'WCXPC'.

Figure 1 .
Figure 1.Phylogenetic analysis and multiple sequence alignment of hot pepper CaTrxh-type proteins.(A) Phylogenetic analysis of the CaTrxh-type family in five plant species.Phylogenetic analysis was performed in MEGA-X, and trees were generated using the neighbor joining method with a bootstrap of 1000 cycles.Trxh proteins are divided into three subgroups as indicated.(B) Protein sequence alignment analysis of hot pepper CaTrxh-type with other Trxh-types.Predicted N-α-acetylation Ala-2 in subgroup I is indicated in blue boxes.In subgroups II and III, Gly-2, which undergoes N-myristoylation, is indicated in red boxes.N-terminal Cys residue in subgroup III, predicted to be palmitoylated, is indicated in purple boxes.The active-site Cys residues and the catalytic motif are indicated by 'WCXPC'.

Figure 2 .
Figure 2. Promoter analysis of CaTrxh-type genes.(A,B) The 1.5 kb promoter of CaTRX h-type genes was analyzed via PlantCARE.Important plant hormone-related motifs and stress-related motifs were placed in the promoters using various shapes.

Figure 2 .
Figure 2. Promoter analysis of CaTrxh-type genes.(A,B) The 1.5 kb promoter of CaTRX h-type genes was analyzed via PlantCARE.Important plant hormone-related motifs and stress-related motifs were placed in the promoters using various shapes.

Figure 3 .
Figure 3. Expression patterns of CaTrxh genes in different developmental stages and tissues.Gene expression of the CaTrxh-type family was analyzed in mature leaves (ML), senescent leaves (SL), flower (F), green fruit (GF), and breaker fruit (BF) by RT-PCR.The CaAct gene was used as a control.

Figure 3 .
Figure 3. Expression patterns of CaTrxh genes in different developmental stages and tissues.Gene expression of the CaTrxh-type family was analyzed in mature leaves (ML), senescent leaves (SL), flower (F), green fruit (GF), and breaker fruit (BF) by RT-PCR.The CaAct gene was used as a control.

Figure 4 .
Figure 4. Analysis of gene expression patterns of the CaTrxh family under low-and high-temperature stress.Peppers grown for 4 weeks were exposed to stress at 4 °C and 42 °C for 12 h and 24 h, respectively, and gene expression was tested by qRT-PCR.The CaAct gene was used as a control.Error bars indicate the standard deviations of three independent qRT-PCR biological replicates.Asterisks indicate significant differences from the control using the unpaired Student's t-test (* p < 0.05 and ** p < 0.01).

Figure 4 .
Figure 4. Analysis of gene expression patterns of the CaTrxh family under low-and high-temperature stress.Peppers grown for 4 weeks were exposed to stress at 4 • C and 42 • C for 12 h and 24 h, respectively, and gene expression was tested by qRT-PCR.The CaAct gene was used as a control.Error bars indicate the standard deviations of three independent qRT-PCR biological replicates.Asterisks indicate significant differences from the control using the unpaired Student's t-test (* p < 0.05 and ** p < 0.01).

Figure 5 .
Figure 5. Analysis of various subcellular localizations of the CaTrxh-type family.Each CaTrxh gene was cloned into a 35S::GFP vector (C-terminal GFP fusion), and Agrobacterium-mediated transient assay was performed in N. benthamiana.After infiltration, the GFP signal was checked using a confocal microscope.(A) Subcellular localization of subgroup I members: CaTrxh1-1-GFP, CaTrxh1-2-GFP, and CaTrxh1-3-GFP.The GFP signal of a member of group I was merged with the chloroplast auto-signal (Red).(B) Subcellular localization of subgroup II members: CaTrxh2-1-GFP and CaTrxh2-2-GFP.The GFP signal of a member of group II was merged with Man49-mCherry, a Golgi marker.(C) Subcellular localization of subgroup III members: CaTrxh9-GFP and CaTrxh10-GFP.The GFP signal of group III member was merged with SAUR63-CFP, a plasma membrane marker.Scale bar: 50 µm.

Figure 5 .
Figure 5. Analysis of various subcellular localizations of the CaTrxh-type family.Each CaTrxh gene was cloned into a 35S::GFP vector (C-terminal GFP fusion), and Agrobacterium-mediated transient assay was performed in N. benthamiana.After infiltration, the GFP signal was checked using a confocal microscope.(A) Subcellular localization of subgroup I members: CaTrxh1-1-GFP, CaTrxh1-2-GFP, and CaTrxh1-3-GFP.The GFP signal of a member of group I was merged with the chloroplast auto-signal (Red).(B) Subcellular localization of subgroup II members: CaTrxh2-1-GFP and CaTrxh2-2-GFP.The GFP signal of a member of group II was merged with Man49-mCherry, a Golgi marker.(C) Subcellular localization of subgroup III members: CaTrxh9-GFP and CaTrxh10-GFP.The GFP signal of group III member was merged with SAUR63-CFP, a plasma membrane marker.Scale bar: 50 µm.

Figure 9 .
Figure 9. Cysteine active motif mutation does not affect homodimer and heterodimer interacti in the CaTrxh family.(A-C) Mutations in the cysteine active motif of CaTrxh family proteins do affect homodimerization and subcellular localization.CaTrxh1-1(C/A)-YFP N and CaTrxh1-1(C/ YFP C or CaTrxh2-1(C/A)-YFP N and CaTrxh2-1(C/A)-YFP C or CaTrxh9(C/A)-YFP N and CaTrxh9(C/ YFP C , in which two cysteine residues of the CXXPC motif were mutated, were transiently expres in tobacco, and were observed via confocal microscopy.(D-F) Intergroup heterodimer format and intracellular localization of CaTrxh family proteins with cysteine active motif mutations w not affected.CaTrxh1-1(C/A)-YFP N and CaTrxh1-1(C/A)-YFP C or CaTrxh2-1(C/A)-YFP N and CaTrx 1(C/A)-YFP C or CaTrxh9(C/A)-YFP N and CaTrxh9(C/A)-YFP C were coexpressed in tobacco plants, a the reconstituted YFP signals were observed using confocal microscopy.Scale bar: 50 µm.

Figure 9 .
Figure 9. Cysteine active motif mutation does not affect homodimer and heterodimer interactions in the CaTrxh family.(A-C) Mutations in the cysteine active motif of CaTrxh family proteins do not affect homodimerization and subcellular localization.CaTrxh1-1(C/A)-YFP N and CaTrxh1-1(C/A)-YFP C or CaTrxh2-1(C/A)-YFP N and CaTrxh2-1(C/A)-YFP C or CaTrxh9(C/A)-YFP N and CaTrxh9(C/A)-YFP C , in which two cysteine residues of the CXXPC motif were mutated, were transiently expressed in tobacco, and were observed via confocal microscopy.(D-F) Intergroup heterodimer formation and intracellular localization of CaTrxh family proteins with cysteine active motif mutations were not affected.CaTrxh1-1(C/A)-YFP N and CaTrxh1-1(C/A)-YFP C or CaTrxh2-1(C/A)-YFP N and CaTrxh2-1(C/A)-YFP C or CaTrxh9(C/A)-YFP N and CaTrxh9(C/A)-YFP C were coexpressed in tobacco plants, and the reconstituted YFP signals were observed using confocal microscopy.Scale bar: 50 µm.

Figure 10 .
Figure10.A model for subcellular localization and protein-protein interactions of the CaTrxh family under heat stress.The CaTrxh-type family is largely divided into three groups, and their intracellular locations are quite specific subcellular localizations.Subgroup I is located in the nucleus and cytoplasm.Subgroup II is predominantly located in the Golgi apparatus and cytosolic regions.Subgroup III is mainly located in the plasma membrane.Interestingly, all CaTrxh family proteins can form homodimers and heterodimers.In the BiFC results of subgroup III and subgroup I or subgroup II present in the plasma membrane, these CaTrxh subgroup III proteins can move subgroup I or subgroup II proteins to the cell membrane.Additionally, when exposed to heat stress, subgroup I showed no change in intracellular location, whereas subgroup II and subgroup III showed a shift toward the tonoplast.This intracellular localization is expected to play a role in regulating the redox form of various target proteins under high-temperature stress or protecting cells through chaperone function.'?' indicates the possibility that CaTrxh directly regulates the activity of a transcription factor (TF).

Table 2 .
Prediction of physicochemical properties of CaTrxh family.