Expression analyses of transgenes and key genes involved in the anthocyanin biosynthetic pathway in transgenic <i>Nicotiana tabacum</i> leaves.

<p><b>(A)</b> qRT–PCR analysis of <i>AmRosea1</i>, <i>AmDelila</i>, <i>MtANR</i> and <i>MtLAR</i> transgenes in transformed leaves of <i>N</i>. <i>tabacum</i>. Error bars correspond to the standard deviation of three replicates. The expression value of <i>AmRosea1</i> in plant Nt#5 was set to 1.00 and the expression levels of the rest of transgenes were plotted relative to this value. To normalize the samples the constitutive <i>NtACT8</i> gene was used. <b>(B)</b> RT-PCR expression analysis of key genes involved in the anthocyanin pathway in <i>N</i>. <i>tabacum</i> Nt#6 and Nt#7 transgenic plants. PCR results were obtained after 30 amplification cycles for all genes and 25 cycles for the housekeeping <i>NtACT8</i> gene.</p

Phenotypes of the <i>AmRosea1</i>:<i>AmDelila</i>:<i>MtANR</i>:<i>MtLAR</i> transgenic tobacco plants.

<p><b>(A)</b> Some of the <i>in vitro</i> regenerated transgenic calli showed an intense purple pigmentation due to accumulation of anthocyanins. <b>(B)</b> <i>In vitro</i> regenerated control plantlet. <b>(C)</b> <i>In vitro</i> regenerated transgenic tobacco plant showing intense purple color in all tissues. <b>(D)</b> Control plant after acclimation in the greenhouse. <b>(E)</b> Transgenic tobacco plant Nt#7 with intense purple pigmentation after acclimation in the greenhouse. <b>(F)</b> Detail of a leaf from the transgenic plant Nt#7 showing intense purple pigmentation in the abaxial side and vascular bundles. <b>(G)</b> Detail of a leaf from the trasngenic plant Nt#5 showing only small patches of purple pigmentation. <b>(H)</b> Entire and disected flower from a control (left) and transgenic plant Nt#7 (right). <b>(I)</b> Carpel and stamens from a disected flower of a control (left) and the Nt#7 transgenic plant (right).</p

Anthocyanin and proanthocyanidin biosynthetic pathways and multigenic construct assembly strategy.

<p><b>(A)</b> Schematic representation of the biosynthetic pathways for anthocyanins and proanthocyanidins. Abbreviations: chalcone synthase (CHS); chalcone isomerase (CHI); dihydroflavonol reductase (DFR); flavanone 3-hydroxylase (F3H); flavonol synthase (FLS); leucoanthocyanidin reductase (LAR); anthocyanidin synthase (ANS); anthocyanidin reductase (ANR); uridine diphosphate glucose-flavonoid 3-<i>O</i>-glucosyl transferase (UFGT). Purple colored arrows represent the catalytic steps that are upregulated by the overexpression of the <i>A</i>. <i>majus</i> transcription factors <i>Rosea1 and Delila</i>. The yellow highlighted area indicates the catalytic steps that are overexpressed in this work by the <i>M</i>. <i>truncatula</i> genes introduced in our multigenic construct, whose catalytic steps are highlighted in darker yellow and blue. <b>(B)</b> Premade GBParts, Modules and vectors used in this work. This includes the parts pCaMV35S promoter (GB0030), pTNos (GB0035), three vectors of the pGreenII-based pDGB1 series (Alfa1, Alfa2 and Omega2), one vector of the pCAMBIA pDGB2 series (Omega1) and two preassembled modules that were previously tested by the GB2.0 developers. The first module (GB0129) expresses the two <i>A</i>. <i>majus</i> transcriptional factors <i>Rosea1</i> and <i>Delila</i> that under the control of the CaMV35S promoter. The second module (GB0235) is the hygromycin resistant cassette that is used to select the transformed plants in the stable transformation process. CaMV35S is the Cauliflower Mosaic Virus 35S Promoter; TNos is the Nopaline synthase terminator; PNos is the Nopaline synthase promoter; K^R and S^R stand for bacterial kanamycin and spectinomycin resistance cassettes; LB and RB represent the Left and Right Borders of the T-DNA. <b>(C)</b> GoldenBraid 2.0 multigenic construct <i>AmRosea1</i>:<i>AmDelila</i>:<i>MtANR</i>:<i>MtLAR</i> generated in this work. The multigenic construct was generated in five steps that include the assembly of the <i>MtANR</i> and <i>MtLAR</i> transcriptional units from its basic parts (Assemblies 1 and 2), the combination of these transcriptional units in a single vector (Assembly 3), the later addition of the <i>A</i>. <i>majus</i> transcriptional factors to the <i>M</i>. <i>truncatula</i> genes (Assembly 4) and finally the incorporation of the hygromycin resistance cassette to generate the multigenic construct that is used in all the experiments of this work. <i>MtANR</i> is the <i>M</i>. <i>truncatula</i> anthocyanidin reductase gene; <i>MtLAR</i> is the <i>M</i>. <i>truncatula</i> leucoanthocyanidin reductase gene.</p

Relationship between purple phenotype, transgene expression and PAs production in three T1 <i>Nicotiana tabacum</i> plants.

<p><b>A-D.</b> Different leaf coloured phenotypes in the T1 of <i>N</i>. <i>tabacum</i> transgenic plants Nt#6.1 (green), Nt#6.8 (purple spots), Nt#6.11 (purple patches) and Nt#7.6 (full purple). <b>E.</b> In the lineage of plants Nt#6 and Nt#7 we analyzed by semi-qRT-PCR three plants with the complete set of transgenes showing a weak (Nt#6.8), a middle (Nt#6.11) and a strong purple phenotype (Nt#7.6). In the three plants the four transgenes were properly expressed. To normalize the samples the constituve <i>NtACT8</i> gene was used. <b>F.</b> The Nt#6.11, Nt#6.8 and Nt#7.6 transgenic plants produced PAs as demonstrated by HPLC-MS analysis of leaf extracts when compared with the WT.</p

Expression analyses of transgenes and key genes involved in the anthocyanin biosynthetic pathway in transgenic <i>Nicotiana tabacum</i> leaves.

<p><b>(A)</b> qRT–PCR analysis of <i>AmRosea1</i>, <i>AmDelila</i>, <i>MtANR</i> and <i>MtLAR</i> transgenes in transformed leaves of <i>N</i>. <i>tabacum</i>. Error bars correspond to the standard deviation of three replicates. The expression value of <i>AmRosea1</i> in plant Nt#5 was set to 1.00 and the expression levels of the rest of transgenes were plotted relative to this value. To normalize the samples the constitutive <i>NtACT8</i> gene was used. <b>(B)</b> RT-PCR expression analysis of key genes involved in the anthocyanin pathway in <i>N</i>. <i>tabacum</i> Nt#6 and Nt#7 transgenic plants. PCR results were obtained after 30 amplification cycles for all genes and 25 cycles for the housekeeping <i>NtACT8</i> gene.</p

Validation of the multigenic construct by transient expression in <i>Nicotiana benthamiana</i> leaves.

<p><b>(A)</b> Agroinfiltrated <i>N</i>. <i>benthamiana</i> leaf with the <i>AmRosea1</i>:<i>AmDelila</i>:<i>MtANR</i>:<i>MtLAR</i> multigenic construct showing purple pigmentation. <b>(B)</b> Infiltrated leaf with <i>DsRed</i> (control). <b>(C)</b> Non-infiltrated wild-type leaf. <b>(D)</b> Expression levels of the <i>AmRosea1</i>, <i>AmDelila</i>, <i>MtANR</i> and <i>MtLAR</i> transgenes in agroinfiltrated leaves of <i>N</i>. <i>benthamiana</i>. <i>NbACT8</i> is used as positive control. PCR results were obteined after 30 amplification cycles for all analized genes. <b>(E)</b> RT-PCR analysis of the anthocyanin biosynthetic pathway genes in agroinfiltrated leaves of <i>N</i>. <i>benthamiana</i>. The PCR results were obteined after 30 amplification cycles for all genes and 25 cycles for the housekeeping <i>NbACT8</i> gene. <b>(F)</b> PA levels in agroinfiltrated leaf extracts of <i>N</i>. <i>benthamiana</i> obteined by the dimethylaminocinnamaldehyde (DMACA) colorimetric reaction. The DMACA reaction showed an increase of the PA’s content in the agroinfiltrated leaf area compared with the non-infiltrated WT leaves. The maximum PA levels were found in the plants infiltrated with the multigenic construct. Statistical T-Test values for (-) catechin (n = 3, t = 11.46, df = 4, <i>p</i> = 0.0003) and (-) epicatechin (n = 3, t = 11.46, df = 4, <i>p</i> = 0.0003).</p

Proanthocyanidin levels in <i>Nicotiana tabacum</i> transgenic plants.

<p><b>(A)</b> Determination of proanthocyanidin levels in leaf extracts of <i>N</i>. <i>tabacum</i> Nt#6 and Nt#7 transgenic plants by dimethylaminocinnamaldehyde (DMACA) assays. Results show a signigicant increase of PA content in the transgenic leaves when compared with the wild-type plant. <b>(B)</b> Determination of PA levels in leaf extracts of transgenic <i>N</i>. <i>tabacum</i> Nt#6 and Nt#7 plants by phloroglucinolysis. Results show a great increase of total PA levels in the transgenic leaves of plant Nt#7 when compared with plant Nt#6 and a wild-type plant. Data represent the average of three replicates and the standard error for each sample.</p