Elsevier

Plant Physiology and Biochemistry

Volume 147, February 2020, Pages 313-321
Plant Physiology and Biochemistry

Research article
Expression of Arabidopsis thaliana Thioredoxin-h2 in Brassica napus enhances antioxidant defenses and improves salt tolerance

https://doi.org/10.1016/j.plaphy.2019.12.032Get rights and content

Highlights

  • Genes encoding Arabidopsis h-type thioredoxins, AtTrx-h2 and AtTrx-h3, were introduced into Brassica napus.

  • AtTrx-h2 B. napus plants exhibited enhanced salt stress tolerance, but AtTrx-h3-overexpressing plants did not.

  • AtTrx-h2-overexpressing B. napus plants had reduced salt-induced oxidative stress.

Abstract

Salt stress limits crop productivity worldwide, particularly in arid and heavily irrigated regions. Salt stress causes oxidative stress, in which plant cells accumulate harmful levels of reactive oxygen species (ROS). Thioredoxins (Trxs; EC 1.8.4.8) are antioxidant proteins encoded by a ubiquitous multigene family. Arabidopsis thaliana Trx h-type proteins localize in the cytoplasm and other subcellular organelles, and function in plant responses to abiotic stresses and pathogen attack. Here, we isolated the Arabidopsis genes encoding two cytosolic h-type Trx proteins, AtTrx-h2 and AtTrx-h3 and generated transgenic oilseed rape (Brassica napus) plants overexpressing AtTrx-h2 or AtTrx-h3. Heterologous expression of AtTrx-h2 in B. napus conferred salt tolerance with plants grown on 50 mM NaCl having higher fresh weight and chlorophyll contents compared with controls in hydroponic growth system. By contrast, expression of AtTrx-h3 or the empty vector control did not improve salt tolerance. In addition, AtTrx-h2-overexpressing transgenic plants exhibited lower levels of hydrogen peroxide and higher activities of antioxidant enzymes including peroxidase, catalase, and superoxide dismutase, compared with the plants expressing the empty vector control or AtTrx-h3. These results suggest that AtTrx-h2 is a promising candidate for engineering or breeding crops with enhanced salt stress tolerance.

Introduction

Oilseed rape (Brassica napus), an important oilseed crop, belongs to the Brassicaceae (mustard family). Brassica crops are severely affected by drought and salt stress because they are cultivated in many arid and semiarid areas (Zhang et al., 2014). In Brassica crops, salt stress reduces nutrient availability (in leaves, stems, and roots) (Chakraborty et al., 2016), chlorophyll contents (Bahrani, 2013), and decreases the total fatty acids in the oils they produce by up to 25% (Bybordi, 2010). Hence, salt stress negatively affects plant development and productivity including seed germination (Mahmoodzadeh, 2008), seedling development (Ahmad, 2010), plant height (Dolatabadi and Toorchi, 2017), biomass (Bybordi, 2010), and grain yields (Mahmoodzadeh, 2008). In addition, with the increasing demand based on the health benefits of canola oil, increased soil salinization due to irrigation and use of chemical fertilizers, and ongoing climate change, reevaluation of the effects of salt stress on Brassica plants and further improvement of salt tolerance in B. napus are urgently needed (Ashraf and McNeilly, 2004; Francois, 1994).

Salinity, characterized by a high concentration of soluble salts in the soil, makes it harder for roots to extract water. The resulting dehydration and osmotic stress retard cell growth and impair metabolic processes (Hasegawa et al., 2000; Park et al., 2016). Ionic toxicity occurs when salts accumulate in the cell, resulting in impaired biochemical reactions (Hasegawa et al., 2000). To reduce the unfavorable effects of salts, plants turn on salt-responsive systems by regulating gene expression, signal transduction, and protein synthesis and modification (Hasegawa et al., 2000; Park et al., 2016). To rectify the ionic imbalance and ionic toxicity, plants increase the cellular K+/Na+ ratio by operating endosomal Na+/H+ antiporters, the plasma membrane-located SALT OVERLY SENSITIVE (SOS) transporter, and H+/K+ transporters. These transporters remove the Na+ from the cytosol and sequester the toxic Na+ into apoplasts or the vacuole. Salt-tolerant plants accumulate osmolytes such as soluble sugars, proline, and glycine betaine, especially in the early stages of exposure to salinity (Hasegawa et al., 2000). Stresses generally increase reactive oxygen species (ROS) levels and therefore, salt tolerance mechanisms include increased antioxidant defenses. ROS-detoxifying enzymes in the antioxidant system include superoxide dismutase (SOD), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and catalase (CAT) (You and Chan, 2015).

Thioredoxins (Trxs, EC 1.8.4.8) also function as part of the antioxidant systems. These small proteins (around 12 kDa) are ubiquitously distributed and highly conserved in all living organisms. Trxs contain two redox-active Cys residues with oxidoreductase activity in a highly conserved redox-active site (Trp-Cys-Gly-Pro-Cys [WCGPC]) (Meyer et al., 2012). Trx proteins regulate the function of their target proteins through oxidoreductase activity (Meyer et al., 2012). Trx-mediated oxidative reactions play an important role in signal transduction under biotic and abiotic stresses (Kneeshaw et al., 2014; Mata-Pérez and Spoel, 2019).

The Arabidopsis thaliana genome encodes 22 Trx isoforms, which are classified into seven subfamilies (h, f, m, z, x, y, and o) based on their amino acid sequence similarity and subcellular localizations (Meyer et al., 2012). Among these groups, h-type Trx proteins are abundant in the cytosol but are also detected in the nucleus, endoplasmic reticulum, and mitochondria (Meyer et al., 2012). H-type Trxs are classified into three subgroups (Gelhaye et al., 2004); subgroups I and II are reduced by NADPH-Trx reductase (NTR), whereas the reduction of subgroup III members is dependent on the glutathione-glutaredoxin system (Gelhaye et al., 2003).

H-type AtTrx proteins are involved in plant disease resistance and tolerance to abiotic stresses such as heat (Kneeshaw et al., 2014; Mata-Pérez and Spoel, 2019). For example, AtTrx-h5 participates in the redox regulation of the transcriptional coactivator NON-EXPRESSOR OF PR1 (NPR1) (Mou et al., 2003; Tada et al., 2008; Cheong et al., 2016). AtTrx-h5 allows active monomeric NPR1 (from inactive cytosolic oligomers) to translocate into the nucleus where it activates redox-sensitive TGACG sequence-specific binding protein (TGA) transcription factors, which are crucial in plant disease resistance. Arabidopsis Trx-h3 was isolated from heat-treated cytosolic extracts of Arabidopsis and conferred enhanced heat shock tolerance (Park et al., 2009). Banana (Musa acuminata) MaTrx12, a homolog of Arabidopsis Trx-h3, is involved in chilling tolerance of harvested banana (Wu et al., 2016). Overexpression of tobacco (Nicotiana tabacum) NtTrx-h3 enhances the resistance to oxidative stress induced by the herbicide paraquat, and exhibits resistance to Tobacco mosaic virus and Cucumber mosaic virus (Sun et al., 2010). In oilseed rape (B. napus), BnTrx-h1 (THL1) and BnTrx-h2 (THL2) are required for successful pollen acceptance (Haffani et al., 2004). Previous reports have suggested that h-type Trxs are useful genetic resources to develop crop plants that are tolerant of abiotic stresses (Yano, 2014). However, the role of Arabidopsis Trx-h2 in abiotic stress responses has not yet been identified. In this study, we used two Arabidopsis h-type Trx genes, AtTrx-h2 and AtTrx-h3. We generated transgenic B. napus plants heterologously overexpressing AtTrx-h2 or AtTrx-h3, and functionally analyzed their salt stress-induced oxidative stress responses. AtTrx-h2 contributed to increased salt tolerance with increased antioxidant enzyme activities, suggesting that the AtTrx-h2-mediated ROS scavenging system is a useful resource to develop salt-tolerant crops.

Section snippets

Plant materials, growth conditions, and salt stress conditions

The wild-type (WT) Brassica napus used in this study is winter-type cv. Youngsan and was used as the background for the transgenic plants. The seeds were washed twice for 10 min each with 2% hypochlorous acid and 0.02% (v/v) Triton X-100 followed by five washes with distilled water, and then vernalized at 4 °C in the dark. Surface-sterilized seeds were sown on ½ strength Murashige and Skoog (MS) medium [3% (w/v) sucrose, and 0.7% (w/v) agar] and grown under a 16-h photoperiod at 23 °C. After

Arabidopsis and B. napus Trx h-type isoforms and heterologous expression of AtTrx-h2 and AtTrx-h3 in B. napus plants

Trxs are encoded by a ubiquitous multigene family with large subfamilies classified by their subcellular localizations including the chloroplast, mitochondria, membranes, nucleus, and cytoplasm (Meyer et al., 2012). The Arabidopsis Trx h-type subfamily is predominantly expressed in cytoplasm, and its members share 26–73% amino acid sequence identity. Phylogenetic analysis clearly divided Trx h-type subgroups I, II, and III, clearly separating subgroups II and III from subgroup I. Interestingly,

Discussion

ROS function as crucial signaling molecules to control a broad spectrum of plant developmental processes via transmitting signals from cell to cell (Mhamdi and Van Breusegem, 2018). However, the accumulation of ROS during environmental stresses, such as UV radiation, heat, drought, and salt, reduces the activities of enzymes; destroys cellular membranes by degrading pigments, proteins, and lipids; and damages nucleic acids, resulting in cell death (Mhamdi and Van Breusegem, 2018). ROS

Authors contributions

MGJ and WYK conceived and designed the study. MGJ, HJP, JAK, GIS, SYJ, and ESL performed the experiments. MGJ, HJP, DJY, SYL, JYC, and WYK analyzed the data. MGJ, HJP, JYC, and WYK wrote the manuscript. All authors critically revised and provided final approval of this manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the Next-Generation BioGreen 21 Program (SSAC, PJ01327301 to WYK and PJ01327303 to JAK), Rural Development Administration (RDA), Republic of Korea.

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