Negative Regulatory Role of Non-Coding RNA Vvi-miR3633a in Grapevine Leaves and Callus under Heat Stress

Abstract

The grapevine, a globally significant fruit and an essential fruit tree species in China, is vulnerable to the adverse effects of high temperatures. Understanding the roles of microRNA and transcription factors in plant development and stress resistance is crucial for mitigating the impact of high temperature on grape growth and yield. This study investigates the response of miRNA to high-temperature stress in grape leaves. The expression level of Vvi-miR3633a was found to be inhibited under heat treatment in both Thompson seedless and Shen yue varieties, while its potential target genes (Vv-Atg36 and Vv-GA3ox2) were induced. Through transgenic overexpression experiments, it was demonstrated that Vvi-miR3633a plays a role in thermal response by affecting the expression of target genes. Furthermore, under heat stress conditions, overexpression of Vvi-miR3633a in grape callus decreased heat resistance compared to the control group (CK). The study also revealed that the target genes of Vvi-miR3633a regulate the expression of oxidase synthesis genes VvSOD and VvCAT, leading to reduced oxidase synthesis which may compromise the oxidation system. Additionally, the expression level of heat shock proteins in the transgenic lines was changed compared to the control (CK). Overall, this research provides valuable insights into understanding the molecular mechanisms involved in different crossing/breeding programs to produce heat-resistant grape varieties. Such varieties can be appropriate to propagate in warm climate areas with high temperature conditions.

1. Introduction

The grapevine (Vitis vinifera L.), as a highly economically valuable fruit tree, is extensively cultivated worldwide [1,2]. In the 21st century, the impact of global warming on the development of the grape industry will be significant [3]. Various abiotic stresses have influenced grape growing and fruit quality [4]. When temperatures exceed 35 °C, changes in sugar and acid content in the grape’s fruit occur, leading to a decrease in quality [5,6]. Therefore, enhancing high-temperature resistance in grapes has become an important focus for development and breeding.

This research discovered that the plant’s reaction to high temperatures is a multifaceted process, encompassing phenotypic, physiological, and molecular regulatory changes [7,8]. In extreme high-temperature conditions, severe tissue damage and cell death can occur, leading to a reduction in photosynthetic rate and the accumulation of harmful substances related to reactive oxygen species [9,10]. Various methods for assessing heat-stress damage in plants were confirmed by the researchers, including Fv/Fm (maximum photosynthetic efficiency of photosystem II), SOD (superoxide dismutase) and CAT (catalase) activity, as well as MDA (malondialdehyde) content [11,12,13]. Due to the complex genetic control of high-temperature response involving phenotypic and physiological changes, traditional breeding techniques are not deemed suitable for producing high temperature-resistant varieties [6,14]. Consequently, it is essential to conduct comprehensive studies on heat resistance mechanisms. A crucial role in plant stress responses and growth development is played by miRNA, a type of non-coding RNA [15,16]; fundamental research into the function of miRNA could facilitate rapid understanding of novel genotype selection.

In the cell nucleus, genomic DNA is transcribed to produce long RNA molecules (up to 1000 nt) that are subsequently cleaved by the ribonuclease Drosha into hairpin structures of approximately 80 bases, which are known as precursor microRNA [16]. These structures undergo further processing by another ribonuclease called Dicer, resulting in the production of mature miRNA products ranging from 21 to 24 nt in size [17]. The biosynthetic pathway of miRNA involves various enzymes such as Dicer-like 1 (DCL-1), SERRATE (SE), Hua Enhancer 1 (HEN1), and Hyponastic Leaves 1 (HYL1) proteins [18]. Generally, miRNA regulates gene expression by cleaving mRNA and inhibiting translation [19,20]. Because some studies on the function of miRNA in plants are limited, further research is necessary.

Numerous research studies have demonstrated the pivotal role of miRNA in regulating high-temperature stress [21,22]. Upon exposure to heat, miR398 downregulates the expression of target genes, thereby enhancing plant heat resistance [23]. Concurrently, tocopherol production (vitamin E) positively influences the accumulation of miR398 under heat stress conditions, thus improving thermal stability [24]. Additionally, miR156, miR160 and miR166 also contribute to heat-resistant responses by modulating the expression of transcription factors [25,26]. In a previous study involving small RNA transcriptome analysis in grapevine leaves, 873 known miRNAs were identified with DEG-seq analysis revealing differential expression of miR3633a under high-temperature stress [27]. Despite being non-conserved in plants, the expression of Vvi-miR3633 was reported by Yin et al. during flower development in camellia [28]. Furthermore, it was observed that in grape fruit, Vvi-miR3633a can facilitate embryo abortion by mediating the synthesis of oxidase SOD and CAT in response to exogenous GA induction [29]. These findings suggest that miR3633a may regulate grape ROS stability in response to high temperatures.

In previous studies, we found differential expression of miR3633a in leaf miRNA libraries. Meanwhile, target gene GO analysis showed that Vvi-miR3633a was involved in grape development and stress resistance [27]. In order to further explore the function of Vvi-miR3633a, we cloned and obtained the sequence of Vvi-miR3633a. The effect of Vvi-miR3633a on callus was then studied experimentally. When Thompson seedless callus mass overexpressed Vvi-miR3633a, it exhibited reduced tolerance to high temperatures compared to the control group. These outcomes suggest that Vvi-miR3633a participates in plant heat resistance and can offer valuable insights for genetic breeding aimed at enhancing resistance in fruit trees.

2. Materials and Methods

2.1. Plant Growth Conditions and Treatments

The Shen yue [3] one-year cuttings were obtained from the Shanghai Academy of Agricultural Sciences and subsequently transplanted into the greenhouse at the College of Agriculture and Biology, Shanghai Jiao Tong University. The Thompson seedless grape plantlets underwent cultivation in a dedicated tissue culture room, with regular replacement every 35 days. The growth medium comprised MS, 30 g/L sucrose, 7 g/L Agar, and 0.25 mg/L IBA, maintaining a pH level around 5.8. The experimental details are illustrated in Supplementary Figure S1. Prior to incubation at a temperature of 25 °C, the grape cuttings were precultured for two days. Leaf samples from Shen yue grapes were collected at both 0 h (CK) and 4 h (HS, 45 °C), while Thompson seedless tissue culture samples were taken at 0 h (CK) and after exposure to HS for three hours (45 °C). All samples were promptly frozen using liquid nitrogen and stored at −80 °C prior to analysis. This treatment process was replicated across three separate biological groups.

The induction of embryonic callus in Thompson seedless explant medium was previously documented [30]. The methods for subculture and heat stress treatment of the callus mass were derived from earlier publications by our research team [31].

2.2. Measurement of Fv/Fm (The Maximal Photochemical Quantum Yield of PSII)

Three grape plantlets (cuttings) were randomly selected from each heat stress treatment. The Fv/Fm value was evaluated, and images were acquired using Imaging-PAM (WALZ, Munich, Germany) (chlorophyll fluorescence software).

2.3. Determination of Catalase and Superoxide Dismutase Activity

Catalase (CAT) and superoxide dismutase (SOD) activities were assessed using the Total Assay Kit (Sangon Biotech, Shanghai, China).

2.4. Extraction of RNA and cDNA Synthesis

The CTAB method [32] was utilized for total RNA extraction from grapevine leaves, while Trizol reagent from Sangon Biotech (Shanghai, China) was employed for total RNA extraction from grapevine mass callus. Initially, 0.2 g samples of grape leaves and callus were pulverized and placed into RNase-free 2 mL tubes. Subsequently, 800 μL preheated CTAB buffer at 65 °C and 50 μL β-mercaptoethanol were swiftly added to the tubes. The CTAB buffer consisted of pH 8.0, CTAB (2%), Tris-HCl (100 mM), EDTA (20 mM), NaCl (2 M). The mixture was thoroughly mixed before being incubated in an oven at 65 °C for 20 min, and shaken every 5 min. Lysate was extracted using vortexing with 850 uL of chloroform/isoamylol (24:1, v/v) followed by centrifugation at 12,000 rpm for 10 min at 4 °C. This chloroform/isoamylol extraction process was repeated twice. Next, a solution containing pre-cooled ethanol (98%) and NaAc (pH 5.2) was prepared and kept in an ice bath for 10 min. Afterward, chloroform/isoamylol alcohol was added, and the mixture was vortexed before centrifugation at 12,000 rpm for 10 min at 4 °C. Then 450 μL of the supernatant was collected and 150 μL LiCl (10 M) was added and mixed. The RNA was precipitated for 12 h at 4 °C and harvested by centrifugation at 12,000 rpm for 10 min at 4 °C. The supernatant was drawn using a pipette and washed with 75% pre-cooled alcohol at −20 °C. Finally, a pipette was used to add 50 mL DEPC ddH₂O for resuspension.

Total genomic DNA removal utilized DNase I (Vazyme, Nanjing, China). Subsequently, the total RNA was then transcribed into cDNA using the Fast King RT Kit (Vazyme, China) and random primers according to the manufacturer’s instructions. The miRNA RT Enzyme Mix (Vazyme, China) was subsequently utilized to synthesize the cDNA.

2.5. Determination of RT-qPCR

The SYBR Green Super-mix enzyme from Vazyme in China was employed to prepare a 10 μL RT-qPCR (real-time quantitative PCR) reaction for the analysis of mRNA and miRNA expression. The gene expression level was determined using the 2^−∆∆Ct method [33]. All primers utilized in the reaction are listed in Supplementary Table S1.

2.6. Vector Construction

The sequence characteristics of Vvi-pri-miRNA were investigated using Thompson seedless and Shen yue varieties as experimental materials for PCR–Sanger sequencing analysis. Initially, fragments of the pri-Vvi-miR3633a sequence were inserted into linear pHB vectors BamH I and Xba I. The plasmid containing the pri-vvi-miR3633a sequence was then introduced into the GV3101 strain through Agrobacterium transformation. Subsequently, they were cultured on LB medium with 25 mg·L⁻¹ rifampicin (Rif) and 50 mg·L⁻¹ kanamycin (Kan) for 2–3 days at a temperature of 28 °C. Mono-clones were selected and incubated in LB liquid medium supplemented with 25 mg·L⁻¹ Rif and 50 mg·L⁻¹ Kan in a shaker for approximately 10 h at a speed of 200 rpm and a temperature of 28 °C.

2.7. Transient Overexpression of Vvi-miR3633a in Thompson Seedless Leaves

The transient overexpression of Vvi-miR3633a was investigated in 5-week-old Thompson seedless grape plantlets. Each experimental group consisted of 3 biological replicates, with 3–6 seedlings per replicate. The leaves were immersed in an Agrobacterium suspension (OD 600 = 0.85) and subjected to vacuum incubation at −0.1 MPa for 30 min. Subsequently, the leaves were removed and placed in a foam box. Co-cultivation was then conducted for 2 days, followed by washing with ddH₂O, drying, freezing with liquid nitrogen, and RNA extraction.

2.8. Thompson Seedless Mass Callus Transformation

Following previously documented procedures, callus formation was initiated from Thompson seedless flower explants. To facilitate callus subculturing, MSTP medium (comprising MS alkali salt, 20 g·L⁻¹ sucrose, 1 mg·L⁻¹ TDZ, 2.2 mg·L⁻¹ picloram, pH5.8) was employed for growth under dark conditions at 26 °C [31]. The transformation process of the callus followed a previously published method. Transgenic callus cultures were maintained in a dark chamber for intervals of 20 days. The expression of miRNA in transgenic callus was validated using various primer pairs through PCR and RT-qPCR analysis (see Supplementary Table S1).

2.9. Statistical Analysis

All biological statistical analysis of the data was conducted with Microsoft Excel (2019) and SPSS (Statistical Package for the Social Sciences) (version, 22.0) software. The images were combined using Microsoft PowerPoint software. The chart was created with TB tools-II software (https://doi.org/10.1016/j.molp.2023.09.010).

3. Results

3.1. Influence of High Temperature on Grape Development

Exposure to high temperatures was observed to hinder grape growth and development. In our investigation of grape response under heat stress conditions, we conducted a comprehensive analysis of both morphological and physiological traits in annual Shen yue grapes during thermal treatments. Consistent with prior studies [6], we designated a temperature of 45 °C for a duration of 4 h as the experimental group while maintaining 0 h exposure as the control group, in order to mitigate potential confounding variables such as environmental factors or soil composition (Figure 1a). Drawing from previous experiments involving high-temperature treatments on Thompson seedless plantlets, we identified 0 h and 3 h intervals as representative time points for our heat treatments (Figure 1b). Post-treatment observations revealed wilted grape leaves and shoots accompanied by browning, a clear indication of suppressed growth resulting from prolonged exposure to elevated temperatures (Figure 1c). Correspondingly, key physiological markers exhibited a notable reduction (p < 0.01, **) in maximum photosynthetic efficiency (Fv/Fm) within Photosystem II (PSII), alongside heightened SOD and CAT enzyme activities (Figure 1e,f). The swift elevation in oxidase activity provided confirmation that plant regulatory mechanisms were activated under heat stress conditions, resulting in an expedited synthesis process converting reactive oxygen species (ROS) into benign compounds.

3.2. The Expression Analyses of Vvi-miR3633a

Leveraging prior research on the miRNA library datasets from Thompson seedless grapes exposed to elevated temperatures [27], we identified a specific miRNA (miR3633a) from among those differentially expressed (p < 0.05). To elucidate the involvement of miR3633a in grape function, we investigated its tissue-specific expression profile within the Shen yue variety. Our observations revealed prominent expression of Vvi-miR3633a in flowers, roots, and leaves while exhibiting lower levels in stems (Supplementary Figure S3a), indicating wide-ranging functionality for miR3633a. Next, we detected the expression level of the primary transcript of miR3633a, and the results showed that pri-miR3633a first increased, and then decreased, under heat stress, and that the expression level was the highest at 4 h. This suggests that the accumulation of miR3633a also has a changing process (Supplementary Figure S3b). High-throughput small RNA sequencing unveiled reduced TPM values for heat-treated Shen yue and Thompson seedless varieties with statistically significant disparities (** p < 0.01) (Figure 2a,b). Additionally, RT-qPCR results corroborated these findings by demonstrating substantial down-regulation of Vvi-miR3633a under high-temperature conditions (** p < 0.01) (Figure 2c,d), suggesting an important role for Vvi-miR3633a in response to heat stress.

3.3. Identification of Vvi-miR3633a and Its Target Genes

The pre-miR3633a sequence of Shen yue and Thompson seedless was obtained by PCR–Sanger sequencing, which was consistent with the sequence of the Pinot Noir reference genome and miRbase 22.0 database (Supplementary Figure S2). Agarose gel electrophoresis showed specific bands approximately 300 bp in size (Figure 3c). At the same time, the sequence of pre-Vvi-miR3633a could be folded to form a stable stem-loop structure (Figure 3b). The secondary structure was located at positions 5918078 to 5918232 of Chr17 (Figure 3a). The mature sequence and star sequence are the red marked fonts in Figure 2b, respectively, and the mature miRNA sequence is 5′-GGAAUGGAUGGUUAGGAGAG-3′.

To further explore the target of miR3633a under high-temperature conditions, we employed the psRNATarget online tool (https://www.zhaolab.org/psRNATarget/, access on 6 June 2023) for predicting the target gene of Vvi-miR3633a. Furthermore, through integration with mRNA transcriptome data from a previous study on heat-stressed grape leaves [34], potential target genes for Vvi-miR3633a were identified as follows: VvGA3ox2 (VIT_219s0002g05300, Gibberellin 3-β double oxygen synthase 2), VvGA2ox3 (VIT_209s0140g00140, GIBBERELLIN 2-BETA-DIOXYGENASE 1), vv-GABA-T3 (VIT_204s0069g00300, Gamma aminobutyrate transaminase 3), vv-Atg36 (VIT_217s0000g04770, Autophagy-related protein 36) and vv-PIF4 (VIT_212s0028g01110, phytochrome interacting factor 4) (see Supplementary Table S2).

3.4. Expression Analyses of Target Genes of Vvi-miR3633a under Heat Stress

To investigate the target genes of Vvi-miR3633a in grape response to heat stress, we conducted a study using the leaves of Thompson seedless plantlets subjected to heat treatment as experimental materials. Our research focused on analyzing the expression patterns of marker genes related to heat stress. The findings indicated that VvHsfA2, VvHSP70, VvSOD and VvCAT, which are heat shock factor genes, were identified as markers for heat stress [34]. Their expression levels showed significant up-regulation (Figure 4a). The thermal response patterns of miR3633a potential target genes were different. Vv-GA3ox2, Vv-GA2ox3 and Vv-PIF4 were significantly induced by heat treatment, while Vv-Atg36 expression was down-regulated and Vv-GABA-T3 expression was not significantly different (Figure 4b). The complementary expression pattern of miRNA–mRNA suggested that Vvi-miR3633a might be involved in the regulation of plant heat tolerance through Vv-GA3ox2, Vv-GA2ox3 and Vv-PIF4.

3.5. Vvi-miR3633a Negatively Regulates Potential Target Genes Expression in Grapevine

To further identify potential target genes for Vvi-miR3633a, a transient expression experiment was conducted in leaves. Agrobacterium carrying the vector pHB-pri-miR3633a was introduced into Thompson seedless grape plantlet leaves, with the empty vector as control (Figure 5). The expression levels of Vvi-miR3633a and its target genes were assessed using RT-qPCR. Compared with empty vector (EV) control (CK), the relative expression of Vvi-miR3633a in the transformed leaves was significantly increased (** p < 0.01), while the relative expression of some predicted target genes was significantly decreased (** p < 0.01) (Figure 5b). For example, the expression of VvAtg36 and VvGA3ox2 was down-regulated in the transient leaves, while the other genes were significantly up-regulated and had no significant differences (Figure 5b). These findings suggest that miR3633a may regulate downstream gene expression by targeting the cleavage of VvAtg36 and VvGA3ox2.

3.6. Overexpression of Vvi-miR3633a in Grape Callus

To decipher the function of Vvi-miR3633a in grapes, we generated a Vvi-pri-miR3633a overexpression vector (OE-miR3633a) and used pHB-GFP as a control (Figure 6a). The high cell division and differentiation capacity of callus is an ideal material for transgene manipulation. The overexpressed callus was validated using RT-qPCR to quantify the expression level of miR3633 in each OE mass callus. For the miR3633a-OE line, 10 mass calluses were randomly selected for assessing the extent of overexpression (Supplementary Figure S4). According to the differences in expression levels, we selected two lines with significant differences (* p < 0.05) and named them OE-Vvi-miR3633a-1 and OE-Vvi-miR3633a-2. To investigate the potential role of Vvi-miR3633a in response to high-temperature stress, we subjected transgenic OE callus to heat treatment at 45 °C. After 24 h, it was observed that the transgenic OE callus exhibited heightened susceptibility to high-temperature stress compared to the WT callus, which displayed reduced thermal damage (browning). Statistical analysis revealed a significantly lower thermal damage rate in WT compared to OE-Vvi-miR3633a-1 and OE-Vvi-miR3633a-2. To validate our findings, we replicated the high-temperature treatment experiment with consistent results (Supplementary Figure S6), indicating that overexpression of Vvi-miR3633a diminished heat resistance in the callus. Subsequent examination of Vv-SOD and Vv-CAT oxidase gene expression levels in the mass callus demonstrated decreased oxidase activity (Supplementary Figure S5) and reduced heat resistance in Vvi-miR3633a transgenic lines following heat treatment. These findings suggest a negative regulatory effect of Vvi-miR3633a on grape under high-temperature stress.

4. Discussion

As an abiotic stress factor, elevated temperature exerts a significant influence on the growth and fruit quality of grape plants [35,36]. Previous studies have demonstrated that heat stress results in decreased photosynthesis and oxidase activity in grapevine. Furthermore, we identified grape varieties resistant to high temperatures based on Fv/Fm values. To ensure the precision of our experimental treatments, we utilized Thompson seedless plantlets for high-temperature treatment to validate relevant physiological indicators. Under heat stress conditions, there was a reduction in Fv/Fm value and an elevation in CAT and SOD activities (Figure 1). These findings indicate that high-temperature environments can impact plant physiological responses, subsequently impeding growth and development.

MicroRNA, as a non-coding RNA, can regulate a variety of physiological responses [16]. Some miRNAs associated with plant development have also been shown to participate in abiotic stress responses [21,37]. For example, miR156 and the SPL module of the target gene not only regulate plant development [38], but also promote the stable expression of HSPs and HsfA2 and enhance heat resistance [26]. MiR166 inhibited the expression of target gene PHB (PHABULOSA) during anthers development, regulated the spatial distribution of downstream genes SPL/NZZ and WUS, and participated in the flower development process [25]. At the same time, the miR165/miR166- PHB module has also been proved to regulate HSFA1 at the transcriptional and translation levels, enhancing plant response to heat stress [39]. With the development of next-generation sequencing (NGS), more miRNAs related to plant growth and development have been identified and their functions studied [40]. Based on the results of previous miRNA transcriptome sequencing studies, a series of differentially expressed miRNAs were identified [27,41,42]. Here, we reveal a non-conserved miRNA, known as miR3633a. RT-qPCR results confirmed that the expression level of Vvi-miR3633a was significantly down-regulated at high temperature (Figure 2). Meanwhile, we examined the grape seedlings at different seedling stages, and the results were consistent. Studies have shown that pre-miRNA sequences can affect the stability of secondary structures, thereby regulating miRNA expression [43]. Further, we amplified the sequence of pre-miR3633a in Shen yue and Thompson seedless varieties, and the results were consistent with those in the miRbase (Figure 3). This may explain the similarity of miR3633a expression trends in high-temperature environments.

In general, promoter cis-acting elements can influence gene expression. To decipher the possible function of VvMIR3633a in grape, we analyzed the promoter sequence. The first 2000 bp of the Vvi-pre-miR3633a sequence was analyzed based on the Thompson seedless genome and Plant Care online web page. A large number of light responsive elements were found, and others were found to respond to external environmental stress, such as the GC-motif, LTP, and ARE elements (Supplementary Table S3). These elements are easily bound by abiotic stress-related transcription factors to regulate miR3633a expression. Under heat stress, the expression of miR3633a is inhibited (Figure 2), while the expression level of its target gene is increased (Figure 4). Bai et al. found that the miR3633a-GA3ox2 module can regulate the expression of downstream vvSOD and vvCAT genes and promote the synthesis of oxidase [29]. Under conditions of high temperature, the synthesis of oxidase can eliminate the accumulation of ROS species in plants. In phenotypic analysis, the heat damage rate of miR3633a-OE callus was significantly higher than that of WT (Figure 6 and Supplementary Figure S6). Therefore, in addition to its regulatory role, miR3633a may target key genes that inhibit oxidase synthesis and participate in the process of heat stress in plants.

Similarly, we previously constructed miRNA libraries of heat-induced grape leaves to analyze potential targets of Vvi-miR3633a. They were VIT_219s0002g05300 (VvGA3ox2) [44], VIT_209s0140g00140 (VvGA2ox3), VIT_204s0069g00300 (Vv-GABAT3) [45], VIT_217s0000g04770 (Vv-Atg36) [46] and VIT_212s0028g01110 (Vv-PIF4) [47]. Most of the functions of these genes are related to plant hormones and light signaling. Under suitable temperature, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) changed the abundance of the flavonoid biosynthesis gene YUCCA8 (YUC8) [48]. Overexpression of Vvi-miR3633a in grape leaves altered the expression of its target genes (Figure 5), indicating the regulatory role of miRNA and its involvement in grape growth and development. Meanwhile, Vv-Atg36 and Vv-GA3ox2, as potential target genes of Vvi-miR3633a, showed significant differences in expression levels under heat stress (Figure 5).

5. Conclusions

Grape thermal response is a complex biological process. In this study, we used morphological characteristics, physiological changes and miRNA–mRNA expression of high-temperature response to construct a model of grapevine interaction response mechanisms to regulate heat stress (Figure 7). Based on these findings, it was observed that elevated temperatures suppressed the manifestation of Vvi-miR3633a, resulting in an elevation of its potential target genes Vv-Atg36 and Vv-GA3ox2. This led to a reduction in SOD and CAT oxidase activity and an increase in heat-induced damage, ultimately weakening the plant’s resistance to high temperatures.