A novel aldo-keto reductase gene, IbAKR, from sweet potato confers higher tolerance to cadmium stress in tobacco

Abstract

High concentrations of Cd can inhibit growth and reduce the activity of the photosynthetic apparatus in plants. In several plant species, aldo-keto reductases (AKRs) have been shown to enhance tolerance to various abiotic stresses by scavenging cytotoxic aldehydes; however, few AKRs have been reported to enhance Cd stress tolerance. In this study, the gene IbAKR was isolated from sweet potato. The relative expression levels of IbAKR increased significantly (approximately 3-fold) after exposure to 200 mmol·L⁻¹ CdCl₂ or 10 mmol·L⁻¹ H₂O₂. A subcellular localization assay showed that IbAKR is predominantly located in the nucleus and cytoplasm. IbAKR-overexpressing tobacco plants showed higher tolerance to Cd stress than wild-type (WT). Transgenic lines showed a significant ability to scavenge malondialdehyde (MDA) and methylglyoxal (MG). In addition, proline content and superoxide dismutase activity were significantly higher and H₂O₂ levels were significantly lower in the transgenic plants than in the WT. Quantitative real-time PCR analysis showed that the reactive oxygen species (ROS) scavenging genes encoding guaiacol peroxidase (GPX), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) and peroxidase (POD) were significantly upregulated in transgenic plants compared to WT under Cd stress. These findings suggest that overexpressing IbAKR enhances tolerance to Cd stress via the scavenging of cytotoxic aldehydes and the activation of the ROS scavenging system.

Keywords

cadmium stress / IbAKR / Ipomoea batatas / sweet potato

Introduction

The heavy metal, Cd, is considered to be biologically nonessential for almost all plants. With rapid industrialization and the widespread use of agrochemicals in agriculture in recent years, very large areas of land have been polluted by Cd. In China, about 7.75% of farmland soil is polluted by Cd^[1]. The effect of Cd toxicity on plants includes the inhibition of growth and a reduction in the activity of the photosynthetic apparatus^[2]. These situations pose a continuously increasing burden to global crop productivity, and hence, there is demand for crop varieties that are adaptive and resistant to various stresses. Genetic engineering has the potential to improve tolerance to Cd stress in plants^[3].

The aldo-keto reductase (AKR) protein superfamily contains>190 members and is found in all phyla. The AKR superfamily comprises 18 families and includes mainly NAD(P)(H)-dependent oxidoreductases. These enzymes reduce carbonyl substrates, such as lipid peroxidation byproducts, keto-steroids, keto-prostaglandins, retinals and quinones^[4]. In plants, AKRs have been reported to enhance tolerance to various abiotic stresses. The overexpression of a novel alfalfa aldose/aldehyde reductase gene in transgenic tobacco plants enhanced tolerance to oxidative damage caused by paraquat, heavy metals, UV-B (280–320 nm) irradiation, drought and low temperature^[5–7]. The overexpression of a rice AKR increased tolerance to oxidative stress, heat and glyphosate in transgenic rice^[8,9]. An aldose reductase from Xerophyta viscosa conferred enhanced drought and salinity tolerance in transgenic tobacco plants^[10]. The expression of an Arabidopsis thaliana-encoded AKR protein in transgenic barley increased tolerance to oxidative stress, Cd stress and freezing stress^[11,12]. Salt tolerance increased in Arabidopsis transformed with PpAKR1, which had been cloned from peach (Prunus persica)^[13]. Recently, a novel member of the AKR superfamily was cloned from a soil Sphingomonas strain and was responsible for detoxifying the Fusarium toxin deoxynivalenol^[14]. Regenerated transgenic tobacco and rice lines expressing PsAKR1, an AKR gene from Pseudomonas, showed improved tolerance to accelerated aging and glyphosate^[9,15]. However, the role of AKRs in Cd stress tolerance has rarely been reported.

Sweet potato, Ipomoea batatas, is an important root crop, and the storage root is widely used in the energy industry^[16]. In addition, the concentrations of Cd is lower in the storage root tissue than in the fibrous root, stem and leaves grown in Cd-contaminated farmland^[17], suggesting that sweet potato, as a source of bioenergy, could be a candidate crop for cultivation in Cd-contaminated soil. Therefore, it is of great significance to explore the Cd-stress-responsive genes in sweet potato to improve its Cd tolerance. In this study, a novel aldo/keto reductase gene, IbAKR, was cloned from sweet potato cv. Lushu 3, and it was found that the overexpression of IbAKR resulted in increased tolerance to Cd stress in transgenic tobacco plants.

Materials and methods

Plant materials

The sweet potato cv. Lushu 3 was used for IbAKR gene cloning and expression analysis. Tobacco (Nicotiana tabacum) cv. Wisconsin 38 was used to study the functions of IbAKR.

Cloning and sequence analysis of the sweet potato IbAKR gene

TransZol UP (TransGen Biotech, Beijing, China) was used to extract total RNA from fresh leaves of 4-week-old in vitro-grown Lushu 3 plants. To amplify the full-length IbAKR sequence, rapid amplification of cDNA ends (RACE) was performed using a 5′/3′ full RACE kit (Takara, Dalian, China). Gene-specific primers (5GSP1 and 5GSP1-2/3GSP1 and 3GSP1-2) were used for 5′ and 3′ RACE. The open reading frame of IbAKR was predicted using ORF finder. The conserved domains of the IbAKR protein were analyzed using NCBI BLAST. For the multiple sequence alignment analysis, the amino acid sequences of IbAKR and other AKR members were retrieved from the AKR superfamily home page and aligned using ClustalW. Phylogenetic analysis was conducted using MEGA6 software. The theoretical molecular weight and isoelectronic point (pI) were calculated at ProtParam. The primers mentioned above are listed in supplementary Table S1.

IbAKR expression analysis

Total RNA was extracted from different tissues as described above. About 1 mg of total RNA was reverse transcribed using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s protocol. The cDNA solution was used as a template for PCR amplification. The sweet potato Ibactin gene was used as an internal control. Quantitative real-time PCR (qRT-PCR) was carried out in a 10 mL volume using SYBR Premix Ex Taq (Takara, Dalian, China) and a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression was quantified using the comparative C_T method^[18]. The primers mentioned above are listed in Table S1.

Subcellular localization assay

The full-length IbAKR coding region without the termination codon was inserted into the pMDC83-GFP vector via PCR with linker primers (pMDC83-AKR-F/R) that contained PacI and AscI sites, generating a C-terminal fusion with the GFP gene under the control of the CaMV 35S promoter. All plasmid constructs were confirmed by sequencing. The vector 35S:GFP, which served as the control, and 35S:IbAKR-GFP were transiently expressed in onion epidermal cells using a biolistic particle delivery system (PDS-1000 Bio-Rad, Hercules, CA, USA). The subcellular localization of the IbAKR-GFP protein was observed with a confocal laser scanning microscope under 488 nm excitation (EZ-C1, Nikon, Tokyo, Japan). The primers mentioned above are listed in Table S1.

Generation of transgenic plants

The expression cassette P_35S-IbAKR-T_NOS was inserted into the plant binary vector pCAMBIA3301 at the XbaI and SacI sites. The sequence-verified plasmid was transfected into Agrobacterium tumefaciens strain EHA105 using the freeze–thaw method. Tobacco leaf explants were used for transformation as described by Horsch et al.^[19]. Both GUS staining and PCR analysis (Bar-F/R) were conducted to confirm positive transgenic plants. The primers mentioned above are listed in Table S1.

Assay for cadmium tolerance

An in vitro assay for Cd tolerance was conducted at 28°C using 1/2 MS medium with 200 mmol·L⁻¹ CdCl₂ and a 16/8 h light/dark cycle. Three plants per line were treated. The growth and rooting ability were continuously observed for 4 weeks. In the pot experiment, 8-week-old tobacco plants were planted in a transplanting box and irrigated with Hoagland solution with 100 mmol·L⁻¹ CdCl₂ every three days. Morphological changes in these lines were observed after 2 weeks.

Analyses of MDA, proline and H₂O₂ content and SOD activity

The malondialdehyde (MDA) content was measured according to the method of Gao et al.^[20]. The proline content and superoxide dismutase (SOD) activity were analyzed as described by He et al.^[21]. H₂O₂ content was measured according to the method of Alexieva et al.^[22].

Leaf disc senescence assay and measurement of chlorophyll content

The leaf disc senescence assay was conducted according to Kumar et al.^[10]. To measure the chlorophyll content, 0.2 g of leaf discs were ground in 80% buffered aqueous acetone. The homogenate was filtered through filter paper and diluted to 25 mL with 80% aqueous acetone. The chlorophyll a and b content of the solution was measured at 663 and 645 nm, respectively, using a spectrophotometer. The chlorophyll concentration was calculated as described by Porra et al.^[23].

Expression analysis of ROS scavenging-related genes

Transgenic and wild-type (WT) plants were treated with 100 mmol·L⁻¹ CdCl₂ for 2 weeks, and the third leaves from the top were used to analyze the expression of reactive oxygen species (ROS) scavenging-related genes guaiacol peroxidase (GPX), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) and peroxidase (POD). Tobacco Ntactin was used as an internal control, and qRT-PCR analysis was conducted as described above. The qRT-PCR primers are listed in Table S1.

Statistical analysis

The experiments were repeated three times, the data were analyzed using Student’s two-tailed t-test, and the results are presented as the mean±SE. A value of P<0.05 or<0.01 was considered statistically significant.

Results

Analysis of IbAKR characteristics

The IbAKR gene was isolated from the sweet potato cultivar Lushu 3. It consisted of five exons and four introns and contained a 1053 bp ORF that encoded a 350-amino-acid polypeptide with a calculated molecular weight of 38.32 kDa and a pI of 5.85. BLAST showed that the AKR protein contained typical AKR signature motifs (Fig. S1a), including an (α/β)₈-barrel motif and catalytic tetrad (Asp-63, Tyr-68, Lys-94, and His-135), which are conserved in the AKR superfamily. The AKR superfamily comprises 18 families and more than 170 members (as of July 2017, families 16–18 had not been included)^[4,14,24]. A phylogenetic analysis of IbAKR showed high amino acid identity with Rauvolfia serpentina AKR13D1 (AY766462, 51.63%) (Fig. S1b). Based on the nomenclature criteria for the AKR family^[25], IbAKR is in family 13 of the AKR superfamily.

Analysis of IbAKR expression

qRT-PCR analysis showed IbAKR expression in all organs of sweet potato examined. It was highly expressed in the roots of 4-week-old in vitro-grown Lushu 3 plants (⁰⁰⁰³⁰¹a). After 4 weeks growth in vitro Lushu 3 plants were treated with 200 mmol·L⁻¹ CdCl₂ and 10 mmol·L⁻¹ H₂O₂ for 0, 3, 6, 12, 24 and 48 h. An increase in IbAKR transcripts was observed after exposure to 200 mM CdCl₂ and peaked at 6 h when the expression level was 3 times higher than that at 0 h (⁰⁰⁰³⁰¹b). Under 10 mmol·L⁻¹ H₂O₂ stress, the expression of IbAKR was induced to a higher level (3.5-fold increase) at 12 h (⁰⁰⁰³⁰¹c).

Subcellular localization of IbAKR

The subcellular localization of the IbAKR protein was investigated by generating 35S_pro-IbAKR-GFP and transiently expressing it in onion epidermal cells. The empty vector 35S_pro-GFP was used as a control. As shown in ⁰⁰⁰³⁰², the GFP fluorescence signal of IbAKR-GFP was detected in the nucleus and cytoplasm, as was the GFP fluorescence signal of the GFP control.

Overexpression of IbAKR conferred strong cadmium stress tolerance to transgenic tobacco plants

To elucidate the function of IbAKR in plants, six transgenic tobacco lines overexpressing IbAKR were generated (L1–L6). The expression level of IbAKR was significantly higher in the transgenic plants than in the WT plants. Two lines (L1, L2) were selected for further functional analysis.

To examine the role of IbAKR in Cd stress, the WT and transgenic plants were cultured on 1/2 MS medium with 0 or 100 mmol·L⁻¹ CdCl₂ for 4 weeks. The growth and rooting of all plants were normal without CdCl₂. With 100 mmol·L⁻¹ CdCl₂, the growth and rooting development of both the WT and transgenic plants were restrained, but the Cd tolerance was significantly higher in the transgenic plants than in the WT (⁰⁰⁰³⁰³a–3c). To further test the role of IbAKR in Cd stress tolerance, the transgenic plants and WT were grown in a transplanting box and irrigated with a 200 mL of 100 mmol·L⁻¹ CdCl₂ every 2 d. After 2 weeks, compared to the two transgenic lines, the WT showed more severe chlorosis and necrosis (indicated by red circle in ⁰⁰⁰³⁰⁴a). The transgenic plants showed significantly lower contents of MDA and H₂O₂ and significantly higher contents of proline than WT (⁰⁰⁰³⁰⁴b–4e).

Overexpression of IbAKR reduced the toxic effects of MG

The levels of methylglyoxal (MG), a cytotoxic compound, increase upon exposing plants to various abiotic stresses. High concentrations of MG are very harmful to plants. The responses of the transgenic lines to MG stress were tested using detached leaf disc senescence assays. Leaf discs from WT and L2 were floated on different concentrations of MG for 2 d. As shown in ⁰⁰⁰³⁰⁵, the chlorophyll contents of WT and L2 showed no difference with 0 and 5 mmol·L⁻¹ MG; however, with 10 mmol·L⁻¹ MG, the leaf discs from L2 retained significantly more chlorophyll than those from WT.

Overexpression of IbAKR upregulated the ROS scavenging-related genes

To investigate the impact of IbAKR-overexpression (IbAKR-OE) on the transcription of genes related to ROS scavenging, the expression levels of GPX, APX, MDHAR and POD under Cd stress were analyzed. The results showed that these genes were significantly upregulated (between 3- and 9-fold) in the transgenic lines compared with the WT after 2 weeks of treatment with 100 mmol·L⁻¹ CdCl₂ (⁰⁰⁰³⁰⁶).

Discussion

AKRs encompass a large superfamily of NAD(P)(H)-dependent oxidoreductases^[24]. In plants, a few AKRs have been reported to enhance tolerance to Cd stress^[5,11]. In this study, IbAKR was cloned from sweet potato. BLAST showed that IbAKR contained typical AKR signature motifs. According to the nomenclature of the AKR superfamily, IbAKR shared 51.63% (>40%) amino acid identity with AKR13D1, suggesting that IbAKR belongs to family 13 of the AKR superfamily; however, its amino acid identity with the other members of family 13 was less than 60%, suggesting that IbAKR is part of a new subfamily of family 13^[26]. These results showed that IbAKR could be a novel member of the AKR superfamily.

The relative expression levels of IbAKR in sweet potato increased 3- and 3.5-fold in response to 200 mmol·L⁻¹ CdCl₂ and 10 mmol·L⁻¹ H₂O₂, respectively, suggesting that IbAKR might have a role in Cd stress tolerance. The overexpression of IbAKR enhanced tolerance to Cd stress in transgenic tobacco plants. Under Cd stress, transgenic plants showed better growth and rooting than WT (Figs. 3a–3c; ⁰⁰⁰³⁰⁴a). This result indicates that the IbAKR gene may be used to improve plant tolerance to Cd stress.

MDA is often considered a major lipid peroxidation marker, and its accumulation beyond a threshold level has been reported to cause irreparable damage to the photosynthetic machinery^[27]. MG is a glycolysis-derived toxic aldehyde, and the accumulation of MG to a high concentration is toxic to cells, as it inhibits cell proliferation^[28]. In a previous study, OsAKR1 exhibited high catalytic activity in the reduction of toxic aldehydes, including lipid peroxidation-derived MDA and glycolysis-derived MG, leading to an increased tolerance to oxidative and heat stress^[8]. In our study, we found that overexpressing IbAKR could significantly reduce the levels of MDA relative to those in WT under Cd stress (⁰⁰⁰³⁰⁴b). Additionally, leaf discs from transgenic plants retained more chlorophyll after MG treatment (⁰⁰⁰³⁰⁵). These results suggest that Cd stress tolerance was enhanced in IbAKR-overexpressing tobacco plants via scavenging MDA and MG and reduction in membrane damage.

In plants, Cd stress leads to the overproduction of ROS, which are highly reactive and toxic. ROS scavenging systems can detoxify ROS to minimize and/or prevent oxidative damage and cells respond to ROS by increasing the activity of ROS-scavenging enzymes^[29]. As a key enzyme of the ROS scavenging system, SOD is usually induced by various abiotic stresses to promote the rapid dismutation of superoxide into oxygen and H₂O₂, which is subsequently removed by GPX, APX and POD through different pathways; moreover, MDHAR is needed to regenerate the active forms of antioxidants^[30,31]. It has been reported that proline acts as a non-enzymatic ROS scavenger and can scavenge singlet oxygen and hydroxyl radicals^[32]. In our study, IbAKR-overexpressing tobacco plants had significantly higher SOD activity and proline levels and significantly lower H₂O₂ levels than WT (⁰⁰⁰³⁰⁴c–4e). qRT-PCR showed that the expression of GPX, APX, MDHAR and POD was significantly upregulated (⁰⁰⁰³⁰⁶), suggesting that the improved Cd stress tolerance of the transgenic tobacco plants is also due to enhanced ROS scavenging.

Conclusions

A novel aldo-keto reductase gene, IbAKR, has been successfully identified and cloned from the sweet potato cv. Lushu 3. The IbAKR-overexpressing tobacco plants possessed higher tolerance than WT to Cd stress. This suggests the transgenic plants have a significant ability to scavenge MDA and MG. In addition, proline content and SOD activity were significantly higher and H₂O₂ levels were significantly lower in the transgenic plants than in the WT. Furthermore, ROS scavenging genes GPX, APX, MDHAR and POD were significantly upregulated in transgenic plants compared to the WT under Cd stress. Our results suggest that the overexpression of IbAKR enhanced Cd tolerance by reducing cytotoxic aldehydes and increasing the capacity for scavenging reactive oxygen species. This study indicates that the IbAKR gene may be useful for improving tolerance to Cd stress in plants.

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The Author(s) 2018. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)