In silico analyses
The National Center for Biotechnology Information (NCBI) Entrez search and retrieval system was used to obtain nucleotide and protein sequences from the Genbank databases (http://www.ncbi.nlm.nih.gov/gquery). Alignments to sequences in the Genbank databases were performed using the relevant Blast algorithm (http://www.ncbi.nlm.nih.gov/BLAST/) . Comparative genomics (i.e. gene structure prediction and homologue/orthologue retrieval) were performed via PLAZA (http://bioinformatics.psb.ugent.be/plaza/) .
The putative sub-cellular localisation of protein sequences were predicted using ProtComp Version 8.0 (http://www.softberry.com/berry.phtml). V. vinifera expressed sequence tags (ESTs) were retrieved from The Institute for Genomic Research (TIGR) Grape Gene Index (http://compbio.dfci.harvard.edu/tgi/) or NCBI. The V. vinifera genomic sequences were retrieved from NCBI or Genoscope (http://www.cns.fr/externe/GenomeBrowser/Vitis/). Protein alignments were performed with Clustal Omega (http://www.ebi.ac.uk/tools/msa/clustalo/), and the resultant phylogenetic trees visualised using MEGA .
Grapevine material for gene isolation, native expression analysis and pigment analysis was harvested from field-grown Vitis vinifera L. cv. Pinotage at Welgevallen experimental farm (Stellenbosch, South Africa). Green, véraison and ripe berries, as well as fully expanded leaf and mature flower material were harvested and flash frozen in the field in liquid nitrogen. The frozen tissue was homogenised in liquid nitrogen and, if not used immediately, stored at -80°C.
Transgenic plants were housed in a greenhouse and grown in a commercial soil mixture supplemented with Nitrosol® every 3 weeks. Analyses of gene expression, pigment concentrations and volatile composition were performed on tissue from fully expanded leaf tissue (leaf position 3 and 4). Leaves were flash frozen in liquid nitrogen immediately upon harvesting and stored in the dark at -80°C. Control plants underwent the same tissue culture, hardening-off and glass house conditions and procedures as the transgenic population.
Isolation, extraction and manipulations of nucleic acids
High molecular weight genomic DNA was isolated from fully expanded V. vinifera leaves as described by Steenkamp et al.. Total RNA from different grapevine leaves was extracted according to the methods described by Reid et al.. Unless otherwise stated, all standard methods for plasmid DNA isolation, manipulations and cloning of DNA fragments, and agarose gel electrophoresis were used as described by Sambrook et al..
Total cDNA was synthesised from 1 μg DNase I-treated (Promega, Madison, WI) total RNA using the Superscript III Platinum first strand synthesis system (Invitrogen) in a 20 μL reaction volume as described by the supplier.
Bacterial strains, media, growth conditions, and transformations
Escherichia coli (DH5α and TOP10F) and Agrobacterium tumefaciens (EHA105) cultures were grown in LB media (1.2% (w/v) tryptone, 1.2% (w/v) NaCl and 0.6% (w/v) yeast extract). Bacterial transformations were performed using the heat-shock method as described in Sambrook et al.. Transformants were selected using the appropriate antibiotic as selection on LB plates. Putative positive colonies were cultured and their plasmids isolated and verified by restriction digest. Unless otherwise stated all E. coli cultures were grown at 37°C and A. tumefaciens cultures at 30°C.
Plasmids, cloning and bacterial transformations
The carotenoid accumulating strains used to perform the functional complementation assays were obtained from F. X. Cunningham (Department of Cell Biology and Molecular Genetics, University of Maryland, MD, USA) and are described in Cunningham  and Cunningham et al.[32, 33].
The primer pair VvCCD1_5’ and VvCCD1_3’ was used to amplify the VvCCD1 gene from V. vinifera L. cv. Pinotage cDNA (Additional file 1). The primer pairs VvCCD4a_5’, VvCCD4a_3’ and VvCCD4b_5’, VvCCD4b_3’ were used to amplify VvCCD4a and VvCCD4b from V. vinifera L. cv. Pinotage cDNA (Additional file 1). The resultant PCR amplicons were cloned into the pGEM-T Easy vector system according to the specifications of the supplier (Promega), to generate the plasmids pGEMt-VvCCD1, pGEMt-VvCCD4a and pGEMt-VvCCD4b (Additional file 2).
For the construction of plasmids for bacterial functional complementation, the 1715 bp VvCCD1 coding region was excised from pGEMt-VvCCD1 as an Ndel/PstI fragment and cloned into the corresponding sites of pTWIN1 to yield pTWIN1-VvCCD1. The 1722 bp VvCCD4a coding region was excised from pGEMt-VvCCD4a as Ndel/BglII fragment and cloned into the compatible Ndel/BamHI sites of pTWIN1 to yield pTWIN1-VvCCD4a. The 1770 bp VvCCD4b coding region was excised from pGEMt-VvCCD4b as an Ndel/BamHI fragment and cloned into the corresponding sites of pTWIN1 to yield pTWIN1-VvCCD4b (Additional file 2).
The binary vector, pART27 was used for both the overexpression and silencing constructs . The 1715 bp VvCCD1 coding region was excised from pGEMt-VvCCD1 as a SalI/SpeI fragment and cloned into the compatible XhoI/XbaI sites of pART7 to yield pART7-VvCCD1. The expression cassette was excised with NotI and cloned into the corresponding site of pART27 to yield pART27-VvCCD1.
The pHANNIBAL vector was used for the construction of a VvCCD1 RNAi/silencing vector . A 148 bp fragment was PCR-amplified from the 3’ untranslated region (UTR) of VvCCD1 from V. vinifera L. cv Pinotage genomic DNA using the primer pair VvCCD1_RNAi_5’ and VvCCD1_RNAi_3’ (Additional file 1). The pGEM®-T Easy vector system was used to clone the PCR amplicon according to the specifications of the supplier (Promega), creating the pGEMt-CCD1(RNAi) plasmid. A 136 bp XhoI and EcoRI fragment was excised from pGEMt-CCD1(RNAi) and ligated into the corresponding XhoI and EcoRI sites in pHANNIBAL. The resultant plasmid was subsequently digested with BamHI and XbaI and the 148 bp BamHI and XbaI fragment from pGEMt-CCD1(RNAi) was ligated into the corresponding sites. The resultant plasmid, pHANNIBAL-CCD1(RNAi), contained a 148 bp inverted repeat of the 3’-UTR of VvCCD1. The expression cassette was excised from pHANNIBAL-CCD1(RNAi) with NotI, and ligated into the corresponding NotI site of pART27 yielding the final VvCCD1 silencing vector, pART27-CCD1(RNAi) (Additional file 2).
Grapevine transformation and regeneration
Somatic embryogenic cultures of V. vinifera L. cv. Sultana were used as source material for the genetic transformation experiments. The somatic embryogenic cultures were obtained and maintained according to the methods described in Vasanth and Vivier . The genetic transformation protocol was essentially according to Franks et al. with some modifications to use liquid cultures as starting material (as described in Vasanth and Vivier ). Briefly, Agrobacterium tumefaciens cells, containing either the overexpression or silencing vector, were harvested by centrifugation at 5,000 rpm for 10 min and resuspended in liquid NN medium, containing 18.5 μg.mL-1 maltose to a final OD600 of 0.8. Acetosyringone (19.7 mg.L-1) was added to the agrobacterial suspension before 2 mL of the somatic embryogenic cell suspensions were added and left for 15 min, with gentle shaking three to five times during this period. The culture was filtered to remove the excess liquid and the callus blotted dry using sterile Whatman no.1 filter paper. Co-cultivation proceeded at 27°C in the dark for 2 days on solid NN medium supplemented with BAP (0.25 μg.mL-1), NOA (1.0 μg.mL-1) and acetosyringone (19.7 μg.mL-1). Subsequently embryos were washed with sterile NN medium containing carbenicillin (200 μg.mL-1), blotted dry on sterilised filter paper and handled according to the protocol of Franks et al. . Selection on kanamycin (100 μg.mL-1) was maintained until in vitro rooted plantlets were obtained, and subsequently hardened off in a greenhouse.
Southern blot analysis
Southern blot analysis was performed using 10–20 μg of genomic DNA extracted from grapevine leaves. The DNA was digested with SpeI, separated in a 0.8% (w/v) TBE agarose gel and transferred to a positively charged Hybond-N nylon membrane as described by the supplier (Amersham-Pharmacia Biotech, Buckinghamshire, UK). Probe labelling, hybridisation, and Biotin detection were performed using the Biotin non-radioactive nucleic acid labelling and detection system according to the specifications of the supplier (Roche Diagnostics, Mannheim, Germany).
Expression analysis of VvCCDs
Primers for qRT-PCR for the expression analysis of VvCCD1, VvCCD4a, VvCCD4b, VvCCD4c, VvCCD4d, VvCCD7 and VvCCD8 were designed using Primer Express 3.0 (Applied Biosystems) (Additional file 1). The V. vinifera elongation factor 1α (VvEF1α) was selected as a “house-keeping” gene to normalise gene expression based on the findings of Reid et al. and Guillaumie et al.; Relative expression analysis of the VvCCD gene family was performed in three different berry developmental stages, corresponding to green, vérasion and ripe berry stages of V. vinifera L. cv Pinotage.
Expression analysis of VvCCD1 in the transgenic grapevine population was similarly performed via qRT-PCR. The grapevine glyceraldehyde-3-phosphate dehydrogenase (VvGAPDH) gene was used as a “house-keeping” gene to normalise gene expression. The expression of VvGAPDH has been shown to be relatively invariant in grapevine berries . Primers were designed to evaluate total VvCCD1 expression (i.e. endogenous and transgene-derived expression), as well as expression derived from only the transgenic VvCCD1 (Additional file 1).
Real-time PCR was performed using an Applied Biosystems 7500 Real-time PCR System. KAPA SYBR® FAST qRT-PCR Kit was used according to the manufacturer’s (Kapa Biosystems, Cape Town, South Africa) instructions. The programme for the PCR reactions was: 50°C for 2 minutes; 95°C for 10 minutes; and 40 cycles of 15 seconds at 95°C and 60 seconds at 58°C. Data were analysed using the Applied Biosystems SDS software (version 1.4). All PCR reactions consisted of at least three technical replicates. Relative expression was calculated using the equation as described by Pfaffl [39
(where E is the PCR efficiency and CP is the cycle number that the florescence crosses the base line).
Bacterial functional complementation and determination of volatile apocarotenoids in bacterial headspace
The extraction and analysis of the volatile apocarotenoids from bacterial cultures were performed based on a method described in Lücker et al.. For clarity the method and relevant modifications are described in detail. pTWIN1-VvCCD1, pTWIN1-VvCCD4a and pTWIN1-VvCCD4b plasmids were introduced into carotenoid accumulating E. coli strains. An empty vector, pTWIN1, was used as a negative control. An overnight culture (5 mL) grown in LB media to saturation was used to inoculate 32 mL of LB containing the appropriate antibiotics (100 μg.mL-1 ampicillin, 34 μg.mL-1 chloroamphenicol and 12.5 μg.mL-1 tetracycline) until an OD600nm of 0.1 was reached. The cultures were incubated in the dark, gently shaking at room temperature until an OD600 nm of 0.6 was reached. To prevent further production of coloured carotenoids the inhibitor diphenylamine (DPA) was added to a final concentration of 100 μM (as described in Cunningham and Gantt ) and the cultures were incubated at room temperature for an additional two hours in the dark. After the two hour incubation with the inhibitor, 8 mL of the 32 mL culture was removed and flash frozen for carotenoid analysis; and the remaining 24 mL of culture was harvested for apocarotenoid analysis. For apocarotenoid analysis the cells were resuspended in 6 mL of LB containing the appropriate antibiotics, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and 1 ppm of α-terpineol (as internal standard, IS). In addition 6 mM ascorbate, 5 μM ferrous sulphate, 200 U/mL catalase were added as described in Baldermann et al. for in vitro CCD1 enzyme assays. The cultures were subsequently transferred to 20 ml SPME vials. The vials were sealed with Bi-metal®crimp seals with 20 mm silicone/polytetrafluoroethylene (PTFE) septa (Brown Chromatographic supplies, Wertheim, Germany). The samples were incubated in the dark, gently shaking at room temperature for 16 hours. After 16 hours, 1 mL of culture was removed for OD600nm determination and 5 mL of 5 M NaCl was added to the 5 mL remaining culture for volatile apocarotenoid extraction.
The HS-SPME extraction of volatile apocarotenoids from the bacterial cultures was performed using a CTC CombiPal auto sampler equipped with the SPME option (CTC Analytics, Switzerland). Extraction conditions were as follows: after incubation at 50°C for 2 minutes SPME extraction was performed for 15 min under constant agitation by exposing a divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fibre (Supelco, Bellefonte, PA) to the headspace. Thereafter the fibre was desorbed and analytes subsequently injected onto the GC column using a split/splitless injector, operated at 240°C, splitless for 2 minutes. The fibre was left in the injector for a further 20 minutes at 270°C for conditioning of the fibre under a purge flow of 60 mL/min. Separation of compounds was achieved on a DB-FFAP column (60 m × 0.25 mm × 0.5 μm) on an Agilent 6890 gas chromatograph coupled to an Agilent 5975C mass spectrometer (MS) (Agilent Technologies, Little Falls, Wilmington, USA). The helium carrier gas flow through the column was 1.2 mL/min and the oven programmed from 40°C (held 5 min), ramped at 10°C/min to 230°C (held for 2 min), with a post run at 240°C (held for 2 min). The total run time was 30 minutes.
The MS was operated in electron impact (EI) mode (70 eV) using Selected Ion Monitoring (SIM), simultaneously acquiring scan data as well. In SIM mode the following m/z fragments were monitored: α-terpineol (IS) (59, 93, 136), 6-methyl-5-hepten-2-one (MHO) (69, 111, 126), geranyl acetone (69, 136, 121), α-ionone (121, 136, 192), β-ionone (136, 177, 192) and pseudo-ionone (81, 109, 135). Compound identification was performed by both comparisons of the retention times with that of authentic standards and the NIST2005 mass spectral library (National Institute of Standards, USA). Peaks of interest were quantified by external standard curves of the authentic standards. Data were normalised to the internal standard concentration (α-terpineol) and to the OD600 of the bacterial cultures before HS-SPME analysis.
Chemical analysis of leaf photosynthetic pigments and volatile apocarotenoids
Photosynthetic pigments of leaves were analysed using the method described in Lashbrooke et al..
Leaf volatiles were extracted according to the method described by Lücker et al. with modifications. Frozen, ground leaf tissue (200 mg) was placed in a 20 mL SPME vial and 10 mL of 20% (w/v) NaCl containing 160 ng 3-octanol (as internal standard, IS). The vial was sealed with a PTFE/silicon septum. Samples in sealed SPME vials were heated to 80°C and incubated for 5 minutes before the injection needle was pierced through the septum exposing the divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 μm coated solid phase microextraction (SPME) fibre (Supelco, Belfonte, PA, USA) to the headspace of the sample. During extraction the sample was stirred at 500 rpm and maintained at 80°C. After 15 minutes the fibre was removed and injected into the GC inlet where it was desorbed for 10 minutes at 260°C.
Extracts were analysed using an Agilent 6890 Gas Chromatograph coupled to a Waters GCT Time of Flight (TOF) Mass Spectrometer (MS) (Waters Corporation, Milford, MA, USA) Waters Masslynx GC/MS workstation software were used to analyse the data. The injector port was heated to 260°C and splitless injection (with a purge time of 3 minutes) was used. Separation was performed on an HP5MS (Agilent Technologies, Palo Alto, CA) column (30 mL × 0.25 mm i.d. × 0.25 μm f.t.) with helium as the carrier gas at a constant flow of 1 mL.min-1. The initial oven temperature was 40°C for 5 minutes, after which the temperature was increased by 5°C.min-1 to 150°C and then at 10°C.min-1 to 280°C (held for 2 minutes). Ionisation was in electron impact mode with an electron energy of 70 eV. The MS detector was set as follows: The transfer line, ion source and trap temperatures were 250, 180 and 150°C, respectively. The mass range was 35 to 650 m/z, with a scan rate of 4 scans.s-1. Compound identification was performed by both comparisons to the retention times of authentic standards and to the NIST05 mass spectral library (National Institute of Standards, USA).