A comparative study of ripening among berries of the grape cluster reveals an altered transcriptional programme and enhanced ripening rate in delayed berries

Summary The developmental programme of grape berries within a cluster is coordinated to synchronize their ripening. Altered transcriptional events and metabolite accumulation are responsible for the differential progress of ripening.

color stage at the ripening rate characteristic of each berry class were obtained by fitting second-order polynomial curves. Using the respective equations we could predict when GH, GS, and PS berries would reach the TSS and color levels observed in RS berries (Supplementary worksheet 2B). The TSS level of RS berries at V, and at 1 and 2 weeks past-V were designated as Brix/color stages of R1, R2, and R3 respectively. Although RS berries took seven days to traverse the increments between the stages, the times calculated for under-ripe berries to traverse the same increments were lower (Table 1).

RNA isolation and NimbleGen Vitis vinifera GeneChip Array Hybridization and data analysis
Total RNA was isolated from the skin, pulp and seed tissues of the pooled berries using the RNeasy Midi Kit (Qiagen Inc., Valencia, CA). Because of the high sugar and phenolic content of the tissues, Qiagen RLC buffer (2% polyethylene glycol (MW 20,000), 0.2 M sodium acetate (pH 5.2), and 1% β-mercaptoethanol) was substituted for lysis buffer. For the reminder of the procedure, the manufacturer's protocol was followed, including on-column DNase digestion (RNase-free DNase, Qiagen, Valencia, CA). First-strand cDNA was prepared from 10 µg total RNA using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) and oligo dT primers. The secondstrand cDNA synthesis reaction contained 3 µg random primer, 5U Klenow fragment (New England Biolabs, Ipswich, MA), Klenow buffer, and 2.5 mM dNTP mixture.
Following incubation at 37 °C for 1.5 h, the double-stranded cDNA was purified using MiniElute PCR Purification Spin Columns (Qiagen Inc., Valencia, CA). The doublestranded cDNA was labeled using the NimbleGen One-Color DNA Labeling Kit Hybridization and washes were performed using NimbleGen reagents and hardware, and microarrays were scanned with an Axon GenePix 4200A Pro Scanner, according to the protocols provided by NimbleGen. Quality control, normalization, and the signal-to-noise ratio for hybridization signals for samples in V and PostV were performed using the webbased ANAIS tools (Simon and Biot, 2010). The Robust Multichip Average (RMA) normalization process involved background adjustment and quantile normalization (Irizarry et al., 2003).
Amplification of the single amplicon per PCR product was verified by analyzing dissociation curves. In this study, six V. vinifera reference genes were evaluated: gamma tonoplast aquaporin, 3 sucrose transporter 11, ERD6-like 16, vacuolar invertase 1, UDP-Glucose glycosyltransferase, and trehalose-6P phosphatase. Peptidyl-prolyl cis-trans isomerase (VIT_06s0004g06610) was used as a reference gene for normalization based on its low M value in our expression data (Vandesompele et al., 2002). All PCR reactions were done with three biological and two technical replicates. The reaction conditions were: heat activation at 95 °C for 5 min (one cycle); 95 °C for 10 s, 60 °C for 30 s (40 cycles). Data were acquired and exported with the 7500 Fast Software version 2.0.6 (Applied Biosystems) and relative gene expression was calculated using the ΔΔ C t method.
Relative fold-expression for each gene was calculated, in which the level of expression in GS berries at V was set at 1.

Identification of transcriptional modules during the ripening transitions
The data integration framework, 'DISTILLER' was used to identify transcriptional modules (co-expressed genes sharing overrepresented cis-regulatory elements or motifs) in the data of differentially expressed genes between the berry ripening stages (Lemmens activity scores were assigned for each motif based on the total number of modules it belongs to and the number of genes in those modules. Similar scores for motifs in each condition were assigned, and integrated with the overall activity score, which was normalized across the motifs. So the final activity scores reflect the relative activity of each motif between tissues, conditions, and across the motifs. Top 100 genes ranked in the order of their reduction in expression variance a (RV) from véraison to maturity among the berry classes. Marked in red are the 15% of the genes that were identified in the top 100 based on their véraison to maturity-expression differences b (Var) alone. Synch stage c denotes the timing of synchronization during the ripening progress assessed from véraison to maturity expression trend as early véraison stage (ES) and late maturity stage (LS) synchronizing genes (Supplementary worksheet 1B). RV is calculated as the ratio between véraison-and maturity-stage expression variances. Very high numbers for RV numbers are due to very low to zero variance at maturity stage.  Numbers in the columns are the percentage of berries assigned to the corresponding times based on berry ripening parameters, Color Index, total soluble solid content, and Elasticity.  Purple color denotes RS berries, and dark green, light green, and orange colors represent GH, GS, and PS berries, respectively. The stages V and 1, 2, and 3-weeks past-V are indicated with plus mark, closed circles, open circles, and closed squares, respectively. (D-G) Discriminant analyses showing the ripening advancement of individual berries within a class. Numbers in the columns are the percentage of berries assigned to the corresponding times based on berry ripening parameters, color (L, a, b, h, and C), total soluble solid content, and elasticity.