Identification and Analysis of the MIR399 Gene Family in Grapevine Reveal Their Potential Functions in Abiotic Stress

Abstract

MiR399 plays an important role in plant growth and development. The objective of the present study was to elucidate the evolutionary characteristics of the MIR399 gene family in grapevine and investigate its role in stress response. To comprehensively investigate the functions of miR399 in grapevine, nine members of the Vvi-MIR399 family were identified based on the genome, using a miRBase database search, located on four chromosomes (Chr 2, Chr 10, Chr 15, and Chr 16). The lengths of the Vvi-miR399 precursor sequences ranged from 82 to 122 nt and they formed stable stem–loop structures, indicating that they could produce microRNAs (miRNAs). Furthermore, our results suggested that the 2 to 20 nt region of miR399 mature sequences were relatively conserved among family members. Phylogenetic analysis revealed that the Vvi-MIR399 members of dicots (Arabidopsis, tomato, and sweet orange) and monocots (rice and grapevine) could be divided into three clades, and most of the Vvi-MIR399s were closely related to sweet orange in dicots. Promoter analysis of Vvi-MIR399s showed that the majority of the predicted cis-elements were related to stress response. A total of 66.7% (6/9) of the Vvi-MIR399 promoters harbored drought, GA, and SA response elements, and 44.4% (4/9) of the Vvi-MIRR399 promoters also presented elements involved in ABA and MeJA response. The expression trend of Vvi-MIR399s was consistent in different tissues, with the lowest expression level in mature and young fruits and the highest expression level in stems and young leaves. However, nine Vvi-MIR399s and four target genes showed different expression patterns when exposed to low light, high light, heat, cold, drought, and salt stress. Interestingly, a putative target of Vvi-MIR399 targeted multiple genes; for example, seven Vvi-MIR399s simultaneously targeted VIT_213s0067g03280.1. Furthermore, overexpression of Vvi_MIR399e and Vvi_MIR399f in Arabidopsis enhanced tolerance to drought compared with wild-type (WT). In contrast, the survival rate of Vvi_MIR399d-overexpressed plants were zero after drought stress. In conclusion, Vvi-MIR399e and Vvi-MIR399f, which are related to drought tolerance in grapevine, provide candidate genes for future drought resistance breeding.

1. Introduction

MicroRNAs (miRNAs) are a class of endogenous, single-stranded, small regulatory RNA molecules, which widely exist in various eukaryotes [1]. In plants, miRNAs range in length from 20 to 24 nt and are processed from longer precursor miRNA (pre-miRNA) molecules with stem–loop structures [2,3]. The pre-miRNA is processed by DICER LIKE1 (DCL1) into mature miRNA, which then regulates gene expression by binding mRNAs and inducing degradation or translation repression [4,5]. Thus, plant miRNAs are thought to mediate almost all plant cellular and metabolic processes via regulating post-transcriptional gene silencing [6,7]. In plants, miRNAs play an important role in a number of physiological processes, such as growth development and biotic and abiotic stress responses, where they can regulate the expression levels of their target genes by cleavage.

With the advent of sequencing technology, multiple miRNAs have been identified in various species, tissues, and physiological conditions [8,9], and they participate in the whole process of plant growth and development. The regulatory module of hpe-miR162a_L-2-ARF19 is involved in early seed development [10]. In Medicago truncatula, overexpression of MtMIR166-insensitive REVOLUTA (MtREV1) led to adaxialized leaves and ectopic leaflets along the dorsoventral axis [11]. The crown root defect 1 (CRD1) regulates crown root development via the miR156 pathway [12]. Rice-specific osa-miR5506 plays an essential role in the regulation of spikelet determinacy, floral organ number, and female gametophyte development [13]. Furthermore, cme-miR2 may modulate the cell growth and fruit size of Hami melon by negatively regulating TIP GROWTH DEFECTIVE 1 (TIP1) [14]. Apple fruit was mostly derived from the hypanthium contributed mostly by sepal tissues, which were positively regulated by APETALA2 (AP2) and over-accumulation of miR172 leading to the silencing of AP2, then leading to dramatic reductions in fruit size and weight [15]. The CsmiR396-CsGRFs/CsGIFs module is diverse in cucumber, and CsGRF3 and CsGRF5 play opposite roles in regulating cell proliferation [16]. The miR156 of tomato targeted SPL/SBP box transcription factors involved in maintenance of the meristematic state of ovary tissues, thereby controlling the initial steps of fleshy fruit development [17]. SlMIR164b is required for flower and shoot boundary specification, and SlMIR164a is required for fruit growth including the expansion of its outer epidermis, which determines the properties of the fruit skin of tomato [18]. Mul-miR477 can repress the expression of the antisense long non-coding RNA (Mul-ABCB19AS) in mulberry and increase the expression of the ATP binding cassette (ABC) transporter B 19 gene (Mul-ABCB19), and it acts as a positive regulator participating in anthocyanin accumulation through the regulatory network of miR477-, Mul-ABCB19AS-, and Mul-ABCB19 [19]. All the above-mentioned studies indicated that miRNAs have extensive regulatory functions in plant growth and development.

Some evidence in plants indicates that the miRNA family might play crucial roles in responses to abiotic and biotic stresses. For example, the miR172-IDS1 module is critically involved in the reactive oxygen species (ROS) scavenging and re-establishment of redox homeostasis in high salinity environments, which further contributes to rice salt tolerance [20]. In maize, the GRMZM2G427404 (CRP04) transcript is targeted by miR190 and may encode a protein that positively regulates drought stress tolerance via an abscisic acid-dependent pathway [21]. Mulberry miR166f targets the transcripts encoding a homeobox-leucine zipper transcription factor whose expression is induced by drought stress and is a positive regulator of drought stress tolerance [22]. Knockout of miR482b and miR482c elicit the expression perturbation of other miRNAs in tomato, suggesting a novel mechanism with cross-regulation of miRNA in tomato [23]. After cotton was infected with Verticillium dahliae, the expression of microRNA 166 (miR166) and miR159 was increased and exported to fungal hyphae for specific silencing [24]. Moreover, miR159 was found to be abundant in Arabidopsis galls after inoculation of root-knot nematodes, and the miR159abc triple mutant was more resistant to root-knot nematodes [25]. Therefore, to more comprehensively recognize the roles of a miRNA family with multiple members, it is necessary and significant to carry out a systematic analysis of each member.

Many plant miRNAs are evolutionarily conserved between species and can be divided into different families that are located at one or more genomic loci [26]. In the miRNA family, even though precursors of different members produce the same or similar mature miRNAs, they exhibit different spatial and temporal expression profiles, resulting in functional diversification that leads to the regulation of different target genes [27]. In recent years, miR399 has been shown to play an important role in plant phosphorus signaling pathways [28]. MiR399s are highly conserved in plants and have been identified in more than 30 species of plants according to the miRBase database [29]. Among them, The Arabidopsis miR399 family contains 7 genes, rice has 11 members, tomato has 2 members, and grapevine (Vitis vinifera L.) has 9 members. It is generally accepted that conserved miRNAs might play crucial roles in regulating fundamentally important biological processes [30].

MiR399 was the first miRNA demonstrated to have the ability to increase plant uptake of phosphorus (P) [31,32,33]. MiR399 has been proposed to be conserved in angiosperms in response to P starvation [34,35,36]. Its target has been identified as UBC24, which encodes a ubiquitin-conjugating E2 enzyme, also known as PHOSPHATE 2 (PHO2 or UBC24) [37]. Meanwhile, miR399 might improve phosphorus absorption and metabolism in rice and Arabidopsis by reducing the transcription level of PHO2. CsUBC24 downregulates the SEPALLATA family, which disrupts the floral meristem identity regulatory network and leads to developmental abnormalities in flowers. By interacting with INDUCER OF CBF EXPRESSION1 (CsICE1), CsUBC24 disturbs stomate function on the anther surface, which inhibits anther dehiscence [38]. In transgenic plants, increasing miR399 transcripts reduces UBC24 transcripts [39]. Under freezing stress, overexpression of tae-miR399 ultimately decreases the expression of AtUBC24, inhibiting the degradation of ICE1 (Inducer of CBF expression 1), which increases the expression of genes involved in the C-REPEAT BINDING FACTOR (CBF) signaling pathway [40]. Although UBC24 was the target of miR399, we did not find literature on the association between UBC24 and specific processes or traits in grapevine.

Grapevine is widely planted worldwide and can be used as a variety for the production of table grapes, juice, and wine. Currently, little information is available on the relationship of miR399 in grapevine. To understand the function of this important miRNA family in grapevine, we conducted a comprehensive analysis to identify Vvi-MIR399 family members. In this study, multiple bioinformatic tools and online websites were combined to identify the Vvi-MIR399 family members of grapevine. Moreover, the cis-elements in the promoter of the Vvi-MIR399 gene family and the target genes predicting the Vvi-MIR399 family were also explored. In the future, miRNAs and their predicted target genes could be used in molecular breeding strategies for disease-resistant or stress-tolerant varieties, offering meaningful views and methods for developing the grapevine industry.

2. Results

2.1. Distribution of miR399 Family Members in Grapevine

The miR399 precursor and mature sequences in all plants were searched for using the miRBase database. A total of 231 precursor miR399 and 275 mature miR399 sequences from 36 species and 18 families were obtained. Among them, there were six, two, eleven, six, and nine members of the precursor miR399 gene family of Arabidopsis, tomato, rice, sweet orange, and grapevine, respectively (Figure 1). Sequences of nine mature miR399 families were also obtained from grapevine. The lengths of the precursor and mature sequences of the Vvi-miR399 gene family were 82~122 nt and 21 nt, respectively (Supplementary Table S1). Nine members of the MIR399 gene family were verified by cloning and sequencing using the grapevine as a cDNA template (Supplementary Figure S1). These results suggested that the number of mature sequences of miR399 in a species was not equal to the number of its corresponding precursors, and different mature sequences might come from the same precursor or multiple precursors. The number of miR399 family members and their precursors was also different in different plants, indicating a high degree of evolutionary complexity in the plant miR399 family.

2.2. Analysis of the Vvi-miR399 Family Precursor and Mature Sequences

To confirm the secondary structures of precursors in the Vvi-miR399 family, all of the precursor sequences were predicted using the website to form a stable stem–loop structure, indicating their ability to produce miRNAs (Figure 2a). Vvi-miR399s were located on the 3′arm of the secondary stem–loop hairpin structure of the Vvi-MIR399s. The minimum free energy of the Vvi-MIR399s ranged from −55.70 kcal/mol to −37.40 kcal/mol, which indicated that the Vvi-MIR399s were relatively stable. The sequences of the MIR399 gene family from grapevine were aligned, and the corresponding phylogenetic tree was constructed. We observed the highest degree of conservation along the arms of the secondary stem–loop structure, especially in the core region of the mature miRNA (GCCaAAGGAGAtTTG), with more variation flanking the mature miRNA (Figure 2b). To further investigate the conserved region in the Vvi-miR399 family, multiple alignment was conducted, and the results showed that the sequences ranging from 2 to 20 nt were relatively conserved, and base preference analysis further confirmed this observation (Figure 2c).

To understand the evolutionary differences, the phylogenetic relationships of precursors in the miR399 members derived from dicot (Arabidopsis, tomato, and sweet orange) and monocot (rice and grapevine) plants were analyzed. The results showed that three major clades were clustered (Figure 2d). Nearly 64.71% (22/34) of the MIR399 gene family from the five plant species were grouped into one clade (Clade Ⅲ), signifying a common ancestry and similar evolutionary path. Most of the Vvi-MIR399 gene family were clustered with the MIR339 genes from dicot plants. Interestingly, Ath-MIR399e does not form part of any specific clade and appears to have diverged independently. Furthermore, we found that all miR399 genes were also divided into three clades in grapevine (Figure 2e). Vvi-miR399a and Vvi-miR399h were grouped together with Vvi-miR399b, Vvi-miR399c, and Vvi-miR399i, while Vvi-miR399g and Vvi-miR399d formed a branch with Vvi-miR399e. In contrast, Vvi-miR399f formed a separate branch, suggesting that the identified Vvi-miR399s might have evolutionary diversification. These results supported the evolutionary conservation and diversification of miR399 family members in grapevine.

2.3. Prediction of Cis-Acting Elements on the Promoters of the Vvi-MIR399 Family

The distribution form and location of genes on chromosomes have important effects on gene expression and genetic linkage. By studying the location and distribution of genes on chromosomes, the functions of genes can be better understood. Based on the chromosomal localization information, the Vvi-MIR399 family members were localized on four chromosomes (Supplementary Table S3 and Figure 3a). Vvi-MIR399a, Vvi-MIR399d, Vvi-MIR399e, Vvi-MIR399f, and Vvi-MIR399h were located in on Chr 10, while Vvi-MIR399b, Vvi-MIR399c, and Vvi-MIR399i were located on Chr 16, Chr 15, and Chr 2, respectively. Vvi-MIR399b and Vvi-MIR399c were located on different chromosomes, whereas they shared the same mature sequence. However, although the maturation sequences of Vvi-MIR399a and Vvi-MIR399h were the same, they were located in different locations on the same chromosome (Figure 3a).

To understand the possible regulatory mechanism of the Vvi-MIR399 family members at the transcriptional level, their promoter sequences were analyzed using the online website. The cis-regulatory elements within 2000 bp of the Vvi-MIR399 promoter sequence upstream of the TSS were predicted. The predicted promoter elements were classified into eight categories, including enhancer and core elements, light-related elements, defense-related elements, phytohormone-related elements, cycle and circadian rhythms, metabolism regulation, seed development, and others. As expected, the core promoter element TATA-box was observed in all the Vvi-MIR399 promoter regions, and all the Vvi-MIR399 gene family members contained CAAT-box, which is a common cis-acting element in promoter and enhancer regions (Supplementary Table S4). The main role of TATA-box was to initiate transcription precisely, while the main role of the upstream promoter element CAAT-box was to control the frequency of transcription initiation, and CAAT-box had the greatest influence on the frequency of transcription initiation. Furthermore, specific promoter elements were analyzed to understand the functional roles of the Vvi-MIR399 family. The Vvi-MIR399 gene family all contained more than 10 cis-acting elements, especially the Vvi-MIR399g member, which contained 32 cis-acting elements in its promoter (Figure 3b). The promoters of Vvi-MIR399a, Vvi-MIR399b, Vvi-MIR399c, Vvi-MIR399d, Vvi-MIR399e, Vvi-MIR399f, Vvi-MIR399h, and Vvi-MIR399i contained 17, 26, 17, 20, 12, 24, 30, and 13 cis-acting elements, respectively. The majority of the elements were related to ABA response, anaerobic response, drought response, GA response, light response, MeJA response, SA response, meristem-specific, protein metabolism regulation, and seed-specific regulation. Additionally, these cis-acting elements could be divided into three classes, including progress-specific, tissue-specific, and stress response. For plant stress response elements, 66.7% (6/9) of the Vvi-MIR399 promoters harbored drought, GA, and SA response elements, with 44.4% (4/9) of the Vvi-MIRR399 promoters also presenting elements involved in ABA and MeJA responses. By contrast, among the nine Vvi-MIR399 promoters, seven, three, two, and one Vvi-MIR399 contained anaerobic, auxin, defense and stress, and low-temperature response elements, respectively. Notably, the light-response element was presented in all Vvi-MIR399 gene family members. These results indicate that the Vvi-MIR399 family members might have a crucial role in responding to various phytohormones and stress, while also reflecting their potential different functions.

2.4. Prediction of Target Genes and Tissue-Specific Expression Analysis

MiRNAs serve functions by regulating target genes. To discover the potential functions of the Vvi-miR399 family members, their target genes were predicted using the miRBase database [29]. The relationships between the Vvi-miR399 family and their predicted target genes are shown (Figure 4a). In total, 31 predicted target genes were found, and each Vvi-miR399 family member had multiple predicted target genes. Notably, some genes were targeted by multiple Vvi-miR399 family members; for example, seven Vvi-miRNA399s (miRNA399a, miRNA399b, miRNA399c, miRNA399e, miRNA399g, miRNA399h, and miRNA399i) simultaneously targeted VIT_213s0067g03280.1, suggesting the functional conservation and diversification of the Vvi-MIR399 family members.

RT-qPCR was performed to further explore the expression patterns of Vvi-MIR399 in the different tissues of grapevine (Figure 4b). The results showed that the Vvi-MIR399 family members had varying degrees of expression in different grapevine tissues. Interestingly, the expression trends of the Vvi-MIR399 family were consistent in different tissues, with the lowest expression level in mature and young fruits and the highest expression level in stems and young leaves. For the Vvi-MIR399b gene, the expression levels in stems were 420-fold higher than those in mature fruits. These results suggest that different Vvi-MIR399 family members might play different roles in various tissues.

2.5. Expression Analysis of Vvi-MIR399 Family Members under Abiotic Stress

In order to explore the function and effect of Vvi-MIR399s in grapevine, the expression patterns of Vvi-MIR399s under low light, strong light, high temperature, cold, drought, and salt stress were investigated (Figure 5). When grapevine plants were exposed to low light stress, Vvi-MIR399a, Vvi-MIR399g, and Vvi-MIR399h showed similar expression patterns, with their lowest levels at 4 h. The expression levels of Vvi-MIR399b, Vvi-MIR399c, Vvi-MIR399d, Vvi-MIR399e, and Vvi-MIR399f were upregulated and increased by more than 2.90-fold at 1 h compared with the control samples at 0 h, followed by a sharp decrease at 4 h and a further increase at 12 h. All nine Vvi-MIR399s showed a rapid decrease in abundance at 4 h (Figure 5a). Therefore, the Vvi-MIR399 gene family members were potentially involved in low light stress during an early stage. The expression level of Vvi-MIR399g increased sharply after 1h with the extension of high light stress time, while the transcripts of Vvi-MIR399a, Vvi-MIR399c, Vvi-MIR399d, and Vvi-MIR399e were barely induced during high light stress. After 12 h of high light treatment, the expression levels of Vvi-MIR399b, Vvi-MIR399f, Vvi-MIR399g, and Vvi-MIR399h increased significantly by 3.36-, 5.05-, 60.28-, and 20.03-fold, respectively, indicating that they might respond to high light stress at a later stage (Figure 5b). When grapevine plants were exposed to heat stress, the expression of Vvi-MIR399b, Vvi-MIR399d, Vvi-MIR399g, Vvi-MIR399h, and Vvi-MIR399i were promptly decreased at 1 h after treatment, indicating that they might be relatively sensitive to heat stress. The abundances of Vvi-MIR399a and Vvi-MIR399f were upregulated at 2 h and 4 h and increased by 5.20- and 2.59-fold, respectively, compared with the control samples at 0 h (Figure 5c). Under cold stress, the expressions of the Vvi-MIR399d, Vvi-MIR399e, Vvi-MIR399f, Vvi-MIR399g, and Vvi-MIR399h genes were rapidly increased at 1 h, suggesting that they might be responsive to cold stress at an early stage. In particular, the expression of Vvi-MIR399d reached its maximum levels at 1 h, 4 h, and 24 h under cold stress, with 11.46-, 9.64-, and 10.08-fold changes, respectively, compared with the control (Figure 5d). In grapevine exposed to drought stress, the nine Vvi-MIR399 gene family members showed similar accumulation patterns, reaching the maximum accumulation at −0.8 MPa, followed by a sharp decrease at −1.0 MPa (Figure 5e). When grapevine was subjected to salt stress, the nine Vvi-MIR399s first decreased, then increased, and then decreased and remained at a low level. The Vvi-MIR399a, Vvi-MIR399d, Vvi-MIR399e, Vvi-MIR399g, and Vvi-MIR399h genes showed similar accumulation patterns, with their levels reaching their peaks at 2 d and their maximal fold changes ranging from 1.10 to 2.12. These results suggest that the Vvi-MIR399 gene family members might respond to salt stress at an early stage (Figure 5f).

2.6. Expression Patterns of Target Genes in Response to Abiotic Stresses

To explore the regulatory function of miR399s in grapevine, the expression patterns of four target genes with a high degree of confidence were also predicted and analyzed using RT-qPCR in response to abiotic stresses (Figure 6). The four genes were promptly increased to their peaks in transcript accumulation at 1 h after low light stress, and their fold changes ranged from 1.76 to 3.56, respectively, compared with the control, indicating that they might be responsive to low light stress at an early stage (Figure 6a). After high light stress, the transcription levels in VIT_214s0006g02510.2 gradually increased by 1.17- and 1.16-fold at 1 and 2 h compared with the 0 h control and then gradually decreased until 12 h. The abundance of VIT_213s0067g03280.1 also increased 24.49-fold at first, then decreased, but then rapidly increased 29.46-fold at 12 h (Figure 6b). VIT_211s0052g01030.1 showed a gradual increase by 1.37-fold in transcript accumulation with prolonged stress, while VIT_213s0067g03280.1 and VIT_214s0006g02510.2 exhibited slight decreases at late stages of heat stress (Figure 6c). In the context of cold stress, VIT_213s0067g03280.1, VIT_214s0006g02510.2, and VIT_217s0000g04480.1 showed similar trends in their abundances, with sharp decreases and reaching their lowest levels at 2 h and modest increases in their levels at 4 and 12 h. The Vvi-miR399 family members might thus promote the accumulation of their target transcripts and play a positive role in stress adaptation (Figure 6d). VIT_213s0067g03280.1 and VIT_217s0000g04480.1 reached their maximum levels at −0.8 MPa drought stress with 6.68- and 3.89-fold changes, respectively, compared with the control (Figure 6e). Compared with the control, the expressions of VIT_213s0067g03280.1, VIT_214s0006g02510.2, and VIT_211s0052g01030.1 were obviously decreased by salt stress. In contrast, the transcript accumulations of VIT_217s0000g04480.1 were increased by 2.76-fold at 2 d after treatment (Figure 6f).

2.7. Overexpression of Vvi-MIR399 Enhanced the Drought Tolerance in Arabidopsis

It is known that the secondary structural determinants of precursors, such as the stem–loops/bulges and sequences surrounding the miRNA/miRNA* region, have an important influence on miRNA processing [41,42]. The different miRNA399 precursors of grapevine show variations in their structures as well as differences in the sequences in the stem parts of their precursors (Figure 2). Therefore, to investigate the biological function of MIR399 in response to drought stress in grapevine according to the upregulation of the Vvi-MIR399 family in grapevine drought stress, three Vvi-MIR399 family members (Vvi-MIR399d, Vvi-MIR399e, and Vvi-MIR399f) were overexpressed in Arabidopsis. Ten-day-old seedlings with consistent growth were grown under drought stress for 30 days: Vvi-MIR399e- and Vvi-MIR399f-overexpressed materials grew normally, whereas the leaves of Vvi-MIR399d were dry and the plants grew slowly (Figure 7a). When these transgenic Arabidopsis thaliana seedlings recovered after water stress, the seeding survival rates of Vvi-MIR399e and Vvi-MIR399f were 88.2% and 89.8%, respectively; i.e., significantly higher than those of WT and Vvi-MIR399d (Figure 7c). However, the plant lengths of these transgenic plants were significantly greater than that of WT (Figure 7d). In order to more directly observe the growth of transgenic plants under drought stress and control the root system, the roots were cleaned and observed. The results showed that the root lengths of the Vvi-MIR399d-, Vvi-MIR399e-, and Vvi-MIR399f-overexpressed materials were significantly greater than that of WT (Figure 7b, e). Vvi-MIR399s played an important role in drought stress, of which Vvi-MIR399e and Vvi-MIR399f were the most drought tolerant.

3. Discussion

3.1. Evolutionary Characteristics of the MIR399 Family

MiR399 acts as a conserved miRNA to be implicated in a wide range of cellular and physiological processes in plants [43]. Based on data from the miRBase, most plant species harbor multiple miR399s. It is known that the existence of multiple copies of miRNA genes within plant species provides one possibility for functional redundancy and specificity [44]. Plant miRNAs are thought to mediate almost all plant cellular and metabolic processes via the regulation of posttranscriptional gene silencing [6]. Conserved miRNAs might play crucial roles in regulating fundamentally important biological processes. A hallmark of RNA silencing was a class of approximately 22-nucleotide RNAs processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer was crucial for the functionality of miRNAs. MiRNAs can regulate target mRNA by disrupting its stability and inhibiting its translation. In this study, the majority of the Vvi-miR399 sequences are highly conserved between 2 and 20 nt, and the diversity lies in their ends (Figure 2c). The 5′-end recognition by Dicer is important for the precise and effective biogenesis of miRNAs [45]. The 5′ variability might have a bearing on the association with different AGOs [46]. Eight mature sequence variants of Vvi-miR399 had ‘T’ at the 5′ terminal nucleotide, suggesting that they primarily load on AGO1. In contrast, one Vvi-miR399 mature variant had ‘C’ at the 5′ terminal base (including Vvi-miR399i), suggesting that it is mainly recruited into AGO5 followed by AGO1. The 3′-end was displayed as a key determinant regulating miRNA activity via 3′-remodeling and/or degradation [47]. Therefore, the diversity and evolutionary conservation of the miR399 gene family are expected to be elucidated to further understand their regulatory roles in plant development and/or stress resistance.

It was proposed that conserved miRNA families have been both conserved and diversified during miRNA evolution [44]. In this study, our data indicated that grapevine miR399s showed evolutionary conservation. We identified nine MIR399 genes in grapevine. In rice, eleven MIR399 exist [43], and in dicot Arabidopsis [48], tomato [49], and sweet orange [50], six, two, and six MIR399 are present, respectively (Figure 2d). The multiple miR399s likely diverged from a common ancestor, as reflected by the evolutionary diversity of the miR399 precursor sequences and most of the Vvi-MIR399 gene family being clustered with the MIR339 genes from dicot plants. According to the phylogenetic tree, Vvi-miR399a, Vvi-miR399b, Vvi-miR399c, Vvi-miR399h, and Vvi-miR399i were all clustered together, signifying a common ancestry and similar course of evolution. Vvi-miR399g and Vvi-miR399d formed a branch with Vvi-miR399e, indicating that the Vvi-miR399s were relatively conservative in their evolution and function. Notably, Vvi-miR399f formed a separate branch (Figure 2e). These observations suggest that the majority of the Vvi-miR399 members had a close evolutionary relationship, while exceptions also existed, which might be related to differences in function. In addition, Vvi-MIR399a and Vvi-MIR399h had the same mature sequence and were located on the same chromosome, indicating that Vvi-miR399a and Vvi-miR399h might have the same function in grapevines (Figure 3a). Numerous previous studies showed that gene expression is controlled by upstream elements such as the promoter [51,52]. Promoters are important parts of genes; their main function is to regulate the initiation time and degree of gene expression (transcription). However, promoters do not control gene activity by themselves but by binding to transcription factors. In addition, analysis of the Vvi-MIR399 promoters showed that Vvi-MIR399 exerted multiple functions. TATA-box and CAAT-box, which were required for transcriptional initiation, and light-response elements were most enriched in the Vvi-MIR399 promoter region, suggesting that these cis-elements were fundamental to the expression of the Vvi-MIR399 family genes (Figure 3b). Drought-response-related elements were found in the promoters of Vvi-MIR399b, Vvi-MIR399c, Vvi-MIR399f, Vvi-MIR399g, Vvi-MIR399h, and Vvi-MIR399i, suggesting that they might be involved in the regulation network of drought stress signals. Low temperature-response-related elements were found only in the promoter of Vvi-MIR399b, which might be involved in the low-temperature stress signal regulation network. Moreover, defense-related elements (TC-rich repeats, LTR, ARE, and MBS) and phytohormone-related elements (TGA-element, TATC-box, TCA-element, ABRE, CGTCA-motif, TGACG-motif, GARE-motif, and P-box) were also present in some Vvi-MIR399 family members, and they regulated the precise initiation and transcriptional efficiency of gene transcription by binding to transcription factors. These results suggest that the Vvi-MIR399s might have a crucial role in responding to various phytohormones and stress, while also reflecting their potential different functions (Supplementary Table S4). Numerous studies showed that miRNAs function as negative regulators for the expression of target genes, playing a crucial role in regulating the growth, development, and stress response of plants. The prediction of Vvi-miR399 target genes showed that most of the Vvi-miR399s had different target genes (Figure 4a). Interestingly, it was found that one gene (VIT_213s0067g03280.1) could be targeted by more than one miRNA. VIT_213s0067g03280.1 harbored a highly conserved miR399 binding site in grapevine, and miR399s might have redundancies in their functions. These results suggest that the miR399s family in grapevine have different functions.

3.2. The MIR399 Family May Participate in Plant Growth and Development

MiR399 was shown to be involved in the growth and development of plants. For example, the silencing of miR399 expression can disrupt the peanut meristem and anther development of citrus [38], and overexpression of miR399 can affect the fruit quality of strawberries [53]. In addition, miR399 is involved in color formation in tomato and apple fruits [54,55]. Overexpressing miR399 in maize (Zea mays L.) was associated with premature senescence after pollination [56]. Enhanced freezing tolerance was observed for tae-miR399-overexpressing Arabidopsis lines [40]. In Arabidopsis, miR399 generated in shoots serves as a long-distance signal that represses PHO2 expression in roots under Pi-limiting conditions, resulting in the activation of Pi uptake and translocation [57]. The results of real-time PCR further showed that Vvi-MIR399s show strong expression in stems, mature leaves, and young leaves, suggesting its role in stem and leaf development (Figure 4b). Meanwhile, the expression degrees of the Vvi-MIR399 were different in different tissues of grapevine. The expression of Vvi-MIR399b in stems was 420-fold higher than that in mature fruits, which indicated its developmental specificity in stems. These results indicate that Vvi-MIR399 family members were expressed in different tissues of grapevine, and the expression trend was consistent, highlighting the developmental specificity.

3.3. The MIR399 Family May Respond to Abiotic Stress in Plants

Previous studies showed that plants respond to abiotic stress by modulating gene expression at post-transcriptional levels via miRNAs [48]. Abiotic stress affects various physiological processes of plant development. Exposure to different abiotic stresses can lead to similar responses in plants. Moreover, different kinds of stresses can trigger responses by the induction of similar types of miRNAs [58]. This indicates that plants share common signaling pathways that act in different abiotic stress responses. After exposure to stress treatments that influenced plant growth and developmental processes, the relevant miRNAs were either down-regulated or up-regulated [59].

Light intensity plays a central role in obtaining energy for plants to survive [60]. Low light stress is a major abiotic stress for the grape industry and can significantly affect plant growth and development [61], especially during the rainy season. The upregulation of Vvi-MIR399b, Vvi-MIR399c, Vvi-MIR399d, Vvi-MIR399e, and Vvi-MIR399f under low light stress indicate that they might act as positive regulators of low light stress (Figure 5a). Plants have evolved sophisticated photoreceptors to control many biochemical and physiological parameters by adapting to different light environments in order to increase their photosynthetic and metabolic performance [62,63]. Light intensity plays a major role in plant germination, cell division, photosynthesis, leaf proliferation, and expansion [64,65,66]. Low light stress reduces inflorescences, resulted in smaller and thinner leaves, shortened flowering periods, and delayed flower bud differentiation [67,68,69]. When grapevine plants were exposed to high light stress, the expression of Vvi-MIR399s (Vvi-MIR399b, Vvi-MIR399f, Vvi-MIR399g, and Vvi-MIR399h) promptly increased at 12 h after treatment, indicating that they might be relatively sensitive to high light stress at a late stage. The Vvi-MIR399 family members harbored light-related elements in promoter regions, which might be involved in light responsiveness and affect the growth and development of grapevines (Figure 3b). The G-box binding factor acts as a positive regulator of lateral root formation, and it differentially regulates the expression of light-inducible genes [70]. AE-box and ATCT-motif are present within the 130-bp fragment of the D2-AtPolλ promoter and regulate the light-mediated activity of the promoter via binding of specific trans-acting factors [71]. Wide- and narrow-leaf plants have distinct frequencies of GATA-motif elements in chloroplast genomes [72]. Indeed, Vvi-MIR399f and Vvi-MIR399g were promptly increased by low and high light stress, implying that Vvi-miR399f and Vvi-miR399g can possibly function as modulators of light-induced signaling pathways.

MiR399 is a kind of miRNA that is sensitive to environmental temperature [73], and miR399b-overexpressing plants and a loss-of-function allele of PHO2 (pho2) exhibited an early flowering phenotype only at normal temperature (23 °C). Interestingly, their flowering time at a lower temperature (16 °C) was similar to that of WT, the expression of TWIN SISTER OF FT (TSF) was increased in miR399b-overexpressing plants and pho2 mutants at 23 °C. In this study, the downregulation of Vvi-MIR399d and the concomitant upregulation of its target gene encoding on VIT_211s0052g01030.1 showed that Vvi-MIR399d plays a critical role in regulating heat stress responses in this species. When grapevine plants were exposed to heat stress, the expression of Vvi-MIR399s (Vvi-MIR399b, Vvi-MIR399d, Vvi-MIR399g, Vvi-MIR399h, and Vvi-MIR399i) were promptly decreased at 1 h after treatment, indicating that they might be relatively sensitive to heat stress at an early stage. Under heat stress, Vvi-MIR399a and Vvi-MIR399f were up-regulated at 2h and 4 h (Figure 5c), while the target genes VIT_213s0067g03280.1 of Vvi-MIR399a and VIT_217s0000g04480.1 of Vvi-MIR399f were up-regulated at 2 h and down-regulated at 4 h (Figure 6c). These results suggested that Vvi-MIR399s mediated the silencing of target genes was under sophisticated regulation. Under freezing stress of transgenic Arabidopsis plants, overexpression of tae-miR399 ultimately decreased the expression of AtUBC24, inhibiting the degradation of AtICE1, which increased the expression of genes involved in the CBF signaling pathway and starch metabolism and promoted the activities of antioxidant enzymes [40]. In Saccharomyces cerevisiae, only overexpression of both GTR1 and PHO84 (VIT_213s0067g03280.1) could restore cold tolerance to an extent equivalent to the genomic fragment, indicating that the original tolerance was due to an additive effect of GTR1 and PHO84 [74]. Vvi-MIR399b harbored the LTR cis-acting element in promoter regions, which can be involved in low-temperature responsiveness (Figure 3b). Indeed, the abundance of Vvi-MIR399b under cold stress reached its lowest levels at 2 h and a modest increase in their levels at 1, 4, and 8 h. Similarly, the accumulation of the Vvi-miR399b target gene VIT_213s0067g03280.1 under cold stress decreased sharply within 2 h and then gradually increased (Figure 6d). After 1 h of cold stress, the expression of some Vvi-MIR399s (Vvi-MIR399d, Vvi-MIR399e, Vvi-MIR399f, Vvi-MIR399g, and Vvi-MIR399h) increased rapidly (Figure 5d), implying that Vvi-miR399b might respond to cold stress at an early stage.

In addition, miR399 was demonstrated to alter in abundance in response to salt stress. MiR399 abundance was altered by the cultivation of Col-0 seedlings for a 7-day period in the presence of 150 mM NaCl [75]. In contrast to the MIM399 transformant line, the rosette of the MIR399 plant line was slightly reduced in size compared with Col-0 rosettes. Furthermore, in the distal tips of mature rosette leaves of the MIR399 plant, areas of chlorotic and necrotic tissue formed. In addition to this vegetative phenotype, the inflorescence stem and its siliques were a pale green to yellow color in contrast to the uniform, healthy green coloration of the reproductive organs of Col-0 plants [76]. The roots from plants overexpressing miR399a exhibited increased sensitivity to salt stress when treated with 75 mM NaCl. Primary root growth in the miR399a-overexpressing roots was reduced by 40%, and the lateral root number per hairy root was decreased by up to 70% under salt stress conditions [77]. In addition, PMI2 (plastid movement impaired) was reported to share sequence and structural similarity with PMI15 (VIT_214s0006g02510.2), another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high light intensities [78]. The expressions of VIT_217s0000g04480.1 from the AP2/ERF family were obviously increased by salt stress. In maize, AP2/ERF family member ZmEREB20 was reported to positively regulate salt tolerance through molecular mechanisms associated with hormone signaling, ROS scavenging, and root-hair plasticity [79]. In our study, the abundances of Vvi-MIR399b, Vvi-MIR399c, Vvi-MIR399f, and Vvi-MIR399i decreased under salt stress (Figure 5f), and the expressions of the Vvi-miR399b, Vvi-miR399c, and Vvi-miR399i target gene VIT_213s0067g03280.1 and the Vvi-miR399f target gene VIT_214s0006g02510.2 were also down-regulated (Figure 6f). Thus, Vvi-miR399b, Vvi-miR399c, Vvi-miR399f, and Vvi-miR399i might act as negative regulators of salt stress.

The expression levels of members of the Arabidopsis thaliana miR399 family under drought stress varied by different fold levels [80], and the down-regulation of ssp-miR399-seq 1 under drought stress in both sugarcane cultivars likely led to increased pyrophosphatase levels [81]. Drought stress treatment of Arabidopsis thaliana seedlings increased miR399 abundances by 2.7-fold [75]. MiR399 was up-regulated 13-fold in tobacco seedlings after exposure to 7.5% PEG [82]. In our study, the levels of seven Vvi-MIR399 family members (Vvi-MIR399a, Vvi-MIR399c, Vvi-MIR399d, Vvi-MIR399e, Vvi-MIR399f, Vvi-MIR399g, and Vvi-MIR399h) were significantly induced at −0.4 MPa and −0.8 MPa. However, Vvi-MIR399b and Vvi-MIR399i decreased at −0.2 MPa and had only one peak (Figure 5e). In addition, we found that Arabidopsis thaliana overexpressing Vvi-MIR399e and Vvi-MIR399f were more drought tolerant than those overexpressing Vvi-MIR399d. The survival rate of Vvi-MIR399e and Vvi-MIR399f after water stress recovery was reported to be significantly higher than those of WT and Vvi-MIR399d, implying that Vvi-MIR399e and Vvi-MIR399f could possibly function as modulators of drought-induced signaling pathways. We presume that different members of the Vvi-miR399 gene family exhibit distinct expression patterns and form a complex regulatory network under drought stress.

4. Materials and Methods

4.1. Plant Materials and Stress Treatments

The tissue-cultured seedlings of the ‘Thompson seedless’ grape variety were maintained in Murashige and Skoog (MS) medium with 0.1 mg/L IAA, which was sub-cultured every 40 d for light intensity treatments. All grapevines used for light intensities treatments were grown at 25 °C under a 16-h-light/8-h-dark photoperiod. Grapevine seedlings were exposed to 1000 lx and 30,000 lx light intensities for 0, 1, 2, 4, 8, and 12 h, respectively.

One-year-old plants from the grapevine cultivar ‘Muscat Hamburg’, grown in a growth chamber, were used for heat, cold, drought, and salt treatments at 45 °C for 0, 1, 2, and 4 h for heat treatments and at 2 °C for 0, 1, 2, 4, 8, 12, and 24 h for cold treatments. For drought treatment, plants were transferred to soil with soil water potential of 0, −0.2, −0.4, −0.6, −0.8, and −1.0 Mpa. Plants were watered with a 300 mM NaCl solution for 0, 1, 2, 3, and 5 h for salt treatments. For all treatments, the 0 h treatment served as the control. The leaves were collected and immediately frozen in liquid nitrogen for further experiments.

4.2. Identification of the miR399 Family in Grapevine

The precursor sequences and mature sequences of miR399 family members in the plants were obtained from the miRbase database (https://www.mirbase.org, accessed on 26 August 2023) (Supplementary Table S1) [29].

4.3. Experimental Verifications of the Vvi-MIR399 Family Members

The primers were designed using the Primer-Blast tools in NCBI (www.ncbi.nlm.nih.gov, accessed on 26 August 2023) (Supplementary Table S2). All Vvi-MIR399 family sequences were amplified and cloned into the pEASY^®-Blunt Cloning vector using the pEASY^®-Blunt Cloning Kit (TIANGEN, Beijing, China). Cloned precursors were further confirmed by Sanger sequencing.

4.4. Structure Prediction of the Vvi-miR399 Precursors

The precursors of Vvi-MIR399s were input on the RNAfoldWebServer website (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi, accessed on 27 August 2023), the secondary structures of the Vvi-MIR399 family member precursors were predicted, and the parameters were set as default.

4.5. Phylogenetic Analyses of the Vvi-MIR399 Family Members

The precursor and mature sequences of the Vvi-MIR399s were entered on MEGA 11 software. Multiple alignments of all Vvi-MIR399s sequences and phylograms were drawn using the ClustalX program with the Maximum Likelihood Estimate method in MEGA 11 with a bootstrap analysis of 1000 replicates.

4.6. Conservation Analysis of the Vvi-miR399 Family Members

Multiple alignment of the Vvi-miR399 family members was conducted using DNAMAN software v6.0.3.99, and the results were submitted to WebLogo (http://weblogo.berkeley.edu, accessed on 27 August 2023) for base preference analysis using default parameters.

4.7. Chromosome Localization of the Vvi-MIR399 Family

To determine the locations of the Vvi-MIR399 family members on grapevine chromosomes, a search was conducted using the miRBase database (https://www.mirbase.org, accessed on 28 August 2023) (Supplementary Table S3), and the results were imported to MG2C_v2.1 (http://mg2c.iask.in/mg2c_v2.1, accessed on 28 August 2023) for visualization.

4.8. Regulatory Element Prediction on the Promoter Regions of the Vvi-MIR399 Family

The website PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html, accessed on 29 August 2023) was employed to predict the cis-acting regulatory elements on the promoters (2000 bp upstream of the precursor sequence) of the Vvi-MIR399 gene family members. The number, distribution, and classification of elements were analyzed (Supplementary Table S4).

4.9. Prediction of Vvi-miR399-Targeted Genes

The target genes of the Vvi-miR399s were predicted using psRNATarget [83]. The psRNATarget parameter settings were set as default with a maximum exception value of 3. The relationship between Vvi-miR399 and the predicted target genes were visualized using Cytoscape [84] (version 3.9.1, created by Ideker et al., San Diego, CA, USA).

4.10. Expression Analysis of the Vvi-MIR399 Family and Target Genes in Grapevine

The total RNA was isolated from grapevine under different tissues and different stress treatments using a modified CTAB method [85] and then transcribed to cDNA, and the results were used as templates for RT-qPCR. The RT-qPCR was performed on a CFX Connect Real-Time Detection System (Bio Rad, Hercules, CA, USA). The program settings were as follows: 95 °C for 150 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The relative expression levels were analyzed using the 2^−∆∆CT method [86]. Actin was used as an internal control. All RT-qPCRs were performed in triplicate. Primer sequences are listed in Supplementary Tables S2 and S5.

4.11. Vector Construction

Vvi-MIR399d, Vvi-MIR399e, and Vvi-MIR399f were constructed onto the hyperexpression vector PHB. The object sequence is shown in Supplementary Table S6. The constructed plasmids were transformed into Agrobacterium tumefaciformis GV3101 and incubated on LB medium supplemented with rifampicin (Rif) and kanamycin (Kan) at 28 °C for 2d. Single clones were incubated in LB liquid medium containing Rif and Kan at 200 rpm and 28 °C for 10–12 h. Positive clones were added to equal volumes of 50% glycerol and stored at −80 °C.

4.12. Arabidopsis Transformation

The transformation of Arabidopsis was performed according to the floral dip method [87]. Glufosinate ammonium 10% solution (dilute 1000×) (Sangon Biotech, Shanghai, China) was used to screen transgenic seedlings. Six biological replicates were selected for each sample as plant length and primary root length measurements.

5. Conclusions

In this study, nine mature members and nine precursor members of the grapevine miR399 family were identified. The precursor sequence of miR399 was highly diverse, while the mature sequence was highly conserved. Promoter analysis identified multiple stress-response elements such as ABA, drought, and low temperature responses, in which the description and number of stress-response elements depended on the specific member of the Vvi-MIR399 family, thereby determining the functional specificity of each member. Moreover, the difference in the transcript level of the Vvi-MIR399 member confirmed the functional diversity under various abiotic stresses. Furthermore, transgenic Vvi-MIR399e and Vvi-MIR399f plants were more drought tolerant than Vvi-MIR399d plants under drought stress, while transgenic plants Vvi-MIR399d and WT had no drought tolerance. This study not only increases the understanding of the grapevine MIR399 family but also increases their regulation and functional differentiation in abiotic stress, as well as their potential applications in crop improvement.