Application of grape VvCHX20 gene in regulating plant fruit growth and quality

Abstract

This invention relates to the field of genetic engineering technology, and in particular to the application of the grape VvCHX20 gene in regulating fruit growth and quality. The VvCHX20 gene, as a cation / hydrogen ion antitransporter located in the plasma membrane, promotes K⁺ accumulation, improves water status, and increases soluble solids content. Transgenic tomato experiments have demonstrated that overexpression of the VvCHX20 gene can increase fruit size, increase the number of locules and peel thickness, and enhance potassium-dependent water retention capacity.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the grape VvCHX20 gene in regulating plant fruit growth and quality. Background Technology

[0002] Gibberellin (GA3), a core member of the gibberellin family, is not only an important endogenous hormone regulating various physiological processes throughout the plant's life cycle, but also a key growth regulator widely used in agricultural production, playing a significant role in fruit trees and economic crops. Besides its classic roles in seed germination, stem elongation, and flowering induction, GA3 has a unique regulatory effect on fruit development, making it an indispensable regulatory tool in horticultural production. Utilizing GA3's inherent properties of relaxing cell walls to promote cell elongation and synergistically amplifying growth signals with auxin (IAA), GA3 treatment is often applied to table grape inflorescences or bunches at specific developmental stages in production. This effectively induces seedlessness, increases berry size, improves bunch compactness, and extends shelf life. Numerous studies on different grape varieties and cultivation environments consistently show that exogenous GA3 treatment significantly increases grape fruit size while improving quality indicators such as soluble solids, titratable acid, and anthocyanin accumulation. These beneficial effects are mainly achieved through regulating cell enlargement-related genes, activating primary and secondary metabolic pathways, and reshaping the hormonal balance within the fruit.

[0003] Fruit development and quality formation are closely related to water and ion transport processes, both of which are regulated by multiple signaling molecules and genetic pathways. For plant cells, especially fruit cells, after cell division ceases, vacuoles continue to expand under the influence of various factors such as osmotic potential, turgor pressure, and water potential, leading to a rapid increase in cell volume. Vacuole expansion depends on water absorption and ion transport, directly affecting cell turgor pressure and thus regulating fruit growth. Studies have shown that aquaporins (AQPs) play a crucial role in regulating plant water transport, and their expression at both the transcriptional and translational levels is finely regulated by external conditions. Furthermore, cation / hydrogen ion antitransporters (CHXs) are essential for maintaining ion homeostasis. The grape CHX gene family contains 19 members involved in cation balance and floral organ development regulation. However, the molecular mechanism by which GA3 regulates water and ion homeostasis during fruit development remains unclear.

[0004] MicroRNAs (miRNAs), as important post-transcriptional regulators, target and regulate mRNAs involved in various physiological processes such as nutrient transport and cell enlargement. In fruit crops, miRNAs participate in regulating multiple aspects of fruit development, including appearance, flavor, texture, and responses to environmental stress. For example, in grapes, the miR156 family and its target genes Vv-SPLs play important roles in berry development and ripening, while the miR172 family regulates berry development by targeting AP2 transcription factors in response to gibberellin signaling. Studies have also identified several GA-responsive miRNAs in grapes, involved in hormone signal transduction, glucose metabolism, and secondary metabolite synthesis. CHXs play a crucial role in promoting cation absorption and transport, thereby significantly regulating various physiological processes such as cell enlargement, volume control, osmotic regulation, pH homeostasis, membrane transport, protein processing, and cellular stress responses. In grapes, the functions of novel miRNAs and their target CHX genes in GA3-regulated fruit growth and ion / water transport remain largely unresolved. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of the grape VvCHX20 gene in regulating plant fruit growth and quality. Overexpression of the VvCHX20 gene can increase tomato fruit size, increase the number of locules and peel thickness, and enhance potassium-dependent water retention capacity.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of the grape VvCHX20 gene in regulating plant fruit growth and quality.

[0008] Preferred results show that overexpression of the grape VvCHX20 gene positively regulates fruit growth and quality in plants.

[0009] Preferably, the nucleotide sequence of the grape VvCHX20 gene is shown in SEQ ID No. 1.

[0010] Preferably, the plant fruit includes tomato fruit.

[0011] Preferably, overexpression of the grape VvCHX20 gene increases the single fruit weight and transverse diameter of the plant.

[0012] Preferably, overexpression of the grape VvCHX20 gene increases the free water content and decreases the bound water content in the plant fruit.

[0013] Preferably, overexpression of the grape VvCHX20 gene increases the soluble solids content of plant fruits.

[0014] Preferably, overexpression of the grape VvCHX20 gene promotes potassium accumulation in plant fruits.

[0015] Preferably, overexpression of the grape VvCHX20 gene increases the number of locules and the thickness of the fruit peel.

[0016] This invention also provides the application of transcription factors in enhancing the activity of the VvCHX20 promoter to positively regulate the transcription of the grape VvCHX20 gene, wherein the transcription factors include one or more of VvAAR3, VvPUB23, VvDPMS1 and VvNH18.

[0017] The beneficial effects of this invention are:

[0018] The VvCHX20 gene, acting as a cation / hydrogen ion antitransporter located in the plasma membrane, promotes K⁺ accumulation, improves water status, and increases soluble solids content. Transgenic tomato experiments have demonstrated that overexpression of the VvCHX20 gene increases fruit size, the number of locules, and peel thickness, and enhances potassium-dependent water retention. This study reveals a novel GA3–miRNA–transcription factor–transporter pathway that optimizes fruit ion and water homeostasis, providing new insights into the mechanisms by which hormones regulate fruit development and offering potential strategies for crop quality improvement. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 (a, b) Dynamic changes in single grape fruit weight at 0 h (2 h), 4, 7 d [early growth stage (stage I)], 22, 37 d [slow growth stage (stage II)], 52 d (color change stage) and 67 d [second swelling stage (stage III)] after gibberellin (GA3) treatment in 2018 and 2019; (c, d) Dynamic changes in total soluble solids (TSS) in grape fruit at 0 h (2 h), 1, 2, 3, 4, 5, 6, 7, 22, 37, 52, and 67 d after GA3 treatment in 2018 and 2019. Different lowercase letters indicate significant differences at the P ≤ 0.05 level, and error bars represent standard errors.

[0021] Figure 2 The dynamic changes of free water content (a, b), bound water content (c, d) and total water content (e, f) of grape berries after GA3 treatment at 0 h (2 h), 1, 2, 3, 4, 5, 6, 7, 22, 37, 52, and 67 days in 2018 and 2019 were shown. Different lowercase letters indicate significant differences at the P ≤ 0.05 level, and error bars represent standard errors.

[0022] Figure 3The dynamic changes in real-time water content (a, b), 24-hour saturated water potential (c, d), osmotic potential (e, f), and turgor pressure (g, h) of grape berries after GA3 treatment in 2018 and 2019 at 0 h (2 h), 1, 2, 3, 4, 5, 6, 7, 22, 37, 52, and 67 days are shown. Different lowercase letters indicate significant differences at the P ≤ 0.05 level, and error bars represent standard errors.

[0023] Figure 4 Co-expression network of miRNAs involved in water / ion transport and their target mRNAs (a); Expression patterns of miRNAs (b) and their target mRNAs (c) in grape berries treated with GA3.

[0024] Figure 5 (a) Phylogenetic analysis and conserved domain prediction of CHX protein sequences from different plants, GenBank accession numbers: AtCAX4 (AF409107), LtNHX1 (EU727217.1), ZjNHX1 (ABY19311), AtCHX23 (At1g05580.2), GsCCHX19.3 (KHN17780.1), AtCHX17 (NP_194101.1), AtCHX20 (AT3G53720.1), VvCHX20 (VIT_08s0007g00020); (b) DELLA protein structure; (c) Transmembrane structure analysis of VvCHX20 protein; (d) Tertiary structure of VvCHX20 protein; (e) Functional domains of VvCHX20 protein: cation / hydrogen ion exchange protein;

[0025] Figure 6 To increase tomato fruit size and regulate fruit water content and ion accumulation by overexpressing VvCHX20, the following phenotypic values ​​were compared: a. Phenotypic values ​​of VvCHX20 transgenic tomatoes and wild-type (WT) tomatoes 45 days after flowering (scale bar = 1 cm); b. Mature fruits of VvCHX20 transgenic tomatoes and wild-type tomatoes 45 days after flowering (scale bar = 1 cm). The following parameters were compared between wild-type and VvCHX20 overexpressing tomatoes 45 days after flowering: single fruit weight (c), fruit transverse diameter (d), fruit longitudinal diameter (e), free water content (f), bound water content (g), total water content (h), total soluble solids content (i), K⁺ content (j), Ca²⁺ content (k), Mg²⁺ content (l), and Na⁺ content (m). Different lowercase letters indicate significant differences at the P < 0.05 level.

[0026] Figure 7 Microscopic observation of cross-sections of VvCHX20 transgenic and wild-type tomato fruits 45 days after flowering. Left: Overall view of the cross-section; Right: Detailed view of each ventricle;

[0027] Figure 8 For screening upstream regulators of the VvCHX20 gene and verifying transcriptional activity, (a) ProVvCHX20 promoter self-activation detection, with the red box indicating the 3AT concentration determined in subsequent interaction verification; (b) Point-to-point interaction verification between the pHis2-ProVvCHX20 promoter and candidate regulators (VvPCK1, VvING1, VvILR3, VvRBRP, VvLR_Like, VvAAR3, VvCBSX1, VvPUB23, VvDPMS1, VvNH18), TLH: -Trp / -Leu / -His; (c) Construction of the pGreenII 0800-LUC vector for the ProVvCHX20 promoter and the pGreenII62-SK vector maps for interacting regulators (VvAAR3, VvPUB23, VvDPMS1, VvNH18); (d) Luciferase (LUC) imaging and LUC / REN activity of SK-VvPUB23 interacting with 0800-ProVvCHX20; d1: 62SK-empty vector + 0800-ProVvCHX20; d2: 62SK-VvPUB23 + 0800-empty vector; d3: 62SK-empty vector + 0800-empty vector; d4: 62SK-VvPUB23 + 0800-ProVvCHX20, (e–g) represent luciferase imaging and LUC / REN activity of 0800-ProVvCHX20 interacting with SK-VvDPMS1, SK-VvNH18, and SK-VvAAR3, respectively, with abbreviations having the same meaning as (d);

[0028] Figure 9 A schematic diagram illustrating how the GA3–novel_miR_130–VvCHX20 regulatory module promotes fruit growth and quality formation.

[0029] Figure 10 Subcellular localization of VvCHX20 in tobacco leaves (scale bar = 50 μm).

[0030] Figure 11 Obtaining VvCHX20 transgenic tomato plants: a. Schematic diagram of key steps in genetic identification (from left to right): VvCHX20 cloning, E. coli bacterial suspension detection, T0 generation transgenic line identification, T1 generation transgenic line identification; bi. Transgenic tomato culture process: tomato planting, leaf explant culture, adventitious bud differentiation, rooting subculture, T0 generation seed planting, obtaining T0 generation transgenic tomato plants, T1 generation seed planting, T1 generation seed transplanting. Detailed Implementation

[0031] This invention provides the application of the grape VvCHX20 gene in regulating plant fruit growth and quality.

[0032] In this invention, overexpression of the grape VvCHX20 gene is preferably used to positively regulate fruit growth and quality. In this invention, the nucleotide sequence of the grape VvCHX20 gene (accession number: VIT_08s0007g00020) is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2, as follows:

[0033] SEQ ID No. 1:

[0034]

[0035] SEQ ID No.2:

[0036] MAFNMTSIKTSSDGAWQGDNPLRFAFPLLIVQTTLVLSISRFLAFLLKPFHQPRVIAEIIGGILLGPSALGRNKDFLHLVFPPWSTPILESVASVGLLFFLFLVGLELDLSSIRRSGKRAFGIALAGISLPFIFGVGITFLLRKAVDGEDKVGFSQCILFIGVSLSITAFPVLARILAELKLLTTDVGQTAMAAAAFNDVAAWILLAPAVALAGNGGSHSSPLASIWILISGVAFVAFMLTIIRPAMNWVGRQCSRKHDSMDEAYICLTLAGVMLSGFTTDLIGIHAIFGGFVFGLTIPKGGEFAQRLIKRIEDFVTGLLLPLYFASSGLKTDVAKIQGVKAWGLLVLVISTACAGKVLGTFVVAMLCMTPVRESLVLGVLMNTKGLVELIVLNIGKEKKVLNDEVFAILVLMALFTTFMTTPAVMAIYKPIRRIATQAQPQIQRESNVAENSSQDKLRILACVHGPANVPSLISLIDSTCNANKSPLKLYVMHLMELTDRTSSILMVQRGRKNGFPFIKGFRRGELKDQVGAAFEPYAHFGRVTVRPTKAISALSTMHEDICHAAKKKRVGMIVLSFHKQWRGEGEEAVENVGHEWRGVNQRVLKNAPCPVGVLVDRGFGGVERRVCILFLGGPDDRYALKLGGSMAEHSAVRVTLVRLVEKGKIDSNSISSQGLAQDGCIDVCCASSTTPIYCGKEKELDEATVAEFRSRWEGSAKHVEKEVEATNVLEEVLLAIGRCREYELIVVGKGGFPPNMVAIAQLSDHQPEHAELGPIGDVLASSGRGITASVLVIQHHSLPHEHHQPLLMPIHDHMDDTITAEEPTV-。

[0037] In this invention, the plant fruit preferably includes tomato fruit. In this invention, overexpression of the grape VvCHX20 gene preferably increases the single fruit weight and transverse diameter of the plant fruit. In this invention, overexpression of the grape VvCHX20 gene preferably increases the free water content and decreases the bound water content of the plant fruit. In this invention, overexpression of the grape VvCHX20 gene preferably increases the soluble solids content of the plant fruit. In this invention, overexpression of the grape VvCHX20 gene preferably promotes potassium ion accumulation in the plant fruit. In this invention, overexpression of the grape VvCHX20 gene preferably increases the number of locules and the peel thickness of the plant fruit.

[0038] This invention also provides the application of transcription factors in enhancing the activity of the VvCHX20 promoter to positively regulate the transcription of the grape VvCHX20 gene, wherein the transcription factors include one or more of VvAAR3, VvPUB23, VvDPMS1 and VvNH18.

[0039] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] 1. Materials and Methods

[0042] 1.1 Experimental materials, growth conditions, GA3 treatment, and fruit sampling

[0043] This embodiment follows established methods and uses Red Globe grapevines with consistent growth conditions. When over 80% of the berries reached a diameter of 8-10 mm, GA3 treatment was applied. The entire bunch was immersed in a GA3 solution containing 2% (v / v) Tween-20 for 10 seconds. The treatment concentrations were 40 mg / L (T1) and 70 mg / L (T2), with a solution containing only 2% (v / v) Tween-20 serving as the control (CK). A randomized block design was used, treating 18 grapevines under standard field management. Immediately after treatment, the bunches were covered with white bags to reduce reagent volatilization and environmental impact. Sampling was conducted at the following time points after treatment: 0 h (approximately 2 h), 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h (Phase I, rapid expansion period); subsequently, sampling was conducted at 22 d, 37 d (Phase II, slow growth period), 52 d (color change period), and 67 d (Phase III, secondary expansion period). Six clusters of fruit were collected at each time point for each treatment, and 20 berries were sampled from the upper, middle, and lower parts of each biological replicate (mixed from two clusters). All sampling was conducted uniformly between 9:00 AM and 10:00 AM, and samples were flash-frozen in liquid nitrogen and stored at -80°C. Berries from the T2 treatment at 0 h (approximately 2 h) were used for RNA extraction and sequencing (3 biological replicates).

[0044] 1.2 Determination of single grape weight and soluble solids content

[0045] One hundred fruits randomly selected from each biological replicate were weighed individually using an analytical balance (accuracy ±0.001 g) to determine the weight of a single fruit. The soluble solids (TSS) content of the fruit homogenate juice was determined using an Abbe refractometer (Shanghai Optical Instrument Factory, model B624853WYA) at 20℃, and the results are expressed as °Brix.

[0046] 1.3 Determination of total water content, free water and bound water content of fruit

[0047] Total water content was determined by gravimetric method: Pretreated weighing bottles were dried at 105℃ to constant weight (difference between two consecutive weighings ≤ 0.002 g), cooled in a desiccator, and weighed; the weight of the empty bottle was recorded (W1). Fresh fruit samples were placed in weighing bottles and weighed; the total weight was recorded (W2). The samples were dried in a 105℃ oven (enzyme inactivation first) to constant weight, cooled in a desiccator, and weighed; the weight of the dried sample plus the weighing bottle was recorded (W3). Free water content was determined according to the Marincek method of Li et al. (2022): Whole fruit samples were weighed in sealed bottles (W5), quickly sliced, mixed with approximately 10 mL of 60–65% (w / w) sucrose solution, immediately sealed, and weighed again (W4). After incubation in the dark with constant temperature and shaking for 6 h, the final sucrose concentration (C) was determined using an Abbe refractometer. f (Initial concentration is Cᵢ). Total water content, free water content, and bound water content are calculated using the following formulas:

[0048] Total moisture content (%) = [(W2 - W3) / (W2 - W1)] × 100%

[0049] Free water content (%) = {[(W4 - W5) × (Cᵢ - C f ) / C f ] / (W5 - W1)}×100%, where (W4 - W5) is the net weight of the added sucrose solution, and (W5 - W1) is the fresh weight of the sample.

[0050] Bound water content (%) = Total water content (%) - Free water content (%)

[0051] 1.4 Determination of water potential, osmotic potential and turgor pressure of grape berries

[0052] Real-time water potential of the fruit was measured at 7:00 AM; for the 24-hour saturated water potential, the fruit bunches were first placed in distilled water in a self-sealing bag and soaked at 4°C for 24 hours before measurement. Following the methods of Nonami and Schulze (1989) and Tyree and Hammel (1972), a plant water potential pressure chamber (Zhejiang Top Cloud Agriculture, model TP-PW-II) was used to measure the two water potentials. Before the measurement, the fruit bunches were thinned to 10 fruits to fit the volume of the pressure chamber. Osmotic potential was calculated using the pressure-volume (PV) curve method, following the approach of Tyree and Hammel (1972): After 24 hours of saturation treatment, the ears of fruit were weighed using a 0.01 g / L balance (W1). The water potential (ψ1, standard: uniform water seepage) was measured using a pressure chamber. The seeped water was absorbed through dry filter paper, and the amount of water seeped (V1) was calculated based on the weight difference of the filter paper. After equilibration for 1 hour, the water potential was measured again (ψ2). This process was repeated until the ears wilted. The ears were then dried at 75°C for 8 hours, and the dry weight was measured (W2). A PV curve was fitted in Excel with the amount of water seeped as the x-axis and the corresponding water potential as the y-axis to calculate the osmotic potential (MPa). The turgor pressure was calculated using the method of Nonami and Schulze (1989), approximately the absolute difference between water potential and osmotic potential: Turgor pressure = |water potential - osmotic potential|.

[0053] 1.5 Analysis of mRNA expression of water / ion transport-related miRNA targets

[0054] The gene expression data used in this embodiment were obtained from previous whole transcriptome sequencing experiments. Using Red Globe grapes as the experimental material, the fruit bunches were immersed in GA3 solutions of different concentrations during the rapid fruit expansion period. Based on the above whole transcriptome sequencing data, preliminary sorting and screening were conducted, with gene function annotation information as a guide, focusing on screening miRNA target mRNAs related to water / ion transport. During the screening process, the functional descriptions of terms such as "water transport" (GO:0006833) and "ion transmembrane transport" (GO:0034220) in the gene ontology (GO) annotations were strictly followed. Simultaneously, information on gene families clearly reported in existing studies to be involved in plant water / ion transport was integrated to ensure that the screened target genes had clear functional relevance. The detailed information of the water / ion transport-related miRNA target mRNAs obtained is shown in Table 1. Heatmaps of the screened target genes were generated using TBtools software (version v1.098765), and their expression patterns under different GA3 concentrations were analyzed.

[0055] Table 1. Annotation information of miRNA target genes

[0056]

[0057] 1.6 Structural and phylogenetic characteristics of VvCHX20 protein

[0058] A phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 replicates. The software used was ClustalX, MEGA 7.0, and EvolView (https: / / www.evolgenius.info / ). DNAMAN software was used to align the sequence of VvCHX20 protein with other CHX family proteins from different plant species. TMHMM 2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) was used to predict the transmembrane structure of VvCHX20 protein, InterProScan (https: / / www.ebi.ac.uk / interpro / about / interproscan / ) was used to predict conserved domains, and SWISS-MODEL (https: / / swissmodel.expasy.org / ) was used to predict the tertiary structure of VvCHX20 protein.

[0059] 1.7 Subcellular localization of VvCHX20: Expression of green fluorescent protein (GFP) fusion protein in tobacco

[0060] The complete open reading frame (ORF) of VvCHX20 was amplified and inserted into the pCAMBIA2300-GFP vector. The pCAMBIA2300-VvCHX20-GFP plasmid was introduced into Agrobacterium GV3101 strain via electroporation, with pCAMBIA2300-GFP serving as a positive control. After centrifugation and resuspending the Agrobacterium culture, the resuspended solution was injected into the lower epidermal cells of tobacco leaves using a 1 mL syringe. After culturing in the dark for 6 h, the cells were placed under normal light conditions (22℃, 16 h light / 8 h dark) and the distribution of fluorescence signals in the tobacco epidermal cells was observed using a laser confocal microscope.

[0061] 1.8 Genetic transformation of tomatoes

[0062] The overexpression vector pCAMBIA2300-VvCHX20-GFP was introduced into Agrobacterium GV3101 strain. The tomato transformation process was as follows: Leaves of 7-day-old 'Micro-Tom' wild-type (WT) tissue culture seedlings were co-cultured with Agrobacterium containing pCAMBIA2300-VvCHX20-GFP at 28℃ for 6 min; transgenic plants were screened in selection medium containing appropriate concentrations of antibiotics, and the transgene integration of T0 and T1 generation plants was verified by PCR technology; fruit samples from wild-type and transgenic plants were collected 45 days after flowering.

[0063] 1.9 Determination of growth indicators and ion content in transgenic tomato fruits

[0064] The weight of a single fruit was determined by direct weighing, and the longitudinal and transverse diameters of the fruit were measured using vernier calipers. Following the method of Samuolienė et al. (2021) with slight modifications, a wet digestion method (H2SO4-H2O2 method) was used for ion content analysis: 0.5 g of dried fruit pulp powder was accurately weighed into a 150 mL Erlenmeyer flask, moistened with ultrapure water, and 5 mL of concentrated sulfuric acid was added. The mixture was allowed to stand for 12 h. The solution was then slowly heated on an electric furnace (heating was increased after the concentrated sulfuric acid emitted white fumes) until it turned dark brown. Then, 30% hydrogen peroxide was added dropwise (initially 10 drops, then gradually reduced to 5-10 drops), with constant shaking. The solution was then heated again to a gentle boil for 5-10 min. This process was repeated 3-5 times until the solution was colorless and transparent. The solution was then heated for another 5-10 min to remove residual hydrogen peroxide. After cooling, the solution was filtered and washed into a 50 mL volumetric flask and diluted to volume. The elemental contents (K, Ca, Mg, Na) were determined using an inductively coupled plasma optical emission spectrometer (ICP-OES, Spektro Genesis, Germany). The standard curve concentration range was 0.01-1.0 mg / L.

[0065] 1.10 Safranin-Fast Green staining for observation of fruit tissue structure

[0066] The fruit tissue structure was observed using the safranin-fast green staining method. The specific steps were as follows: First, the fruit was harvested and fixed in 4% paraformaldehyde for 24 h. Then, tissue samples were prepared according to the paraffin sectioning method: the samples were embedded in paraffin (Leica 39601006) at 56-58℃ and cut into 3 μm thick sections. The sections were stained with safranin-fast green staining solution (PH1852). The stained sections were observed and photographed using an upright microscope (Nikon CI-S), and the sections were scanned using a scanner (Hamamatsu NanoZoomer-SQ). The digital images were viewed and acquired using SlideViewer software.

[0067] 1.11 Yeast library screening and yeast one-hybrid (Y1H) experiment

[0068] Yeast library screening and Y1H experiments were performed according to the method of Cao et al. (2024). The brief steps are as follows: The promoter region (upstream -1 to -1500 bp) of VvCHX20 was cloned from the grape genome, inserted into the phis2 vector, and self-activation was detected; then, the Phis2 plasmid with the promoter linked and the PGADT7 library were co-transformed into Y187 yeast for library screening; the co-transformed yeast was plated on -Trp / -Leu medium, and DNA products were amplified and sequenced using positive clones as templates and PGADT7 as amplification primers; the obtained sequences were compared with the grape database on the NCBI website to identify the complete target gene sequence and predict its function (Table 2); the coding sequence of the target gene was cloned into the pGADT7 vector and Y1H verification experiments were performed.

[0069] Table 2 NCBI annotations for candidate regulators

[0070]

[0071] 1.12 Dual-luciferase reporter gene assay

[0072] Dual-luciferase reporter gene assays were performed following the method described by Wu et al. (2025). The VvCHX20 promoter was amplified and ligated into the pGreenII 0800-LUC vector, while candidate genes for point-to-point interaction verification were cloned into the empty vector pGreenII 62-SK. The recombinant vector was transformed into Agrobacterium GV3101 strain (containing the pSoup helper plasmid). The interaction between transcription factors and promoters was evaluated by the ratio of firefly luciferase (LUC) activity to Renilla luciferase (REN) activity. Luminescence detection was performed using a gel imaging system (VILBER LOURMAT, Fusion FX6 XT, France). The methods for measuring the activities of firefly luciferase (LUC) and Renilla luciferase (REN) were as reported by Cao et al. (2024). All primer sequences are shown in Table 3.

[0073] Table 3 Primers used in this embodiment

[0074]

[0075] 1.13 Statistical Analysis

[0076] The relevant data were statistically analyzed using SPSS 21.0 software ANOVA. The Duncan multiple comparison method was used to compare the differences in means, and the significance level was set at P<0.05.

[0077] 2 Results

[0078] 2.1 Effects of GA3 treatment on grape berry weight and soluble solids content

[0079] Exogenous GA3 treatment significantly increased grape berry fresh weight (FW) and total soluble solids (TSS). From 0 to 4 days post-treatment, there were no significant differences in berry fresh weight among treatments (including the control); significant differences began to appear from day 7, with the T2 treatment showing significantly higher berry fresh weight than the control and T1 treatments in the 2019 experiment. The accumulation rate of berry fresh weight accelerated significantly from days 22 to 37 and from 52 to 67, while the accumulation rate was slower from day 37 to 52. By day 67, the berry fresh weight distribution in both years was T2 > T1 > control, with significant differences among treatments.

[0080] GA3 treatment significantly affected the accumulation pattern of TSS. Although the initial TSS content varied slightly between years (e.g., the T2 treatment had the lowest initial content in 2018), fruits treated with GA3 (especially the T2 treatment) showed a significantly higher accumulation rate. During the critical early stage (0–7 days), the T2 treatment accumulated TSS the fastest, with a significantly higher accumulation rate than other treatments (e.g., 41.4% for T2 treatment, 28.6% for T1 treatment, and 20% for the control in 2018; 15.9% for T2 treatment, 13.4% for T1 treatment, and 10.1% for the control in 2019, 0–67 days), resulting in TSS content exceeding that of other treatments in the T2 treatment at 5–7 days. TSS accumulated steadily throughout development in all treatments, but the accumulation rate of GA3 treatments (T1 and T2) was significantly higher than the control, especially after 22 days (an average increase of 12.3%). Therefore, by day 67, the TSS content in both years of the experiment showed that T2 > T1 > control, and the differences between treatments were significant.

[0081] 2.2 Effects of GA3 treatment on grape fruit moisture parameters

[0082] Exogenous GA3 treatment significantly altered the water state of the fruit. Water composition analysis showed that GA3 treatments (T1, T2) rapidly and significantly increased free water content within 0–1 day, while decreasing bound water content, whereas the control showed no significant change. From 7 to 67 days, the free water content of all treatments increased significantly, with T2 consistently showing the highest free water content, significantly higher than T1 and the control. Conversely, bound water content decreased significantly during this period, with T2 and T1 treatments reaching their lowest bound water content, while the control showed the highest final bound water content. The consistent trend in the two-year trial results indicates that GA3 treatment (especially T2 concentration) effectively promotes free water accumulation and reduces the proportion of bound water, and this effect persists throughout the entire fruit development cycle. In the two-year trial, the total fruit water content generally increased from 0 to 7 days after treatment. The increase in total water content in GA3 treatments (T1, T2) within 1 day after treatment was significantly higher than that in the control. From 1 day onwards, the total water content in T2 treatment remained the highest, significantly higher than T1 and the control by 67 days. During the transition from the first exponential growth phase to the first stagnant phase (7–22 days), the total water content of all treatments decreased significantly, but the fruits treated with GA3 (T1, T2) still maintained a higher total water content. From the first stagnant phase to the second exponential growth phase (22–52 days), the total water content of all treatments decreased significantly further, and the differences between treatments were significant (T2>T1>control). In the later stage of the second exponential growth phase (52–67 days), the rate of decrease in total water content slowed down.

[0083] 2.3 Effects of GA3 treatment on grape berry water potential

[0084] Exogenous GA3 treatment significantly affected the water relationship of the fruit. Over the two-year trial, the real-time water potential (Ψ) of the fruit generally showed a downward trend across all treatments. The water potential of the GA3-treated fruit (T1, T2) showed a brief but significant increase within 0–1 day, while the control remained stable. From 7 to 67 days, the water potential of all treatments decreased significantly, with a plateau occurring between 37 and 52 days. By day 67, the water potential of the T2 treatment remained the highest, significantly higher than that of T1 and the control (T2>T1>Control).

[0085] 24-hour saturation water potential (Ψ_sat) and osmotic potential (Ψ_π) showed similar response patterns. Fruits treated with T1 and T2 showed a significant transient increase in both Ψ_sat and Ψ_π within 0–1 day, while the control showed no significant change. Subsequently, up to 7 days, Ψ_sat and Ψ_π gradually decreased. From 7 to 67 days, both Ψ_sat and Ψ_π decreased significantly in all treatments, and a plateau was observed between 37 and 52 days. Ψ_sat and Ψ_π remained at their highest levels throughout development in the T2 treatment, with significant differences among treatments at 67 days (T2 > T1 > control).

[0086] The variation patterns of fruit turgor pressure (Ψ_p) differed. Fruits treated with T1 and T2 showed a significant increase in turgor pressure from 0 to 7 days, reaching a peak around 7 days, significantly higher than the control; then it slowly decreased until 37 days; from 37 to 52 days, the turgor pressure of all treatments decreased sharply and significantly; thereafter (52 to 67 days), the turgor pressure of all treatments slowly recovered, with the turgor pressure of the T2 treatment generally significantly higher than that of T1 and the control.

[0087] 2.4 Regulatory network and expression patterns of water / ion transport-related miRNAs and mRNAs

[0088] The constructed regulatory network revealed that 11 target mRNAs involved in water / ion transport are posttranscriptionally regulated by 12 different miRNAs. These regulatory miRNAs include conserved families (such as vvi-miR394a-c, vvi-miR167c, vvi-miR172a-d, and vvi-miR396b) and novel miRNAs (novel_miR_130, novel_miR_55, novel_miR_142, novel_miR_64, novel_miR_128, novel_miR_13, and novel_miR_31).

[0089] Notably, GA3 treatment significantly downregulated the expression of novel_miR_130 in grape berries, while simultaneously upregulating the expression of its potential target mRNA—VvCHX20 (VIT_08s0007g00020), which encodes the cation / hydrogen ion antitransporter 20. This inverse expression pattern suggests a potential negative regulatory relationship between the two, meaning that novel_miR_130 may inhibit VvCHX20 expression under basal conditions, while GA3-mediated miRNA downregulation relieves this inhibition. Given the crucial role of CHX family proteins in ion homeostasis and pH regulation, this study will focus on VvCHX20 for subsequent functional validation to elucidate its molecular mechanism in GA3-regulated water / ion dynamics.

[0090] 2.5 Structure, phylogenetic characteristics, and subcellular localization of VvCHX20 in tobacco leaves

[0091] The full-length coding sequence (CDS) of VvCHX20 was successfully cloned and sequenced. Bioinformatics analysis predicted that VvCHX20 encodes an 826-amino acid protein with a molecular weight of approximately 89 kDa. Phylogenetic reconstruction showed that VvCHX20 closely clusters with identified cation / hydrogen ion antitransporters such as Arabidopsis thaliana AtCHX2 and AtCHX17, and soybean GsCHX19.3, indicating functional conservation within this transporter family. Structural analysis further revealed that VvCHX20 contains 10 predicted transmembrane domains, primarily located in the highly hydrophobic N-terminal region. Notably, amino acids 35–424 contain a conserved cation / hydrogen ion antitransporter domain, supporting its presumed function in ion transport.

[0092] To experimentally determine the subcellular localization of VvCHX20, this study constructed a 35S::VvCHX20-GFP fusion expression vector and transiently expressed it in tobacco leaves. Laser confocal microscopy revealed that the fluorescence signal of VvCHX20-GFP was specifically localized to the plasma membrane, co-localizing with the autofluorescence of the plasma membrane; while the control expressing only GFP showed diffuse fluorescence signals in both the nucleus and cytoplasm. These results confirm that VvCHX20 is a transporter located on the plasma membrane, consistent with its predicted function of participating in ion exchange in the pericellular space.

[0093] 2.6 Phenotypic, moisture state, and ion content analysis of tomato fruits overexpressing VvCHX20

[0094] Eight independent VvCHX20 overexpressing tomato lines were identified, and lines #2, #4, and #8 were selected for subsequent phenotypic and physiological evaluation. Compared with the wild type, the mature fruits of these transgenic lines were significantly larger and rounder. Quantitative measurements confirmed that the single fruit weight and transverse diameter of the transgenic lines were significantly increased, but the longitudinal diameter showed no significant difference. Moisture state analysis showed that the free water content of the VvCHX20 overexpressing fruits was significantly increased, while the bound water and total water content were lower than those of the wild type. Regarding soluble solids, the content of line #8 was significantly higher than that of the wild type, and the content of lines #2 and #4 was also higher than that of the two sublines of the wild type (WT-1 and WT-3). Ion content analysis showed that the potassium (K⁺) content of strains #4 and #8 was significantly higher than that of the wild type, while there were no significant differences in the calcium (Ca²⁺), magnesium (Mg²⁺), and sodium (Na⁺) content between all transgenic strains and the wild type. This indicates that VvCHX20 overexpression specifically promotes the accumulation of K⁺ in tomato fruits without broadly affecting other cations.

[0095] Cross-sectional analysis of fruits from wild-type WT-1 and VvCHX20-#8 lines revealed that WT-1 fruits had two locules, while the transgenic lines had three locules. Furthermore, the pericarp thickness corresponding to each locule in the VvCHX20-#8 line was increased compared to WT-1. These morphological changes, combined with alterations in water and ion content, suggest that VvCHX20 overexpression may affect fruit development by regulating cell enlargement and potassium-dependent water distribution.

[0096] 2.7 Screening of VvCHX20-regulated transcription factors

[0097] To investigate the transcriptional regulatory mechanism of VvCHX20, this study constructed the Phis2-proVvCHX20 recombinant vector. Self-activation assays confirmed that 300 mM 3-aminotriazole (3AT) effectively inhibited background reporter gene activity; this concentration was used in subsequent yeast one-hybrid experiments. Candidate transcription factors were cloned into the pGADT7 vector and co-transformed with Phis2-proVvCHX20 into yeast cells for point-to-point interaction verification. The experiments identified four proteins that specifically bind to the VvCHX20 promoter: VvAAR3 (CULLIN ubiquitination-deficient protein 1), VvPUB23 (U-Box E3 ubiquitin ligase), VvDPMS1 (polyterpene phosphate mannose synthase 1), and VvNH18 (nudix hydrolase 18), indicating that they may function as direct transcriptional regulators.

[0098] To verify transcriptional activation activity, the VvCHX20 promoter was inserted into the pGreenII 0800-LUC vector, and each candidate gene was cloned into the pGreenII 62-SK vector. Dual-luciferase assays showed that VvAAR3, VvPUB23, VvDPMS1, and VvNH18 significantly enhanced the activity of the VvCHX20 promoter. These results indicate that these functionally diverse proteins involved in ubiquitination, glycosylation, and hydrolysis can positively regulate VvCHX20 transcription by directly binding to the promoter.

[0099] In summary, this study demonstrates that GA3 improves grape fruit growth and quality by downregulating novel_miR_130 and promoting the transcriptional activation of VvCHX20 through specific transcription factors such as VvAAR3, VvPUB23, VvDPMS1, and VvNH18. VvCHX20, as a cation / hydrogen ion antitransporter located on the plasma membrane, promotes K⁺ accumulation, improves water status, and increases soluble solids content. Transgenic tomato experiments confirmed that VvCHX20 overexpression increases fruit size, increases the number of locules and peel thickness, and enhances potassium-dependent water retention. This study reveals a novel GA3–miRNA–transcription factor–transporter pathway that optimizes fruit ion and water homeostasis, providing new insights into the mechanisms by which hormones regulate fruit development and offering potential strategies for crop quality improvement.

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of the grape VvCHX20 gene in regulating plant fruit growth and quality.

2. The application according to claim 1, characterized in that, Overexpression of the grape VvCHX20 gene positively regulates fruit growth and quality in plants.

3. The application according to claim 1, characterized in that, The nucleotide sequence of the grape VvCHX20 gene is shown in SEQ ID No.

1.

4. The application according to claim 1, characterized in that, The plant fruit includes tomato fruit.

5. The application according to claim 1, characterized in that, Overexpression of the grape VvCHX20 gene increases the single fruit weight and transverse diameter of the plant.

6. The application according to claim 1, characterized in that, Overexpression of the grape VvCHX20 gene increases the free water content and decreases the bound water content in the fruit.

7. The application according to claim 1, characterized in that, Overexpression of the grape VvCHX20 gene increases the soluble solids content of plant fruits.

8. The application according to claim 1, characterized in that, Overexpression of the grape VvCHX20 gene promotes potassium accumulation in plant fruits.

9. The application according to claim 1, characterized in that, Overexpression of the grape VvCHX20 gene increases the number of locules and the thickness of the fruit skin.

10. The application of transcription factors in enhancing VvCHX20 promoter activity to positively regulate the transcription of the grape VvCHX20 gene, characterized in that, The transcription factors include one or more of VvAAR3, VvPUB23, VvDPMS1, and VvNH18.

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