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MAP  >  Fruit Research
2024 Volume 4
Article Contents
Abstract
Introduction
Materials and methods
Plant materials, growth conditions, and stress treatment
Vector construction
Chlorophyll and anthocyanin content determination
Physiological analysis
RNA isolation and qRT-PCR analysis
Results
Screening and characterization of lncRNAs
Generation of lncRNA overexpression lines
Effect of lncRNA overexpression on heat tolerance in Arabidopsis
Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis
Effect of lncRNA overexpression on drought tolerance in Arabidopsis
Discussion
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
Rights and permissions
References
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ARTICLE   Open Access    

MbbHLH93, a transcription factor associated with cold and drought tolerance in Malus baccata

  • 1.

    The Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs/National-Local Joint Engineering Research Center for Development and Utilization of Small Fruits in Cold Regions, College of Horticulture & Landscape Architecture, Northeast Agricultural University, Harbin 150030, Heilongjiang, China

  • 2.

    Horticulture Branch of Heilongjiang Academy of Agricultural Sciences, Harbin 150040, Heilongjiang, China

  • # Authors contributed equally: Lihua Zhang, Yu Xu

More Information
  • Received: 04 September 2024
    Revised: 25 September 2024
    Accepted: 27 September 2024
    Published online: 02 December 2024
    Fruit Research  4,  Article number: e038 (2024)  |  Cite this article
  • The bHLH transcription factor is known to regulate cold signals and stress tolerance. In the present study, a new bHLH gene MbbHLH93, located in the nucleus, was isolated from Malus baccata, whose up-regulated expression were strongly induced by cold and drought treatment, and MbbHLH93-overexpressed heterologous lettuce plants displayed cold and drought stress-tolerant phenotypes. Determination of physiological and biochemical indexes associated with abiotic stress responses showed that overexpression of MbbHLH93 increased the activities of antioxidant enzymes superoxide dismutase, peroxidase, and catalase in lettuce plants treated with cold and drought stress, and decreased the contents of H2O2, O2·−, and malondialdehyde, which contributed to reducing cell membrane lipid peroxidation. Meanwhile, the accumulation of proline in transgenic plant cells increased, regulating cell osmotic pressure. Furthermore, quantitative expression analysis indicated that overexpression of MbbHLH93 improved the expression levels of LsCBFs, which were positive functional genes in response to cold and drought stress, enhancing plant tolerance. This research demonstrates that the MbbHLH93 is a key regulator in plant tolerance to cold and drought stresses, providing new knowledge for plant tolerance regulation.

  • Introduction

    Plants are continuously subjected to unpredictable environmental conditions and encounter a multitude of stressors throughout their growth and development, posing a significant challenge to global crop production and food security[1]. Heat and drought are undoubtedly the two most important stresses that have a huge impact on crops. Both elicit a wide array of biochemical, molecular, and physiological alterations and responses, impacting diverse cellular processes and ultimately influencing crop yield and quality[2].

    A primary physiological consequence of both stresses is the diminished photosynthetic capacity, partially resulting from the degradation of chlorophyll due to leaf senescence under stress conditions. Chlorophyll accumulation was diminished in numerous plants subjected to drought or heat stress conditions[3,4]. Various environmental stresses prompt excessive generation of reactive oxygen species (ROS), initiating oxidative damage that compromises lipids, and proteins, and poses a serious threat to cellular functions[2]. To mitigate oxidative stress and minimize damage, plants have developed various protective mechanisms to neutralize ROS. Several antioxidant enzymes, such as SOD, POD, and CAT, are integral to cellular antioxidative defense mechanisms. Additionally, antioxidants such as anthocyanins and proline serve as crucial ROS scavengers[5,6]. The elevation in temperature typically induces the transient synthesis of heat shock proteins (Hsps), which function as molecular chaperones in protecting proteins from denaturation and aggregation, with their activity primarily regulated at the transcriptional level by heat shock factors (Hsfs)[7]. The significance of Hsps and Hsfs in all organisms, including plants, has been assessed in various stress conditions that could disrupt cellular homeostasis and result in protein dysfunction[7]. Drought stress can also trigger the transcription of a suite of marker genes, including RD29A, RD29B, NCED3, AREB1, Rab18, etc., which assist plants in mitigating cellular damage during dehydration and bolstering their resilience to stress[8−10].

    Previous research efforts focusing on the regulatory control of stress-related genes have largely centered around protein-coding genes. In recent years, non-protein-coding transcripts have emerged as important regulatory factors in gene expression. Among them, long non-coding RNAs (lncRNAs) lncRNAs have been identified as implicated in various abiotic stresses[11,12]. LncRNAs are a class of non-coding RNAs (ncRNAs) exceeding 200 nucleotides in length. They possess minimal or no protein-coding potential[13]. In plants, lncRNAs are specifically transcribed by RNA polymerases Pol IV, Pol V, Pol II, and Pol III[14,15]. LncRNAs exhibit low abundance and display strong tissue and cellular expression specificity relative to mRNAs. Moreover, sequence conservation of lncRNAs is was very poor across different plant species[13,16,17]. The widespread adoption of high-throughput RNA sequencing technology has revealed lncRNAs as potential regulators of plant development and environmental responses. In cucumber, RNA-seq analysis has predicted 2,085 lncRNAs to be heat-responsive, with some potentially acting as competitive endogenous RNAs (ceRNAs) to execute their functions[18]. In radish, a strand-specific RNA-seq (ssRNA-seq) technique identified 169 lncRNAs that were differentially expressed following heat treatment[19]. In Arabidopsis, asHSFB2a, the natural antisense transcript of HSFB2a was massively induced upon heat stress and exhibited a counteracted expression trend relative to HSFB2a. Overexpression of asHSFB2a entirely suppressed the expression of HSFB2a and impacted the plant's response to heat stress[20]. For drought stress resistance, 244 lncRNAs were predicted in tomatoes to be drought responsive probably by interacting with miRNAs and mRNAs[21]. Under drought stress and rehydration, 477 and 706 lncRNAs were differentially expressed in drought-tolerant Brassica napus Q2 compared to drought-sensitive B. napus, respectively[22]. In foxtail millet and maize, 19 and 644 lncRNAs, respectively, were identified as drought-responsive[23,24]. Despite the identification of numerous lncRNAs by high-throughput sequencing, which suggests their potential involvement in various abiotic stress processes, only a minority have been experimentally validated for function.

    In our previous study, we characterized 1,229 differentially expressed (DE) lncRNAs in Chinese cabbage as heat-responsive, and subsequent bioinformatics analysis reduced this number to 81, which are more likely associated with heat resistance[25]. lnc000283 and lnc012465 were selected from among them for further functional investigation. The findings indicated that both lnc000283 and lnc012465 could be promptly induced by heat shock (HS). Overexpression of either lnc000283 or lnc012465 in Arabidopsis plants enhanced their capacity to tolerate heat stress. Additionally, both lnc000283 and lnc012465 conferred drought tolerance to transgenic Arabidopsis.

    Materials and methods

    Plant materials, growth conditions, and stress treatment

    The lncRNA sequences examined in this study were from Chiifu-401-42 Chinese cabbage and all Arabidopsis plants were of the Col-0 background. Transgenic plants expressing lnc000283 and lnc012465 were generated using the Agrobacterium tumefaciens-mediated floral dip method[26]. Single-copy and homozygous T3 plants were identified through genetic segregation on an agar medium supplemented with kanamycin. The T3 generation plants, or their homozygous progeny, were utilized in the experiments.

    For phenotypic assessment, Arabidopsis seeds were initially sown on filter paper moistened with ddH2O and placed in a 4 °C freezer for 2 d. Subsequently, they were evenly planted in nutrient-rich soil and transferred to a growth chamber operating a 16-h day/8-h night cycle, with day/night temperatures of 22 °C/18 °C and a light intensity of 250 μmol·m−2·s−1. After 10 d of growth, Arabidopsis plants with uniform growth were transferred to 50-hole plates. Arabidopsis plants grown in Petri dishes were firstly seed-sterilized and then sown on 1/2 MS medium supplemented with 10 g·L−1 sucrose. The seeds were then placed in a 4 °C refrigerator for 2 d in the dark before transferring them to a light incubator. The day/night duration was set to 16 h/8 h, the day/night temperature to 21 °C/18 °C, and the light intensity to 100 μmol·m−2·s−1.

    For heat treatment, 3-week-old seedlings were subjected to 38 °C for 4 d within a light incubator, subsequently transferred to their original growth conditions under the same light/dark cycles. For drought treatment, 3-week-old Arabidopsis seedlings were deprived of water for 10 d, followed by rehydration to facilitate a 2-d recovery period. Plants were photographed and surveyed both before and after treatment.

    Vector construction

    The lncRNA sequences (lnc000283 and lnc012465) were chemically synthesized based on RNA-seq data, with restriction sites for BamH1 and Kpn1 engineered upstream and downstream. The resultant lncRNA constructs were subcloned into the pCambia2301 binary vector, incorporating a cauliflower mosaic virus (CaMV) 35S promoter. The recombinant vectors were transformed into Escherichia coli TOP10 competent cells (Clontech), incubated at 37 °C overnight, after which single clones were selected for PCR verification, and the confirmed positive colonies were submitted for sequencing. Following verification, the correct plasmids were introduced into A. tumefaciens strain GV3101 using the freeze-thaw method and subsequently transformed into Arabidopsis wild-type (Col) plants.

    Chlorophyll and anthocyanin content determination

    To quantify the chlorophyll content, the aerial portions of wild-type and transgenic Arabidopsis plants, grown in Petri dishes were weighed, minced, and then subjected to boiling in 95% ethanol until fully decolorized. Aliquots of 200 μL from the extract were transferred to a 96-well plate and the absorbance at 663 nm and 645 nm was measured via spectrophotometry by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Three biological replicates were analyzed for WT and each transgenic line. Chlorophyll content was determined according to the formula of the Arnon method[27]: Chlorophyll a = (12.72A663 − 2.59A645) v/w, Chlorophyll b = (22.88A645 − 4.67A663) v/w, Total chlorophyll = (20.29A645 + 8.05A663) v/w.

    The quantification of anthocyanin was performed as follows: aerial parts of wild-type and transgenic Arabidopsis plants, cultivated in Petri dishes, were weighed and ground to powder in liquid nitrogen. Subsequently, the samples were incubated in 600 μL of acidified methanol (containing 1% HCl) at 70 °C for 1 h. Following this, 1 mL of chloroform was added, and the mixture was vigorously shaken to remove chlorophyll. The mixture was then centrifuged at 12,000 rpm for 5 min, after which the absorbance of the aqueous phase was determined at 535 nm using a spectrophotometer (Shimadzu, Kyoto, Japan). Three biological replicates were analyzed for WT and each transgenic line. The relative anthocyanin content was calculated according to anthocyanin concentration and extraction solution volume. One anthocyanin unit is defined as an absorption unit at a wavelength of 535 nm in 1 mL of extract solution. In the end, the quantity was normalized to the fresh weight of each sample.

    Physiological analysis

    Three-week-old transgenic and WT A. thaliana plants, subjected to normal conditions or varying durations of heat or drought stress, were utilized for subsequent physiological assessments. All assays were performed in accordance with the method described by Chen & Zhang[28]. In brief, 0.1 g of fresh leaf tissue was homogenized in 500 μL of 100 mM PBS (pH 7.8) while chilled on ice. The homogenate was then centrifuged at 4 °C, and the resultant supernatant was employed for further analysis. For the determination of MDA content, 100 μL of the supernatant was combined with 500 μL of a 0.25% thiobarbituric acid (TBA) solution (which was prepared by dissolving 0.125 g of TBA in 5 mL of 1 mol·L−1 NaOH before being added to 45 mL of 10% TCA) and boiled for 15 min. Following a 5 min cooling period on ice, the absorbance was measured at 532 nm and 600 nm. The activity of POD was determined as follows: initially, 28 μL of 0.2% guaiacol and 19 μL of 30% H2O2 were sequentially added to 50 mL of 10mM PBS (pH 7.0), after thorough heating and mixing, 1 mL was transferred into a cuvette, then 50 μL of the supernatant was added to the cuvette and the absorbance at 470 nm was monitored every 15 s for 1 min. To determine the proline content, a reaction solution was prepared by mixing 3% sulfosalicylic acid, acetic acid, and 2.5% acidic ninhydrin in a ratio of 1:1:2, then 50 μL of the supernatant was added to 1 mL of the reaction solution, which was then subjected to a boiling water bath for 15 min (the solution turned red after the boiling water bath). Following cooling on ice, the absorbance at 520 nm was recorded. For the quantification of proline, an L-proline standard curve was prepared by dissolving 0, 5, 10, 15, 20, 25, and 30 μg of L-proline in 0.5 mL of ddH2O, followed by the addition of 1 mL of the reaction solution and measuring the absorbance at 520 nm. The proline content in the samples was then determined based on the L-proline standard curve.

    RNA isolation and qRT-PCR analysis

    Total RNA was isolated from the aerial parts of Arabidopsis using the TaKaRa MiniBEST Plant RNA Extraction Kit, followed by purification and reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (Takara). The cDNA product was diluted 10 times and real-time PCR was conducted in triplicate for each biological replicate using SYBR PCR Master Mix (Applied Biosystems) on the ABI 7500 system under the following conditions: 98 °C for 3 min, followed by 40 cycles of 98 °C for 2 s and 60 °C for 30 s. The relative expression levels of each gene were normalized against the transcript abundance of the endogenous control UBC30 (At5g56150) and calculated using the 2−ΔCᴛ method. The specific primers employed for qRT-PCR are detailed in Supplemental Table S1.

    Results

    Screening and characterization of lncRNAs

    In our prior investigation, dozens of lncRNAs associated with the heat stress response in Chinese cabbage were identified through informatics analysis. Two lncRNAs (lnc000283 and lnc012465) were chosen for genetic transformation in Arabidopsis to elucidate their functions comprehensively. Transcriptome data analysis indicated that the expression of lnc000283 and lnc012465 in Chinese cabbage were both induced by HS. To verify the accuracy, the expression patterns of lnc000283 and lnc012465 were confirmed through quantitative real-time PCR (qRT-PCR), and the results from qRT-PCR were consistent with those obtained from RNA-seq (Fig. 1a). The corresponding homologous genes in Arabidopsis were identified as CNT2088434 and CNT2088742, exhibiting sequence similarities of 88% and 87%, respectively (Supplemental Fig. S1). Subcellular localization predictions using the lnclocator database (www.csbio.sjtu.edu.cn/bioinf/lncLocator) suggested that both lncRNAs are localized within the nucleus (Supplemental Table S2). Bioinformatics analysis was conducted using the CPC tool (http://cpc.cbi.pku.edu.cn/) indicated that lnc000283 and lnc012465 are noncoding sequences, with coding probabilities of 0.0466805 and 0.0432148, respectively comparable to the well-characterized lncRNAs COLDAIR and Xist, but significantly lower than those of the protein-coding genes UBC10 and ACT2 (Fig. 1b).

    Figure 1.  Characteristics of lnc000283 and lnc012465. (a) Expression level of lnc000283 and lnc012465 in Chinese cabbage leaves treated at 38 °C at different time points, as determined by qRT-PCR and RNA-seq. CK is a representative plant before heating, and T1, T4, T8, and T12 denote plants that were subjected to 38 °C for 1, 4, 8, and 12 h, respectively. The expression levels were normalized to the expression level of Actin. (b) Analysis of coding potential for lnc000283 and lnc012465. The coding potential scores were calculated using the CPC program. UBC10 (At5g53300) and ACT2 (At3g18780) are positive controls that encode proteins. COLDAIR (HG975388) and Xist (L04961) serve as negative controls, exhibiting minimal protein-coding potential.
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    Generation of lncRNA overexpression lines

    To elucidate the role of lnc000283 and lnc012465 in response to abiotic stress, overexpression vectors were constructed for these lncRNAs, driven by the CaMV 35S promoter, and they were introduced into Arabidopsis thaliana (Col-0 ecotype). Through PCR identification and generational antibiotic screening, two homozygous positive lines for lnc012465 and lnc000283 were obtained. The relative expression levels of these lncRNAs were assessed using qRT-PCR (Fig. 2a). When plants were grown in 1/2 MS medium, with the consumption of nutrients, and reduction of water, the leaves of WT began to turn yellow, but the lnc000283 and lnc012465 overexpression lines developed a deep purple color of leaf veins (Fig. 2b). Examination of chlorophyll and anthocyanin contents in the plants revealed that both overexpression lines had higher levels of chlorophyll and anthocyanin compared to the WT, suggesting that the transgenic plants might possess enhanced resistance to nutritional or water stress (Fig. 2c, d).

    Figure 2.  Arabidopsis plants overexpressing lnc000283 and lnc012465 had higher anthocyanins and chlorophyll content. (a) The relative expression level of lnc000283 and lnc012465 in WT and different transgenic lines. UBC10 (At5g53300) was used as an internal control. Each value is mean ± sd (n = 3). (b) The phenotype of WT and Arabidopsis overexpressing lnc000283 or lnc012465 grown on 1/2 MS medium 50 d after sowing. The (c) anthocyanin and (d) chlorophyll content of WT and transgenic Arabidopsis overexpressing lnc000283 or lnc012465. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01).
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    Effect of lncRNA overexpression on heat tolerance in Arabidopsis

    Given that lnc000283 and lnc012465 were highly induced by heat, the thermotolerance of the overexpressing (OE) plants were compared to that of the wild type. Arabidopsis plants were initially exposed to a an HS treatment at 38 °C for 4 d, followed by recovery at room temperature. The death caused by HS was processive. Post-severe HS challenge for 4 d, OE plants initially appeared similar to WT, but upon recovery, their leaves started to fold or curl, followed by a transition to yellow, white, and eventually drying out (Fig. 3a). OE lnc000283 and OE lnc012465 plants exhibited enhanced thermotolerance compared to WT, with lnc012465 showing particularly strong tolerance (Fig. 3a; Supplemental Fig. S2a). After 5 d of recovery, leaf coloration indicated that transgenic plants maintained a significantly higher percentage of green leaves and a lower percentage of bleached leaves compared to WT (Fig. 3b; Supplemental Fig. S2b). Under non-heat-stress conditions, WT and OE plants possessed comparable water content. However, following heat stress, the fresh-to-dry weight ratio of OE lnc000283 and lnc012465 lines was significantly greater than that of WT (Fig. 3c; Supplemental Fig. S2c). Abiotic stresses frequently trigger the production of excessive reactive oxygen species (ROS), which are believed to cause lipid peroxidation of membrane lipids, leading to damage to macromolecules. Leaf MDA content is commonly used as an indicator of lipid peroxidation under stress conditions; therefore, the MDA content in both transgenic and WT plants was assessed. Figure 3d shows that the MDA content in WT plants progressively increased after heat treatment, whereas in the two lines overexpressing lnc012465, the MDA content increased only slightly and remained significantly lower than that in WT at all time points. In plants overexpressing lnc000283, the MDA content did not significantly differ from that of WT before heat stress. However, after 4 d of heat treatment, the MDA content was significantly lower compared to WT (Supplemental Fig. S2d). The results suggested that the expression of both lnc012465 and lnc000283 can mitigate injury caused by membrane lipid peroxidation under heat-stress conditions. Peroxidase (POD) is a crucial antioxidant enzyme involved in ROS scavenging. Figure 3e and Supplemental Fig. S2e demonstrate that POD activity increased in both transgenic and WT plants after heat treatment. However, the increase in WT plants was modest, whereas OE lnc000283 and OE lnc012465 plants exhibited consistently higher POD activity. As anticipated, proline levels were induced in response to stress in all studied plants (Fig. 3f; Supplemental Fig. S2f). However, under normal conditions and 2 d post-heat stress treatment, the proline content in OE lnc000283 and OE lnc012465 plants did not exhibit significant changes compared to WT (Fig. 3f; Supplemental Fig. S2f). Moreover, after 4 d of heat stress, the proline content in OE lnc012465 lines was significantly lower than in WT, and the OE lnc000283 transgenic line 12-6 also showed a marked decrease in proline content compared to WT (Fig. 3f; Supplemental Fig. S2f). The results indicated that the thermotolerance of plants overexpressing either lnc000283 or lnc012465 was independent of proline accumulation.

    Figure 3.  Overexpressing lnc012465 lines are more tolerant to heat stress. (a) Phenotypes of WT and OE lnc012465 plants were assessed before and after exposure to heat stress. The heat treatment was applied to 25-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh-to-dry weight ratio of Arabidopsis leaves was measured before and after 38 °C heat treatment. (d)−(f) depict the MDA content, POD activity, and proline content in Arabidopsis leaves at varying durations of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis

    To elucidate the molecular mechanisms by which lncRNAs enhance thermotolerance in Arabidopsis, the expression of the Hsf gene HsfA7a and three Hsps (Hsp25.3, Hsa32, and Hsp18.1-CI) in OE lnc000283, OE lnc012465, and WT Arabidopsis plants were investigated at various time points following heat treatment. As shown in Fig. 4 and Supplemental Fig. S3, both Hsf and Hsps exhibited a rapid response to heat stress with strong induction. Notably, the transcripts of HsfA7a and Hsp25.3 were significantly upregulated at 1 h after heat exposure, then experienced a sharp decrease. Hsa32 and Hsp18.1-CI were highly induced at 1 h and, unlike the other proteins, sustained high expression levels at 3 h (Fig. 4; Supplemental Fig. S3). At 1 h post-heat treatment, the transcript levels of Hsa32 and HsfA7a in OE lnc000283 did not significantly differ from those in WT. However, by 3 h, Hsa32 expression was roughly 50% of the WT level, while HsfA7a expression was approximately double that of WT (Supplemental Fig. S3). The overexpression of lnc000283 did not significantly affect the transcript level of Hsp25.3 at any of the tested time points. Notably, Hsp18.1-CI expression in both lines overexpressing lnc000283 was significantly induced at all three detection points post-heat treatment, reaching approximately 4-9-fold higher levels than in the WT (Supplemental Fig. S3). In Arabidopsis plants with elevated expression of lnc012465, the expression patterns of all Hsp and Hsf genes were similar to those in plants overexpressing lnc000283, with the notable exception of Hsa32. Unlike the WT, Hsa32 did not show a trend of down-regulation at 3 h post-heat treatment (Fig. 4). The findings suggest that the substantial induction of Hsp18.1-CI may play a role in enhancing the thermotolerance of Arabidopsis plants overexpressing lnc000283 and lnc012465.

    Figure 4.  The expression of HSF and HSP genes in lnc012465 overexpressing lines before and after different heat treatment times. Gene expression levels were quantified using RT-qPCR and normalized to UBC10 (At5g53300). Each value represents the mean ± standard deviation (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Effect of lncRNA overexpression on drought tolerance in Arabidopsis

    Prior research has implicated a significant proportion of genes in conferring resistance to various abiotic stresses. To elucidate the functions of lnc000283 and lnc012465 more thoroughly, WT and transgenic plants were subjected to drought stress by depriving them of water for 9 d. It was noted that the majority of leaves in WT plants withered and dried, whereas the OE lnc000283 and OE lnc012465 plants exhibited reduced withering, with only a minority displaying dryness (Fig. 5a; Supplemental Fig. S4a). Eight days post-rewatering, a negligible fraction of WT seedlings exhibited recovery, whereas the overwhelming majority of transgenic plants regained vigorous growth (Fig. 5a; Supplemental Fig. S4a). The transgenic plants demonstrated a significantly higher survival rate compared to the WT plants. Following 9 d of water deficit treatment, less than 40% of the WT plants survived, whereas the OE 012465 lines 8-7 and 9-1 exhibited survival rates of 100% and 95%, respectively, and the OE 000283 lines 11-10 and 12-6 had survival rates of 87% each. (Fig. 5b; Supplemental Fig. S4b). Water loss serves as a critical metric for assessing plant drought tolerance, hence the fresh-to-dry weight ratio of excised leaves was assessed via desiccation analysis. Following 4 d of drought treatment, the fresh-to-dry weight ratio for WT plants was reduced to 43%, whereas for OE lnc000283 lines 11-10 and 12-6, it was reduced to 73% and 75%, respectively. For OE 012465 lines 8-7 and 9-1, the ratios were reduced to 67% and 62%, respectively (Fig. 5c; Supplemental Fig. S4c). The findings indicated that lnc000283 and lnc012465 endow the transgenic plants with drought tolerance.

    Figure 5.  Overexpressing lnc012465 lines are more tolerant to drought stress. (a) Phenotype of WT and OE lnc012465 plants before and after subjecting to drought stress. Drought treatment was carried out on 20-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh weight to dry weight ratio of Arabidopsis leaves before and after undergoing 38 °C heat treatment. (d)−(f) MDA content, POD activity, and proline content in Arabidopsis leaves under different times of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001)
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    MDA content in leaves is a standard biomarker for assessing the extent of drought stress-induced damage. Prior to drought stress exposure, MDA levels in WT and transgenic plants were comparable. However, following 7 and 9 d of water deficit, the MDA content in the transgenic plants was markedly reduced compared to the WT, suggesting a less severe degree of membrane lipid peroxidation in the transgenic plants (Fig. 5d; Supplemental Fig. S4d). Oxidative stress frequently coincides with drought stress, hence the activity of POD was assessed to evaluate the ROS scavenging ability. The findings indicated that as the duration of drought treatment increased, POD activity progressively rose. Before drought exposure, the POD activity in lines 11-10 and 12-6 of OE 000283 was 2.4-fold and 2.2-fold higher than that of the WT, respectively (refer to Supplemental Fig. S4e). Following drought treatment, the POD activity in the transgenic lines remained significantly elevated compared to the wild type, although the enhancement was less pronounced than before the treatment (Supplemental Fig. S4e). In the OE 012465 plants, the POD activity in lines 8-7 and 9-1 significantly surpassed that of the wild type, with the discrepancy being more pronounced during drought stress (Fig. 5e). The proline content in WT and OE 000283 plants exhibited no significant differences before and after 7 d of treatment. However, after 9 d of drought, the proline content in OE 000283 plants was significantly lower compared to that in the WT (Supplemental Fig. S4f). OE 000465 plants showed no significant difference from the wild type before and after drought treatment (Fig. 5f). The findings were consistent with those under heat stress, indicating that the enhanced stress resistance due to the overexpression of lnc000283 and lnc012465 in Arabidopsis is not reliant on proline accumulation.

    Following drought stress treatment, the expression levels of drought-related genes such as RD29A, RD29B, NCED3, AREB1, and Rab18 were significantly elevated in plants overexpressing lnc000283 and lnc012465 compared to WT plants. These findings suggest that lnc000283 and lnc012465 modulate Arabidopsis drought tolerance by regulating the expression of genes associated with the drought stress response (Fig. 6; Supplemental Fig. S5).

    Figure 6.  The expression of drought-responsive genes in lnc012465 overexpressing lines before and after different drought treatment time. Gene expression levels were determined by qRT-PCR normalized against UBC10 (At5g53300). Each value is mean ± sd (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Discussion

    The integrity of global food security is under threat due to the confluence of rapid population expansion and profound climatic shifts[29]. Amidst the shifting climatic landscape, heat and drought stress have emerged as primary limitations to crop yield and global food security. Understanding how plants detect stress cues and acclimate to challenging conditions is a pivotal biological inquiry. Moreover, enhancing plant resilience to stress is essential for maintaining agricultural productivity and fostering environmental sustainability[2]. Concurrently, the advancement of next-generation sequencing (NGS) technology has led to the identification of a substantial number of lncRNAs that participate in diverse stress responses, with functional analyses having been conducted on several of these molecules.[30] For instance, in the case of potatoes, the lncRNA StFLORE has been identified to modulate water loss through its interaction with the homologous gene StCDF1[31]. LncRNA TCONS_00021861 can activate the IAA biosynthetic pathway, thereby endowing rice with resistance to drought stress[32]. In wheat, the expression of TalnRNA27 and TalnRNA5 was upregulated in response to heat stress[33]. Our prior investigation identified a total of 81 lncRNAs in Chinese cabbage that engage in intricate interactions with their respective mRNA targets across various phases of heat treatment[25]. Two lncRNAs, lnc000283 and lnc012465, were chosen for subsequent functional analysis. Findings confirmed that these lncRNAs endow transgenic Arabidopsis plants with enhanced tolerance to both heat and drought, thereby offering novel resources for enhancing stress resistance through genetic engineering.

    Abiotic stresses frequently trigger the synthesis of anthocyanins, serving as natural antioxidants that mitigate oxidative damage by neutralizing surplus reactive oxygen species (ROS), thereby protecting plants from growth inhibition and cell death, allowing plants to adapt to abiotic stresses[34,35]. For instance, during chilling stress, the accumulation of anthocyanins within leaves can mitigate oxidative damage, thereby enhancing the photosynthetic rate[36]. Consequently, the level of abiotic stress tolerance can be inferred from the concentration of anthocyanins. The reduction of photosynthetic ability is one of the key physiological phenomena of stresses, which is partly due to the degradation of chlorophyll caused by leaf senescence during stress. The reduced accumulation of chlorophyll in the plants was seen in many plants when exposed to drought or heat stress conditions. The current investigation revealed that lncRNA-overexpressing plants cultivated in Petri dishes exhibited increased accumulation of both chlorophyll and anthocyanins in advanced growth phases, indicating that these transgenic plants, overexpressing lnc000283 and lnc012465, demonstrated enhanced stress tolerance and superior growth performance relative to WT (Fig. 2c, d).

    Upon exposure to heat stress, there is a marked induction of transcription for numerous genes that encode molecular chaperones in plants, with the vast majority of these genes contributing to the prevention of protein denaturation-related damage and the augmentation of thermotolerance[37−39]. The present investigation identified multiple heat-inducible genes in plants overexpressing lnc000283 and lnc012465, as well as in WT (Fig. 4; Supplemental Fig. S3). The findings indicated that of the four HSP or HSF genes examined, Hsp18.1-CI exhibited a significantly greater abundance in both OE lnc000283 and OE lnc012465 plants compared to the WT following heat treatment for several days. Hsp18.1-CI, formerly referred to as Hsp18.2 has been the subject of investigation since 1989.[40] Following the fusion of the 5' region of Hsp18.2 in frame with the uidA gene of Escherichia coli, the activity of GUS, serving as the driver gene was observed to increase upon exposure to HS[40]. The Arabidopsis hsfA2 mutant exhibited diminished thermotolerance after heat acclimation, with the transcript levels of Hsp18.1-CI being substantially reduced compared to those in wild-type plants following a 4-h recovery period[41]. The findings revealed that the upregulation of Hsp18.1-CI protein is a critical mechanism by which plants achieve enhanced protection against heat stress in adverse environmental conditions, thereby bolstering their thermotolerance.

    Plants cultivated in natural settings are often subjected to concurrent multiple abiotic stresses, which can exacerbate threats to their routine physiological functions, growth, and developmental processes[42,43]. Elucidating the molecular mechanisms underlying plant responses to abiotic stress is crucial for the development of new crop varieties with enhanced tolerance to multiple abiotic stresses. Previous research has indicated that the overexpression of certain protein-coding genes can endow plants with resistance to a variety of abiotic stresses. For instance, tomatoes with robust expression of ShCML44 demonstrated significantly enhanced tolerance to drought, cold, and salinity stresses[44]. Overexpression of PeCBF4a in poplar plants confers enhanced tolerance to a range of abiotic stresses[45]. With respect to lncRNAs, transgenic Arabidopsis plants that overexpress lncRNA-DRIR displayed marked increased tolerance to salt and drought stresses compared to the wild-type[46]. In the present study, both overexpression lines of lnc000283 and lnc012465 exhibited resistance to heat and drought stresses, thereby contributing to the enhancement of plant resilience against multiple stresses (Figs 3, 5; Supplemental Figs S2, S4).

    The number of genes implicated in plant drought resistance is regulated by both ABA-dependent and ABA-independent pathways[47,48]. It is well established that the expression of RD29A exhibits a high level of responsiveness to drought stress, operating through both ABA-dependent and ABA-independent mechanisms[49]. RD29B, AREB1, and RAB18 are governed by an ABA-dependent regulatory pathway[10,49,50]. NCED3 is involved in ABA biosynthesis[51]. In the present study, the transcript levels of RD29A, RD29B, NCED3, AREB1, and RAB18 were significantly elevated in OE lnc000283 and OE lnc012465 plants compared to those in the WT plants (Fig. 6; Supplemental Fig. S5). The findings indicated that the drought tolerance imparted by OE lnc000283 and OE lnc012465 plants is contingent upon an ABA-dependent mechanism.

    Prior research has indicated that certain long non-coding RNAs (lncRNAs) can assume analogous roles across diverse biological contexts. For example, the lncRNA bra-miR156HG has been shown to modulate leaf morphology and flowering time in both B. campestris and Arabidopsis[52]. Heterogeneous expression of MSL-lncRNAs in Arabidopsis has been associated with the promotion of maleness, and similarly, it is implicated in the sexual lability observed in female poplars[53]. In the present study, lnc000283 and lnc012465 were induced by heat in Chinese cabbage, and their heterologous expression was found to confer heat tolerance in Arabidopsis. Additionally, sequences homologous to lnc000283 and lnc012465 were identified in Arabidopsis (Supplemental Fig. S1). The data suggest that these sequences may share a comparable function to that of heat-inducible sequences, potentially accounting for the conservation of lnc000283 and lnc012465'os functionality across various species.

    Conclusions

    In conclusion, the functions of two heat-inducible lncRNAs, lnc000283 and lnc012465 have been elucidated. Transgenic Arabidopsis lines overexpressing these lncRNAs accumulated higher levels of anthocyanins and chlorophyll at a later stage of growth compared to the WT when grown on Petri dishes. Furthermore, under heat and drought stress conditions, these OE plants exhibited enhanced stress tolerance, with several genes related to the stress resistance pathway being significantly upregulated. Collectively, these findings offer novel insights for the development of new varieties with tolerance to multiple stresses.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and supervision: Li N, Song X; experiment performing: Wang Y, Sun S; manuscript preparation and revision: Wang Y, Feng X, Li N. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article and its supplementary information files.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (32172583), the Natural Science Foundation of Hebei (C2021209019), the Natural Science Foundation for Distinguished Young Scholars of Hebei (C2022209010), and the Basic Research Program of Tangshan (22130231H).

  • The authors declare that they have no conflict of interest.

  • Supplementary Table S1 Primers designed in the present study.
    Supplementary Table S2 Physiological and biochemical properties of test proteins. CDS, coding sequence; MW, molecular weight of the amino acid sequence; GRAVY, grand average of hydropathicity; pI, theoretical isoelectric point.
    Supplemental Fig. S1 The expression patterns of MbbHLH93 in root, stem, new leaf and mature leaf of Malus baccata. Relative expression levels of MbbHLH93 were calculated via the via 2−△△Ct method and values were shown as the mean ± standard deviation (SD) based on the three repetitions. Lowercase letters displayed the significant differences at p < 0.05.
    Supplemental Fig. S2 Genetic transformation and screening of MbbHLH93 overexpressed lettuce. (a) the genetic transformation process of MbbHLH93 overexpressed lettuce; including coculture stage, calli acquisition, resistant bud screening, rooting stage, transplanting of transgenic seedlings, and seed acquisition stage. (b) Identification of MbbHLH93-overexpressed lettuce lines by semi-quantitative RT-PCR analyses.
    Supplemental Fig. S3 Relevant physiological indicators of MbbHLH93 transgenic lettuces (L1, 7, 8) under cold and drought stress. Values were shown as the mean ± standard deviation (SD) based on the three repetitions. Lowercase letters displayed the significant differences at p < 0.05 (one-way ANOVA followed by Tukey's multiple range test).
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  • Cite this article

    Zhang L, Xu Y, Lv L, Wang T, Liu W, et al. 2024. MbbHLH93, a transcription factor associated with cold and drought tolerance in Malus baccata. Fruit Research 4: e038 doi: 10.48130/frures-0024-0032
    Zhang L, Xu Y, Lv L, Wang T, Liu W, et al. 2024. MbbHLH93, a transcription factor associated with cold and drought tolerance in Malus baccata. Fruit Research 4: e038 doi: 10.48130/frures-0024-0032
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MbbHLH93, a transcription factor associated with cold and drought tolerance in Malus baccata

  • 1.

    The Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs/National-Local Joint Engineering Research Center for Development and Utilization of Small Fruits in Cold Regions, College of Horticulture & Landscape Architecture, Northeast Agricultural University, Harbin 150030, Heilongjiang, China

  • 2.

    Horticulture Branch of Heilongjiang Academy of Agricultural Sciences, Harbin 150040, Heilongjiang, China

  • Received: 04 September 2024
  • Revised: 25 September 2024
  • Accepted: 27 September 2024
  • Published online: 02 December 2024
Fruit Research  4,  Article number: e038  (2024)  |  Cite this article

Abstract: The bHLH transcription factor is known to regulate cold signals and stress tolerance. In the present study, a new bHLH gene MbbHLH93, located in the nucleus, was isolated from Malus baccata, whose up-regulated expression were strongly induced by cold and drought treatment, and MbbHLH93-overexpressed heterologous lettuce plants displayed cold and drought stress-tolerant phenotypes. Determination of physiological and biochemical indexes associated with abiotic stress responses showed that overexpression of MbbHLH93 increased the activities of antioxidant enzymes superoxide dismutase, peroxidase, and catalase in lettuce plants treated with cold and drought stress, and decreased the contents of H2O2, O2·−, and malondialdehyde, which contributed to reducing cell membrane lipid peroxidation. Meanwhile, the accumulation of proline in transgenic plant cells increased, regulating cell osmotic pressure. Furthermore, quantitative expression analysis indicated that overexpression of MbbHLH93 improved the expression levels of LsCBFs, which were positive functional genes in response to cold and drought stress, enhancing plant tolerance. This research demonstrates that the MbbHLH93 is a key regulator in plant tolerance to cold and drought stresses, providing new knowledge for plant tolerance regulation.

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    Introduction

    • Cold and drought stresses are key abiotic environmental factors that strongly affect plant growth, development, and yield[1,2]. When plants are subjected to cold and drought stresses, it will cause an imbalance of free radical metabolism in cells, leading to the excessive accumulation of reactive oxygen species (ROS)[3]. And then leads to oxidative stress, which can cause oxidative damage to proteins, lipids, and pigments[4], and even lead to plant death. Therefore, it is of significance to research the effects of cold and drought stresses on plant growth and development and improve plant tolerance to cold and drought stresses.

      A series of plant responses to environmental stress were regulated by transcription factors, which receive stress signals and modulated the expression of stress-related functional genes. The pathway 'CBF-COR' among many stress regulation pathways was the focus of researchers. CBF (C-repeat binding transcription factor), also known as DREB1, is a class of plant-specific transcription factors activated by cold stress or ICE. Plant cold-responsive (COR) genes can produce cold regulatory proteins to improve plant cold tolerance. The CCGAC sequence of CBF can bind to the CRT/DRE cis-element in the COR gene promoter to activate the cold-resistance response. Tobacco NtbHLH123 confers tolerance to cold stress by regulating the NtCBF pathway and reactive oxygen species homeostasis[5]. The bHLH transcription factor ICE1 induced the expression of CBF3/DREB1A and COR genes under cold stress by combining with the cis-acting element MYC (CANNTG) of their promoter regions, to improve the tolerance of transgenic plants to stress[6]. MaNAC1 from banana (Musa acuminata) is directly bound to the MaICE1 promoter to target MaCBF1 and enhance the cold tolerance of banana plants through a CBF-dependent pathway[7]. Overexpression of wild rice OrbHLH2, which is highly homologous to ICE1, induces the up-regulation of salt stress-related genes such as DREB1A/CBF3, COR15A, and enhances the tolerance to salt stress in transgenic plants[8]. Members of the bHLH gene family might regulate environmental stress response via the CBF-dependent pathway. However, it is not clear whether bHLH93, which belongs to the same bHLH IIIb subgroup is also involved in the CBF pathway playing a role in defense against abiotic plant stresses.

      Apple is one of the most important economic fruit trees in the world. So far, there has been a certain basis for the research on the stress tolerance regulation mechanism of the bHLH gene in cultivated apple (Malus domestica). The MdCIbHLH1 protein binds to the promoter of MdCBF2 to up-regulate the expression of MdCBF2, which contributes favorably to the cold tolerance of transgenic apple plants[9]. Under cold conditions, MdbHLH4 represses the expression of MdCBF1 and MdCBF3 by directly binding to their promoters. MdbHLH4 also interacts with MdCICE1L, a homolog of AtICE1 in apple, and represses the binding of MdCCE1L to the MdCBF1/3 promoter, which inhibits its expression negatively regulating cold tolerance in plants[10]. MdABI4 positively regulated apple cold tolerance by interacting with MdICE1, which activated the downstream cold stress-crucial gene MdCBF1. While MdJAZ1/2 interferes with the interaction between MdABI4 and MdICE1 to negatively regulate apple cold tolerance. Therefore, MdABI4 accurately regulates cold tolerance of apple plants by integrating JA and ABA signals to form a JAZ-ABI4-ICE1-CBF cascade pathway[11]. Similarly, overexpression of bHLH transcription factor MdbHLH33 increased the expression levels of cold stress-related genes MdCBF2, MdCOR15A-1, and MdCOR15A-2 in apple calli, and MdbHLH33 could bind to LTR cis-acting elements of MdCBF promoter to positively regulate low-temperature stress[12].

      Wild apple (Malus baccata) as an important germplasm resource of apple tolerance is rich in valuable resistance genes, which is of great significance for apple resistance breeding. MxNAS1 contributes to transgenic tobacco plants' tolerance to Fe stress by increasing the plant's antioxidant capacity and MbCBF1 also contributes to plant tolerance against cold and salt stress[13,14]. In the present study, another cold- and drought-induced transcription factor MbbHLH93 was identified and cloned. Phylogenetic and structural analysis revealed that MbbHLH93 is a typical bHLH family protein. To determine the function of MbbHLH93 in plant tolerance to stress, the MbbHLH93-overexpressed heterologous lettuce (Lactuca sativa) was obtained to investigate its effect on plant tolerance. The results confirm that MbbHLH93 functions to enhance the cold and drought tolerance of lettuce. Heterologous expression of MbbHLH93 up-regulated the expression levels of its downstream stress response crucial genes LsCBFs and increased the contents of chlorophyll and proline as well as the activities of antioxidant enzymes but reduced the accumulation of MDA, H2O2, and O2·− in lettuce leaves, alleviating cold and drought damage to lettuce plants. The present study provides important candidate genes for further study of plant tolerance regulation and lays a foundation for the genetic improvement of tolerance plants.

    Materials and methods

      Plant materials

    • Apple plants (Malus baccata) from previous research[15] were precultured in Hoagland hydroponic solution at 25 °C and 80% relative humidity for a 16-h light/8-h dark cycle. The nutrient solutions were replaced every 3 d during the experiment. A total of 50 seedlings of M. baccata displaying similar growth (well-developed roots and 10-12 leaves) were separated into five groups for control, cold (4 °C), heat (37 °C), salt (200 mM NaCl), and drought (20% PEG6000) treatments for 12 h[16]. Roots and new leaves of various treatments were sampled at 0, 1, 3, 6, 9, and 12 h.

      The Hong Kong iceberg lettuce (Lactuca sativa L.) was used in the present study. Lettuce seeds were disinfected with 75% ethanol and 10% sodium hypochlorite solution and seeded in 1/2 MS (Murashige & Skoog) solid medium. After 1 d of vernalization at 4 °C, then grown at 25 °C and 70% relative humidity for a 16-h light/8-h dark cycle.

      Cloning and characterization of MbbHLH93

    • Total RNA was extracted from the leaves of M. baccata using an OminiPlant RNA Kit (Kangweishiji, Beijing, China) and the cDNA synthesis was conducted with HiFiScript gDNA Removal RT MasterMix (Kangweishiji, Beijing, China). MbbHLH93 was cloned with the specific primer shown in Supplementary Table S1.

      The protein sequences of MbbHLH93 (M. baccata) were obtained according to the MdbHLH93 (Malus × domestica), and were retrieved from the Genome Database for Rosaceae (GDR) (www.rosaceae.org). MbbHLH93 were used as query proteins to identify their homologous proteins in pear (Pyrus communis), peach (Prunus persica), and black raspberry (Rubus occidentalis) with a protein-protein BLAST (BLASTp) in GDR[17,18]. Structural motif annotation of MbbHLH93 was analyzed via the MEME program (http://meme-suite.org/). Phylogenetic analyses of MbbHLH93 orthologous proteins from these four Rosaceae species were conducted via MEGA6.0 software (www.megasoftware.net) by the maximum likelihood method based on 1,000 bootstrap replicates. Subsequently, the physiological and biochemical properties of tested proteins were conducted with the ProtParam online website (https://web.expasy.org/protparam/).

      Subcellular localization of MbbHLH93

    • The coding sequences (CDS) of MbbHLH93 without stop codon were inserted into the BamH I and Sal I sites downstream of GFP in the pSAT6-GFP-N1 vector, obtaining the fusion expression vector pro35S::MbbHLH93::GFP. Subsequently, the fusion expression vector and pSAT6-GFP-N1 empty vector were respectively transformed into tobacco leaves for 24 h according to a previous study[19]. 0.5 μg/mL 4,6-diamidino-2-phenylindole (DAPI) (Solarbio, China) served as cell nuclear dye. GFP and conjugate of DNA and DAPI fluorescence were observed respectively via a confocal laser scanning microscope (LSM 710; Carl Zeiss) at the wavelengths of 488 nm and 405 nm.

      Expression analysis of MbbHLH93

    • The expression analysis of MbbHLH93 was performed via qRT-PCR reactions based on a previous description[20]. Each reaction involved three biological replicates for error minimization, and the transcript level of MbbHLH93 was determined via the 2−ΔΔCᴛ method with Actin (EB127077) as an internal reference. Primer sequences crafted for this experiment are shown in Supplementary Table S1.

      Gene transformation

    • To obtain MbbHLH93 transgenic lettuce, the CDS of MbbHLH93 was introduced into pCAMBIA2300 vectors under the control of the CaMV35S promoter, constructing the MbbHLH93 overexpression vector. Genetic transformation of the lettuce plant was performed by the Agrobacterium-mediated leaf disk method[21]. The positive transgenic lettuce plants were screened in a selection medium containing kanamycin and identified by semi-quantitative RT-PCR analyses. The homozygous T2 generation plants were used for subsequent experiments.

      Stress treatments of lettuce plants and physiological measurements

    • A total of 30 seedlings of 25-day-old lettuce displaying similar growth were separated into three groups for control, cold (−7 °C for 6 h), and drought (no-watered for 7 d) treatments and lettuce plants were subjected respectively to 6 h and 7 d of low temperature and drought stress were collected for subsequent determination of genes and physiological indexes related to low-temperature response. The plant phenotype and survival rates were analyzed after returning the stressed lettuces to normal growth for 15 d.

      The content of chlorophyll, free proline, malondialdehyde (MDA), relative electrolyte leakage (EL), active oxygen (H2O2 and O2·−), and the enzyme activity of superoxide dismutase (SOD), peroxidase POD, and catalase (CAT) were measured respectively based on previous research[22,23]. Each reaction involved three biological replicates for error minimization.

      Detection of key stress-related genes

    • Total RNA extraction and cDNA synthesis from the leaves of cold- and drought-stressed lettuce refer to the methods above. The qRT-PCR was performed to analyze the expression levels of stress-related LsCBF genes in control and transgenic lettuce, with LsIPP2 and LsEIF2 as internal references[24,25]. Primer sequences crafted for this experiment are shown in Supplementary Table S1.

      Statistical analysis

    • The experimental data were processed using IBM SPSS Statistics 21 software. One-way ANOVA followed by Tukey's multiple range test (p < 0.05) was used to compare significant differences between all the groups of each data set, and values are shown as the mean ± standard deviation (SD) of biological triplicates.

    Results

      Characterization and phylogenetic analysis of MbbHLH93

    • The coding region of MbbHLH93 contains 1,428 bases encoding a predicted protein of 498 amino acids (aa). Physiological and biochemical properties of MbbHLH93 indicated that MbbHLH93 protein is probably an acidic hydrophilic protein (Supplementary Table S2). MbbHLH93 contained conserved bHLH and ACT-like domains at the C-terminal, which were consistent with other tested family members in Rosaceae species, indicating MbbHLH93 belongs to the bHLH family gene (Fig. 1a). Further, phylogenetic analysis showed that bHLH93 of apple and pear are closely related (Fig. 1b).

      Figure 1. 

      Sequence alignment and phylogenetic analysis of bHLH93 in Rosaceae species. (a) Sequence alignment of MbbHLH93. The conserved amino acid residues are shown in purple. The bHLH and ACT-like domain are displayed respectively with rectangles in dark blue. (b) Phylogenetic analysis of MbbHLH93. The MbbHLH93 orthologous proteins from Arabidopsis and four Rosaceae species were obtained to build a phylogenetic tree via the maximum likelihood method based on the 1,000 bootstrap analyses of the MEGA6.0 software.

      Subcellular localization of MbbHLH93

    • To characterize the biological role of MbbHLH93 in plant stress response, a MbbHLH93::GFP construct was transiently transformed into tobacco leaves to generate a fluorescently tagged MbbHLH93 protein for observation of protein subcellular localization. Green fluorescence was present in the whole cell of positive control, while only distinct fluorescence was observed in the nucleus (Fig. 2b, f), and the DAPI (4,6-diamidino-2-phenylindole) staining also confirmed that MbbHLH93 was targeted to the nucleus in vivo (Fig. 2a, e).

      Figure 2. 

      Subcellular localization of MbbHLH93. 35Spro::MbbHLH93::GFP was expressed transiently into tobacco leaves with 35Spro::GFP as positive control. (a), (e) DAPI dyeing; (b), (f) GFP signals; (c), (g) bright field; (d), (h) merge. Scale bars: 50 μm.

      Expression patterns of MbbHLH93 in various tissues of Malus baccata

    • To understand the expression patterns of MbbHLH93 genes in Malus baccata plants, qRT-PCR analysis was conducted for evaluating MbbHLH93 expression level in root, stem, new leaves, and mature leaves. The MbbHLH93 showed significantly different expression levels in different tissues, and a high expression amount was observed in new leaves and roots (Supplementary Fig. S1). Subsequently, the variation trend of MbbHLH93 gene expression was observed in roots and new leaves during the cold, drought, salt, and hot treatments. The results indicated that all four abiotic stresses could induce the expression of MbbHLH93, and with the extension of stress treatment time, the expression level of MbbHLH93 in new leaves and roots showed a trend of first increasing and then decreasing (Fig. 3). It was worth noting that cold and drought treatments could rapidly induce large amounts of expression of MbbHLH93 in the early stage of stress treatment. The highest expression abundance was detected in the roots induced by 6 h of low temperature and 3 h of drought, as well as in the new leaves induced by 9 h of low temperature and 9 h of drought (Fig. 3), indicating MbbHLH93 might play an essential role in plant low temperature and drought tolerance.

      Figure 3. 

      The expression patterns of MbbHLH93 in Malus baccata under various stress treatments. (a) The expression levels of MbbHLH93 in new leaf under stress treatments. (b) The expression levels of MbbHLH93 in root under stress treatments. The leaf and root samples were collected 0, 1, 3, 6, 9, and 12 h after treatments. Relative expression levels of MbbHLH93 were calculated via the 2−ΔΔCᴛ method and values were shown as the mean ± standard deviation (SD) based on three repetitions. Lowercase letters displayed the significant differences at p < 0.05 (one-way ANOVA followed by Tukey's multiple range test).

      Functional verification of MbbHLH93 in enhancing cold tolerance of lettuce

    • To determine the function of MbbHLH93 in affecting plant stress tolerance, the MbbHLH93 overexpression vector pCAMBIA2300-MbbHLH93 was transformed into lettuce plants. The lettuce exhibits a short life cycle and the necessity of cold-resistant production in northeast China[25]. Six MbbHLH93-overexpressed lettuce lines (L1, L2, L3, L7, L8, and L11) were identified via kanamycin tolerance and semi-quantitative RT-PCR analyses (Supplementary Fig. S2). Among them, three lines (L1, L7, L8) were screened for following research with the wild type (WT) and empty vector line (VL) transformed lettuces as controls.

      Subsequently, the phenotype of control and MbbHLH93-overexpressed lettuce lines after being treated with low temperature (−7 °C for 6 h, and recovered at room temperature for 15 d) were analyzed. The results showed control and transgenic lettuces suffered from freezing injury to different extents under longer duration of cold stress, while, obviously more severe damage was observed in control plants. Most of the transgenic lettuce lines rather than control lines after cold stress were able to resume normal growth when they were transferred to room temperature (Fig. 4a). These results confirmed that overexpression of MbbHLH93 significantly improved plant adaptability to cold stress.

      Figure 4. 

      MbbHLH93 enhances cold resistance of lettuce plants. (a) Phenotype of wild line (WT), vector line (VL), and MbbHLH93-overexpressed lettuce (L1, L7, L8) under cold for 0 h (control), cold for 6 h, and recovery for 15 d. Scale bar: 3 cm. (b) Determinations of physiological indices associated with cold stress in wild line (WT), vector line (VL), and MbbHLH93-overexpressed lettuces (L1, L7, L8) under cold for 0 h (control) and cold for 6 h. Values are shown as the mean ± standard deviation (SD) based on three repetitions. Lowercase letters show the significant differences at p < 0.05 (one-way ANOVA followed by Tukey's multiple range test).

      Cold tolerance is closely related to a series of physiological parameters in plants[26,27]. To analyze the basis for the altered tolerance to stress in MbbHLH93-overexpressed transgenic materials, cold-related physiological indicators were analyzed. Under normal growth conditions, the relative electrolyte leakage of control and transgenic lettuce plants was similar (Supplementary Fig. S3). After low-temperature stress, electrolyte leakage of control was significantly higher than that of MbbHLH93-overexpressed lettuce lines, indicating that control plants were more subjected to cell membrane damage during low-temperature stress than transgenic plants. Proline content increased and chlorophyll content declined significantly in lettuce plants after experiencing low-temperature stress, while the contents of proline and chlorophyll in MbbHLH93-overexpressed strains were both significantly higher than that of control after cold stress (Supplementary Fig. S3). Similar to the change in chlorophyll, the overexpression of MbbHLH93 also significantly increase the antioxidant enzyme (SOD, POD, CAT) activities under cold stress (Fig. 4b). Additionally, low temperature caused an increased accumulation of H2O2, O2·− as well as MDA in lettuce cells, but the heterologous expression of MbbHLH93 in lettuces relieved their accumulation to some extent (Fig. 4b).

      Functional verification of MbbHLH93 in enhancing drought tolerance of lettuce

    • To further explore the function of MbbHLH93 in affecting plant drought tolerance, a waterless stress assay was conducted for 7 d to assess the phenotype and survival rate of lettuces. In keeping with the results of cold stress, after drought treatment, control lines were subjected to more serious damage than transgenic lettuces. Most of the control seedlings nearly died when they were returned to room temperature for 7 d, while MbbHLH93-overexpressed transgenic lettuces could resume growth (Fig. 5a). These results displayed the drought tolerance function of MbbHLH93.

      Figure 5. 

      MbbHLH93 enhances drought resistance of lettuce plants. (a) Phenotype of wild line (WT), vector line (VL), and MbbHLH93-overexpressed lettuces (L1, L7, L8) under drought for 0 h (control), drought for 7 d and recovery for 15 d. Scale bar: 3 cm. (b) Determinations of physiological indices associated with drought stress in wild line (WT), vector line (VL), and MbbHLH93-overexpressed lettuce lines (L1, L7, L8) under drought for 0 h (control) and drought for 7 d. Values are shown as the mean ± standard deviation (SD) based on three repetitions. Lowercase letters indicate the significant differences at p < 0.05 (one-way ANOVA followed by Tukey's multiple range test).

      Moreover, drought-related physiological indicators were analyzed in control and transgenic lines. The results showed that drought stress led to an increase in the relative electrolyte leakage and proline, MDA, H2O2, and O2·− content as well as antioxidant enzyme activity, while a decline of chlorophyll content was seen. After drought treatment, MbbHLH93-overexpressed lettuces showed a lower relative electrolyte leakage and MDA content but higher proline and chlorophyll contents compared to the control (Supplementary Fig. S3). In addition, drought stress contributed to the activity of antioxidant oxidase increased more and the accumulation of reactive oxygen species (ROS) decreased in transgenic lettuce compared to the control (Fig. 5b).

      Over-expression of MbbHLH93 enhanced the expression of the stress response gene

    • To genetically clarify if the effect of MbbHLH93 overexpression on plant tolerance is dependent on the classical CBF pathway, the expression levels of LsCBF genes were determined in transgenic lines. Low temperature induced the up-regulated expression of most LsCBF genes, except for LsCBF2 and LsCBF13. After low-temperature stress, the expression level of most LsCBFs in transgenic lettuce was significantly higher than that in control. Similarly, the expression levels of most CBF genes were positively induced after drought treatment, and drought treatment also significantly increased the expression level of LsCBFs in transgenic lettuce compared to the control (Fig. 6). These results indicated that MbbHLH93 could positively regulate the expression of LsCBF genes, thereby improving the cold and drought tolerance of plants.

      Figure 6. 

      Expression of LsCBF genes in wild line (WT), vector line (VL), and MbbHLH93-overexpressed lettuce lines (L1, L7, L8) after control, cold and drought stresses. LsCBF8, LsCBF9, and LsCBF10 were quantitatively amplified with the same set of primers. Relative expression levels of LsCBFs were calculated via the 2−ΔΔCᴛ method and values are shown as the mean ± standard deviation (SD) based on three repetitions. Lowercase letters indicate the significant differences at p < 0.05 (one-way ANOVA followed by Tukey's multiple range test).

    Discussion

    • Abiotic stress seriously damages plant growth and crop yield[28]. Transgenic technology has become a mature and rapid method to cultivate resistant crops with the development of biotechnology, which highly relies on the extraction and functional identification of key genes associated with stress regulation[29,30]. To date, researchers have demonstrated that bHLH family proteins play an important role in abiotic stress responses, but there are few studies on the anti-stress application of bHLH family proteins in wild apple (Malus baccata). In the present study, the wild apple bHLH family gene MbbHLH93 was cloned and transferred into lettuce to identify its biological function of regulating plant cold and salt tolerance. The analyses of gene structure and phylogenetics showed that MbbHLH93 was a bHLH family gene (Fig. 1a). Under low temperature and drought stress, the MbbHLH93 was significantly induced and the survival rate of MbbHLH93 transgenic lettuces was significantly higher than that of controls (Figs 4a & 5a), indicating that MbbHLH93 functioned in significantly enhancing plant low temperature and drought tolerance.

      The bHLH transcription factors play crucial roles in various biological processes, such as plant flowering, pollen fertility, plant stomata, embryo and seed development, and abiotic stress response[31,32]. AtbHLH093, belongs to the ICE1 family bHLH-LZs, has a role in controlling flowering time and is required for apical meristem function[33,34]. Furthermore, overexpression of AtbHLH093 leads to a weak decrease in the number of mature stomatal phenotype[35]. Some studies have also pointed out that bHLH93 plays an important role in tolerance to plant stress. Ding et al. predicts that Prunus mume PmbHLH06 (bHLH93) can directly interact with PmbHLH38 (FBH4) thereby forming a dimer to function under low temperature stress[36]. Knockdown of bHLH93 in tobacco impaired disease tolerance by reducing the expression of the defense gene PDF1.2[37]. MdbHLH093 increases H2O2 accumulation and activates the SA signaling pathway in interaction with MdMYB116 to improve apple tolerance to powdery mildew[38]. Another recent study showed that MdbHLH093 in apple positively regulates dopamine accumulation through transcriptional regulation of MdTyDC conferring drought tolerance[39]. To further understand the function of MbbHLH93 in plant stress response, the expression levels of LsCBF genes, and a series of stress-related physiological indicators in MbbHLH93-overexpressed lettuce were analyzed, indicating that MbbHLH93 changed the stress-related physiological indicators in lettuce cells by up-regulating the expression of LsCBFs, thereby positively regulating the cold and drought tolerance of plants (Fig. 7).

      Figure 7. 

      Working model of MbbHLH93 function on the cold and drought adaptation in lettuce. (a) There is no MbbHLH93 in wild lettuce lines, and a large number of ROS accumulate in the cells under low temperature and drought treatment, threatening the normal growth of plants. (b) Cold and drought stress treatments rapidly induced large expression of transcription factor MbbHLH93 in MbbHLH93-overexpressed lettuce lines, and regulated lettuce plants' adaptation to stress through the classic CBF pathway. Primarily, membrane lipid peroxidation caused by large accumulation of reactive oxygen species in cells was reduced via improving the activities of antioxidant enzymes in transgenic lines, increasing plant stress resistance.

      CBF cold response pathway plays a crucial role in the cold regulatory network[14,40]. Heterologous expression of AtCBF3 or AtCBF1 could increase the low-temperature tolerance of transgenic eggplant[41], potato[42], and petunia[43]. At low temperature, ICEs can bind to the MYC recognition site of the CBF gene promoter to enhance the expression of the CBF gene and its downstream stress-related target gene[44]. Heterologous overexpression of grapevine VabHLH1 and VvbHLH1 in Arabidopsis could induce the expression of AtCBF1, AtCBF2, AtCBF3, and other cold response genes[45]. In this study, MbbHLH93 could not induce the expression of CBF genes in lettuce plants grown at room temperature, which was consistent with previous findings[6]. After low temperature and drought treatment, the overexpression of MbbHLH93 induced significantly the up-regulated expression of a large number of LsCBFs gene in lettuce (Fig. 6), suggesting that MbbHLH93 could enhance cold tolerance by activating LsCBFs expression. Research conducted on different plants has shown variations in the response of CBF proteins to different environmental stresses. Cold-treated Arabidopsis plants showed higher expression of CBF1, CBF2, and CBF3 genes, in contrast to salinity and drought treatments[46]. While CBF1, CBF2, and CBF3 are more tolerance to drought, the CBF4 gene in grapes is typically activated by cold treatment[47]. In MbbHLH93 transgenic lines, LsCBF1/3/5/6 were significantly induced by both cold and drought, whereas LsCBF2/7 and LsCBF8/9/10/12 responded better to a single drought or cold (Fig. 6). This result suggested functional differences among different LsCBF genes in stress response and provided potential CBFs for the MbbHLH93 transgenic lines in response to drought and cold stress.

      Abiotic stress will cause many physiological and biochemical changes in plant cells, such as electrolyte leakage and MDA content are indexes to assess membrane damage[15,48], and proline content affects osmotic regulation to protect proteins and cell membranes in response to environmental stresses[49,50]. SlbHLH96 and a bHLHm1 transcription factor gene MdSAT1 caused declined electrolyte leakage and MDA contents, and increased cold tolerance[51]. The overexpression of MdbHLH130 in tobacco also enhanced the plant tolerance via regulating the level of physiological indexes[52]. In accordance with the above results, overexpression of MbbHLH93 also decreased the electrolyte leakage and contents of MDA, but increased proline contents in lettuce plants after cold and drought treatment (Supplementary Fig. S3). It suggests that the higher tolerance of MbbHLH93 overexpression lines to low-temperature and drought may be attributed to the regulation of osmotic homeostasis, which may be one of the mechanisms of tolerance to abiotic stresses.

      In addition, ROS accumulation was used to evaluate stress damage[53]. Excessive accumulation of ROS led to cell oxidative damage, affecting plant growth and development. The present study found that cold and drought stress induced excessive ROS accumulation in lettuce plants (Figs 4b & 5b). The lower H2O2 and O2·− accumulation in MbbHLH93 overexpressed lines indicated that these transgenic materials suffered less stress damage. These results were consistent with lower electrolyte leakage and MDA accumulation in transgenic plants[54]. Plants have evolved essential defense mechanisms, such as enzyme scavenging systems, aimed at preventing the excessive accumulation of ROS[55]. The enzyme activities of CAT, POD, and SOD in MbbHLH93 transgenic lettuces were significantly higher than those of the control under cold and drought stress (Figs 4b & 5b), indicating that MbbHLH93 can improve the activity of antioxidant enzymes in lettuce cells under stress, which is the main reason for less ROS accumulation in transgenic materials. SOD, as an important protective enzyme for ROS scavenging in plants, scavenged excess O2·− and directly mitigated O2·− induced damage to cell membranes in the MbbHLH93 transgenic line. CAT and POD were able to act synergistically to break down H2O2, and the increase in their activity in MbbHLH93 lines maximized the scavenging of potentially harmful ROS and protected the structure of the cell membrane. These results also indicated that MbbHLH93 could enhance the scavenging ability of ROS by regulating cell osmotic potential and enhancing plant tolerance to low temperature and drought.

    Conclusions

    • In summary, the present study demonstrates the function of transcription factor gene MbbHLH93 in enhancing plant tolerance to cold and drought. MbbHLH93 was rapidly induced under low temperature and drought stress, and its overexpression up-regulated the expression of a series of LsCBFs, the key genes that respond to abiotic stress, in lettuce and probably regulated the physiological and biochemical changes of cells via the 'CBF' pathway to cope with stress response. Specifically, the overexpression of MbbHLH93 increased the content of intracellular osmotic regulatory substances and maintained the balance of cellular and external osmosis. Moreover, membrane lipid peroxidation caused by a large accumulation of ROS in cells was reduced via improving the activities of antioxidant enzymes in transgenic lines, increasing plant stress tolerance. These findings enrich our understanding of tolerance regulation of Malus baccata and lay a foundation for plant tolerance regulation and genetic improvement.

      Acknowledgments

      • This research was supported by the National Natural Science Foundation of China (32172521), the Outstanding Youth Science Foundation of Heilongjiang Province (YQ2023C006), the China Postdoctoral Science Foundation (2023MD744175), and Modern Agricultural Industrial Technology Collaborative Innovation and Promotion System of Heilongjiang Province. The authors would like to thank Mr. Li Dalong from Northeast Agricultural University for providing the instruments.

      Author contributions

      • The authors confirm contribution to the paper as follows: study conception and design: Han D, Huo J; data collection: Zhang L, Xu Y, Lv L, Wang T; analysis and interpretation of results: Zhang L, Liu W, Li X, Li W; draft manuscript preparation: Zhang L, Xu Y. All authors reviewed the results and approved the final version of the manuscript.

      Data availability

      • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Lihua Zhang, Yu Xu

      Supplementary information
      • Supplementary Table S1 Primers designed in the present study.
      • Supplementary Table S2 Physiological and biochemical properties of test proteins. CDS, coding sequence; MW, molecular weight of the amino acid sequence; GRAVY, grand average of hydropathicity; pI, theoretical isoelectric point.
      • Supplemental Fig. S1 The expression patterns of MbbHLH93 in root, stem, new leaf and mature leaf of Malus baccata. Relative expression levels of MbbHLH93 were calculated via the via 2−△△Ct method and values were shown as the mean ± standard deviation (SD) based on the three repetitions. Lowercase letters displayed the significant differences at p < 0.05.
      • Supplemental Fig. S2 Genetic transformation and screening of MbbHLH93 overexpressed lettuce. (a) the genetic transformation process of MbbHLH93 overexpressed lettuce; including coculture stage, calli acquisition, resistant bud screening, rooting stage, transplanting of transgenic seedlings, and seed acquisition stage. (b) Identification of MbbHLH93-overexpressed lettuce lines by semi-quantitative RT-PCR analyses.
      • Supplemental Fig. S3 Relevant physiological indicators of MbbHLH93 transgenic lettuces (L1, 7, 8) under cold and drought stress. Values were shown as the mean ± standard deviation (SD) based on the three repetitions. Lowercase letters displayed the significant differences at p < 0.05 (one-way ANOVA followed by Tukey's multiple range test).
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      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
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  • About this article
    Cite this article
    Zhang L, Xu Y, Lv L, Wang T, Liu W, et al. 2024. MbbHLH93, a transcription factor associated with cold and drought tolerance in Malus baccata. Fruit Research 4: e038 doi: 10.48130/frures-0024-0032
    Zhang L, Xu Y, Lv L, Wang T, Liu W, et al. 2024. MbbHLH93, a transcription factor associated with cold and drought tolerance in Malus baccata. Fruit Research 4: e038 doi: 10.48130/frures-0024-0032
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  • Introduction
  • Materials and methods

  • Plant materials

  • Cloning and characterization of MbbHLH93

  • Subcellular localization of MbbHLH93

  • Expression analysis of MbbHLH93

  • Gene transformation

  • Stress treatments of lettuce plants and physiological measurements

  • Detection of key stress-related genes

  • Statistical analysis

  • Results

  • Characterization and phylogenetic analysis of MbbHLH93

  • Subcellular localization of MbbHLH93

  • Expression patterns of MbbHLH93 in various tissues of Malus baccata

  • Functional verification of MbbHLH93 in enhancing cold tolerance of lettuce

  • Functional verification of MbbHLH93 in enhancing drought tolerance of lettuce

  • Over-expression of MbbHLH93 enhanced the expression of the stress response gene

  • Discussion
  • Conclusions
  • Acknowledgments
  • Author contributions
  • Data availability
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
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