Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance

<div><p>Flavan-3-ols contribute significantly to flavonoid content of tea (<i>Camellia sinensis</i> L.). Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA <i>CsDFR</i> and <i>CsANR</i> encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of <i>CsDFR</i> and <i>CsANR</i> produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of <i>CsDFR</i> and <i>CsANR</i> transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing <i>CsDFR</i> and <i>CsANR</i> also showed resistance against infestation by a tobacco leaf cutworm <i>Spodoptera litura</i>. Results suggested that the overexpression of <i>CsDFR</i> and <i>CsANR</i> cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance.</p></div

Physical Modelling of Flow and Dispersion in Urban Canopy

Katedra fyziky atmosféryDepartment of Atmospheric PhysicsFaculty of Mathematics and PhysicsMatematicko-fyzikální fakult

General outline of anthocyanins and flavan-3-ols biosynthetic pathway in plants.

<p>The enzymes are: PAL, Phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl CoA-ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H flavanone-3-hydroxylase; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanin synthase; ANR1, anthocyanin reductase1; ANR2, anthocyanin reductase2; GT, Glucosyl transferase.</p

Generation of <i>CsDFR</i> and <i>CsANR</i> transgenic tobacco.

<p>A, Graphic representation of pCAMBIA 1302 vector with cDNA of <i>CsDFR</i> and <i>CsANR</i>. <i>CsDFR</i> and <i>CsANR cDNA</i> was inserted in-between the <i>NcoI</i> and <i>BglII</i> restriction site of pCAMBIA 1302. B, Genomic DNA PCR confirmed the insertion of <i>CsDFR</i> and <i>CsANR cDNA</i> in plant genome of transgenic lines. C, Semi-quantitative PCR documented the transcript expression levels of <i>CsDFR and CsANR</i> in transgenic tobacco lines. Housekeeping gene 26S rRNA was used as internal control for expression study and experiments were repeated at least three times with similar results.</p

<i>CsDFR</i> and <i>CsANR</i> overexpression provided anti-herbivores effect against <i>S. litura</i> in transgenic tobacco.

<p>picture showed less feeding by <i>S. litura</i> on leaf discs of <i>CsDFR</i> transgenic tobacco line (A) and <i>CsANR</i> transgenic tobacco line (B) as compared to leaf discs of control tobacco plants. The relative percentage growth inhibition of <i>S. litura</i> feeding on leaf discs of selected <i>CsDFR</i> lines (C) and <i>CsANR</i> lines (D) as compared to relative percentage growth inhibition on leaf discs of control tobacco plants. Data is the mean of three replications with <i>error bars</i> indicating ± SD.</p

Additional file 2: of Strategies for high-altitude adaptation revealed from high-quality draft genome of non-violacein producing Janthinobacterium lividum ERGS5:01

Table S1. Whole genome sequence-based in silico comparison of strain ERGS5:01 and other related Janthinobacterium strains in database for DDH and ANI. (DOCX 17 kb

Transcript expression level of <i>NtCHS</i> and <i>NtANR2</i> gene enhanced in <i>CsDFR</i> and <i>CsANR</i> transgenic tobacco.

<p>A, Transcript expression level of <i>NtCHS</i> in <i>CsDFR</i> transgenic lines. B, Transcript expression level of <i>NtCHS</i> in <i>CsANR</i> transgenic lines. C, Transcript expression level of <i>NtANR2</i> in <i>CsDFR</i> transgenic lines. D, Transcript expression level of <i>NtANR2</i> in <i>CsANR</i> transgenic lines. Expression of 26S rRNA was used as internal control and experiment was repeated at least three times. Below gel pictures relative level of expression is shown with bar diagram and values are mean of three replications with <i>error bars</i> indicating ± SD. Statistical significance is indicated as (*) for P<0.05.</p

Additional file 3: of Strategies for high-altitude adaptation revealed from high-quality draft genome of non-violacein producing Janthinobacterium lividum ERGS5:01

Figure S2. Certificate of deposition of strainERGS5:01 at Microbial Culture Collection (MCC) at National Centre for Cell Science, Pune, India. (PDF 377 kb

The <i>CsDFR</i> and <i>CsANR</i> overexpressing transgenic lines show higher contents of total flavonoids and increased flavan-3-ols content in transgenic tobacco compared to control tobaccos.

<p>Three flavan-3-ols namely catechin, epicatechin and epigallocatechin were measured in <i>CsDFR</i> and <i>CsANR</i> transgenic lines vis-à-vis control tobacco. Total flavonoids in <i>CsDFR</i> and <i>CsANR</i> overexpressing transgenic tobaccos (A). The catechin (B), epicatechin (C) and epigallocatechin (D) contents in <i>CsDFR</i> and <i>CsANR</i> transgenic lines as well as control tobacco plants. Data is the mean of three replications with <i>error bars</i> indicating ± SD. Statistical significance is indicated as (*) for P<0.05.</p

Additional file 4: of Strategies for high-altitude adaptation revealed from high-quality draft genome of non-violacein producing Janthinobacterium lividum ERGS5:01

Table S2. Genes predicted to encode industrially important enzymes in the genome of J. lividum ERGS5:01. (DOCX 14 kb