Functional Characterization of Sugar Beet M14 Antioxidant Enzymes in Plant Salt Stress Tolerance

Salt stress can cause cellular dehydration, which induces oxidative stress by increasing the production of reactive oxygen species (ROS) in plants. They may play signaling roles and cause structural damages to the cells. To overcome the negative impacts, the plant ROS scavenging system plays a vital role in maintaining the cellular redox homeostasis. The special sugar beet apomictic monosomic additional M14 line (BvM14) showed strong salt stress tolerance. Comparative proteomics revealed that six antioxidant enzymes (glycolate oxidase (GOX), peroxiredoxin (PrxR), thioredoxin (Trx), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase3 (DHAR3)) in BvM14 were responsive to salt stress. In this work, the full-length cDNAs of genes encoding these enzymes in the redox system were cloned from the BvM14. Ectopic expression of the six genes reduced the oxidative damage of transgenic plants by regulating the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), ascorbic acid (AsA), and glutathione (GSH), and thus enhanced the tolerance of transgenic plants to salt stress. This work has charecterized the roles that the antioxidant enzymes play in the BvM14 response to salt stress and provided useful genetic resources for engineering and marker-based breeding of crops that are sensitive to salt stress.


Introduction
Wild sugar beet (Beta corolliflora Zoss.) has excellent characteristics of drought resistance, frost resistance, salt tolerance, cold tolerance, and apomixis. In the early stage of the study, diploid cultivated beet (B. vulgaris L.) and tetraploid wild sugar beet (B. corolliflora Zoss.) were crossed by distant hybridization. After obtaining allotriploid, they were further backcrossed with cultivated sugar beet, and the M14 with the wild sugar beet chromosome 9 (BvM14) was selected for apomixix and high salt tolerance. It is a rare germplasm for studying plant salt stress tolerance mechanisms [1][2][3][4][5].
Reactive oxygen species (ROS), including superoxide anions (O 2 •− ), hydroxyl radicals (HO 2 • ), hydrogen peroxide (H 2 O 2 ), and singlet oxygen ( 1 O 2 ), play an important role in plant metabolism, signal transduction, photosynthesis regulation, bacterial defense, and cell apoptosis [6][7][8]. Salinity affects plants growth and development through osmotic stress, ion toxicity, overproduction of ROS, and oxidative stress [9]. It is known that chloroplasts, mitochondria, peroxisomes, apoplast, and plasma membranes are the main sites of cellular ROS generation [10]. Overproduction of ROS can lead to severe damage of protein, membrane lipid, DNA, and other cellular components [11]. To cope with this challenge, plants have antioxidative mechanisms that consist of enzymatic and non-enzymatic components to regulate ROS synthesis and scavenging. The antioxidant enzymes of ROS scavenging in plants mainly include superoxide dismutase (SOD), ascorbate perxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and thioredoxin (Trx) [12][13][14]. Some antioxidative genes have been cloned from rice [12], Arabidopsis [15], maize [14], and soybean [16], while only partial sequences of peroxisome APX gene coding region and GOX gene have been obtained from salt-tolerant sugar beet [17]. However, there are few reports about the different roles of these enzymes under salt stress.
In the past years, the salt tolerance characteristics of BvM14 have been wellstudied [1,2,4,[18][19][20]. The differentially expressed proteins (DEPs) under salt stress (0, 200, 400 mM) have been studied by iTRAQ LC-MS/MS. A total of 76 DEPs have been identified in leaves of the BvM14. These proteins involve photosynthesis, metabolism, protein synthesis, protein folding and degradation, stress and defense, cell structure, transcription, and transport processes. Among them, six main proteins (GOX, PrxR, Trx, APX, DHAR3, and MDHAR) of the antioxidant enzymes system changed most significantly in the ROS scavenging system under salt stress [4]. However, to the best of our knowledge, salt tolerance of the GOX, PrxR, Trx, APX, DHAR3, and MDHAR genes and their relationships have not been characterized in sugar beet M14.
In this work, we aimed to investigate the following related questions. What are the reactions that are directly responsible for these six genes in the antioxidant enzyme system during salt stress? What is the relationship between these six genes in antioxidant enzyme system under salt stress conditions? How important are the six genes in antioxidant enzyme system during salt stress? To answer these questions, molecular biological methods were used to evaluate the functions of the six major antioxidant enzymes in BvM14 under salt stress [3,4]. We generated a complete set of single mutants for the six key genes in Arabidopsis and analyzed the function of the six genes under salt stress. Understanding the mechanism of ROS scavenging allows for a powerful strategy to enhance crop salt tolerance.

Isolation and Sequence Analysis of Genes
Total RNA was isolated and subjected to reverse transcription by using the Super-ScriptTM III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA). The full-length coding regions were PCR-amplified from the sugar beet M14 cDNA with gene specific primers (Supplementary Table S1).

Quantitative Real-Time PCR Analysis
Quantitative real-time PCR analysis (qRT-PCR) analyses were performed using SYBR Premix ExTaqTM II Mix (TaKaRa, Shiga, Japan). GAPDH (glyceraldehyde-3-phosphate dehydrogenase, accession no. DQ355800) was used as a reference. The expression levels of all candidate genes were analyzed by the 2 −∆∆CT CT method. The primers used for qRT-PCR were listed in Supplementary Table S1 [1,2,22].

Generation and Phenotypic Analyses of Transgenic A. thaliana
At present, the genetic transformation system in sugar beet is not successful, so the gene function research will be carried out in A. thaliana. Currently, our team is experimenting with various approaches to the genetic transformation system in sugar beet.
The fresh weight and dry weight and physiological indicators of leaves were measured in the soil. The one-month wild type (WT) and transgenic plants were treated with 150 mM NaCl for 7 days.

Cis-Regulatory Elements(CREs) Analysis
The promoters of the six stress-responsive genes in BvM14 were analyzed for putative cis-elements using available genomic sequences and our transcriptomic data [19]. In addition, the 2000 bp genomic sequences located on the 5 upstream of the Transcriptional Start Site (TSS) of the six stress-responsive genes sequences were extracted and analyzed with PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 15 November 2022) and TB tools. The Promoter binding of transcription factors was predicted by PlantTFDB (http://planttfdb.gao-lab.org/prediction.php, accessed on 3 June 2022) analysis [16].

Statistical Analysis
For all the experiments, three biological replicates with three technical replicates of each treatment were measured. All data were analyzed using GraphPad Prism 8 (Dr. Harvey Motulsky, San Diego, CA, USA). For multiple comparions, one-way analysis of variance (ANOVA) was used to determine statistical significance among treatments at p < 0.05.

Ectopic Expression of BvM14 Antioxidant Enzymes Enhanced Plant Salt Tolerance
Ectopic expression of the six genes encoding the antioxidant enzymes promoted root elongation of transgenic plants under salt stress. With WT plants as control, the root length of plants overexpressing (OX) the BvM14-Trx, BvM14-PrxR, and BvM14-APX increased sig-nificantly by 1.3-fold, 1.2-fold, and 1.2-fold after 150 mM NaCl treatment. Compared with knock-out (KO) mutant plants of Trx, PrxR, and APX genes, the root length of complementation transgenic lines (CO) was restored by complementation with the BvM14-Trx, BvM14-PrxR, and BvM14-APX genes to the levels of OX plants ( Figure 1). Plants overexpressing other enzyme-coding genes also showed similar phenotypes (Supplementary Figure S1).

Ectopic Expression of Genes Encoding the Antioxidant Enzymes of BvM14 Improved Plant Antioxidant Capacity
The

Antioxidant Enzyme Activities of WT, Different KO, and Transgenic Plants under Salt Stress
Compared with WT plants, overexpression of BvM14-GOX gene increased significantly in gene expression levels and the antioxidant enzyme activities under 150 mM NaCl stress. The Trx and PrxR activities in BvM14-GOX gene OX lines were significantly increased by 1.4-fold and 1.2-fold relative to WT under salt stress ( Figure 5).  The Trx and PrxR enzyme activities in BvM14-PrxR gene OX lines and BvM14-Trx gene OX lines were significantly higher by 1.5-fold and 1.3-fold than WT under salt stress, respectively ( Figure 6 and Supplementary Figure S5). Therefore, it is inferred that the two genes have synergistic effects in plant tolerance to salt stress.    Both in BvM14-DHAR3 and BvM14-MDHAR OX lines, the APX enzyme activity was significantly higher by 1.5-fold and 1.3-fold than WT, in DHAR3 and MDHAR KO lines, the APX enzyme activity was significantly reduced by 0.3-fold and 0.7-fold relative to CO under salt stress (Supplementary Figure S6). Increased DHAR and MDHAR activities were reported in different plants subjected to abiotic stresses [24][25][26].

Discussion
Based on the previous transcriptome database of BvM14 under salt stress, the openreading frames (ORF) of the coding genes BvM14-GOX, BvM14-PrxR, BvM14-Trx, BvM14-APX, BvM14-MDHAR, BvM14-DHAR3 encoding major enzymes in the antioxidant system were cloned and analyzed. Arabidopsis KO mutants of the homologous genes, CO lines, OX lines, and WT plants were used to characterize the roles of these enzymes in plant salt stress tolerance. Enzyme activity and transcriptional level of six major enzymes in transgenic lines were detected.
ROS (mainly H 2 O 2 and O 2 •− ) accumulation can be used as an important indicator for cellular oxidative stress [27]. MDA content can reflect the degree of membrane lipid peroxidation in plant [28]. The results showed that overexpression of the six genes in Arabidopsis increased the tolerance of the OX plants to salt stress, which may be attributed to enhanced antioxidante capacity and reduced oxidative damage by decreasing the contents of H 2 O 2 and MDA and increasing the AsA and GSH contents. Further analysis showed that overexpression of the BvM14-GOX led to significant increases in the expression levels and enzyme activities of other key enzymes in the antioxidant enzyme system. DHAR recycles ascorbic acid (AsA), which is then oxidized to form MDHAR. MDHAR is further converted to dehydroascorbate (DHA). AsA is essential to main the cellular redox state under abiotic stresses. MDHAR accompanies APX and scavenges H 2 O 2 in the mitochondria and peroxisome [29][30][31][32][33][34]. For example, the BvM14-Trx and the BvM14-PrxR mutually promote each other; the expression level and enzyme activity of BvM14-APX were positively correlated with the expression levels and enzyme activities of BvM14-DHAR3 and BvM14-MDHAR, but the expression level and enzyme activity of BvM14-MDHAR were negatively correlated with the expression level and enzyme activity of BvM14-DHAR3; the PrxR/Trx pathway had no significant interaction with the CAT pathway or the AsA/GSH pathway and participated in the plant antioxidant process independently. Based on these results, a salt stress response regulatory network of BvM14 antioxidant system was constructed.
GOX plays an important role in the glycolate-glyoxylate conversion during photorespiration, which catalyzes the oxidation of glycolate to generate glyoxylate and H 2 O 2 [35][36][37][38]. BvM14-GOX regulates the activity of other key enzymes in the antioxidant enzyme system and the expression of corresponding genes by catalyzing the production of H 2 O 2 from glycolic acid, the regulation of H 2 O 2 may occur in a fluctuating manner because the association-dissociation of GOX and CAT could take place dynamically and transiently in response to environmental stresses or stimuli. Consistently, related research showed that in spite of the constant and high production of ROS caused by the transgenic GOX in rice, but it can assist innate antioxidative systems in modulating ionic and redox homeostasis for salt stress tolerance [39], so as to improve the antioxidant capacity of plants and reduce the inhibition of salt stress on plant growth and development [35].
PrxR/Trx pathway-related coding genes PrxR and Trx have a vital function in cellular antioxidative defense via eliminating excessive ROS [15,[40][41][42]. BvM14-Trx gene and BvM14-PrxR gene act synergistically to eliminate excess of H 2 O 2 in plants, but they do not participate in MDA and GSH metabolic pathways since OX plants did not show differences in AsA/GSH pathway and CAT pathway. In Arabidopsis, AtTrxh2 overexpressing transgenic plants exhibited higher activities of antioxidant enzymes including peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), compared with the plants expressing the empty vector control [43]. In tomato, SlTrxh enhanced nitrate stress tolerance with decreased oxidative damage by increased antioxidant enzyme activities and interacted with SlPrx [42]. The AsA-GSH cycle is one of the important antioxidant systems in plants [44,45]. APX is involved in the initial step of the AsA-GSH cycle that scavenges excess ROS and protects the plant from salt stress [46,47]. In AsA/GSH pathway, BvM14-APX expression and enzyme activity were positively correlated with BvM14-DHAR3 and BvM14-MDHAR expression and enzyme activity, while BvM14-MDHAR expression and enzyme activity were negatively correlated with BvM14-DHAR3 expression and enzyme activity. In tobacco, overexpression of MnSOD, MDHAR, DHAR, and CAT in transgenic plants exhibited the improvement of salt tolerance [29,48]. The improved MnSOD, CAT, POD, APX, DHAR, MDHAR, and GR expression was also detected in wheat lines and two Chrysanthemum species under cold acclimation [49], which was similar with our results. PrxR/Trx pathway has no obvious interaction with CAT pathway and AsA/GSH pathway in plant antioxidant enzymes system.
The distribution and type of CREs in promoters affect the activities and functions of genes. In this study, through a systematic analysis of CRE in the promoter regions of the six genes, we identified various types of CRE (Figure 8c). Related to salt stress, previous studies have shown that abscisic acid (ABA) responsive element binding protein (AREB)/ABRE binding factors (ABFs) in bZIP transcription factors were involved in salt stress [50,51]. Other research indicated that the GT-1 element directly controls the salt response of OsRAV2. The study provided a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress [52,53]. In this work, we found the CREs in promoters of the six genes can bind many transcription factors, it contains transcription factors (e.g., MYB, C2H2, ERF) related to salt stress (Figure 8b). Recent studies suggested that the AP2/ERF TF family are involved in abiotic stress adaptation [54,55]. Functional analysis of the SmAP2-17 gene confirmed its role in plant salt tolerance [56]. It has also been demonstrated that C2H2 zinc finger proteins and MYB transcription factors play vital roles in biotic and abiotic stress tolerance [57].

Conclusions
In conclusion, we characterized the key genes encoding the major antioxidant enzymes of ROS scavenging system in plant salt stress tolerance. In different pathways, the key enzymes synergistically or antagonistically play important role in plant salt stress tolerance. In the PrxR/Trx pathway, the BvM14-Trx and the BvM14-PrxR mutually promote each other, but the BvM14-Trx gene and BvM14-PrxR gene do not affect AsA/GSH pathway and CAT pathway. In the AsA/GSH pathway, BvM14-APX expression and enzyme activity were positively correlated with BvM14-DHAR3 and BvM14-MDHAR expression and enzyme activity. Meanwhile, the CREs in promoters contain salt stress-responsive elements ABRE, GT-1 motifs and the CREs in promoters of the six genes can bind transcription factor MYB, C2H2, and ERF related to salt stress. Based on the working model, it became clear that multiple levels of regulations, including transcription and translation, are important in controlling plant salt stress tolerance. Future efforts in improving crop salt stress tolerance can benefit from the results from this study and need to consider multiple genes and markers to achieve optimal outcomes. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox12010057/s1, Table S1: List of the primer sequences for the six genes tested by RT-PCR; Figure S1: Effects of salt stress on the root length of seedlings with different genotypes (WT, OX, KO, and CO).