Article Figures & Data
Figures
- Figure 1.
Scheme of branch pathways of phenylpropanoid metabolism in plants leading to the synthesis of anthocyanins, flavonols, PAs, and lignin. Enzymes that function in multiple or specific pathways are indicated. Abbreviations are as follows: ANR, anthocyanidin reductase; ANS/LDOX, anthocyanidin synthase; CAD, cinnamyl alcohol dehydrogenase; C4H, cinnamate 4-hydroxylase; CCR, cinnamyl-CoA reductase; C3H, 4-coumarate 3-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; COMT, caffeic acid O-methyltransferase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F5H, ferulate 5-hydroxylase; FLS, flavonol synthase; 3GT (UFGT), UDPG-flavonoid-3-O-glucosyltransferase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; PAL, Phe ammonia-lyase; 3RT, anthocyanidin-3-glucoside rhamnosyl transferase.
- Figure 2.
Features of VvMYB5a protein. A, Schematic representation of the major functional domains in the VvMYB5a protein based on data derived from sequence comparisons. R2 and R3 are the two repeats of the MYB DNA-binding domain (DBD). The IR domain corresponds to the residues involved in putative interaction with MYC proteins (glutamine-rich domain [GRD]). B, Phylogenetic analysis of a selection of plant MYB proteins was performed by using a BLAST algorithm on the GenBank and EMBL databases. The VvMYB5a protein sequence was aligned to known plant MYB sequences, and their distance relationships were resolved using the neighbor-joining tree-building method. The human protein c-MYB is used as an outgroup. See “Materials and Methods” for accession numbers.
- Figure 3.
VvMYB5a expression in grapevine tissues. Southern blots of semiquantitative RT-PCR products blotted onto nylon membrane and hybridized with the radiolabeled VvMYB5a 3′-UTR probe. Elongation factor EF1-γ was used as a control. A, Expression of VvMYB5a during grape berry development (berry flesh and skin). B, Expression of VvMYB5a in berry tissues. C, Expression of VvMYB5a in grapevine tissues harvested from fruit cutting.
- Figure 4.
Analysis of general phenylpropanoid gene expression in leaves of transgenic tobacco overexpressing VvMYB5a. A, Transcripts for three biosynthetic genes of the general phenylpropanoid metabolism (PAL, C4H, and 4CL) were detected by semiquantitative RT-PCR in leaves from three VvMYB5a independent lines (A–C) and compared to wild-type lines. VvMYB5a indicates the transgene expression level. UBI was used as a quantitative control. B, Quantification of the RT-PCR results shown in A was performed by measuring the intensity per millimeters squared (Quantity One software; Bio-Rad) of the bands and calculating the ratios between the bands in the gene-specific blot and its corresponding band in the UBI control blot (transcript ratio). Data are means ± se of two semiquantitative RT-PCR experiments from two independent RNA extractions. Asterisk (*) indicates a significant difference from wild-type plants (P ≤ 0.01; Student's t test).
- Figure 5.
Accumulation of phenylpropanoid compounds in VvMYB5a-overexpressing plants. Flowers of transgenic plants (B) showed a clear phenotypic change in petal and stamen pigmentation compared to wild-type flowers (A). Increased pigmentation is observed in petal epidermal cells of transgenic plants (F) compared to control lines (E). A strong red pigmentation is also observed in transgenic stamen epidermal cells (D and H) compared to wild-type stamens (C and G). DMACA staining of petal cells (J) and seed coats (L) from transgenic lines show CT accumulation compared to control petals (I) and seeds (K). E to J, Bars = 50 μm.
- Figure 6.
HPLC analysis of methanolic extracts from control and VvMYB5a petals and stamens. A, HPLC chromatograms at 521 nm from transgenic (VvMYB5a) and control (wild-type) petals. B, HPLC chromatograms at 521 nm from transgenic (VvMYB5a) and control (wild-type) stamens. Peaks 1, 2, and 4 are unknown. Peak 3 corresponds to cyanidin-3-rhamnoglucoside and peak 5 corresponds to quercitin-3-rhamnoglucoside. Each chromatogram has been obtained from the same injected quantity of sample (0.375 mg dry weight).
- Figure 7.
Analysis of transcript accumulation in flowers of transgenic tobacco overexpressing VvMYB5a. A, Transcripts for four flavonoid biosynthetic genes (CHS, CHI, F3H, and DFR) were detected by semiquantitative RT-PCR in petals from three VvMYB5a independent lines (A–C) and compared to wild-type lines. VvMYB5a indicates the transgene expression level. UBI was used as a quantitative control. B, Quantification of the RT-PCR results shown in A was performed by measuring the intensity per millimeters squared (Quantity One software; Bio-Rad) of the bands and calculating the ratios between the bands in the gene-specific blot and its corresponding band in the UBI control blot (transcript ratio). Data are means ± se of two semiquantitative RT-PCR experiments from two independent RNA extractions. Asterisk (*) indicates a significant difference from wild-type plants (P ≤ 0.01; Student's t test). C, Transcripts for four flavonoid biosynthetic genes (CHS, CHI, F3H, and DFR) were detected by semiquantitative RT-PCR in stamens from three VvMYB5a independent lines (A–C) and compared to wild-type lines. VvMYB5a indicates the transgene expression level. UBI was used as a quantitative control. D, Quantification of the RT-PCR results shown in C was performed by measuring the intensity per millimeters squared (Quantity One software; Bio-Rad) of the bands and calculating the ratios between the bands in the gene-specific blot and its corresponding band in the UBI control blot (transcript ratio). Data are means ± se of two semiquantitative RT-PCR experiments from two independent RNA extractions. Asterisk (*) indicates a significant difference from wild-type plants (P ≤ 0.01; Student's t test). E, Northern-blot analysis of ANS gene expression in petals and stamens from wild-type and VvMYB5a lines. Total RNA was extracted from tissues harvested at the opening of flowers. Experiments were repeated with three VvMYB5a and wild-type lines with similar results.
- Figure 8.
Modification of monolignol metabolism in transgenic anthers. A, Cross sections of anthers at stage 10 from wild-type (I–III) and VvMYB5a-overexpressing tobacco plants (IV–VI). Arrows indicate endothecial cells developing complete (II) or incomplete (V) lignified fibers (shown more enlarged in III and VI, respectively). ep, Epidermis; end, endothecium. Bars = 500 μm (I and IV), 100 μm (II and V), and 20 μm (III and VI). B, Transcripts for monolignol biosynthetic genes (COMT, CAD, and CCoAOMT1, 5, and 6) were detected by semiquantitative RT-PCR in stamens from three VvMYB5a independent lines (A–C) and compared to wild-type lines. VvMYB5a indicates the transgene expression level. UBI was used as quantitative control. C, Quantification of the RT-PCR results shown in B was performed by measuring the intensity per millimeters squared (Quantity One software; Bio-Rad) of the bands and calculating the ratios between the bands in the gene-specific blot and its corresponding band in the UBI control blot (transcript ratio). Data are means ± se of two semiquantitative RT-PCR experiments from two independent RNA extractions. Asterisk (*) indicates a significant difference from the wild-type plants (P ≤ 0.01; Student's t test).
Tables
- Table I.
Epicatechin and catechin content (milligrams per gram fresh weight tissue) of wild-type and transgenic plants from petal extracts
Quantitative determination of catechin and epicatechin content in tobacco flowers has been performed by HPLC. The eluate was monitored at 280 nm. Purified standards of catechin and epicatechin were used as controls. Data are the means ± sd of two replicates. N.D., Not detected.
Sample Catechin Epicatechin VvMYB5a line A N.D. 10.05 ± 0.56a VvMYB5a line B N.D. 11.22 ± 0.47a VvMYB5a line C N.D. 9.35 ± 0.64a Wild-type plants N.D. 1.18 ± 0.43 ↵a Significantly different from wild type (P < 0.005; Student's t test).
- Table II.
Total anthocyanin content (milligrams per gram dry weight tissue) of wild-type and transgenic plants from stamen and petal extract
Quantitative determination of total anthocyanin content in tobacco flowers has been performed by spectrophotometry at 535 nm. A purified standard of keracyanin was used as a control. Data are the means ± sd of four replicates. N.D., Not detected.
Sample Stamens Petals VvMYB5a line A 4.93 ± 0.07 3.51 ± 0.12a VvMYB5a line B 4.20 ± 0.09 3.42 ± 0.07a VvMYB5a line C 3.86 ± 0.08 3.24 ± 0.08a Wild-type control N.D. 1.48 ± 0.26 ↵a Significantly different from wild type (P < 0.001; Student's t test).
- Table III.
Keracyanin content (milligrams per gram dry weight tissue) of wild-type and transgenic plants from stamen and petal extract
Quantitative determination of keracyanin content in tobacco flowers has been performed by HPLC. The eluate was monitored at 521 nm. A purified standard of keracyanin was used as a control. Data are the means ± sd of four replicates. N.D., Not detected.
Sample Stamens Petals VvMYB5a line A 3.81 ± 0.41 2.20 ± 0.05a VvMYB5a line B 3.54 ± 0.39 2.02 ± 0.04a VvMYB5a line C 3.42 ± 0.31 1.99 ± 0.12a Wild-type plants N.D. 1.18 ± 0.42 ↵a Significantly different from wild type (P < 0.01; Student's t test).
Genes Accession No. Sense Primers Antisense Primers Nt-PAL D17467 5′-GCCAACAGGATAAAAGAATGC 5′-TCCCACAATATAAGCCCAAGC Nt-4CL U50845 5′-CACACTGGCCACATTGGGTTCATT 5′-TTCGCCCGTTGCCAAACTGGAAAT Nt-CHS AF311783 5′-CCTTTGGGAATTTCTGATTGG 5′-TCCCACAATATAAGCCCAAGC Nt-F3H AF036169 5′-AGCTAGAGACTACTCCAGGTG 5′-AACCGTGATCCAAGTTTTGCCA Nt-COMT X74453 5′-CTTGGAGGATTAAGCAATATA 5′-AAGCTTTTTCCTAACACACTGC Nt-CAD X62343 5′-ACTGCAATGGAGAGGTTTGGAG 5′-TACATAAACGTCACTCTCGATCAC Nt-CCoAOMT-1 U38612 5′-AACCGGGAAAACTACGAGATTGGT 5′-TCTTGGTTGCCACAAATGACGAT Nt-CCoAOMT-5 AF022775 5′-GGGTACTCCCTCCTTGCTACTGCC 5′-ATATTGGAATGATCAGGTGATGCG Nt-CCoAOMT-6 Z56282 5′-GACAACACCCTATGGAATGG 5′-ATAGCGATAATCATGAGATAC Nt-UBI U66264 5′-TCCAGGACAAGGAGGGTAT 5′-GAGACCTCAGTAGACAAAGC VvMYB5a-3′-UTR AY555190 5′-CCCCACCCAGCAATTTCTGTG 5′-CCATTTCACATACGATATTCACAC Vv-EF-γ AF176496 5′-GCGGGCAAGAGATACCTCAA 5′-TCAATCTGTCTAGGAAAGGAAG
Supplemental Data
Supplemental Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Table I
- Supplemental Data - Supplemental Table II
