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Chemical and Biological Technologies in Agriculture
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Functional characterization of malate dehydrogenase, HcMDH1, gene in enhancing abiotic stress tolerance in kenaf (Hibiscus cannabinus L.)

Chemical and Biological Technologies in Agriculture volume 11, Article number: 141 (2024) Cite this article

Abstract

Drought and salt stress are two important environmental factors that significantly restrict plant growth and reproduction. Malate dehydrogenase is essential to life as it is engaged in numerous physiological processes in cells, particularly those related to abiotic stress reactions. However, a complete understanding of MDH family members in kenaf is not clear yet. In this study, subcellular localization analysis and a yeast transcriptional activation assay revealed that HcMDH1 was localized in chloroplasts but had no transcriptional activation activity. When exposed to salt or drought stress, yeast cells expressing the HcMDH1 gene exhibit an increased survival rate. Overexpression of HcMDH1 in Arabidopsis increased seed germination rate and root growth when transgenic lines were exposed to varying concentrations of mannitol and NaCl. Subsequent physiological studies revealed that transgenic lines had higher concentrations of soluble carbohydrates, proline, and chlorophyll and lower concentrations of malondialdehyde (MDA) and reactive oxygen species (ROS). Furthermore, inhibiting HcMDH1 in kenaf using virus-induced gene silencing (VIGS) decreased salt and drought tolerance due to elevated ROS and MDA levels. In these silenced lines, the expression of six essential genes engaged in stress-resistance and photosynthesis, namely HcGAPDH, HcGLYK, HcFBA, HcFBPase, HcPGA, and HcLSD, is significantly altered under salt and drought stress. In summary, HcMDH1 is a complex and positive regulatory gene that plays a key role in regulating chlorophyll content, antioxidant enzyme activity and osmotic regulation under salt and drought stress, which may have implications for kenaf transgenic breeding.

Graphical Abstract

Highlights

  1. 1.

    HcMDH1 is located in the chloroplasts and could be induced by salt and drought stresses.

  2. 2.

    Expression of the HcMDH1 in yeast improved its survival rate under salt and drought conditions.

  3. 3.

    HcMDH1 can improve salt and drought tolerance ability by changing antioxidant activity.

  4. 4.

    Silencing HcMDH1 reduced salt and drought tolerance by altering the expression of some stress-responsive genes.

Introduction

Salt stress is a devastating abiotic stress that has significant global implications, with salinization-related losses estimated to account for over 20% of annual crop losses [1]. Drought, another critical abiotic stress, severely limits agricultural expansion, adversely affects crop yield and quality, and can even result in crop death [2]. Soil salinity and drought are important factors that hinder plant growth and development, posing a threat to agricultural productivity [1, 3]. Throughout the process of evolution, plants have developed many mechanisms to resist and adapt to drought stress. These mechanisms encompass a wide range of physiological, biochemical, metabolic, and tolerance processes, which are facilitated by alterations in the expression patterns of numerous genes [2,3,4]. Therefore, enhancing drought and salt tolerance is a primary objective of molecular breeding in crops [2, 4].

In addition to phenotypic characteristics malate, an important metabolite in plants, is produced by malate dehydrogenase (MDH) from oxaloacetate. Malate, a four-carbon dicarboxylic acid, plays a key role in several cellular metabolic processes, such as transferring energy between the cytoplasm and organelles, facilitating energy exchange among organelles, and maintaining reducing power [5]. MDH is a kind of oxidoreductase widely distributed in plants, animals, and microorganisms. It mainly catalyzes the reversible transformation of malic acid and oxaloacetic acid and is an essential intermediate product in the life of animals and plants [6, 7]. MDH is involved in several biochemical and physiological activities in plants, such as TCA cycle, C4 cycle, oxidation of fatty acids, respiration, nitrogen assimilation, etc. [8]. MDH, an enzyme associated with the tricarboxylic acid (TCA) cycle, shows a significant increase under salinity stress, along with other enzymes, such as succinate dehydrogenase (SDH), isocitrate dehydrogenase (IDH), acetate hydratase 2, carbonic anhydrase, and citrate synthase [9]. This enzyme complex maintains normal glycolysis and Krebs cycle functions, providing plants with sufficient energy to support diverse metabolic processes under salinity stress [10].

Previous studies reported that MDH is a highly active enzyme involved in respiration, energy metabolism and reactive oxygen species (ROS) metabolism in plants [11,12,13,14], which play an important role in response to abiotic stress. In Arabidopsis, overexpression of ZmNADP–MDH results in increased chlorophyll and protein content, while reducing the production of H2O2 and membrane lipid peroxidation [15]. Studies on Stylosanthes guianensis suggest that SgMDH in the stigma may participate in the response to metal stress [16]. In alfalfa, MDH gene overexpression promotes the synthesis of organic acids, thereby enhancing resistance to aluminum (Al) toxicity [17]. The GhmMDH1 gene in cotton is involved in plant and root growth under condition of phosphorus deficiency [18]. In addition, the expression of the NAD-dependent MDH gene in apple cytoplasm is positively correlated with growth and metabolic activity, contributing to plant growth and salt stress response [12]. Overexpressing MdcyMDH under salt stress conditions significantly increases reduction activity of chMDH and cyMDH. Furthermore, mMDH has been shown to have higher oxidation activity than wild-type plants [19]. A study was conducted in rice to examine the correlation between natural variation and salt tolerance phenotypes in 12 rice MDH family genes. The results showed that substantial SNPs in OsMDH genes (OsMDH8.1 and OsMDH12.1) were linked to salt tolerance at the seedling stage [20]. In addition, OsMDH1 in mutant rice also decreases vitamin B6 concentration, which inversely regulates salt tolerance [21]. The increased expression of NADP–MDH in Rice cultivars with different salt tolerance under salt stress, suggesting a positive role for MDH in salt tolerance [22]. The expression of PtMDH2 in poplar was significantly upregulated in response to salt stress [23]. Similarly, the RNA-seq and qRT-PCR results revealed that PvMDH-3, PvMDH-6, and PvMDH-8 play an important role in response to salt stress in common bean [24]. Although MDH family genes hold promise as candidate genes for abiotic stress tolerance, many of their functions in other plants remain unknown and require further verification through subsequent experiments.

Kenaf (Hibiscus cannabinus L.) is a salt- and drought-resistant annual crop that belongs to the genus Hibiscus in the family Malvaceae [25, 26]. It is known for its high fiber content and has various applications, such as textile and paper production, as well as cosmetics and medicine [27]. Kenaf-derived products are natural, renewable, and biodegradable, making them environmentally friendly [28]. Therefore, understanding the stress resistance mechanism of kenaf is important for its large-scale cultivation and improvement of climate and ecological conditions. Although some MDHs have been studied in other plants, their role in kenaf remains unclear. This study focuses on HcMDH1, which was isolated from kenaf to investigate its molecular mechanism in drought and salt tolerance. The expression profiles of HcMDH1 were analyzed under drought and salt stress conditions. Functional analysis was conducted through overexpression in yeast and Arabidopsis, as well as silencing using virus-induced gene silencing (VIGS) in kenaf. The results demonstrate that HcMDH1 plays a crucial role in enhancing kenaf's resistance to salt and drought stress.

Materials and methods

Plant materials and growth conditions

The seeds of the kenaf cultivar "CP085", which was obtained from Guangxi University (Nanning, China) were sterilized with a 3% hydrogen peroxide solution for 10 min. Subsequently, they were germinated in an incubator at a temperature of 27 °C/25 °C (day/night) with a light/dark cycle of 10/14 h and a light intensity of 280 μmol·m−2·s−1 for 5 days. On the sixth day of germination, the seedlings were transferred to a tray containing a 1/2 strength Hoagland solution. After 7 days, the seedlings were exposed to either 200 mM NaCl or 15% PEG6000. Leaves were collected at 0, 2, 6, 12, 24, and 48 h after treatment. The samples were promptly cryopreserved in liquid nitrogen and stored at − 80 °C temperature for total RNA extraction.

Gene bioinformatics analysis

Using our previous transcriptome data [29], we obtained and cloned the coding sequence (CDS) of the HcMDH1 gene in kenaf. The three-dimensional structures and transmembrane regions were predicted by the Phyre2 server (http://www.sbg.bio.ic.ac.uk/ ~ phyre2/html/page.cgi?id = index); The evolutionary relationship between the HcMDH1 protein and MDH proteins from other plants was analyzed using MEGA 7.0 software and a phylogenetic tree was constructed. The MDH proteins amino acid sequence were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/).

Transcriptional activation and yeast activity assay

To construct the plasmid BD-HcMDH1, the coding sequence of HcMDH1 was introduced into the GAL4-binding domain of the pGBKT7 vector (BD). This was accomplished using gene-specific primers (Table S1).The recombinant BD-HcMDH1 plasmid was then inserted into the yeast strain Y2H and cultured in SD/-Trp medium. The pGBKT7-53 and pGADT7-LargeT interactions were employed as positive controls; however, the empty BD vector was utilized as a negative control. Following PCR detection, the solution containing positive transformants was diluted by factors of 0, 10, and 100, and then spread onto plates containing SD/-Trp and SD/-Trp/-Leu/-Ade/X-a-Gal. The plates were incubated at 30 °C for 3 days.

The HcMDH1 gene sequence was used to design specific primers (Table S1). HcMDH1 was inserted into the pYES2 protein expression vector and obtained, pYES2–HcMDH1 was introduced into the yeast strain BY4741 following the manufacturer's guidelines (Clontech). An empty pYES2 vector was transferred into BY4741 as a control. After measuring the absorbance of strain culture at 600 nm, the liquid culture was diluted to various concentrations. The treatment and control fluids were diluted by factors of 10, 100, 1000, and 10,000 after the initial dilution. To test for drought and salinity stress, 3 μL of the solution was transferred into 50 mL of 200 mM NaCl or 15% PEG6000 and incubated at 30 °C for 24 h. In addition, the solutions were cultured at 30 °C for 4 days. The experiment was replicated thrice. Photographs were captured to document the growth of yeast strains on the plate.

Subcellular localization analysis

The CDS of HcMDH1 was inserted into the pBI121–EGFP vector at Kpn I and Xba I recognition sites, and the resulting pBI121–EGFP–HcMDH1 recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101. The bacterial solution was then injected into the tobacco leaves and were examined under a confocal laser microscope following the detailed protocol outlined by Luo et al. [29].

Overexpression of HcMDH1 gene in Arabidopsis plants

Wild-type (WT) Arabidopsis thaliana ecotype Columbia Col-0 plants were transformed with Agrobacterium tumefaciens GV3101 containing the pBI121–EGFP–HcMDH1 recombinant vector by infesting their inflorescences. The T1 and T2 seeds were subjected to cultivation on a 1/2 MS medium supplemented with 100 mg/L kanamycin for selecting positive plants. Following this, the plants that successfully survived were subsequently transplanted into soil to obtain T3 seeds. Selecting WT and HcMDH1 high expression lines seeds to determine germination rate by 1/2 MS medium supplemented with 100 or 200 mM NaCl, or 150 or 300 mM mannitol for 7 days. To measure root length, 4-day-old seedlings of Arabidopsis thaliana were transferred to 1/2 MS medium, supplemented with different concentrations of NaCl and mannitol, and allowed to grow for 7 days. In addition, 2-week-old seedlings from the T3 generation were subjected to a 7-day treatment with a 200 mM NaCl solution or natural drought for 7 days in soil, and then rewatered for 3 days.

HcMDH1 silenced plants by virus-induced gene silencing (VIGS) technology

For VIGS analysis, the optimal target was selected, and specific primers were created using the SGN–VIGS online tool (https://vigs.solgenomics.net/). The forward and reverse primer sequences can be found in Table S1. A 431 bp gene fragment was cloned into the pTRV2 vector to construct pTRV2–HcMDH1 recombinant and then introduced into Agrobacterium GV3101 strain. The pTRV1 (auxiliary vector), pTRV2, pTRV2–HcMDH1 and pTRV2–HcTrx cell suspension of the Agrobacterium was injected into the cotyledon of kenaf seedlings accordingly to the method previously reported by Chen et al. [30]. The effectiveness of VIGS was evaluated by targeting HcTrx gene as a positive control, which manifested as an albino leaf phenotype [31].

The silencing efficacy of the third true leaf of pTRV2–HcMDH1 seedling was assessed by qRT-PCR, and positive seedlings were selected to stress treatments (1/2 strength Hoagland solution, as well as 1/2 strength Hoagland solution containing 150 mM NaCl or 15% PEG for 7 days). And then, agronomic traits and relevant physiological indices were measured.

Gene expression and promoter cis-acting elements analysis

Total RNA of kenaf was extracted using the quick extraction reagent for total RNA (YFXM0011, YI FEI XUE Biotech Co., Ltd, Nanjing, China). The HiScript III Reverse Transcription Kit (Vazyme Biotech Co., Ltd, Nanjing, China) was used to synthesise first-strand cDNA from total RNA. TransStart Top Green qPCR SuperMix (TransGen Biotech) and the Bio-Rad CFX96 (Bio-Rad Laboratories) were used to perform qRT-PCR. To design primer in accordance with the specifications for quantitative real-time fluorescent primer design, Primer Premier 5.0 was utilized (Table S1). The reaction protocol involved a pre-denaturation stage at 94 ℃ for 3 min, followed by 40 cycles of 94 ℃ for 5 s and 60 ℃ for 30 s. The housekeeping gene HcActin3 from soybeans was used as a tick. The 2−ΔΔCT method [30] was used to calculate changes in gene expression levels for each response, with three replicates.

The 2 kbp upstream promoter sequence from the start codon of the HcMDH1 gene (gene ID Hc.04G016350.t1 in kenaf genome and annotation files) was obtained from the National Genomics Data Centre using accession number GWHACDB00000000.1 [32]. The PlantCARE web (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) were used to search for cis-acting elements in the promoter region.

Physiological stress indexes

Relative water content (RWC) of kenaf leaf was determined by using the methodology described by Luo et al. [26]. The RWC calculation carried out using the following formula RWC = [FW–DW]/FW × 100%.The assays for superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were conducted using the protocols outlined in Luo et al. [33]. In addition, we analyzed the levels of soluble sugar, malondialdehyde (MDA), proline, and chlorophyll using the methodology described by Luo et al. [29]. We employed 3, 3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining to determine the leaf contents of H2O2 and O2, respectively, following the method published by Luo et al. [33]. The experiments were performed in three independent biological duplicates.

Statistical analysis

Statistical analyses were conducted using one-way ANOVA in SPSS 20.0. The Duncan's multiple range test was employed to assess the impact of various treatments, with a significance level of p < 0.05. The data are displayed as the mean value ± the standard deviation (SD).

Results

The expression of HcMDH1 and its promoter analysis

A total of nine MDH genes, named HcMDH1–HcMDH9 (Table S2), were identified from previous transcriptome data of kenaf under salt and drought stress (SRA database under accession number PRJNA498212 and PRJNA857555). Among these, HcMDH1 was found to be significant in both drought and salt conditions, showing the most pronounced change in expression (Fig. S1). Therefore, we selected HcMDH1 gene for subsequent study.

This work involved the cloning of HcMDH1 and its utilization in the construction of a vector for gene function analysis. In this study, the CDS of HcMDH1 was amplified and sequenced. The findings of the bioinformatics analysis are presented in Fig. 1A. According to the Phyre2 prediction, HcMDH1 is comprised of eight transmembrane α-helices, as illustrated in Fig. 1B. The phylogenetic tree analysis indicated a close relationship between the HcMDH1 protein and various plant species, such as Hibiscus trionum, Hibiscus syriacus, Gossypium, Theobroma cacao, and Durio zibethinus (Fig. 1C). This suggests that HcMDH1 has been conserved throughout the evolution of plants, specifically in kenaf.

Fig. 1
figure 1

Sequence, subcellular localization, and transcript expression levels analysis of HcMDH1. A Nucleotide sequence and coding amino acid sequence of HcMDH1. The black shading represents the malate dehydrogenase domain; B three-dimensional structure of HcMDH1 protein; C Phylogenetic tree analysis of HcMDH1 for various plants. Changes in expression levels of candidate genes under D salt stress (150 mM NaCl) and E drought stress (15% PEG6000). Data are the mean values ± SE (n = 3). * (p < 0.05) or ** (p < 0.01) indicated significant difference in Duncan's test

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To analyze the changes in the expression pattern of HcMDH1 in the stress response of kenaf, the transcript level of HcMDH1 was detected using qRT-PCR under salt and PEG-6000 treatments. The results indicated that the expression level of the genes remained relatively stable for the first 2 h following NaCl treatment, but it substantially increased from 6 to 48 h (Fig. 1D). The results of the PEG6000 treatment showed that the expression of HcMDH1 was significantly elevated after 2 h and remained elevated until 12 h, and then, it slightly decreased (Fig. 1E). These findings indicate that HcMDH1 is crucial in mediating the effects of salt and drought stress.

To ascertain the molecular mechanism of HcMDH1 gene function, cis-acting elements in the promoter region of HcMDH1 gene was identified. The 5′ flanking region of the HcMDH1 promoter, which is approximately 2 kb in length, was cloned. The cis-acting elements present in this region were analyzed. Figure 2 illustrates the presence of several light-responsive elements, including GT1-motif, G-box, TCT-motif, Box-4, MRE, and AE-box elements. Other cis-acting factors that were involved in the response to abiotic stress included gibberellin (GA), auxin (IAA), methyl jasmonate acid (MeJA), abscisic acid (ABA), STRE, MBS, and ARE elements (Table 1). Therefore, based on these data, we deduced that HcMDH1 might be involved in stress tolerance, as well as in the response to photoperiod and circadian rhythm in kenaf plants.

Fig. 2
figure 2

Cis-acting elements in the promoters of HcMDH1 genes. The different colored wedges represent different cis-acting elements. The functions of the cis-acting elements are annotated separately under the legend

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Table 1 Important cis-acting elements of HcMDH1 promoter
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Subcellular localization and transcriptional activation activity assay of HcMDH1 protein

It was predicted by the plant–mPLoc programme that the HcMDH1 protein is located in the chloroplast. Furthermore, the subcellular localization analysis revealed that the red autofluorescence of chlorophyll was coincident with the GFP fluorescence of the fusion protein, suggesting that HcMDH1 is located in the chloroplast (Fig. 3A). Moreover, transcriptional activation analysis showed that only positive control clones were able to grow normally on a synthetic drop-out medium containing 5-bromo-4-chloro-3-indolyl-α-D-galactoside (X-α-gal) that was deficient in leucine, tryptophan, histidine, and adenine (SD/-Leu/-Trp/-His/-Ade). This outcome indicates that HcMDH1 lacks transcriptional activation activity (Fig. 3B).

Fig. 3
figure 3

Analysis of Subcellular localization and transcriptional self-activation activity of HcMDH1 protein. A Subcellular localization of HcMDH1 protein. Images of cells expressing GFP; B transcription activity analysis of HcMDH1. Positive, pGADT7-p53 + pGBKT7-T; BD, pGADT7 + pGBKT7; HcMDH1, pGADT7 + pGBKT7–HcMDH1; SD/-Trp, synthetic drop-out medium lacking tryptophan; SD/-Trp-His-Ade (X-α-gal), synthetic drop-out medium containing X-α-gal and lacking histidine, tryptophan, and adenine

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Heterologous expression of the HcMDH1 in a yeast system enhanced salt and drought tolerance of yeast strains

The pYES2–HcMDH1 plasmid was introduced into yeast cells and subjected to stress conditions. Under typical growth conditions, there were no significant differences in the survival rate and growth rate between the yeast strains transformed with pYES2–HcMDH1 and those transformed with the empty pYES2 vector on galactose-induced solid medium. In addition, there were no notable variations in cell size. However, under drought stress, the growth rate of both yeast strains, those transformed with pYES2–HcMDH1 and the control cells transformed with the empty pYES2 vector, was inhibited. Notably, the yeast strains transformed with pYES2–HcMDH1 exhibited a considerably lower growth rate and a much higher cell number than the control cells. A similar phenomenon was observed when the yeast strains were subjected to salt stress (Fig. 4).

Fig. 4
figure 4

Growth of yeast strains transformed with pYES2–HcMDH1 and empty vector pYES2 under different stress treatments. A Normal growth conditions; B NaCl stress; C PEG stress

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Overexpressing HcMDH1 enhanced Arabidopsis thaliana germination rate

To examine the function of HcMDH1 in response to abiotic stress, the CDS sequence of HcMDH1 was inserted into the PBI121 vector, which is driven by the CaMV 35S promoter. Subsequently, T3 seeds were planted on 1/2 MS medium, 1/2 MS medium supplemented with 50 mg/L Kanamycin, respectively, and high-expressing HcMDH1 lines were identified using qRT-PCR analysis. Based on the higher expression levels of strains OE-3, OE-5, and OE-7 (Fig. S2), these transgenic lines of HcMDH1-OEs were chosen for subsequent studies.

To determine salt and drought stress sensitivity of HcMDH1 in Arabidopsis plants, the germination rate of HcMDH1-OEs and WT was tested under various concentrations of NaCl and mannitol. The germination rates of HcMDH1-OEs and WT did not differ significantly in the absence of treatment. However, when treated with 100 mM and 200 mM NaCl, HcMDH1-OE outperforms WT in terms of germination rate. Similarly, HcMDH1-OEs has a greater germination rate than WT when treated with 150 mM and 300 mM mannitol (Fig. 5). Specifically, from day 2 forward, the germination rate of HcMDH1-OEs was consistently higher than WT when exposed to varying doses of NaCl and mannitol (Fig. S3). This result was in accordance with the expression level of HcMDH1 gene in Arabidopsis.

Fig. 5
figure 5

Germination percentage evaluation. A Visual representation showing the percentage of germination in normal, drought, and salinity conditions for both the WT and overexpressed lines. B Determination of germination percentage in 1/2 MS media without any additives, 1/2 MS media with 100 mM NaCl, and 1/2 MS media with 200 mM NaCl. C Determination of germination percentage in 1/2 MS media without any additives, 1/2 MS media with 150 mM mannitol, and 1/2 MS media with 300 mM mannitol. Data are the mean values ± SE (n = 3). * (p < 0.05) or ** (p < 0.01) indicated significant difference in Duncan's test

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HcMDH1 enhanced root length in overexpressed lines

No differences in root length were observed between HcMDH1-OEs and WT seedlings under control conditions as well as at varying NaCl and mannitol concentrations. However, after 7 days of treatment, the root length of HcMDH1-OEs was significantly greater than that of WT seedlings under both salt stress (100 or 200 mM NaCl) and drought stress (150 or 300 mM mannitol) (Fig. 6A, B and C). Those data suggest that HcMDH1 enhances the resistance of Arabidopsis to salt and drought stress.

Fig. 6
figure 6

Root length determination of WT and overexpressed lines in salinity and drought stress treatment. A Root length of WT and overexpressed lines in normal, salinity, and drought conditions; B root length determination in 1/2 MS media and 1/2 MS media with 100 mM and 200 mM NaCl; C Root length determination in 1/2 MS media and 1/2 MS media containing 150 mM and 300 mM mannitol. Data are the mean values ± SE (n = 3). * (p < 0.05) or ** (p < 0.01) indicated significant difference in Duncan's test

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HcMDH1 overexpression enhanced salt and drought stress tolerance in Arabidopsis plants

Our study revealed that the leaves of the transgenic lines maintained their green color even under conditions of salt and drought stress, but the leaves of the WT exhibited a color change to purple and yellow. Evaluation of drought resistance in non-irrigated conditions revealed that transgenic strain outgrew WT seedlings in terms of growth, while WT seedlings displayed severe wilting symptoms. After being irrigated again, the transgenic lines regained its vitality and its leaves maintained their green color, exhibiting a higher recovery compared to the WT (Fig. 7A). There was no discernible difference between WT and transgenic lines under normal growth conditions. Nevertheless, when exposed to salt and drought conditions, the transgenic lines exhibited a notably higher survival rate, relative water content, and chlorophyll content (Fig. 7B–D).

Fig. 7
figure 7

Salt and drought tolerance analysis of WT and HcMDH1 transgenic seedlings. A Figurative illustration of the WT and overexpressed lines during normal, salinity, and drought conditions; B survival rates; C relative water content; D chlorophyll content. Different lowercase letters indicated significant differences in Duncan's test (p < 0.05)

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Silencing of HcMDH1 compromised kenaf tolerance to salt and drought

We employed VIGS method to further examine the functionality of the HcMDH1 gene. The positive control pTRV2–HcTrx transformed plants had albino leaves 2 weeks following the VIGS experiment (Fig. S4A, B). The qRT-PCR method was used to determine the relative expression level of HcMDH1 silenced and WT plants. The results showed that the HcMDH1 gene was indeed silenced, as the relative expression level of HcMDH1 in pTRV2–HcMDH1 plants was much lower than that of pTRV2 plants (Fig. S4C). Following exposure to normal, salt, and drought conditions, we observed that plants with silenced HcMDH1 gene exhibited a notable decrease in plant height, fresh weight, root length, and root surface area compared to the control plants (Fig. 9G–J). This decline was observed regardless of whether the stress was caused by salt or drought. When submerged in DAB staining solution, HcMDH1-silenced plants displayed more dark brown color spots under salt or drought stress than those of control, suggesting a higher concentration of H2O2. Similarly, the rise in dispersed dark blue patches suggested that the silenced plants had a higher O2− content than the control group (Fig. S4D, E). Simultaneously, the quantification of H2O2 and O2− content in leaves further verified the staining results (Fig. 9K, and L). In addition, pTRV2 plants showed significantly higher water content and a lower water loss rate than pTRV2–HcMDH1 plants (Fig. S5). These findings demonstrated that kenaf resistance to drought and salt stress was decreased when HcMDH1 was silenced.

Expression of stress‑response and photosynthesis genes in HcMDH1-silenced plants

The results of phenotypic analysis revealed that kenaf is less tolerant to salt and drought stress when HcMDH1 is silenced. qRT-PCR was performed to monitor the changes in transcription levels of stress-related and photosynthesis-related genes in pTRV2 and pTRV2–HcMDH1 lines under salt and drought stress. These genes include Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), D-glycerate 3-kinase (GLYK), fructose-bisphosphate aldolase, class I (FBA), fructose-1, 6-bisphosphatase I (FBPase), 3-Phosphoglycerate (3-PGA), and the lesion simulating disease (LSD). The results showed that the expression levels of HcGAPDH significantly increased, while five others genes were significantly downregulated in kenaf leaves of the pTRV2–HcMDH1 lines (Fig. 10). These findings suggest that silencing the HcMDH1 gene may alter the expression of these endogenous abiotic stress-related genes in response to salt and drought stress, hence either directly or indirectly lowering the plant's stress tolerance.

Discussion

The escalating problems of salt and drought stress in agriculture pose a significant danger to crop production and food security. Plants have developed certain adaptation mechanisms to cope with challenges in order to ensure their survival. These mechanisms include the ability to detect stress signals and rapidly modify their biological responses [34]. The investigation into the mechanism of gene stress resistance has garnered significant interest. Kenaf, a crop known for its high stress resistance, can serve as a valuable tool for understanding the underlying mechanisms of its stress response through the analysis of its gene function. In this experiment, HcMDH1 was found to contain malate dehydrogenase conserved domains (Fig. 1A), and the phylogenetic analysis demonstrated that HcMDH1 exhibited the closest evolutionary relationship to other MDHs within the Malvaceae family (Fig. 1C). This suggests that HcMDH1 in kenaf has been highly conserved throughout the species' evolution. These malate dehydrogenases may have distinct biological roles due to variations in subcellular localization [8, 11, 35]. The subcellular localization experiment indicated that HcMDH1 is mainly located in chloroplasts (Fig. 3A), leading us to hypothesize that it is primarily involved in photosynthesis. The promoter region of a gene often comprises cis-elements which have a direct impact on the regulation of stress responsive gene expression that have significant functions in responding to various stress stimuli [36]. Transcriptional initiation events are determined by the molecular switches that are created by the interactions between transcription factors and cis-acting elements [37]. Hence, the identification of cis-acting elements in the promoter region may help to determine the molecular mechanism of HcMDH1 gene function. Furthermore, the study conducted bioinformatics analysis on the promoter region of HcMDH1 gene found that not only the HcMDH1promoter region contained various types of cis-acting elements, including those associated with photoresponse, hormonal response, development, and abiotic stress response (Fig. 2, Table 1), but also its transcriptional level was significantly induced by salt and drought stress (Fig. 1D, and E). In summary, HcMDH1 is likely to be closely related to kenaf stress resistance.

To study the function of HcMDH1 stress resistance, we first found by yeast resistance assay, heterologous expression of HcMDH1 gene into a yeast system improves the ability of yeast strains to withstand high salt and drought conditions, preliminary providing evidence of its capacity to boost stress resistance (Fig. 3B). To further confirm the role of HcMDH1 in response to abiotic stress in plants, overexpressing HcMDH1 Arabidopsis thaliana seed and seedlings subjected to salt or drought stress on 1/2 MS medium, showed reduced sensitivity to seed germination rate and root growth (Figs. 5 and 6). In addition, under salt and drought stress, transgenic Arabidopsis HcMDH1 can greatly increase survival rate and relative water content (Fig. 7). This study provide evidence that HcMDH1 gene is strongly associated to kenaf signal transduction in response to salt and drought and plays an important regulatory role. As we all know, plants have developed several strategies and advanced mechanisms to cope with drought stress, which include both morphological and physiological alterations [38, 39]. Exposure of plants to abiotic stress leads to the accumulation of ROS within their bodies. The antioxidant enzyme system, which is comprised of SOD, CAT, and POD, is enhanced to a certain extent to eliminate toxic compounds from the plant, mitigate oxidative stress caused by ROS, and preserve normal development [40,41,42]. SOD, POD, and CAT are important indicators reflecting cell oxidative damage [43]. Yao et al. (2011) found that the overexpression of MdcyMDH gene is involved in salt and cold stress resistance, as it enhances the activity of SOD and CAT in transgenic apple callus and tomato, reduces the production of ROS, and improves the resistance of transgenic plants to cold and salt stress [12]. Our study had similar results, overexpression of HcMDH1 significantly increased Arabidopsis thaliana seedlings SOD, POD, and CAT activity (Fig. 8). Chlorophyll, a key component of chloroplasts, captures and transmits sunlight during photosynthesis [44]. In higher plants, chlorophyll plays a crucial role in multiple processes that are essential for plant growth, development, and responses to abiotic stress [45]. Overexpression of HcMDH1 significantly increased the chlorophyll content in the leaves of Arabidopsis thaliana seedlings (Fig. 7). It is consistent with the mMDH1 mutant of soybean showed the phenotype of yellow leaf flower, and the photosynthetic efficiency decreased significantly [13], it may be closely related to the localization of HcMDH1 in chloroplasts. In addition, soluble sugars and proline are important substances for plant osmotic regulation under stress [46]. Soluble sugars are an important source of energy in organisms [47]. Soluble sugars and proline enhance the water solubility in plants, which helps plants to bind proteins with water molecules, ultimately improving osmotic balance and the overall plant resistance towards water stress [47, 48]. The content of H2O2, O2− and malondialdehyde (MDA) also can be used to indicate the degree of damage caused by stress [49]. Our study observed reduced accumulation of H₂O₂ and O₂⁻ in the leaves of HcMDH1-OEs Arabidopsis plants subjected to salt and drought stress (Fig. 8A, B and D). This was accompanied by a significant enhancement in the content in proline and soluble sugar levels (Fig. 8A, J). These results were similar to the overexpression of ZmNADP–MDH in Arabidopsis, which also resulted in improved resistance physiology, including a decrease in ROS and an increase in soluble substance and chlorophyll content [15]. Conversely, the HcMDH1-silenced lines were more sensitive to salt and drought stress than CK, due to more ROS accumulation in the HcMDH1-silenced kenaf (Fig. 9). To sum up, the HcMDH1 gene is closely associated with antioxidant enzyme activity and osmotic regulation in kenaf under salt and drought stress.

Fig. 8
figure 8

Osmoregulation and antioxidant enzyme activity in WT and HcMDH1 transgenic plants. A DAB staining; B H2O2 content; C NBT staining; D O2− content; E–G SOD, POD, CAT activity; H–J MDA, proline, and soluble sugar content, respectively. Different lowercase letters indicated significant differences in Duncan's test (p < 0.05)

Full size image
Fig. 9
figure 9

VIGS analysis in HcMDH1-silenced kenaf plants under salt and drought stresses. A–C Phenotypes of pTRV2 and pTRV2–HcMDH1 plants under 0.5 × Hoagland’s solution, 0.5 × Hoagland’s solution containing 150 mM NaCl, and 15% PEG6000 stresses, respectively; D–F root scans of pTRV2 and pTRV2–HcMDH1 plants under 0.5 × Hoagland’s solution, 0.5 × Hoagland’s solution containing 150 mM NaCl, and 15% PEG6000 stresses, respectively; G plant height; H fresh weight; I root length; J root surface area; K H2O2 content; L O2− content. Different lowercase letters indicated significant differences in Duncan's test (p < 0.05)

Full size image

Glucose, being the primary carbon source in cells, supplies energy to many cellular components and has a vital function in intracellular regulation. Gluconeogenesis and glycolysis are the main pathways involved in the metabolism of glucose. One of the essential enzymes in the processes of glycolysis and gluconogenesis is glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It facilitates the process of oxidative phosphorylation of glyceraldehyde-3-phosphate, resulting in the production of 1, 3-diphosphoglyceric acid. This enzyme is essential for sustaining vital biological functions. Research has demonstrated that GAPDH is present in a wide range of tissues and cells. It has a role in plant immune response and stress response, and is tightly linked to several cellular activities including DNA repair, apoptosis, and cell homeostasis [50]. In this study, under salt or drought conditions, the expression of HcGAPDH either in pTRV2 or in pTRV2–HcGAPDH lines. These results indicated that HcGAPDH gene was involved in stress resistance response in kenaf. The expression levels of pTRV2–HcGAPDH lines were significantly higher than those of pTRV2 lines. Therefore, we hypothesized that the expression of HcGAPDH is negatively regulated by HcMDH1. Fructose-1, 6-bisphosphate aldolase (FBA) and fructose-1, 6-biphosphatase (FBPase) are metabolic enzymes that play crucial roles in glycolysis, gluconeogenesis, and the regulation of photosynthetic rate [51, 52]. Photorespiration interacts with numerous metabolic processes in plants and is thus an integral aspect of plant metabolism [53]. The process of photorespiration is facilitated by 3-phosphoglycerate (3-PGA) and D-glycerate 3-kinase (GLYK) [54, 55]. GLYK catalyzes the phosphorylation of glycerate to produce 3-PGA, which is the final step in the photorespiration pathway [56]. The genes responsible for lesion simulating disease (LSD) encode a group of zinc finger proteins that have a role in programmed cell death (PCD) and serve as a crucial regulatory factor for plants in their response to biotic and abiotic stress [57]. The study found that HcMDH1-silenced kenaf plants had lower transcript levels of those five stress-related and photosynthesis genes relative to the control under salt and drought stress (Fig. 10). These findings suggested that HcMDH1 may function as an integrator of the responses of these stress-related genes to salt and drought stresses. It is possible that HcMDH1 interacts with these stress-responsive genes, photosynthesis-related genes, and metabolic pathways, contributing to salt and drought tolerance.

Fig. 10
figure 10

Relative transcript levels of stress‑response and photosynthesis genes in HcMDH1-silenced kenaf plants. A HcGAPDH; B HcGLYK; C HcFBA; D HcFBPase; E HcPGA; F HcLSD. Different lowercase letters indicated significant differences in Duncan's test (p < 0.05)

Full size image

In summary, this study demonstrates that HcMDH1 positively regulates plant salt tolerance and drought resistance under drought stress by regulating antioxidant enzyme activity and osmotic regulation, as well as some stress-response and photosynthesis genes. However, its specific molecular regulatory mechanisms remain complex and require further investigation. Future studies should focus on elucidating the exact molecular mechanisms by which HcMDH1 regulates salt stress and drought stress, including: on the one hand, analyzing the cis-elements in the HcMDH1 promoter to identify upstream transcription factors that bind to these specific cis-elements, and determining how these factors regulate HcMDH1 expression. On the other hand, identifying the genes that interact with HcMDH1 and elucidating their roles in stress signaling pathways and mechanisms of response to salt and drought stress will provide deeper insights. These studies will enhance our understanding of how HcMDH1 can be enhanced to improve abiotic resistance through genetic engineering.

Conclusion

In the present study, we isolated the HcMDH1 gene from kenaf and performed an in-depth analysis of its molecular mechanism in regulating salt and drought stressors. Subcellular localization showed that the HcMDH1 protein is located in the chloroplast. The expression of HcMDH1 increased in response to salt or drought stress. HcMDH1 enhanced salt and drought tolerance in Arabidopsis through ROS scavenging, reducing lipid oxidative damage, and maintaining osmotic pressure balance. Meanwhile, silencing of HcMDH1 gene resulted in reduced ability of kenaf plants to tolerate high salt levels and drought conditions. Further analysis showed that HcMDH1 notably reduced expression of six genes associated with stress and photosynthesis under salt and drought treatments. In summary, these results provide a foundation for deeper insights into the role of MDHs in plant adaptation to environmental stress. In addition, they offer valuable data for applying HcMDH1 function in plants, potentially leading to advancements in molecular breeding and crop selection for improved stress resistance.

Availability of data and materials

Data are provided within the manuscript or supplementary information files.

Abbreviations

MDH:

Malate dehydrogenase

RNA-Seq:

RNA sequencing

CAT:

Catalase

MDA:

Malondialdehyde

POD:

Peroxidase

SOD:

Superoxide dismutase

qRT-PCR:

Quantitative real-time PCR

DAB:

3, 3′-Diaminobenzidine

NBT:

Nitroblue tetrazolium

H2O2 :

Hydrogen peroxide

O2 − :

Superoxide anion radical

PEG:

Polyethylene glycol

ROS:

Reactive oxygen species

VIGS:

Virus-induced gene silencing

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Acknowledgements

We offer great thanks to Prof. Ru Li for revising the manuscript.

Funding

This research work was supported by the National Natural Science Foundation of China (Grant No. 31960368).

Author information

Authors and Affiliations

  1. College of Agriculture, Key Laboratory of Crop Genetic Breeding and Germplasm Innovation; Guangxi Key Laboratory of Agro-Environment and Agric-Products Safety, Guangxi University, Nanning, 530004, China

    Dengjie Luo, Zengqiang Li, Samavia Mubeen, Muzammal Rehman, Shan Cao, Caijin Wang, Jiao Yue, Jiao Pan & Peng Chen

  2. Henan Collaborative Innovation Center of Modern Biological Breeding, Henan Institute of Science and Technology, Xinxiang, 453003, China

    Zengqiang Li

  3. College of Life Science & Technology, Guangxi University, Nanning, 530004, China

    Dengjie Luo & Ru Li

  4. Guangxi Subtropical Crops Research Institute, Nanning, 530001, China

    Gang Jin & Tao Chen

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Contributions

P.C. conceived the project. D.L. performed the experiment and wrote the main manuscript text. Z.L., S.M. and M. R. revised the manuscript. S.C. and C.W. constructed pGBKT7, pBI121 and VIGS vector. J.Y. and J.P. performed qRT-PCR analysis. G.J., R.L. and T.C. assisted in plant materials management. All the authors have read and approved the manuscript. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Correspondence to Peng Chen.

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Supplementary Information

40538_2024_670_MOESM1_ESM.docx

Additional file 1: Table S1 Primers for qPCR, pGBKT7, pYES2, pBI121 and VIGS vector construction. Table S2 Basic information of MDH genes in kenaf. Fig. S1 Expression of HcMDH1–HcMDH8 genes. Fig. S2 Screening of positive HcMDH1 plants and analysis of HcMDH1 expression. Fig. S3 Germination rate of HcMDH1-OEs and WT seeds under different concentrations of NaCl and mannitol treatment. Fig. S4 Silencing HcMDH1 detection of kenaf VIGS. Fig. S5 Relative water content and water loss rate of pTRV2 and pTRV2–HcMDH1 plants.

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Luo, D., Li, Z., Mubeen, S. et al. Functional characterization of malate dehydrogenase, HcMDH1, gene in enhancing abiotic stress tolerance in kenaf (Hibiscus cannabinus L.). Chem. Biol. Technol. Agric. 11, 141 (2024). https://doi.org/10.1186/s40538-024-00670-1

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