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

Transcriptome analysis unveils a potential novel role of VvAP1 in regulating the developmental fate of primordia in grapevine

  • 1.

    Grape Genetics Research Unit, USDA-Agricultural Research Service, Geneva, NY 14456, USA

  • 2.

    Institutional Research and Engagement Office & Department of Natural Science College of Arts and Science, University of St. La Salle, Bacolod City 6100, Philippines

  • 3.

    Plant Biotechnology Resource and Outreach Center, Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA

  • 4.

    E&J Gallo Winery, Modesto, CA 95358, USA

  • 5.

    Appalachian Fruit Research, USDA-Agricultural Research Service, Kearneysville, WV 25430, USA

More Information
  • Received: 27 September 2023
    Revised: 02 December 2023
    Accepted: 26 December 2023
    Published online: 04 March 2024
    Fruit Research  4,  Article number: e011 (2024)  |  Cite this article
  • The grapevine shoot meristem contains undifferentiated primordia known as anlagen, which can develop into either inflorescences or tendrils depending on vine age, growth status, hormone balance, and other factors. Interestingly, a gain-of-function mutation in the DELLA domain of VvDELLA1 in the dwarf mutant grape, Vitis vinifera L. cv. Pixie, virtually disrupts the normal developmental course of anlagen and reroutes tendril-bounded anlagen toward inflorescence development even at the juvenile stage. To understand the underlying mechanism(s), we compared the transcriptome profiles of V. vinifera cv. Pinot Meunier (from which Pixie was derived), Pixie, and three other V. vinifera grape cultivars (Dena, Gina, and Tia) which were derived from crosses involving Pixie and carry the same DELLA mutation. Our findings revealed significant mis-regulation of hundreds of genes, profoundly reshaping both transcriptome landscapes and regulatory pathways in the mutant grapes. Interestingly, VvAP1, a central positive flower regulator in annuals, was unexpectedly co-downregulated with VvTFL1a, a flowering repressor. We also found several other key flower regulators which were either upregulated (e.g., VvFT, VvLFY) or downregulated (e.g., VvSOC1s) in all mutant grapes, although the overall effect was moderate. These findings, along with the previous identification of tendril-specific expression of VvAP1 and inflorescence-specific expression of VvLFY, support that VvAP1 promotes anlagens to develop tendrils, whereas VvLFY favors inflorescences formation. The balance between these factors, particularly the abundance of VvAP1 transcripts, ultimately dictates whether anlagens develop into tendrils or inflorescences.

  • Introduction

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

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

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

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

    Materials and methods

    Plant materials, growth conditions, and stress treatment

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

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

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

    Vector construction

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

    Chlorophyll and anthocyanin content determination

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

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

    Physiological analysis

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

    RNA isolation and qRT-PCR analysis

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

    Results

    Screening and characterization of lncRNAs

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Conclusions

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

    Author contributions

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

    Data availability

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

    Acknowledgments

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

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

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  • Cite this article

    Arro J, Yang Y, Song GQ, Cousins P, Liu Z, et al. 2024. Transcriptome analysis unveils a potential novel role of VvAP1 in regulating the developmental fate of primordia in grapevine. Fruit Research 4: e011 doi: 10.48130/frures-0024-0004
    Arro J, Yang Y, Song GQ, Cousins P, Liu Z, et al. 2024. Transcriptome analysis unveils a potential novel role of VvAP1 in regulating the developmental fate of primordia in grapevine. Fruit Research 4: e011 doi: 10.48130/frures-0024-0004
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Transcriptome analysis unveils a potential novel role of VvAP1 in regulating the developmental fate of primordia in grapevine

  • 1.

    Grape Genetics Research Unit, USDA-Agricultural Research Service, Geneva, NY 14456, USA

  • 2.

    Institutional Research and Engagement Office & Department of Natural Science College of Arts and Science, University of St. La Salle, Bacolod City 6100, Philippines

  • 3.

    Plant Biotechnology Resource and Outreach Center, Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA

  • 4.

    E&J Gallo Winery, Modesto, CA 95358, USA

  • 5.

    Appalachian Fruit Research, USDA-Agricultural Research Service, Kearneysville, WV 25430, USA

  • Received: 27 September 2023
  • Revised: 02 December 2023
  • Accepted: 26 December 2023
  • Published online: 04 March 2024
Fruit Research  4,  Article number: e011  (2024)  |  Cite this article

Abstract: The grapevine shoot meristem contains undifferentiated primordia known as anlagen, which can develop into either inflorescences or tendrils depending on vine age, growth status, hormone balance, and other factors. Interestingly, a gain-of-function mutation in the DELLA domain of VvDELLA1 in the dwarf mutant grape, Vitis vinifera L. cv. Pixie, virtually disrupts the normal developmental course of anlagen and reroutes tendril-bounded anlagen toward inflorescence development even at the juvenile stage. To understand the underlying mechanism(s), we compared the transcriptome profiles of V. vinifera cv. Pinot Meunier (from which Pixie was derived), Pixie, and three other V. vinifera grape cultivars (Dena, Gina, and Tia) which were derived from crosses involving Pixie and carry the same DELLA mutation. Our findings revealed significant mis-regulation of hundreds of genes, profoundly reshaping both transcriptome landscapes and regulatory pathways in the mutant grapes. Interestingly, VvAP1, a central positive flower regulator in annuals, was unexpectedly co-downregulated with VvTFL1a, a flowering repressor. We also found several other key flower regulators which were either upregulated (e.g., VvFT, VvLFY) or downregulated (e.g., VvSOC1s) in all mutant grapes, although the overall effect was moderate. These findings, along with the previous identification of tendril-specific expression of VvAP1 and inflorescence-specific expression of VvLFY, support that VvAP1 promotes anlagens to develop tendrils, whereas VvLFY favors inflorescences formation. The balance between these factors, particularly the abundance of VvAP1 transcripts, ultimately dictates whether anlagens develop into tendrils or inflorescences.

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    Introduction

    • Transition from vegetative to reproductive growth is a pivotal juncture in the development of plants, orchestrated by a combination of intrinsic developmental signals and extrinsic cues[1]. Photoperiod is a major external factor, serving as a cue to trigger the transition from vegetative to floral meristem in many annual plants. In winter annuals with a life cycle spanning fall, winter, and into the next spring and summer seasons, vernalization induced by cold temperatures is a major external factor responsible for the transition[2−6]. Unlike annuals, grapevine is a perennial woody species and has unique and intricate biological processes for flower formation and development. The switch from juvenile to adult phase in grapevine is accompanied by the appearance of the first tendril along with the change of spiral to alternate leaf arrangement. Then vine's shoot apical meristem (SAM) in the main branch's apex continues producing both leaf primordia and uncommitted primordia, known as anlagen. Depending on genetic and environmental cues, an uncommitted primordium can develop into either inflorescence or tendril. Usually, the first two or three undifferentiated axillary primordia in latent buds give rise to inflorescences after they have undergone dormancy while those buds emerging in the current season's shoots are destinated to become tendrils, which serve as support structures in grapevine[7−9]. Tendrils are regarded as intermediate or modified inflorescences due to their inability to initiate floral meristem, as evidenced by their interchangeable nature and specific expression of key floral meristem identity APETALA1 (VvAP1) and LEAFY (VvLFY). Studies showed that application of cytokinins and gibberellic acid (GA) in shoots could promote anlagens to develop into inflorescences and tendrils, respectively[9−12]. Interestingly, even young tendrils treated with cytokinin can be transformed into inflorescences, whereas GA treatment effectively reverts young inflorescences back to tendrils[13,14]. This underscores the dynamic, hormone-responsive and reversable features of anlagens, tendrils and inflorescences, in a stark contrast to the terminal, irreversible nature of highly differentiated floral tissues in other woody fruit crops such as apples and peaches.

      A grapevine mutant derived from a somatic mutation in the L1 cell layer of Vitis vinifera cv. Pinot Meunier manifests dominant dwarfism and insensitive to GA[15], similar to the gain-of-function GA-insensitive (gai) mutants found in Arabidopsis, rice and wheat, collectively referred to 'green revolution' dwarfism[16−18]. In-depth scrutiny revealed that the L1 dwarf mutant bears a mutation in the DELLA domain of VvDELLA1 or VvGAI1[15]. This mutation induces an amino-acid substitution in the DELLA domain of VvDELLA1 and disrupts interaction of the VvDELLA1 protein with GA signals. Consequently, VvDELLA1 proteins evade GA-mediated breakdown, resulting in highlighted accumulation of the VvDELLA1 factor and its growth-inhibitory impact on stem elongation, as evidenced by the dominant dwarf phenotype in the L1 mutant[15] and in the transgenic Arabidopsis with ectopic expression of the mutated VvDELLA1 (Vvgai1)[19]. Unexpectedly, the same mutation in the L1 dwarf mutant drastically enhanced the conversion of tendril-bound anlagens to inflorescences even in the current shoots or in juvenile seedlings[15]. This suggests that VvDELLA1 functions distinctively from its counterparts in annuals, which typically repress flowering or delay the transition from vegetative to reproductive phases[20−23].

      DELLA proteins play a pivotal role as central hubs, translating hormonal stimuli, physiological cues and environmental inputs into specialized regulatory networks that govern plant growth and development. In the GA pathway characterized in Arabidopsis and other annual crops, the interactions between GA and its receptor, GIBBERELLI INSENSITIVE DWARF1 (GID1), facilitate binding to the DELLA domain of the DELLA proteins. Consequently, these interactions render DELLAs highly susceptible to polyubiquitination, marking them for subsequent degradation by the 26S proteasome[24]. This orchestrated process results in diminished DELLAs' levels, along with their growth-repressive function, leading to increased stem elongation and the promotion of flowering. Conversely, the loss of this interaction results in elevated DELLAs' levels, reinforcing their inhibitory actions, thereby yielding general outcomes of dominant dwarfism and delayed flowering[20−23].

      Despite their lack of direct DNA binding capacity, DELLAs exert control over gene function by physically engaging with specific partner proteins. For example, in Arabidopsis stem tissue, TCP transcription factors interact with promoters and activate core cell-cycle genes to foster stem growth. In contrast, DELLAs directly interfere with the DNA binding activity and functions of these TCPs, ultimately suppressing stem growth and elongation[25]. In leaves, CONSTANS (CO), a key factor of flowering under long-day conditions, requires association with nuclear factor NF-YB2 to activate FLOWERING LOCUS T (FT). However, DELLAs disrupt this association, resulting in the delay of flowering[26]. In shoot apices, DELLAs have been shown to upregulate KRP3, a gene coding for a cell cycle inhibitor that curbs meristem size and inflorescence development[27]. Evidently, DELLAs enforce growth restraint across various tissues by reconfiguring distinct regulatory pathways.

      A crucial inquiry revolves around the mechanism by which VvDELLA1 orchestrates and advances flowering, as well as the specific genes it targets in the grapevine. In Arabidopsis, the transition from vegetative meristem to inflorescence, followed by floral formation and development, is underpinned by an intricate network of genes. Remarkably, these genes demonstrate functional conservation across annuals, perennials, and even in woody plants like grapevine[28−38]. Plausibly, VvDELLA1 could potentially exert its influence on some of these orthologs or others that assume novel roles in shaping the developmental fate of anlagens in the grapevine. To elucidate this intricate regulation, we compared the transcriptome profiles of shoot tissues from four V. vinifera grape cultivars of Pixie, Dena, Gina and Tina containing the same gain-of-function mutated VvDELLA1 and the wild-type grape Pinot Meunier from which the gain-of-function VvDELLA1 (Vvgai1) was originally discovered[15]. Our investigation uncovered that the gain-of-function mutation in VvDELLA1 caused extensive mis-regulation of multiple genes, resulting in a collective transformation of the transcriptomic landscapes and the regulatory frameworks in the mutant vines. We identified at least two orthologs of flower regulators, VvAP1 and VvTFL1a, substantially downregulated due to the influence of the mutated VvDELLA1. This downregulation is closely linked to the significant conversion of vegetative anlagens into inflorescences. Our study examined potential interactions between identified gene candidates and VvDELLA1, as well as the plausible mechanisms underlying the regulation of flowering in grapevine.

    Material and methods

      Plant materials

    • Five V. vinifera cultivars were used in this study. Pinot Meunier is the cultivar from which the original L1 dwarf mutant was discovered. Pixie is a GA-insensitive dwarf mutant recovered via tissue regeneration from L1 layer cells of Pinot Meunier[39] and is phenotypically and genotypically identical to the L1 dwarf mutant as previously reported[15]. Pixie shows a monopodial growth, producing few, if any, lateral branches from the axillary buds (Fig. 1) and has a precocious flowering habit, producing inflorescences and bunches starting in the first year of its growth, and even in the younger/upper portion of its main branch (Fig. 1). Pixie is a hermaphrodite and was used as a pollen donor to cross with two grapevine rootstock cultivars of 187G and Freedom. Three dwarf mutant grape cultivars were selected from the F1 progenies. Two of them, named as Dena and Gina, were derived from the cross of Pixie with 187G and the third one, named as Tia, was from the cross of Pixie with Freedom (Cousins, unpublished). Dena, Gina, and Tia share the same dwarf and flowering phenotypes with their paternal parent Pixie.

      Figure 1. 

      Pixie shoot trait characteristics. (a) Pixie shows a monopodial growth, producing few, if any, lateral branches from the axillary buds. (b) Pixie has a precocious flowering habit, producing inflorescences and bunches starting in the first year of its growth, and even in the younger/upper portion of its main branch.

      All GA-insensitive dwarf mutant materials were grown in pots at the same time, and then maintained through a hydroponics system under greenhouse conditions for at least two years at the time of sampling. Nine shoots, each with 1−3 cm portion of the shoot tips that include young and unfolded leaves, were obtained from nine separately potted vines, and pooled in groups of three to make three biological replicates per genetic material. At the time of sampling, Pixie and its derived cultivars were in stages of reproductive growth, bearing inflorescences and fruit bunches along their singular main canes, but the wild-type control Pinot Meunier, the cultivar whose L1 meristem Pixie was developed from, remained in the juvenile phase. Shoot tissues of the control were likewise pooled to obtain three biological replicates. All collected/pooled tissues were taken fresh, flash frozen and stored at −80°C until further processing.

      RNA-Seq library preparation and sequence reads processing

    • RNA extraction and RNA-Seq library preparation were performed as previously described[40]. RNA-Seq libraries were multiplexed for 100-bp paired-end sequencing using Illumina HiSeq 2000 at the Cornell University Biotechnology Resource Center, Ithaca, NY, USA. The RNA-seq library read qualities were assessed using FASTQC ver 0.11.9[41]. Removal of sequence adapters was done using Trimmomatic ver 0.32 (Illumina, San Diego, CA, USA). The artifact-free sequences were then individually aligned to the 12X ver 2 Vitis reference genome sequences[42] using STAR aligner ver 2.7.3[43] following a paired-end alignment protocol, with default parameters for paired-end data, which includes a standard mismatch allowance and considers canonical junctions for splice-aware alignment, as well as with an auto generation of gene counts needed for differential expression analyses. Gene expression quantification was performed using HTSeq ver 0.11.1[44] to count reads aligned to genes.

      Expression analyses

    • Differential expression analyses were performed using edgeR package in R[45]. After an initial assessment, the significance threshold was set at a false discovery rate (FDR) of at least 0.05 (p < 0.001) to include all DEGs of hormone and flowering genes. Gene Ontology (GO) analysis was done using AgriGO ver 2[46], with a few modifications of the accompanying R script for visualization. Motif scan was facilitated using PlantTFDB[47]. All other visualizations such as plot and bar graph were done in R and MS excel.

      Hormone and flowering genes curation

    • We evaluated the expression profiles of hormone and flowering genes. Hormone genes were retrieved from RIKEN Plant Hormone Research Network (http://hormones.psc.riken.jp/). Flowering regulator genes and pathway were retrieved from flowering interactive database (FLOR-ID; www.phytosystems.ulg.ac.be/florid/). Grapevine homologues to the retrieved Arabidopsis genes were identified and verified from the annotation in the grape genome database using their BLAST feature[48].

    Results

      Differentially expressed genes (DEGs)

    • To identify the genes that were subjected to differential regulation by the gain-of-function VvDELLA1 in the L1 dwarf mutants, we collected and conducted RNAseq profiling of shoot apices from the four mutant grape cultivars. These collected shoot apices encompass a range of structures, including the shoot apical meristem (SAM) responsible for continuous shoot growth, axially positioned primordial anlagen directed towards inflorescences or tendrils, and leaf primordia which develop into leaves. At the time of sampling, these genetic materials had been thriving in the hydroponic-fed pots for a minimum of two years, featuring both inflorescences and berries in the woody basal branches, as well as in the upper sections (Fig. 1). Simultaneously, we collected shoot apices from Pinot Meunier vines, the wild-type control (WT). WT remained in its juvenile stage at the time of sampling, indicated by their anlagens only processing into tendrils.

      We conducted pairwise comparisons of Pixie, Dena, Gina, and Tia with WT, respectively. The numbers of differentially expressed genes (DEGs) at FDR ≤ 0.05 were 4,726 for Pixie, 2,744 for Dena, 2,731 for Gina and 3,210 for Tia. Our further analysis revealed that 723 DEGs were shared by at least three of the four mutants and 317 DEGs were shared by all four. Because the four grape mutants have very diverse genetic background, DEGs shown consistently across three or four of the mutants were most likely real. Among the 723 DEGs, 373 were up-regulated while 350 were down-regulated (Fig. 2; Table 1). As depicted in Fig. 3, the read abundance of these 723 DEGs spans a spectrum ranging from as low as 0.5 counts per million reads (CPM) to approximately 2,500 CPM. Remarkably, the fluctuations in expression change between the mutant and WT are considerably diverse, varying from a mere 1-fold to 550-fold difference. Notably, in line with many gene expression profiles, approximately 50% of the DEGs manifested low to moderate expression levels, usually within the range of 1−32 CPM. Within this segment, the most substantial changes in responses were observed, reaching up to a 500-fold alteration. It is intriguing to observe that the up-regulated DEGs tended to exhibit slightly more pronounced response changes, particularly those expressed at moderate to abundant levels which could soar up to 250 folds. In contrast, the changes for down-regulated genes sharing similar expression abundance fell between 30 to 60 folds. For the DEGs that were highly abundant (> 100 CPM), the alteration in their expression was relatively conservative, at around 2 folds. Genes involved in either hormone production and signal transduction, or flower formation, development, and flower regulation exhibited relatively low levels of expression or abundance. This observation underscores their vital functional significance as even minor alterations can trigger substantial physiological, metabolic, developmental, or phenotypic changes.

      Figure 2. 

      A Venna diagram showing the overlaps of DEGs in 'Pixie', 'Dena', 'Gina' and 'Tia' each compared to the WT 'Pinot Meunier'. FDR ≤ 0.05.

      Table 1.  Numbers of DEGs that were of consistent responses in the shoots of four Pixie mutant background.

      Expression changeNo. of DEGs1
      Up-regulated373
      Down-regulated350
      Total723
      1 ≥ 1.5-fold change, FDR ≤ 0.05 in at least three of the four mutants.

      Figure 3. 

      Expression profiles of 723 DEGs that were consistently up-regulated or down-regulated in terms of average fold changes vs average expression levels.

      Gene Ontology analysis of the shared DEGs

    • The GO enrichment analyses of the 373 up-regulated DEGs showed that most of these genes were linked to fundamental cellular activities, including responses to abiotic stimulus and regulation of cell size (Table 2 & Fig. 4). Notably, the up-regulated DEGs with substantial fold-changes played pivotal roles in biosynthesis processes, encompassing cellulose synthase, 3-ketoacyl-CoA synthase, trehalose-phosphatase/synthase, and Deoxyxylulose- 5-phosphate synthase, among others. Additionally, several significant gene families related to hormones and regulation, such as GRAS, MYB, AUX/IAA and ethylene, as well as various heat-shock proteins, 30S ribosomal, and response regulators of cytokinin, auxin response factors, and several homologs within the DOF, ERF and TCP transcription factor families were observed (data not shown). Conversely, among the 350 down-regulated DEGs, a considerable portion were also associated with fundamental cellular processes (Fig. 4). This category encompassed genes like protein kinases, along with an abundance of defense response-related genes such as lacasse and LRR-bearing genes. The down-regulated genes with notable fold changes displayed an enrichment of GO terms primarily associated with reproduction, transport, and localization processes (Table 2). These terms are exemplified by numerous carrier genes responsible for cation, potassium, auxin, and mate effluxes, as well as genes linked to floral development, including sucrose and peptide transporters. Noteworthy addition to this list were genes associated with meristem development: JAR1, a JA signaling gene; PRR7, a major gene in the temperature-sensitive circadian pathway; and KNAT1, a significant homeobox gene governing meristem cell fate determination. Evidently, these DEGs play a substantial role in influencing various pathways, culminating in a remodeling of the overall regulatory landscape, and ultimately giving rise to mutated phenotypes within the L1 mutants.

      Table 2.  Enriched GO terms among the 373 up-regulated and 350 down-regulated DEGs observed in the mutants.

      GO termsNumber
      UP-regulated DEGs
      Response to abiotic stimulus27
      Regulation of cell size3
      Down-regulated DEGs
      Anatomical structure development16
      Reproduction11
      Response to stimulus36
      Biological regulation46
      Transport44
      Establishment of localization44
      Localization44
      Cellular metabolic process112

      Figure 4. 

      GO analysis of the 373 up-regulated and 350 down-regulated DEGs observed in the mutants.

      Expression of key GA pathway genes

    • Considering that GA represses the shift from vegetative growth to inflorescence in grapevine[13], investigating the expression patterns of genes linked to GA production and signal transduction in the L1 dwarf mutants becomes pertinent to discern whether these genes undergo feedback regulation by VvDELLA1. Among the GA signaling homologs, GID1a exhibited the most remarkable upregulation in shoot apices, with its transcript surpassing others by 100−400 folds (Fig. 5a). This suggests GID1a plays an important role in shoot apices. Nonetheless, it was not differentially expressed between WT and any of the four mutant cultivars. In terms of the two biosynthesis GA families, GA5 (GA20ox with about eight members) and GA4 (GA3ox with about three members), vital in the final stages, differential regulation was observed. Only GA20ox5 displayed consistent upregulation across all four mutants, albeit insignificantly. The remaining maintained consistent expression between WT and mutants (Fig. 5a). Similarly, VvDELLA2, one of the three DELLAs, exhibited upregulation in all mutants, yet insignificantly. Conversely, in the GA deactivation GA2ox gene family, both GA2ox1 genes and GA2ox8, particularly the latter, showed significant (FDR ≤ 0.05) downregulation across all mutants (Fig. 5b). This highlights the substantial impact of GA2ox8 downregulation, indicating that VvDELLA1 potentially targets and negatively regulates GA deactivation GA2ox family (five members), subsequently influencing GA accumulation.

      Figure 5. 

      Notable expression of GA pathway genes in the L1 dwarf mutants. (a) Normalized transcript levels (counts per million, CPM) derived from aligned reads from three biological replicates for WT and dwarf mutants of genes involved in GA biosynthesis, including GA5 (GA20ox) with eight members and GA4 (GA3ox) with three members, and GA signal transduction, featuring GID and DELLA homologs. (b) Relative expression changes of GA deactivation genes, log2 fold change scale as calculated using edgeR at significance threshold set at FDR ≤ 0.05. The graph shows the average log2 fold change for three biological replicates between WT and the dwarf mutants. *: statistical significance at p ≤ 0.05.

      VvAP1 and VvTFL1a were substantially down-regulated in the L1 dwarf mutants

    • While a myriad of genes spanning biochemical, physiological, metabolic, and regulatory pathways exhibited differential regulation in the mutants (Fig. 2; Tables 1, 2), the direct connections between these regulations and the heightened flowering phenotype in the L1 dwarf mutants remain elusive. This ambiguity arises likely due to the presence of multiple groups of meristem or primordia (e.g., SAM, anlagens, leaf primordia, and others) in the shoot tips utilized for this study, thereby complicating the analysis. Hence, we directed our attention toward genes pertinent to flowering regulation and hormone metabolism with a focus on signal transduction. Among a pool of 37 potential candidates examined, the majority of them maintained consistent expression levels between WT and the four mutants (Table 3). Only a handful of flower-positive regulators, including VvLFY, and the orthologs of FLOWERING LOCUS T (VvFT) and LATE MERISTEM IDENTITY1 (VvLMI1), were consistently upregulated in the mutants. Their transcript abundance increased by at least 2 folds on average compared to that in WT (Table 3), aligning well with their established positive flower-regulatory roles in Arabidopsis and other plants. Although these up-regulations did not reach statistical significance, even subtle changes in their expression could potentially exert significant influence on regulatory cascades and flower phenotypes. Likewise, the orthologs of Type-B ARABIDOPSIS RESPONSE REGULATOR1 (VvARR1), VvARR2b, and VvARR12, involved in cytokinin signal transduction, exhibited upregulation. This mirrors cytokinin's role in promoting the tendril-to-inflorescence transition[9−12]. As expected, the flower repressor VvTFL1a experienced a substantial and statistically significant downregulation of nearly 3 folds (p < 0.05), which implies its vulnerability to VvDELLA1 regulation. Interestingly, VvAP1, whose ortholog acts as both a floral integrator and a regulator of floral meristem identity, exhibited a significant downregulation of at least 3 folds in the L1 dwarf mutants (p < 0.05, Table 3). VvAP1 and VvTFL1a are the only two function-opposite floral regulators that were found significantly regulated in the L1 dwarf mutants, suggesting their functional importance (Table 3). Interestingly, they both were co-downregulated instead of being regulated in opposite directions. Additionally, the orthologs of another flowering integrator, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (VvSOC1), showed moderate downregulation across all three copies: VvSOC1a, VvSOC1b and VvSOC1c. This suggests that the transformation of a positive flower regulator into a flowering-repressive factor may not be limited to VvAP1, further indicating the intricate regulatory complexity underpinning unique floral development in grapevine.

      Table 3.  Differential regulation of key shoot and flower regulator genes with qRT-PCR rating for selected genes.

      Gene namePathwayGrapevine gene
      ID ver 2      		Grapevine gene
      ID ver 3      		Arabidopsis gene IDAverage folds of changes between mutants and WTAverage expression across all libraries
      VvFTMeristem identityVIT_00s0203g00080−AT1G654802.710.26 ± 0.18
      VvTFL1VIT_06s0080g00290−AT2G27550(−2.81) *1.38 ± 0.88
      VvLFYVIT_08s0007g04200−AT5G618502.2218.18 ± 8.99
      VvTFL1BFT gene familyVIT_08s0007g03450−AT5G038401.771.05 ± 0.54
      VvTFL1CVIT_16s0100g00700−1.160.1 ± 0.18
      VvMFTVIT_17s0000g02630−AT1G18100(−2.46)0.08 ± 0.06
      VvAP1VIT_01s0011g00100−AT1G69120(−3.03) *5.9 ± 4.45
      VvCALaVIT_01s0010g03890Vitvi01g01673AT1G26310(−1.47)42.91 ± 18.08
      VvCALbVIT_17s0000g04990Vitvi17g004701.093.2 ± 1.23
      VvFULVIT_14s0083g01030Vitvi14g01341AT5G60910(−1.56)6.73 ± 3.53
      VvLMI1VIT_08s0007g04200−AT5G037902.2218.18 ± 8.99
      VvWUSVIT_04s0023g03310−AT2G179503.20.17 ± 0.19
      VvFDaVernalizationVIT_00s0349g00050−AT4G359002.452.89 ± 1.69
      VvFDbVIT_18s0001g14890Vitvi18g01165(−1.13)13.85 ± 1.95
      VvFLCVIT_15s0048g01270Vitvi15g00776AT5G101401.143.16 ± 3.44
      VvAGL24Agamous / MADS MIKC gene familyVIT_18s0001g07460Vitvi18g00517AT4G245401.3438.16 ± 10.25
      VvSVPaVIT_00s0313g00070Vitvi07g01441AT2G22540(−1.01)32.78 ± 8.94
      VvSVPbVIT_03s0167g00070−(−1.47)28.24 ± 11
      VvSVPcVIT_15s0107g00120Vitvi15g002251.2518.23 ± 4.9
      VvSVPdVIT_18s0001g07460Vitvi18g005171.3438.16 ± 10.25
      VvSOC1.1VIT_15s0048g01250−AT2G45660(−1.38)32.46 ± 9.71
      VvSOC1.2VIT_16s0022g02380−AT2G45660(−1.51)8.7 ± 2.8
      VvSOC1.3VIT_15s0048g01240−(−1.67)74.18 ± 26.71
      VvSPL3aSPL/ Ageing pathwayVIT_04s0210g00170Vitvi04g01556AT2G33810(−1.03)60.11 ± 53.82
      VvSPL3bVIT_10s0003g00050Vitvi10g00481(−1.14)30.95 ± 7.85
      VvSPL9VIT_08s0007g06270Vitvi08g01720AT2G422001.01136.75 ± 49.19
      VvSPL4VIT_12s0028g03350Vitvi12g00280(−1.11)74.42 ± 31.95
      VvSPL13VIT_01s0010g03910Vitvi01g01678(−1.54)48.51 ± 15.96
      Vvlog5CytokininVIT_06s0004g02680−(−1.73)22.17 ± 17.55
      VvRRVIT_05s0077g01480−1.5383.62 ± 20.74
      VvARR12VIT_11s0206g00060−1.2621.69 ± 4.58
      VvARR11VIT_01s0010g02230−1.412.09 ± 1.71
      VvARR2VIT_02s0012g00570−(−1.13)152.22 ± 31.65
      VvARR2bVIT_01s0011g05830−1.4169.94 ± 13.7
      VvARR12VIT_04s0008g05900−1.3723.28 ± 6.45
      VvyabbyVIT_15s0048g00550−1.39186.31 ± 79.49
      * Significant at p ≤ 0.05.
    Discussion

    • Grapevine has evolved a unique flower developmental programming, as characterized by specialized but versatile anlagen primordia that can give rise to tendrils in current shoots or inflorescences when emerging from latent buds. However, the fate of these anlagen, whether vegetative or reproductive, is susceptible to hormonal fluctuations influenced by vine age and seasonal changes[7,8,37]. This intricate regulation, while complex, is completely disrupted in the L1 dwarf mutants due to a gain-of-function mutation in VvDELLA1. This mutation leads to a pronounced de-repression of the transition from anlagens to inflorescence, a phenotype indicating the positive role of the mutated VvDELLA1 in promoting this transition[15]. Strikingly, this contradicts the repressive functions of its counterparts in annual plants[20−23]. The specific mechanism by which VvDELLA1 diverges and facilitates anlagen-to-inflorescence development in grapevine remains unclear. This study delves into transcriptome profiles of both WT and four L1 dwarf mutants to shed light on this phenomenon. Our analyses underscores VvDELLA1 as a central regulatory node, and its dominant mutation leads to the mis-regulation of several hundred genes. This comprehensive shift significantly alters the transcriptome and regulatory landscapes in the L1 dwarf mutants (Fig. 2; Tables 1 & 2).

      Although various genes associated with biological processes, metabolism pathways, and regulatory circuits exhibit mis-regulation, their direct roles in governing anlagen-to-inflorescence development remain elusive. This is further complicated by the presence of multiple meristematic cells and tissues (e.g. SAMs, leaf primordia, and anlagens) in the shoot apices used for RNA-seq analysis in this study. Through a meticulous investigation focused on genes involved in the regulation of vegetative-to-floral meristem transition, floral meristem identity, and cytokinin signal transduction, we have identified VvTFL1a and VvAP1 as potential pivotal flower regulators. These findings suggest potential regulatory modules orchestrating the developmental trajectory of anlagens in grapevine.

      The mis-regulation of flower-regulators and cytokinin signal genes in the L1 dwarf vines may be responsible for the remarkable augmentation of tendrils transitioning into inflorescences. At least seven genes, including VvLMI1, VvFT and VvLFY, which are known to positively regulate flowering in Arabidopsis, and VvARR1, VvARR2b and VvARR12, which are associated with cytokinin signaling, showed moderate upregulation in the L1 mutants (Table 3). These genes are functionally conserved across plant species, including grapevine[29], and cytokinin has been documented to induce the tendril-to-inflorescence transition in grapevine[9−11]. Thus, even a slight increase in expression of these genes could have a profound regulatory impact and lead to a noteworthy phenotypic response. In contrast, VvTFL1a, a known flower repressor, was one of two flower regulators that underwent significant downregulation in the L1 dwarf mutants (Table 3). This observation is consistent with the fact that TFL1 in apple has been demonstrated to repress the juvenile-to-adult transition and flowering[49], and ectopic expression of one of the three VvTFL1s in Arabidopsis results in delayed flowering[50]. Consequently, VvTFL1a likely assumes a similar repressive role in impeding the anlagen-to-inflorescence development or promoting the anlagen-to-tendril transition. This notion is further supported by its specific expression during the initial stages of tendril development[29].

      An unexpected revelation emerges as VvAP1, akin to VvTFL1a, showed significant downregulation in the L1 dwarf mutants, a departure from the roles its counterparts play in annual plants. In a parallel manner, the three VvSOC1s genes were also moderately downregulated in these mutants. In model species such as Arabidopsis, both AP1 and SOC1 collaborate with FT and LFY to consolidate signals arising from photoperiod fluctuations, temperature changes, vernalization, and hormonal balance. This concerted effort aims to initiate the transition from vegetative meristem to inflorescence meristem transition and the subsequent formation of floral meristems. In this intricate process, FT is initially synthesized in leaves and subsequently migrates to the shoot apex, where it engages with FLOWERING LOCUS D (FD). This interaction subsequently triggers the upregulation of AP1, CAULIFLOWER (CAL), and FRUITFUL (FUL), collectively driving the transformation of the shoot apical meristem into the inflorescence meristem (IM)[28,30,33,34,38]. IM sustains its indeterminate inflorescence growth and eventually gives rise to flower meristem. The dynamics of these two developmental events are delicately managed through an intricate interplay involving TFL1, LFY and AP1. At the heart of IM, TFL1 is expressed and migrates from the core to the outer layer, where it exerts transcriptional repression over LFY and AP1. This repression is pivotal in preserving indeterminate growth[31,32]. Simultaneously, AP1 and LFY proteins generated in floral meristem suppress the transcription of TFL1, ensuring the progression of floral formation and development[35,36]. This reciprocal repression elegantly synchronizes the two spatiotemporally intertwined developmental events in Arabidopsis. However, co-downregulation of VvAP1 with VvTFL1a in the L1 dwarf mutant apparently seems to challenge this reciprocal repression relationship and the flower-positive regulatory role attributed to AP1 in Arabidopsis.

      The divergent regulation of VvAP1 and VvLFY in the L1 dwarf mutants point to distinct functional roles, where the former fosters the transition of anlagen to tendrils, while the latter enhances anlagen's development into inflorescences. This interpretation is further supported by their specific expression patterns within tissues[51]. In grapevine, tendrils and inflorescences represent vegetative and reproductive growth states, respectively, yet they are homologous structures/organs as indicated by their reciprocal homeotic transformations, shared meristematic origin, co-existence in intermediate structure, and the activation of floral meristem regulator genes VvFT, VvLFY, VvAP1, and VvFUL [7,9,51−53]. This evidence strongly suggests that tendril is, in essence, a modified inflorescence lacking floral meristems. Consequently, the primary distinction between these tissues lies in their capacity to generate floral meristems, a process primarily regulated by AP1 and LFY in Arabidopsis[35,36]. As anticipated, the upregulation or downregulation of VvAP1 or VvLFY, or both, exerts direct control over the formation of floral meristems and developmental fate of anlagens.

      Supporting this prediction, both VvLFY and VvAP1 are actively transcribed in inflorescences across five Vitaceae species examined[51]. However, VvAP1's expression is only limited to tendrils[51]. These unforeseen tissue-specific expression patterns challenge the notion that both VvAP1 and VvLFY play, like their Arabidopsis counterparts, similar roles in grapevine. Instead, they suggest that these two genes have functionally diverged and play contrasting roles in regulating floral meristems in tendrils and inflorescences: VvAP1 likely promotes and maintains tendrils by inhibiting floral meristem formation, while VvLFY enhances inflorescence development by promoting the floral meristem. However, the relative balance or abundance of these factors ultimately governs whether tendrils or inflorescences are formed. This is reinforced by the observation that the extensive conversion of the tendril-bound anlagens into inflorescences in the L1 dwarf mutant is correlated with the downregulation of VvAP1 (Table 3). Consequently, VvAP1 seems to have adopted a novel but negative regulatory role in relation to floral meristems. Conceivably, genes coregulated with VvAP1 (VvSOC1a, VvSOC1b and VvSOC1c) or with VvLFY (VvLMI1, VvFT) may functionally interact with VvAP1 and VvLFY, respectively. Taken together, our findings, combined with previously documented tissue-specific expression data, uncover a novel role for VvAP1 and elucidate a potential mechanism underlying the regulation of floral meristems, tendrils, and inflorescence development in grapevine, as illustrated in Fig. 6.

      Figure 6. 

      A schematic illustration of the possible opposing roles of VvAP1 and VvLFY in regulating the developmental fate of anlagens in grapevine. (a) At the anlagen-to-inflorescence transition, the lateral primordial meristem gives rise to the latent bud in the leaf axil, which contains the shoot apical meristem (SAM), leaf primordium, and uncommitted anlagen. Expression or upregulation of VvFT, VvAP1, and VvLFY is observed in this stage. The anlagen is then differentiated into an inflorescence primordium in late seasons and eventually develops into a mature inflorescence in the following spring. (b) The anlagen-to- tendril developmental course is associated with increased expression of VvAP1 and VvTFL1a. The shoot apex produces lateral anlagens with the expression or activation of VvAP1, VvLFY, and VvTFL1a. These anlagens are then destined to become tendril primordia, followed by the development of tendrils in current growing shoots. The ratio between VvLFY and VvAP1 likely controls the developmental route of the anlagens to either inflorescences or tendrils. It is noted that anlagens at any stage or derived primordia are sensitive to hormone regulation, with cytokine (CK) promoting inflorescences and GA favoring tendrils. The red arrows indicate the anlagen in either shoot apex or latent bud, or inflorescence primordium in the latent bud. Up or down arrows indicate the upregulation and downregulation, respectively.

      The noteworthy observation that VvTFL1a and VvAP1 among flower regulator genes were significantly modulated in the L1 dwarf mutant implies their central roles in grapevine regulation. However, their simultaneous downregulation challenges the conventional view, indicating that they actually exert negative control over flowering, which appears to contradict their reciprocal repression observed in their Arabidopsis orthologs[35,36]. Plausibly, internal physiological cues such as vine age, growth state, and hormone equilibrium, or external signals including temperature, photoperiod, and chilling, could directly or indirectly target VvTFL1a, VvAP1, or both, possibly through the intermediary of the VvDELLA1 factor. This orchestration determines when and where anlagen evolve into tendrils or inflorescences. Given that GA-mediated degradation of the VvDELLA1 factor results in the de-repression of both VvTFL1 and VvAP1, it becomes evident why GAs repress flowering in grapevine. The fact that GA2ox1 and GA2ox8, constituents of the GA2ox family responsible for GA deactivation, experienced substantial downregulation in the L1 mutants (Fig. 5b), suggests that VvDELLA1 is a negative regulator of these two genes. This regulation likely leads to an accumulation of more GAs, which, in turn, triggers the degradation of DELLA1. This intricate regulatory interplay among GA2ox1 and GA2ox8, GAs and the VvDELLA1 could perpetually maintains VvTFL1a and VvAP1 in repression, thus steering the developmental trajectory of anlagen toward tendril formation in growing shoots and juvenile vines.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and supervision: Zhong GY; data collection: Arro J; analysis and interpretation of results: Arro J, Liu Z, Zhong GY; RNAseq library preparation: Yang Y; development of the genetic materials: Cousins P; manuscript revision and discussion: Song G; draft manuscript preparation: Arro J, Liu Z; finalization of the manuscript: Liu Z, Zhong GY. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data generated or analyzed during this study are included in this published article.

      Acknowledgments

      • We wish to thank Debra Johnston of USDA-ARS Grape Genetics Research Unit for providing her assistance in maintaining the L1 DELLA mutant vines in hydroponic tanks in the greenhouse. Jie Arro is a participant of the ORISE-ORAU Education and Training Program.

      Conflict of interest

      • The authors declare that they have no conflict of interest. Guo-qing Song is the Editorial Board member of Fruit Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (3) References (53)
  • About this article
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    Arro J, Yang Y, Song GQ, Cousins P, Liu Z, et al. 2024. Transcriptome analysis unveils a potential novel role of VvAP1 in regulating the developmental fate of primordia in grapevine. Fruit Research 4: e011 doi: 10.48130/frures-0024-0004
    Arro J, Yang Y, Song GQ, Cousins P, Liu Z, et al. 2024. Transcriptome analysis unveils a potential novel role of VvAP1 in regulating the developmental fate of primordia in grapevine. Fruit Research 4: e011 doi: 10.48130/frures-0024-0004
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  • Introduction
  • Material and methods

  • Plant materials

  • RNA-Seq library preparation and sequence reads processing

  • Expression analyses

  • Hormone and flowering genes curation

  • Results

  • Differentially expressed genes (DEGs)

  • Gene Ontology analysis of the shared DEGs

  • Expression of key GA pathway genes

  • VvAP1 and VvTFL1a were substantially down-regulated in the L1 dwarf mutants

  • Discussion
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
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