Potential Role of WIP Family Genes in Drought Stress Response in Rubus idaeus

Abstract

Rubus idaeus is one of the primary cultivated species of raspberries, renowned for its appealing color, distinctive flavor and numerous health benefits. WIP proteins, which contain three conserved amino acids (W: Tryptophan, I: Isoleucine, P: Proline) and four zinc finger motifs in a highly conserved C-terminal region, are members of the A1d subgroup of C2H2 zinc finger proteins. Drought is one of the main limiting factors of plant growth and development, which restricts the cultivation and utilization of raspberry in northwest China. In this study, to obtain candidate genes for drought resistance, we identified key related genes, RiWIPs, from R. idaeus and analyzed their bioinformation and tissue stress response expression to drought. We found that there are three RiWIPs in R. idaeus and they are located on chromosomes 3, 4 and 6 of R. idaeus, respectively. The open reading frames (ORFs) of the RiWIPs ranged from 870 to 1056 bp in length, encoding 289 to 372 amino acid residues. The proteins were highly conserved and feature diverse conserved motifs. The promoters of the RiWIPs contained abundant cis-elements related to growth, development and stress response. Tissue-specific expression analysis revealed that the RiWIPs were expressed in the leaves, stems and roots of both drought-susceptible and drought-tolerant cultivars, except for RiWIP2, which was only expressed in the roots of the drought-tolerant one. Under drought stress, the transcriptional activity of the RiWIPs was increased to different degrees with specificity in the leaves, stems and roots. Our study demonstrated the role of WIP genes in raspberry drought response and provided a marker gene, RiWIP2, for drought resistance and candidate genes for subsequent drought-resistant breeding of R. idaeus.

1. Introduction

Raspberries are one of the third-generation fruits, which have significant value for research and utilization. Rubus idaeus, a plant in the Rosaceae family, belongs to the genus Rubus, and it is one of the primary cultivated raspberries species [1]. The fruit of R. idaeus is famous for its unique nutritional and medicinal properties, earning the moniker ‘life fruit’ [2]. It is used not only for fresh consumption but also in the production of juices, fruit rolls, preserves and cold beverages. It is also valued in medicine for its beneficial effects, such as nerve protection, vision improvement, kidney nourishment and diuretic properties [3,4]. At present, the United States, Russia and the United Kingdom are the major producers and consumers of raspberries, with a long history of cultivation and utilization. Meanwhile, this is accompanied by many years of experience in the artificial breeding of raspberries, focusing mainly on fruit quality [5], yield [6], extended cropping season [7] and studies on pests and diseases [8]. Apart from the above breeding goals, there are relatively few studies on the local environmental adaptability of R. idaeus [9].

Since raspberries were introduced to China in the 1990s, raspberry cultivation has primarily developed in the northeastern regions and Xinjiang, mainly for export to countries such as Russia, the United States and South Korea [10,11]. Currently, raspberries are widely distributed and cultivated in northern China, demonstrating significant adaptability, especially in arid areas like Inner Mongolia, Gansu and Ningxia. Reports of raspberry cultivation in these regions have been steadily increasing, with growing recognition of the economic and ecological benefits of raspberry cultivation. However, recent years there has been an increase in extreme weather events globally, which has exacerbated drought conditions in northwest China, negatively impacting plant growth, crop yield and economic returns [12,13]. Consequently, there is an urgent need to select drought-resistant germplasm and understand its resistance mechanisms and to further explore and create drought-resistant germplasm [9].

Plant drought response involves the regulation of genes, cells, tissues, organs, metabolism, signaling and so on. Among these, transcription factors (TFs) are protein molecules with a specific structure and function that regulate gene expression [14]. The WIP family is one of the TF families in plants, belonging to the A1d subgroup of C2H2 zinc finger proteins. WIPs contain three conserved amino acids (W: Tryptophan, I: Isoleucine, P: Proline), from which they derive their name, and possess four zinc finger motifs in a highly conserved C-terminal region [15,16]. In Arabidopsis thaliana, there are six WIP members. Among them, AtWIP1 (TRANSPARENT TESTA 1, TT1) was reported to be involved in seed coat pigmentation and the differentiation of the seed coat cell layer [17]. AtWIP2 relate to pollen tube growth, replum development and fruit lignification [18]. AtWIP2, AtWIP4 and AtWIP5 act redundantly in cell fate determination during primary root development [15,19]. The mutations of AtWIP6 lead to alterations in leaf vein patterning [15,20].

Furthermore, previous studies of WIP genes showed JrTT1 (a WIP TF) was involved in plant growth, development and drought response [21] and the expression activity of JrTT1 promoter region is related to WRKY recognition elements [22]. Moreover, in another study, it was shown that overexpression of the Brassica napus TT1 gene in cotton can improve its drought resistance [23]. These reports drew our attention to the WIP gene family and raised questions as to whether the WIP transcription factor in R. idaeus is highly conserved as in other plants and related to drought response. To answer these questions, we identified the key WIP genes using existing high-quality whole-genome sequencing data and analyze their bioinformatics profiles and upstream promoter sequences to elucidate their potential roles in drought stress resistance. Additionally, their tissue expression and drought stress response expression in R. idaeus were analyzed to verify gene function.

2. Materials and Methods

2.1. Plant Materials and Treatments

Plant materials were collected from 1-year-old R. idaeus cultivars WC (relatively drought-susceptible) and DQS (relatively drought-tolerant), which were cultivated in the greenhouse of the Chinese academy of forestry (located in Haidian district, Beijing, China). Eighteen seedlings (nine from the WC cultivar and nine from the DQS cultivar) were selected for PEG₆₀₀₀-induced drought stress treatment. The leaves, stems and roots from controls (0 d) and after 3 d and 5 d of treatment were collected and frozen in liquid nitrogen, and then stored at −80 °C for subsequence RNA isolation. Three additional seedlings from each line were maintained under normal watering conditions and used as controls. Each time point included three plants with a similar height and biomass. Three biological replicates were conducted for each test sample.

2.2. Identification and Chromosomal Locations of WIP Genes in R. idaeus

Based on the reported WIPs of A. thaliana, six AtWIP sequences were retrieved from the TAIR database (https://www.arabidopsis.org/ (accessed on 3 October 2023)). These sequences were then locally blasted against the transcriptome of R. idaeus ‘Joan J’ genome v2.0 (https://www.rosaceae.org/Analysis/14031373 (accessed on 18 November 2022)) using the BLAST tools provided by NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 18 November 2022)), resulting in the identification of homologous WIP proteins in R. idaeus [24]. The ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/ (accessed on 18 November 2022)) was used to locate the open reading frame (ORF) of potential R. idaeus WIP genes, and the corresponding protein sequences were queried and validated in the NCBI protein database. Additionally, ExPasy (https://web.expasy.org/protparam/ (accessed on 19 April 2023)) was utilized to analyze the number of amino acids, molecular weight and theoretical isoelectric points (pI) of RiWIP proteins. The protein subcellular localization was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/ (accessed on 27 September 2024)). The secondary structure was predicted using PredictProtein (https://open.predictprotein.org/ (accessed on 27 September 2024)). The third structure was predicted using SWISS-MODEL (https://swissmodel.expasy.org/interactive (accessed on 27 September 2024)). Phosphorylation sites were predicted using netphos 3.1 (https://services.healthtech.dtu.dk/service.php?NetPhos-3.1 (accessed on 28 September 2024)). The chromosomal location information for the RiWIPs was obtained from the R. idaeus ‘Joan J’ genome v2.0 and visualized using TBtools.

2.3. Analysis of Evolutionary Relationship and Gene Structure

To analyze the evolutionary relationship of the three WIPs of R. idaeus, six protein sequences of A. thaliana WIPs were downloaded from the TAIR database, while six Vitis vinifera WIPs and nine Populus trichocarpa WIPs were obtained from the Phytozome v13 (https://phytozome-next.jgi.doe.gov/ (accessed on 27 June 2024)) database. The WIPs from R. idaeus, A. thaliana, V. vinifera and P. trichocarpa were aligned using Clustal X2 software, and a phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates. The resulting phylogenetic tree was modified using Adobe illustrator CC 2018 version 22.1 software.

The gene structure maps were generated using GSDS (Gene Structure Display Server 2.0, https://gsds.gao-lab.org/ (accessed on 3 November 2024)). The intron scale parameter was shrunk to 5. The conserved motifs were predicted using MEME Suite (https://meme-suite.org/ (accessed on 31 March 2023)) with the following parameters: the number of motifs was 15, any number of repetition was allowed, and the motif width was between 6 and 50. The motif domains and chromosomal locations were visualized using TBtools v2.136 software [25].

2.4. Cis-Acting Elements Analysis of the Promoter Region of RiWIP Genes

The promoter regions of the RiWIP genes were identified using Gtf/GFF3 Sequences Extract and Fasta Extract or filter tools in TBtools [25]. PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 3 April 2023)) was used to analyze the cis-acting elements within the promoter regions of RiWIP genes. The results were visualized using TBtools.

2.5. Expression Analysis of RiWIPs

Total RNA was extracted from each sample using the CTAB method [26]. The RNA concentration and purity (A260/280 ratio) were detected, and RNA with a purity of 1.8–2.0 was used for the subsequent assays. The RNA was then reverse-transcribed into cDNA using Evo M-MLV RT Mix Kit (Accurate Biotechnology, Changsha, China). The cDNA was diluted 10 times and used as the template for Quantitative Reverse Transcription PCR (qRT-PCR). The qRT-PCR was conducted used SYBR Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) with an internal reference gene of R. idaeus 18S (KP125886.1) [27]. The primers used are shown in Table S1. The 20 μL reaction mixture included 10 μL 2X SYBR Green Pro Taq HS Premix, 2 μL cDNA, 0.4 μL forward primers, 0.4 μL reverse primers and 7.2 μL ddH₂O. The reaction procedures were set following the qRT-PCR kit instructions. The procedures were performed three times on each gene. The data were analyzed using IBM SPSS Statistics 23 software. The sample variability is expressed as a standard deviation. The expression differences between the different time points and 0 d were test by Student’s t-test (p < 0.05). The results were visualized using Origin 2021 v9.8 software.

3. Results

3.1. Sequence Characteristics and Chromosomal Locations of R. idaeus WIP Genes

Four putative RiWIP genes were identified from the transcriptome of ‘Joan J’ R. idaeus. Among these genes, one was found to be missing a complete WIP domain and four C2H2 zinc fingers (ZFs). The conserved domain of the other RiWIPs were confirmed by CD-Search tools in NCBI, InterProScan and the PROSITE of Expasy (Figure S1). Consequently, three genes in R. idaeus were classified as WIP family members and named RiWIP1, RiWIP2 and RiWIP3, respectively, according to their order on the chromosomes (

Table 1. Sequence characteristics of WIP gene in R. idaeus.

Gene Name	Gene ID	Chromosome Site	Number of Amino Acids/aa	Molecular Weight/Da	Theoretical Isoelectric Points	ORF	Subcellular Localization
RiWIP1	Rid.03g116370.m1	Chr3	316	77,649.77	5.09	951	nucleus
RiWIP2	Rid.04g154500.m1	Chr4	289	71,436.91	5.07	870	nucleus
RiWIP3	Rid.06g261630.m1	Chr6	372	89,055.49	5.03	1056	nucleus

, Figure 1A). The open reading frames (ORFs) of the RiWIPs ranged from 870 to 1056 bp in length, encoding 289 to 372 amino acid residues. The molecular weights of these proteins ranged from 71,436.91 to 89,055.49 Da, and their theoretical pIs were between 5.03 and 5.09. The protein subcellular localization prediction showed that the RiWIPs were all located in the nucleus. The number of phosphorylation sites of the three RiWIPs were 32, 28 and 42, respectively (Table S2). Among the three kinds of proteins, the content of serine was higher than those of threonine and tyrosine, indicating that serine is a common phosphorylation site in the R. idaeus WIP family. The prediction of the RiWIPs’ secondary structure showed that the content of components in the secondary structure from large to small is as follows: loop > helix > strand (Figure S2). In RiWIP1, the loop was as high as 77.85%, the second was the helix, accounting for 11.71%, while the strand accounted for 10.44%. In RiWIP2, the loop, helix and strand accounted for 87.54%, 6.57% and 5.88%, respectively. In RiWIP3, the proportion of the loop, helix and strand was 81.18%, 11.02%, 7.80%, respectively (Figure S2A–C). It can be inferred that the spatial structure of the RiWIPs is extensive, primarily caused by interactions between the main chains, and is less influenced by interactions between the side chains. The result of tertiary structure modeling showed that the RiWIPs mainly contained random coils and extended strands, and this further verified their extensiveness (Figure S3).

Chromosome localization analysis showed that RiWIP1, RiWIP2 and RiWIP3 were located on chromosomes 3, 4 and 6 of the R. idaeus, respectively, with chromosomes 1, 2, 5 and 7 containing no RiWIPs (Figure 1A).

3.2. Sequence Conservation and Evolutionary Relationship of RiWIP Proteins

To analyze the conservation of the RiWIPs, the protein sequences of the RiWIPs were aligned with the AtWIPs from A. thaliana. The result showed that the RiWIPs contained the WIP domain, followed by four zinc fingers (ZFs), ZF1, ZF2, ZF3 and ZF4 (Figure 1B). Additionally, a conserved domain sequence logo was generated using MEME tools, revealing that the RiWIPs contained conserved motifs. Motif1-4 were components of the WIP domain, with sequence similarity reaching 100% at 44 sites in Motif1, 35 sites in Motif2, 17 sites in Motif3 and 17 sites in Motif4 (Figure 1C).

To investigate the evolutionary relationships of the RiWIPs in comparison with other plants, protein sequences from three WIPs of R. idaeus, six WIPs of A. thaliana, six WIPs of V. vinifera and nine WIPs of P. trichocarpa were aligned to construct an unrooted phylogenetic tree. The analysis showed that the RiWIPs clustered into distinct branches (Figure 2). Specifically, RiWIP1 was closely related to AtWIP3 and grouped with VIT 206s0080g00270.1, Potri.009G143700.1.p and Potri.004G183900.1.p. RiWIP2 exhibited the highest homology with AtWIP6, Potri.010G129000.1.p and VIT 201s0011g03100.1, while RiWIP3 was closed to AtWIP2, VIT_208s0007g08070.1 and Potri.016G052700.1.p.

3.3. Gene Structure and Conserved Motif Composition of WIPs

The genomic DNA sequences of the RiWIPs were analyzed using the GSDS online tool to examine the gene structure. The results revealed that each RiWIP gene has two coding sequences (CDS) regions and two introns. The gene structures of RiWIP1, RiWIP2 and RiWIP3 were all interrupted by introns (Figure 3A).

The conserved motifs within the RiWIPs were identified using the MEME Suite. The results showed that each motif contains between 6 and 50 amino acids (Table S3), with each gene sequence containing 11 to 15 motifs (Figure 3B). Among these, Motif1, 2, 3, 4, 5, 7 and 14 were the most common conserved motifs and were present in all the RiWIPs.

3.4. The Cis-Acting Elements in Promoters of WIP Genes

Three 2000 bp upstream promoter regions of the RiWIPs were obtained from the NCBI database and cis-acting elements were predicted using PlantCARE tools. The analysis identified several core cis-acting regulatory DNA elements. These included CAAT-box, found 20 to 42 times, and TATA-box, found 24 to 64 times, across all the RiWIPs. Additionally, the AT~TATA-box appeared six times in RiWIP1 and eight times in RiWIP2. Stress response-related elements were abundant in all the RiWIP promoters (Figure 4), such as MYB-related elements (including MYB, MYB recognition site, Myb-binding site, MYB-like sequence, MBS), MYC elements, TC-rich repeats (defense and stress responsive-related elements), LTR (low-temperature responsive elements) and ARE (anaerobic induction-related elements). Moreover, various light-responsive elements were present, including the GA-motif, ATC-motif, ATCT-motif, TCCC-motif, GT1-motif, AE-box, I-box, G-box, Box 4 and chs-CMA1a. Some elements involved in hormone regulation were also identified. The SA responsiveness element (TCA-element) was found in all the RiWIPs, while MeJA responsiveness elements (TGACG-motif, CGTCA-motif) were present in the promoters of RiWIP1 and RiWIP3, but not in RiWIP2. The ABA responsiveness element (ABRE) was detected in the RiWIP2 promoter, and the GA responsiveness element (GARE-motif) was found in RiWIP3, with the P-box present only in RiWIP2 (Figure 4).

3.5. Tissue Expression Specificity of RiWIPs

To investigate the tissue-specific expression of the RiWIPs, we measured the transcriptional activity of RiWIP1, RiWIP2 and RiWIP3 in the leaves, stems and roots of two lines, the drought-susceptible line WC and the drought-tolerant line DQS (Figure 5A). The results are summarized below.

In the drought-susceptible WC cultivar, RiWIP1 and RiWIP3 were expressed in all the tissues. Among them, RiWIP1 exhibited high expression in the stems and roots, with particularly prominent expression in the stems. The expression of RiWIP1 in the leaves was only 2.48% of that in the stems. RiWIP3 showed significant expression in the roots, with levels 1.78- to 6.41-fold of that in the leaves and stems. RiWIP2 was not detected in any of the tissues.

In the drought-tolerant DQS cultivar, RiWIP1 and RiWIP3 showed the highest expression in the roots, followed by the stems, and were the lowest in the leaves. The expression of RiWIP1 in the roots was 58.29-fold of that in the leaves and 2.01-fold of that in the stems, while the RiWIP3 expression in the roots was 12.08-fold of that in the leaves and 1.63-fold of that in the stems. RiWIP2 was not detected in the leaves and stems, but trace amounts of RiWIP2 were detected in the roots, suggesting a possible correlation between RiWIP2 and drought response in R. idaeus.

3.6. Expression Levels of RiWIPs in Response to Drought Stress

To investigate the potential role of RiWIPs in drought stress response, we measured the expression of RiWIP1, RiWIP2 and RiWIP3 under 5 d of PEG₆₀₀₀ stress in the drought-sensitive line WC (Figure 5B). We found that, after drought stress, the expression of the RiWIPs was upregulated by different degrees in the leaves, stems and roots. Among these, RiWIP1 increased to 1.42~2.26-fold in the leaves, stems and roots, and the upregulation of expression in the roots was the most obvious. For RiWIP2, the expression was not detected before the drought stress, while after the drought treatment, only a trace expression was detected in the roots, and the expression level was only 0.16. RiWIP3 showed a significantly upregulated level after 5 d drought stress in stems and the expression of RiWIP3 in stems at 5 d was 1.83-fold of that before treatment, while the expression in the leaves and roots was 0.92~1.05-fold of that before the treatment.

To verify the response ability of the RiWIPs to drought stress, the transcript abundance was assessed using qRT-PCR for 3 and 5 d of PEG₆₀₀₀ stress in the drought-susceptible line WC (Figure 6). The results showed that there were significant changes in the relative expression levels of RiWIP1, RiWIP2 and RiWIP3. All the RiWIPs were suppressed in the leaves but upregulated in the stems and roots under drought stress. Notably, after drought stress, RiWIP1 reached its highest relative expression level in the stems at 5 d, while RiWIP2 and RiWIP3 peaked in the roots at 3 d. The expression of all the RiWIPs peaked after 3 d of PEG₆₀₀₀ treatment.

In detail, in the leaves, the expression of RiWIP1, RiWIP2 and RiWIP3 was suppressed by PEG₆₀₀₀ stress, with expression values of −3.79, −2.60 and −2.08 at 3 d and −12.40, −10.89 and −10.48 at 5 d, respectively. In the stems, all the RiWIPs were upregulated under PEG₆₀₀₀ stress, with peak expression levels occurring at 3 d. Particularly for RiWIP3, the relative expression value reached 9.93, which was 1.96-fold that of the value at 5 d. RiWIP1 and RiWIP2 showed expression levels of 1.53-fold and 1.00-fold compared to the levels at 5 d, respectively. In the roots, all the RiWIPs were upregulated, with expression levels ranging from 0.22~2.80 after 5 d of PEG₆₀₀₀ stress, and the relative expression of RiWIP2 reached 2.80 (Figure 6). These results suggest that RiWIP2 functions as a drought stress response gene.

4. Discussion

R. idaeus is classified as one of the third-generation fruits, and is rich in essential nutrients for human health and particularly abundant in anthocyanins, thus holding significant economic value [4,28]. In China, raspberries (R. idaeus) are primarily distributed in northern regions. However, in recent years, frequent extreme weather conditions globally have increasingly impeded the development and utilization of economic plants due to drought, especially in the northwest region. Therefore, it is crucial to implement drought-resistant breeding programs [12,29]. Previous studies have indicated that WIP1 was involved in drought response and the expression activity of the promoters was related to the length of the WRKY element [21,22]. Furthermore, many studies have demonstrated that WRKYs play a role in drought tolerance [30,31,32]. Therefore, in this study, we focused on the identification of RiWIPs in R. idaeus to clarify the fundamental biological roles of WIPs and predict their potential role in stress resistance.

We used six WIP protein sequences of A. thaliana (AtWIP1, AtWIP2, AtWIP3, AtWIP4, AtWIP5, AtWIP6) to identify homologous proteins in the transcriptome library of the R. idaeus ‘Joan J’ genome data using local BLAST. As a result, a total of nine WIP homologous proteins were initially discovered. After removing duplicates, four unique sequences remained. The gene sequences were then queried against the CDS library using sequence codes, and the ORFs were identified with the ORF Finder tool of the NCBI database, followed by translation into proteins. Sequence accuracy was confirmed through re-sequencing comparisons with the WIP family of genes of A. thaliana and conserved domain analysis (Figure S1). Notably, one gene (Rid.03g141820.m1) displayed a WIP domain according to NCBI, InterProScan and Expasy analyses; however, it had fewer than four zinc finger structures at the C-terminus, indicating that did not belong to the A1d subfamily. Consequently, three WIP genes were selected for further analysis, named RiWIP1, RiWIP2 and RiWIP3 according to their chromosomal positions (Figure 1A). Gene structure analysis revealed that all the RiWIPs contained introns (Figure 3). The analysis of gene location and structure in chromosomes provides the basis for further revealing the interaction between related genes in drought response. The three RiWIPs contained seven motifs in common, with Motif1, Motif2, Motif3 and Motif4 representing parts of the conserved domain. This analysis highlighted the high conservation among WIP members in R. idaeus.

Plant promoters act as transcriptional switches that regulate the initiation and extent of gene expression in specific tissues or under physiological stress conditions [33]. For instance, in Arabidopsis, the MYC and MYB cis-acting elements in the rd22 promoter played the role in the drought- and ABA-induced gene expression of rd22 [34]. rd22BP1 (AtMYC2) and AtMYB2 specifically bound the MYC and MYB cis-acting elements and activated the transcription of the reporter gene [35]. In Paeonia ostia, PoWRKY69 could bind the W-box element of the PoFBA5 promoter and contribute to activating the PoFBA5 expression [36]. In this study, the upstream regions of the RiWIPs contain a wealth of functional elements, such as the MYB, MYC and WRKY binding sites (Figure 4), including MYB, CCAAT-box, MBS, MYB recognition site, Myb-binding site, MYB-like elements, MRE, W-box, etc., indicating that RiWIPs may be crucial genes involved in plant drought stress response [34,35,36,37]. In addition, the upstream regions of the RiWIPs include elements responsive to light, anaerobic induction, low temperature and hormone regulation, as well as TC-rich repeats related to defense and stress responses (Figure 4). Above all, these findings further support our hypothesis that RiWIPs may play a significant role in plant growth, development and stress responses, especially in drought response.

To clarify the potential function of WIP, we analyzed WIP gene expression in different tissues. We accessed their expression levels in the leaves, stems and roots of R. idaeus drought-susceptible (WC cultivar) and drought-tolerant (DQS cultivar) lines (Figure 5A). The results indicated that RiWIP1 and RiWIP3 were expressed in all the tissues of both the drought-susceptible and drought-tolerant cultivars, with particularly strong expression in the stems and roots, while only trace amounts of RiWIP2 were detected in the roots of the drought-tolerant line DQS. This expression pattern is similar to the expression pattern of other genes. For example, in A. thaliana, the representative independent GUS reporter lines pWIP1, pWIP2 and pWIP3 are expressed in the cotyledon vasculature, pWIP2, pWIP3 and pWIP4 are expressed in the shoot apical meristem region, and pWIP2 and pWIP3 are expressed in young aerial tissues [15]. In walnut, the expression patterns of WIP TFs JrTT1-1, JrTT1-2 and JrTT1-3 were different in the roots and stems, and showed tissue specificity in response to drought [21]. In sweet potato, IbKNOXs in Class M were specifically expressed in the stem tip and hardly expressed in other tissues, suggesting that they might play an important role in the development of meristem tissue [38]. In a study of the Dof gene family genome-wide identification and molecular evolution in Camellia oleifera, 21 ColDof genes were expressed in all 221 seed species, while the other 24 ColDof gene members were not expressed [39]. All the above reports further illustrate that RiWIPs may play a role in R. idaeus drought response with tissue-specific expression.

To find out whether RiWIPs respond to drought stress, we analyzed the changes in RiWIP expression in the drought-resistant line WC under 5 d of PEG₆₀₀₀ stress. The findings revealed that RiWIP1 and RiWIP3 genes were expressed in the leaves, stems and roots, while RiWIP2 showed only slight expression in the roots after drought stress (Figure 5B). Based on these observations, we hypothesize that there may be a potential correlation between RiWIP2 and the drought response in R. idaeus. Further, we designed primers and carried out the qRT-PCR assay. The result revealed that the transcript abundance of all the RiWIPs was induced with different tendencies, also reflected the tissue expression specificity of the RiWIPs (Figure 6). Our results were consistent with the previous studies exploring response to drought. The transcript abundance of five JrMYBs in walnut (Juglans regia) was induced with different tendencies in the leaves and roots, as well as reflecting the tissue expression specificity of JrMYBs [40]. In Cui et al.’s study, five SlPML genes showed different expression levels, and their different expression pattern in different tissues indicated various functions in drought response [41]. Therefore, we conclude that RiWIP1, RiWIP2 and RiWIP3 play different roles in drought response. It is worth noting that RiWIP2 was expressed in the roots of the drought-tolerant line DQS (Figure 5A) and the drought-resistant line WC after 5 d of PEG₆₀₀₀ stress (Figure 5B). These findings indicate that RiWIP2 may be used as a key gene in the screening of the drought-resistant germplasm of raspberry. In the future, further research on the RiWIP genes is needed to clarify its pathway of drought response. In addition, the effect of RiWIP2 in screening drought-resistant germplasm needs to be verified further.

5. Conclusions

Three RiWIP genes were identified, which belong to the A1d subgroup of C2H2 zinc finger proteins from R. idaeus. RiWIP1, RiWIP2 and RiWIP3 were located on chromosomes 3, 4 and 6, respectively, and the ORFs ranged from 870 to 1056 bp in length, encoding 289 to 372 amino acid residues. Their sequences were highly conserved and their proteins contained diverse conserved motifs. The promoters of the RiWIPs contained abundant cis-acting elements related to growth, development and drought response. The RiWIPs could express in the leaves, stems and roots of both the drought-susceptible cultivar and drought-tolerant cultivar, except for RiWIP2, which was only expressed in the roots of the drought-tolerant cultivar. The qRT-PCR assay showed that all the RiWIPs were clearly induced by the drought treatment, with specificity in the leaves, stems and roots. In brief, RiWIP genes may be potential candidates for improving R. idaeus drought response. This study provides important information for future breeding programs and enhancing the drought resistance of the species.