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

Abstract

Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis 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 CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR 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 CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR 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.

Figure 1. General outline of anthocyanins and flavan-3-ols biosynthetic pathway in plants.

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.

Figure 2. Generation of CsDFR and CsANR transgenic tobacco.

A, Graphic representation of pCAMBIA 1302 vector with cDNA of CsDFR and CsANR. CsDFR and CsANR cDNA was inserted in-between the NcoI and BglII restriction site of pCAMBIA 1302. B, Genomic DNA PCR confirmed the insertion of CsDFR and CsANR cDNA in plant genome of transgenic lines. C, Semi-quantitative PCR documented the transcript expression levels of CsDFR and CsANR 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.

Figure 3. Morphological characterization and yield parameters of CsDFR and CsANR overexpressing transgenic lines vis-à-vis mock tobacco plants.

CsDFR and CsANR overexpressing transgenic lines were longer than mock tobacco plants (A, B). Close up views showing early flowering in CsDFR and CsANR transgenic lines compared to mock tobacco (C). The CsDFR and CsANR overexpressing transgenic lines produced early flowering as compared to control tobacco plants (D). The capsule yield (E), seed yield (F) and seed weight per thousand seed (G) in CsDFR and CsANR overexpressing transgenic lines as compared to mock tobacco plants. Mean ± SD from three replications are shown. Statistical significance is indicated as (*) for P<0.05.

Figure 4. Transcript expression level of NtCHS and NtANR2 gene enhanced in CsDFR and CsANR transgenic tobacco.

A, Transcript expression level of NtCHS in CsDFR transgenic lines. B, Transcript expression level of NtCHS in CsANR transgenic lines. C, Transcript expression level of NtANR2 in CsDFR transgenic lines. D, Transcript expression level of NtANR2 in CsANR 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 error bars indicating ± SD. Statistical significance is indicated as (*) for P<0.05.

Figure 5. The CsDFR and CsANR overexpressing transgenic lines show higher contents of total flavonoids and increased flavan-3-ols content in transgenic tobacco compared to control tobaccos.

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

Figure 6. CsDFR and CsANR overexpression increased free radicals scavenging activity and lowered oxidative stress induced cell death.

A, The scavenging activity measured by DPPH method in CsDFR and CsANR overexpressing transgenic lines. B, H₂O₂ induced cell death in CsDFR and CsANR overexpressing transgenic lines. Data is the mean of three replications with error bars indicating ± SD. Statistical significance is indicated as (*) for P<0.05.

Figure 7. CsDFR and CsANR overexpression provided anti-herbivores effect against S. litura in transgenic tobacco.

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

Figure 8. Modulation in phytohormones level in CsDFR and CsANR overexpressing transgenic lines.

Endogenous GA₃ was decreased (A) and free indole acetic acid (IAA) was increased (B) in transgenics. Data is the mean of three replications with error bars indicating ± SD. Statistical significance is indicated as (*) for P<0.05.