Abnormal Endogenous Repression of GA Signaling in a Seedless Table Grape Cultivar with High Berry Growth Response to GA Application

Abstract

Gibberellin (GA) application is routinely used in the table grape industry to increase berry size and cluster length. Although grapevine cultivars show a wide range of growth responsiveness to GA₃ application, the reasons for these differences is unclear. To shed light on this issue, two commercial grapevine cultivars with contrasting berry response to GA were selected for comparative analysis, in which we tested if the differences in response: (1) is organ-specific or cultivar-related; (2) will be reflected in qualitative/quantitative differences in transcripts/proteins of central components of GA metabolism and signaling and levels of GA metabolites. Our results showed that in addition to the high response of its berries to GA, internodes and rachis of cv. Black finger (BF) presented a greater growth response compared to that of cv. Spring blush (SB). In agreement, the results exposed significant quantitative differences in GA signaling components in several organs of both cultivars. Exceptionally higher level of all three functional VvDELLA proteins was recorded in young BF organs, accompanied by elevated VvGID1 expression and lower VvSLY1b transcripts. Absence of seed traces, low endogenous GA quantities and lower expression of VvGA20ox4 and VvGA3ox3 were also recorded in berries of BF. Our results raise the hypothesis that, in young organs of BF, low expression of VvSLY1b may be responsible for the massive accumulation of VvDELLA proteins, which then leads to elevated VvGID1 levels. This integrated analysis suggests causal relationship between endogenous mechanisms leading to anomalous GA signaling repression in BF, manifested by high quantities of VvDELLA proteins, and greater growth response to GA application.

Keywords: DELLA proteins; Vitis vinifera; VvSLY1; gene expression; gibberellin; gibberellin signaling.

FIGURE 1

Effect of GA₃ and PAC on size of organs of V. vinifera cv. Black finger (BF) and cv. Spring blush (SB). Altered response of organs of BF (A,B,E,F,I,J) and SB (C,D,G,H,K,L) to GA₃ and GA biosynthesis inhibitor, PAC treatments during the 2010 growing season (similar results were obtained in experiments carried out in 2011 growing season). GA₃ and PAC (0.8 mM) included Triton X-100 (0.025%). Internodes and rachises were treated with 121 μM GA₃, while berries were treated with 90 μM GA₃. Tissues/organs were dipped or sprayed until run-off. Increase in size was monitored at specific time intervals. Young shoots and inflorescences with tightly packed flowers (stage 15, E-L 15, on the Modified Eichhorn and Lorenz system) were selected for internodes and rachis experiments, respectively. Clusters with berries of 2–3 mm diameter (E-L 27) were selected for berry experiments. (A,C) Gross morphology of representative internodes of BF and SB after 20 days of treatment. (E,G) Gross morphology of representative rachises of BF and SB after 20 days of treatment. (I,K) Gross morphology of representative berries of BF and SB after 30 days of treatment. (B,D) Average per cent increase in length of new internodes arising after treatment. Increase in length of internode is expressed as per cent increase of initial length, which was assumed to be 0.5 mm. (F,H) Average per cent increment (as a factor of pre-treatment length) in length of rachises of BF and SB. (J,L) Average per cent increase in berry weight relative to mean weight at time of treatment (0 day). Data points with different letters indicate significantly different values according to Tukey HSD LSMean test at α = 0.05 and 25 measurements, except for berries with 150 measurements. Bar = 5 cm.

FIGURE 2

Different alleles of VvDELLAs isolated from cv. Black finger (BF) and cv. Spring blush (SB) interact with VvGID1s and VvSLY1s in Y2H assays. (A) Interaction between VvDELLAs and VvGID1s proceed in a GA-dependent manner. The addition of 100 μM GA₃ to the medium enhanced GID1–DELLA interactions. (B) Interaction between VvDELLAs and VvSLY1s. VvDELLA3_SB1 and VvDELLA3_SB2 represent the two alleles of VvDELLA3 isolated from SB. The bars represent the mean ± SE of at three replicates.

FIGURE 3

Spatio-temporal expression profile of VvDELLA paralogs in V. vinifera cv. Black finger (BF) and cv. Spring blush (SB). Spatial and temporal expression profiles of VvDELLA1 (A), VvDELLA2 (B), and VvDELLA3 (C) in BF and SB organs sampled during the 2010 growing season. Total RNA was extracted from pooled samples, and the absolute mRNA levels of each gene were determined by real-time quantitative RT-PCR (qRT-PCR) and normalized against VvGAPDH. To ensure accurate quantitation of transcript levels, primers of similar efficiencies were used, and calibration curves determined from known copy numbers of single plasmid containing all qRT-PCR amplicons. The bars represent the mean ± SE. of three biological repeats with two technical repeats each. Asterisks (^∗) indicates relative expression levels that are significantly different (Student’s t-test; P < / > 0.05) between both cultivars. Statistical significance of relative expression values among organs of individual cultivars were calculated using Tukey HSD LSMean test at α = 0.05, and presented in Supplementary File S2. In, internodes; Ra, rachis; Le, leaves; Te, tendrils; Pi, pistils; Be, berries; 0 d, berries sampled at 2–3 mm diameter (E-L 27); 10 d, berries sampled 10 days after E-L 27; 30 d, Berries sampled 30 days after E-L 27; Y, young; M, mature. The experiment was repeated during 2011 growing season.

FIGURE 4

Spatio-temporal profile of VvDELLA proteins in V. vinifera cv. Black finger (BF) and cv. Spring blush (SB). Blots of total protein extracted from internodes (A), rachises (B), leaves (C), tendrils (D), pistils and berries (E) at different developmental stages. Sampling of the tissues was carried out in 2010. Blots were incubated with affinity-purified, gene-specific, anti-VvDELLA polyclonal antibodies. Recombinant full-length proteins (R.P.) (3.75 ng each of VvDELLA1 and VvDELLA2 and 37.5 ng of VvDELLA3) were used as sizing controls. Coomassie Brilliant Blue-stained (CBB) proteins were used as loading control. In all lanes except R.P., solid black arrows show band of interest, and asterisked-bands (^∗) indicate non-specific proteins detected by the anti-VvDELLA antibodies. Differences in sizes of R.P. and endogenous VvDELLA proteins result from V5 and 6xHis tags on the R.P. 0 d, berries sampled at 2–3 mm diameter (E-L 27); 10 d, berries sampled 10 days after E-L 27; 30 d, berries sampled 30 days after E-L 27. Similar results were obtained when samples collected in 2011 growing season were analyzed.

FIGURE 5

Effect of GA₃ application on VvDELLA accumulation in V. vinifera cv. Black finger (BF) and cv. Spring blush (SB). GA₃-induced degradation of VvDELLA1 and VvDELLA2 proteins in internodes (A), rachis (B), and pistils (C) of BF and SB collected during the 2010 growing season. Immunoblot analyzes of VvDELLA proteins in organs were carried out using protein-specific, affinity-purified, anti-VvDELLA polyclonal antibodies. Total proteins were extracted from organs treated for 6 h with GA₃ (G, 121 μM for rachis, and 90 μM for pistils). Control (C) samples were treated with Triton X-100 (0.025%). Physiological stage at which organs were treated is detailed in “Materials and Methods.” Recombinant full-length proteins (R.P.) (3.75 ng each of VvDELLA1 and VvDELLA2) were used as size controls. In all lanes except R.P., solid black arrows show band of interest, and Asterisked-bands indicate non-specific proteins detected by the anti-VvDELLA antibodies. Differences in sizes of R.P. and endogenous VvDELLA proteins result from tags on the R.P. Underlined numbers indicate intensity of bands relative to GA₃-treated samples as determined by ImageJ. Consistent results were obtained when the experiment was repeated during the 2011 growing season.

FIGURE 6

Spatio-temporal expression profiles of VvGID1 and VvSLY1 paralogs in V. vinifera cv. Black finger (BF) and cv. Spring blush (SB). Spatial and temporal expression profiles of VvSLY1a (A), VvSLY1b (B), VvGID1a (C), and VvGID1b (D) in BF and SB organs collected during the 2010 growing season. Total RNA was extracted from pooled samples, and the absolute mRNA levels of each gene were determined by real-time quantitative RT-PCR (qRT-PCR) and normalized against VvGAPDH. To ensure accurate quantitation of transcript levels, primers of similar efficiencies were used, and calibration curves determined from known copy numbers of single plasmid containing all qRT-PCR amplicons. The bars represent the mean ± SE. of three biological repeats with two technical repeats each. Asterisks (^∗) indicates relative expression levels that are significantly different (Student’s t-test; P < / > 0.05) between both cultivars. Statistical significance of relative expression values among organs of individual cultivars were calculated using Tukey HSD LSMean test at α = 0.05, and presented in Supplementary File S2. In, internodes; Ra, rachis; Le, leaves; Te, tendrils; Pi, pistils; Be, berries; 0 d, berries sampled at 2–3 mm diameter (E-L 27); 10 d, berries sampled 10 days after E-L 27; 30 d, berries sampled 30 days after E-L 27; Y, young; M, mature. Similar results were obtained from samples collected during the 2011 growing season.

FIGURE 7

Spatio-temporal accumulation of endogenous bioactive GAs in V. vinifera cv. Black finger (BF) and cv. Spring blush (SB). Quantification of endogenous GA₁ (A) and GA₄ (B) in organs of BF (black-filled bars) and SB (gray-filled bars) at different developmental stages. Bioactive GAs were extracted from 0.5 g fresh weigh of homogenized tissue, and quantified using triple quadrupole mass spectrometer coupled to an Ultra High Performance Liquid Chromatography (UHPLC) system equipped with an octylphenyl column. The bars represent the mean ± SD. of three biological replicates of extractions. In, internodes; Ra, rachis; Le, leaves; Te, tendrils; Pi, pistils; Be, berries; 0 d, berries sampled at 2–3 mm diameter (E-L 27); 10 d, Berries sampled 10 days after E-L 27; 30 d, berries sampled 30 days after E-L 27; Y, young; M, mature. Samples were collected during the 2010 growing season.

FIGURE 8

Spatio-temporal expression profiles of GA metabolism genes in V. vinifera cv. Black finger (BF) and cv. Spring blush (SB). Spatial and temporal expression profiles of VvGA2ox (A), VvGA3ox (B), and VvGA20ox (C) paralogs in organs of BF and SB collected during the 2010 growing season. Y-axis is the normalized relative expression (NRE) (Giacomelli et al., 2013) of the respective paralog, expressed as a per cent of the total NRE of that paralog in all organs analyzed. NREs were calculated, as described by Giacomelli et al. (2013), from the average of three biological replicates of relative transcripts normalized against the expression of VvGAPDH, which is unaffected by GA. Transcripts were measured by qRT-PCR using EvaGreen DNA-binding dye on the 96.96 Dynamic Array Integrated Fluidic Circuits (IFCs). In, internodes; Ra, rachis; Le, leaves; Te, tendrils; Pi, pistils; Be, berries; 0 d, berries sampled at 2–3 mm diameter (E-L 27); 10 d, berries sampled 10 days after E-L 27; 30 d, berries sampled 30 days after E-L 27; Y, young; M, mature. Full description of experimental procedure is given in Section “Materials and Methods.” Graphs of the spatial and temporal expression profiles of the individual metabolism genes are presented in Supplementary Figure S5, and detailed statistical significance provided in Supplementary File S2.

FIGURE 9

Seed traces in berries of V. vinifera cv. Black finger (BF) and cv. Spring blush (SB). Anatomy of representative berries of BF (A) and SB (B), harvested at 30 days after fruit set, and showing presence or absence of seed trace. Yellow circles indicate position of seed trace. Bar = 500 μm.