Effect of GA₃ Treatment on Berry Development in the Large Berry Mutant of ‘Delaware’ Grapes

Abstract

The large berry mutant (LBM) of the ‘Delaware’ (V. vinifera × (V. labrusca × V. aestivalis)) is known to produce larger berries than ‘Delaware’ when subjected to traditional double gibberellic acid-3 (GA₃) treatment. In this study, we first compared ‘Delaware’ and the LBM in terms of their berry and cluster quality to reevaluate previous findings on LBM. Next, we compared berry size without GA₃, histological characteristics of the early developmental berry, and then GA-related gene expression to reveal the characteristics of the LBM. The previous finding that LBM yielded larger berry sizes than ‘Delaware’ with double GA₃ treatment was verified, but without GA₃ treatment, LBM did not produce larger berries than ‘Delaware’. This highlights the prerequisite of exogenous GA for larger berry size in LBM. Histological analysis revealed an increase in cell numbers of the inner and outer mesocarp walls in the early developmental stage of LBM berries. Gene expression analysis of the flower buds and berries indicated an increased expression of VvGID1A, encoding the GA receptor, in LBM than in ‘Delaware’ 3 h after the first GA₃ treatment. Additionally, the expression of VvGID1A and VvGID1B was higher in LBM than that in ‘Delaware’ before and after the second GA₃ treatment. The expression of VvGID2A, which interacts with the GA receptor and promotes GA signaling, was also higher in LBM than in ‘Delaware’ before the first GA₃ treatment. However, the expression of VvSLR1, VvGAI1, and VvGAI2 that encode DELLA proteins, essential negative regulators for GA reactions, mostly showed no significant changes. These results suggest that LBM berries had higher GA sensitivity than the ‘Delaware’. Based on these results, we believe that the larger berry size in LBM may be due to increased cell numbers resulting from high GA sensitivity.

Introduction

Grape (Vitis spp.) is a horticultural crop cultivated worldwide. In Japan, table grapes are in greater demand than wine grapes; among table grapes, seedless cultivars are preferred over seeded ones owing to their ease of consumption. Most seedless grape cultivars in Japanese markets are genetically seeded cultivars, rendered seedless by treatment with the plant hormone gibberellic acid (GA). Seedless grape cultivation was developed in the 1960s in Japan using cultivars such as ‘Delaware’ (V. vinifera × (V. labrusca × V. aestivalis)) (Maul et al., 2022; Saito, 2011; Senno, 1981), and now extends to ‘Kyoho’ (V. labruscana × Vitis vinifera), ‘Pione’ (V. labruscana × Vitis vinifera), and ‘Shine Muscat’ (V. labruscana × Vitis vinifera). The seedless cultivars of which were generated by immersing the buds or flowers in GA (Enya, 2018; Saito, 2011; Udo, 2014). In general, the cultivars are subjected to double gibberellic acid 3 (GA₃) treatment; the first treatment facilitates seedless fruit production, while the second facilitates berry enlargement (Saito, 2011). In the case of the ‘Delaware’ cultivar, the first GA₃ treatment (100 ppm) is applied 14 days before full bloom (DBFB14), while the second (75–100 ppm) is applied 10 days after full bloom (DAFB10). In addition, combined use of the cytokinin, forchlorfenuron (CPPU), during the first treatment stabilizes fruit set (Nakagomi, 1993; Saito, 2011). However, a single GA₃ treatment can be implemented to minimize labor, but does not necessarily yield the same berry size as conventional double GA₃ treatments (Enya, 2018; Saito, 2011; Udo, 2014).

GIBBERELLIN INSENSITIVE DWARF 1 (GID1) is a GA receptor that plays a significant role in GA signaling (Nakajima et al., 2006; Ueguchi-Tanaka et al., 2005). Arabidopsis has three GID1-like genes, GID1A, GID1B, and GID1C (Nakajima et al., 2006). The interaction between GID1 and DELLA, a sub-family of GRAS proteins that negatively regulates GA signaling, is activated upon GA binding to GID1 (Achard et al., 2007; Bolle, 2004; Daviére et al., 2014; Guo et al., 2017; Peng et al., 1997; Silverstone et al., 1998; Ueguchi-Tanaka et al., 2007; Wen and Chang, 2002). Consequently, DELLA interacts with the E3 ubiquitin ligase SCF^SLEEPY1, which leads to its ubiquitination and degradation by the 26S proteasome (Fu et al., 2004; Griffiths et al., 2006; McGinnis et al., 2003; Ueguchi-Tanaka et al., 2008).

Five DELLA proteins (GA INSENSITIVE (GAI), REPRESSOR OF gal-3 (RGA), and RGA-Like (RGL1, RGL2 and RGL3)) are reported in Arabidopsis (Bolle, 2004; Silverstone et al., 1998; Wen and Chang, 2002). Together, RGA, RGL1 and RGL2 modulate GA-regulated floral development (Lee et al., 2002; Tyler et al., 2004), with RGL2 playing a major role during seed germination in Arabidopsis (Tyler et al., 2004). GAI and RGA in Arabidopsis are orthologues of SLENDER1 (SLR1) in Oryza Sativa, RHT-B1/Rht-D1 in wheat, D8 in maize, and GAI1 in grapes (Boss and Thomas, 2002; Ikeda et al., 2001; Peng et al., 1999). AtSLY1, encoding SLY1, the F-box protein, which determines the specificity of parts of SCF complexes, is a homolog of OsGID2 (McGinnis et al., 2003; Sasaki et al., 2003).

GA signaling for fruit set and growth is studied in table grapes, owing to the completion of V. vinifera genome sequencing in 2007 (Velasco et al., 2007). In ‘Kyoho’ grapes, the effect of GA was studied using transcriptome analysis of flowers (Cheng et al., 2015). Comprehensive gene expression analysis revealed that VvmiRNA159 regulates GA signaling in floral development (Wang et al., 2018). VvmiR156a negatively regulated VvAGL80 after GA₃ treatment, which promoted grape blooming (Su et al., 2021). Pre-bloom GA and auxin treatment facilitated grape berry parthenocarpy by downregulation of VvIAA9 and VvARF7 via DELLA (Jung et al., 2014). The transcriptomic comparison between ‘Shine Muscat’ and ‘Honey Venus’, which have differences in seeded berry rates under GA₃ treatment, showed dynamic differential expression of many phytohormone related genes in ovules (Nishiyama et al., 2022). Despite the diverse studies exploring GA signaling in the grape berry, detailed mechanisms underlying the parthenocarpy and berry development of grapes in response to GA are still unclear; furthermore, higher GA sensitivity grapevine mutants are yet to be analyzed (Gallego-Giraldo et al., 2014; Griffiths et al., 2006).

The large berry mutant of ‘Delaware’ (LBM), found in the Shimane prefecture in Japan, is a bud mutation of ‘Delaware’ (Togano, 2019). LBM berry flesh weight is approximately 1.3 times higher than that of ‘Delaware’ under double GA₃ treatment (Togano, 2019). Additionally, the berry flesh weight of LBM with single GA₃ treatment at 200 ppm is similar to that of ‘Delaware’ that was subjected to conventional double GA₃ treatment at 100 ppm (Togano, 2019). This may be attributed to differences in GA sensitivities between the two lines; moreover, GA signaling in both lines are yet to be reported. Accordingly, this study investigated the characteristics of berry development in ‘Delaware’ and LBM, and identified differences in GA signaling mechanisms between them.

Materials and Methods

Plant materials

Grapevine ‘Delaware’ (V. vinifera × (V. labrusca × V. aestivalis)) and LBM were cultivated at the Shimane Agricultural Technology Center, Shimane Prefecture, Japan. The 6-year-old ‘Delaware’ and LBM vines were cultivated under rain-shelter conditions, and these berries were used to compare the quality of the berries and clusters at harvest time. The berry number per cluster was adjusted to around 90. The 5- to 6-year-old ‘Delaware’ and LBM vines were cultivated under rain-shelter conditions, and these clusters, which were adjusted to approximately 20 berries each by eliminating berries at the fruit set period, were used to compare the pollinated berry size at harvest time. The 4- to 5-year-old ‘Delaware’ and LBM vines were cultivated under rain-shelter conditions, and these berries were used for histological analysis during their early developmental stages. The berries were soaked in about 50–100 volumes of FAA solution (100% ethanol: acetic acid: formalin: ultrapure water in a 12:1:1:5 ratio), depressurized, and stored in glass bottles until further use at room temperature. The flower buds, which were collected from three 8-year-old ‘Delaware’ and LBM vines under rain-shelter conditions with heating, and the berries, which were collected from three 4-year-old potted ‘Delaware’ and LBM vines under rain-shelter conditions, were used for gene expression analysis. The spike shaping was the same between the two lines in all experiments.

Quality assessment of berries and clusters

For quality assessment of the berries and clusters, five clusters per vine were each collected and measured from three ‘Delaware’ and LBM vines (n = 3). For berry weight measurement, the weight of the cluster without the peduncle was measured and divided by the number of berries. For °Brix and total acid measurement, seven berries were collected from each cluster and their juice analyzed. °Brix was measured by refractometer PAL-1 (Atago Co., Ltd., Tokyo, Japan). Total acid was determined by titration and expressed as tartaric acid equivalents.

Plant hormone treatment

All first treatments were applied 14 days before full bloom (DBFB14), while the second treatments were applied 10 days after full bloom (DAFB10). Except for the flower buds used in gene expression analysis, the first treatment of all other samples was carried out by soaking the clusters in 100 ppm GA₃ (Kyowa Hakko Bio Co., Ltd., Tokyo, Japan) + 0.1% (v/v) Approach BI (Maruwa Biochemical Co., Ltd., Tokyo, Japan) as a spreading agent + 5 ppm CPPU (Sumitomo Chemical Co., Ltd., Tokyo, Japan). The second treatment was carried out by soaking the clusters in 100 ppm GA₃ + 0.1% (v/v) Approach BI. Samples for flower bud gene expression analysis were treated with either 100 ppm GA₃ (Tokyo chemical industry Co., Ltd., Tokyo, Japan) + 0.1% (v/v) polyoxyethylene (20) Sorbitan Monolaurate (tween 20; Fujifilm Wako Pure Chemical Co., Ltd., Osaka, Japan) as a spreading agent or 0.1% (v/v) tween 20 as a spreading agent for the GA or MOCK treatments, respectively. Flower buds were collected at 0 h, and after 1 h, 3 h, 6 h, and 24 h of GA₃ treatment; berries were collected before the second GA₃ treatment at DAFB10 and DAFB12, respectively. Samples were frozen immediately and stored at −80°C until RNA extraction.

Histological analysis

The grape berry samples stored in FAA were embedded in a resin prepared using the Technovit^® 7100 (Kulzer, Hanau, Germany) following the method by Kuroiwa (1991). The embedded samples were then cut into 5–10 μm thick sections near the equatorial plane using a rotary microtome (RV-240; YAMATOKOHKI Co. Ltd., Saitama, Japan). Sections were then stained using safranin and observed under a biological microscope (ECLIPSE E100v; Nikon Co., Tokyo, Japan). For each berry specimen, the pericarp and the mesocarp inner and outer walls were evaluated at 20 locations.

RNA isolation and gene expression analysis using qRT-PCR

Total RNA was extracted from flower bud and berry samples using a Cica genius^®︎ RNA Prep Kit (for Plant) (Kanto Chemical Co. Inc., Tokyo, Japan) following the manufacturer’s instructions. A modified buffer was used for berry RNA extraction (McQuinn et al., 2018). First strand cDNA was synthesized by reverse transcription using the extracted RNA as a template. Quantitative RT-PCR was performed using the KOD SYBR^® qPCR Mix (Toyobo Co., Ltd., Osaka, Japan) to quantify target gene expression. Thermal Cycler Dice Real Time System TP800 (Takarabio, Co., Ltd., Shiga, Japan) was used for the real-time RT-PCR. VvUBI, encoding the housekeeping gene ubiquitin (UBI), was used as the endogenous control; the target genes were: VvGID1A, VvGID1B, VvGID2A, VvGID2B, VvGAI1, VvGAI2, VvSLR1, and VvWRKY33. Primers for qRT-PCR were designed using Primer3 Plus <http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi> with primer sequences listed in Table 1. The reaction conditions for qRT-PCR include initial denaturation at 98°C for 2 min, followed by 40 cycles of denaturation at 98° C for 10 s, annealing at 60°C for 10 s, and extension at 68°C for 30 s.

Table 1

Primers used for qPCR.

Results and Discussion

Growth and development of berries and clusters

Previous literature reports larger berries for LBM than for ‘Delaware’ under double GA₃ treatment (Togano, 2019). We first reevaluated berry and cluster quality of ‘Delaware’ and LBM under double GA₃ treatment. The berry weight of LBM was significantly greater than that of ‘Delaware’ under double GA₃ treatment, with an average difference of 24% (Table 2). The larger berry size for LBM than ‘Delaware’ observed is consistent with previous literature (Togano, 2019). However, there were no significant differences between ‘Delaware’ and LBM in terms of °Brix, total acid, cluster weight, and the number of berries per cluster under double GA₃ treatment (Table 2). These results show that, other than size, there is no difference in berry quality. We speculate that the characteristics exhibited by LBM berries are attributable to differences in GA sensitivities between the two lines. Consequently, seeded berries at harvest time were evaluated to examine whether the presence or absence of exogenous GA₃ resulted in a larger berry size in LBM. No significant differences were observed in berry diameter, length, or weight between the two lines at harvest time (Fig. 1a, b). This ruled out the possibility of endogenous plant hormones, such as indole acetic acid (IAA) and GA, affecting LBM berry size. Accordingly, the larger LBM berry size appears as a response to exogenous GA; moreover, it is possible that LBM has a high GA sensitivity.

Table 2

Quality of berry and cluster of ‘Delaware’ and LBM under double GA₃ treatment.

Fig. 1

Seeded berry growth of ‘Delaware’ and LBM. Berry size (a) and berry weight (b). LBM, the large berry mutant of ‘Delaware’. Error bars indicate standard error (SE, n = 4–5). NS, not significant (Welchʼs t-test).

Observation of pericarp tissue during early berry development

We examined differences between LBM and ‘Delaware’ in early berry development under GA₃ treatment. If GA sensitivity differences exist between LBM and ‘Delaware’, the first GA₃ treatment likely affects early berry development (Figs. 2 and 3). Thus, the pericarp tissue of early developmental stage berries was analyzed to determine the cause of the large berry size in LBM. As active cell division in pollinated grape berries ends by the tenth day after pollination (DAP10; Ojeda et al., 1999), we observed the tissues at 5 days after treatment (DAT5) and 12 days after treatment (DAT12), which fall around DAP10. At DAT5, the inner wall length of the LBM berry was significantly greater than that of ‘Delaware’, while at DAT12, no significant difference was observed (Fig. 3a). At DAT5 no significant difference was observed for the inner and outer walls cell numbers and density between LBM and ‘Delaware’, but at DAT12 they were significantly higher in LBM than ‘Delaware’ (Fig. 3c, d, e, f). There was no significant difference in outer wall lengths between the two lines at DAT5 and DAT12 (Fig. 3b).

Fig. 2

Berry tissue during early development in ‘Delaware’ and LBM grapevines. Schematic diagram of the berry fresh structure (a). Structure of pericarp around berry equator at DAT5 (b) and DAT12 (c). LBM, the large berry mutant of ‘Delaware’; DAT5, 5 days after GA₃ treatment; DAT12, 12 days after GA₃ treatment.

Fig. 3

Histological analysis of early developmental berry in ‘Delaware’ and LBM grapevines. Length of inner wall (a), length of outer wall (b), cell number of inner wall (c), cell number of outer wall (d), cell density of inner wall (e), cell density of outer wall (f). LBM, the large berry mutant of ‘Delaware’; DAT5, 5 days after GA₃ treatment; DAT12, 12 days after GA₃ treatment. Error bars indicate SE (n = 10). *, **, *** indicate significant differences at P < 0.05, P < 0.01 and P < 0.001 respectively, using Welchʼs t-test. NS, not significant.

In grapes, GA₃ treatment induces parthenocarpy via auxin signaling related genes (Jung et al., 2014). In the early stage of berry development, just after fruit set stimulation, there is a period of active cell division followed by a period of active cell enlargement (Coombe and Mccarthy, 2000; Ojeda et al., 1999). In this study, DAT5–12 corresponds to a period of active cell division. Consequently, the increase in cell numbers of the inner and outer walls in LBM berries indicates more active cell division in the early developmental stage than that observed in ‘Delaware’ (Figs. 2 and 3). It is known that in ‘Delaware’ the inner wall of the pericarp constitutes the majority of the mature berry (Nakagawa and Nanjo, 1965), but early in the berry development at DAT5 and DAT12, the outer wall was still thicker than the inner wall (Fig. 3a, b). As one might assume from the differences observed in cell number and cell density between the two lines at the early berry developmental phase (Fig. 3c, d, e, f), the cause of the large berry size was expected to be either cell division alone or both cell division and cell expansion. Therefore, the increased cell number in the mesocarp of the LBM berries may related to the larger berry size. However, as we did not compare the berry structures without exogenous GA, the possibility that differences in berry development at DAT5 and DAT12 were not due to GA₃ treatment cannot be ruled out. In future studies, histological analysis of the mature berry is necessary to determine whether differences in the cell numbers of early developing berries ultimately affect the berry size.

Gene expression analysis following the first GA₃ treatment

We investigated gene expression analysis just after the first GA₃ treatment to identify differences in the cultivar’s response to GA. As ‘Delaware’ has a genetic makeup consisting of genes from multiple species, gene expression analysis in this study was performed with reference to the genomic information of V. vinifera, for which this cultivar would have at least one set of V. vinifera genomes. The grape genome has two homologs of GID1—VvGID1A and VvGID1B—which encode the GA receptor (Acheampong et al., 2015; Yoshida et al., 2018). VvSLY1a and VvSLY1b have multiple partial homologies with AtSLY1 and OsGID2, which induce the degradation of DELLA (Acheampong et al., 2015). In this study, we assigned VvSLY1a and VvSLY1b as VvGID2A and VvGID2B, respectively. VvGID1A showed higher expression in LBM than that in ‘Delaware’ at 3 h after GA treatment (Fig. 4a). There was no comparative difference in VvGID1B expression between these lines, except at 6 h after GA treatment in ‘Delaware’ (Fig. 4b). VvGID2A showed significantly higher expression in LBM than in ‘Delaware’ at 0 h (Fig. 4c). VvGID2B showed higher expression in LBM than in ‘Delaware’ at 3 h after the MOCK treatment (Fig. 4d). However, the expression of all VvDELLAs (VvSLR1, VvGAI1 and VvGAI2) showed no significant differences between the two lines (Fig. 4e, f, g). The expression of VvWRKY33, a GA-responsive gene, was likely more activated by GA in LBM than in ʻDelawareʼ at 6 h after GA treatment. However, no significant difference in its expression between GA₃-treated ‘Delaware’ and LBM was observed (Fig. 4h). From these results, VvGID1A and VvGID2A in flower buds just after or around the first GA treatment may contribute to GA sensitivity.

Fig. 4

Gene expression analysis after GA₃ treatment of flower buds 14 days before full bloom (DBFB14) in ‘Delaware’ and LBM grapevines. The flower buds were treated with 100 ppm GA₃ + 0.1% (v/v) polyoxyethylene (20) Sorbitan Monolaurate (Tween 20) as a spreading agent as the GA treatment, or 0.1% (v/v) Tween20 as a spreading agent as the MOCK treatment. Relative expression levels of VvGID1A (a), VvGID1B (b), VvGID2A (c), VvGID2B (d), VvSLR1 (e), VvGAI1 (f), VvGAI2 (g), and VvWRKY33 (h). VvSLR1, VvGAI1, and VvGAI2 were collectively referred to as VvDELLAs. LBM, the large berry mutant of ‘Delaware’; GA, GA₃ treatment; MOCK, MOCK treatment; N, ʻDelawareʼ, L, the large berry mutant of ‘Delaware’. Error bars indicate SE (n = 3). * indicates significant difference at P < 0.05 by Welchʼs t-test and different letters indicate significant differences at P < 0.05 by Tukeyʼs HSD test. NS, not significant.

GID1, the GA receptor, and GID2, promote DELLA degradation in the presence of GA. Both are essential for GA signaling, as their loss of function reduces GA sensitivity in Arabidopsis and rice (Ariizumi et al., 2008; Sasaki et al., 2003; Ueguchi-Tanaka et al., 2008). It is reported that the expression of GID1 is subject to negative feedback from GA signaling (Hou et al., 2008), and this mechanism may suppress excessive GA signaling. Hence, the GID1 expression level may be a limiting factor for GA signaling under the saturated GA conditions caused by external treatment. This study observed high expression levels of VvGID1A or VvGID2A in LBM during GA₃ treatment, which may raise GA sensitivity in the early GA response. Thus, the degradation of DELLA proteins, which are negative regulators of GA signaling, may be promoted in GA sensitive LBM relative to ‘Delaware’, resulting in the positive regulation of GA signaling in LBM. These results further indicate that the larger berry size in LBM was caused by exogenous GA, which may induce active cell division in LBM berry pericarp more than in ‘Delaware’. This is supported by the fact that VvWRKY33, a homolog of WRKY33 induced by GA in almond anthers (Li et al., 2021), showed a greater response to GA in LBM than in ‘Delaware’.

Gene expression analysis following the second GA₃ treatment period

Furthermore, we analyzed gene expression around the second GA₃ treatment period. The expression of VvGID1A and VvGID1B in LBM was higher than that in ‘Delaware’ at DAFB10 and DAFB12 (Fig. 5a, b). There was no significant difference in the expression of VvGID2A and VvGID2B between the two lines at DAFB12 (Fig. 5c, d). The expression of VvGAI2 was higher in LBM than in ‘Delaware’ at DAFB10, and there was no significant difference in the transcript expressions of VvSLR1 and VvGAI1 between LBM and ‘Delaware’ at DAFB10 (Fig. 5e, f, g). The expression of all DELLAs showed no significant differences between LBM and ‘Delaware’ at DAFB12 (Fig. 5e, f, g). The high GA sensitivity of LBM may be attributed to the increased expression of VvGID1s in LBM around the second GA₃ treatment period. These results suggest that, just after the first and around the second GA₃ treatment period, the LBM berries have a higher sensitivity than the ‘Delaware’ berries. Although previous studies on seedless grapes with GA₃ treatment reported differential downstream gene expression of the DELLA proteins in GA signaling (Arro et al., 2019; Cui et al., 2020; Su et al., 2021), no study has yet focused on the high GA sensitivity of grape berries. LBM has characteristics of high GA sensitivity, therefore this study revealed that the high expression of the GA receptor gene may be responsible for the increased GA sensitivity.

Fig. 5

Expression analysis of genes related to the GA response of ‘Delaware’ and LBM grapevine berries subjected to GA₃ treatment 10 and 12 days after full bloom (DAFB10 and DAFB12, respectively). Relative expression levels of VvGID1 (a), VvGID1B (b), VvGID2A (c), VvGID2B (d), VvSLR1 (e), VvGAI1 (f), and VvGAI2 (g). Error bars indicated SE (n = 3). *, ** indicate significant difference at P < 0.05 and P < 0.01, respectively (Welchʼs t-test). NS, not significant.

Conclusion

In the present study, it was demonstrated that, with GA₃ treatment, the cell number in LBM increased during the early berry development, which may cause the larger berry size. Based on the comparative results of seeded berry development and GA-related gene expression, it is evident that LBM is highly sensitive to GA. However, the accumulation of DELLA proteins was not evaluated in this study. As such, future studies are necessary to investigate the roles of the DELLA proteins during GA signaling in LBM. The genetic mutation of LBM is beneficial as it produces larger berries without compromising on berry quality. If the genetic mutation related to berry size and/or GA sensitivity in LBM can be identified, it may be possible to apply similar traits to other cultivars through genetic transformation or genome editing. For this reason, LBM is an important genetic resource for further research on GA sensitivity in grapes.

Acknowledgements

The authors thank Dr. Katsumi Ohta, Dr. Akira Nakatsuka, Dr. Tomoya Esumi and Dr. Sokichi Shiro for their advice. The authors would like to thank Editage (www.editage.com) for English language editing.

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