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Pyramiding of tea Dihydroflavonol reductase and Anthocyanidin reductase increases flavan-3-ols and improves protective ability under stress conditions in tobacco

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Abstract

Tea (Camellia sinensis) is one of the richest sources of flavan-3-ols, an important class of flavonoids. The expression level of gene-encoded key regulatory enzymes of flavan-3-ol/anthocyanin biosynthetic pathway, dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR), has been highly correlated with the flavan-3-ol contents and antioxidant activity in tea plant. In the present study, pyramiding of CsDFR and CsANR in tobacco was achieved. However, single transgenic tobacco overexpressing either CsDFR or CsANR was documented earlier. In continuation, pyramided transgenic lines were evaluated for the possible, either same or beyond, effect on flavan-3-ol accumulation and protective ability against biotic and abiotic stresses. The pyramided transgenic lines showed early flowering and improved seed yield. The transcript levels of flavan-3-ol/anthocyanin biosynthetic pathway and related genes in pyramided transgenic lines were upregulated as compared to control tobacco plants. The accumulations of flavan-3-ols were also found to be higher in pyramided transgenic lines than control tobacco plants. In contrast, anthocyanin content was observed to be decreased in pyramided transgenic lines, while DPPH activity was higher in pyramided transgenic lines. In pyramided transgenic lines, strong protective ability against feeding by Spodoptera litura was documented. The seeds of pyramided transgenic lines were also found to have better germination rate under aluminum toxicity as compared to control tobacco plants. Interestingly, the synergistic effect of these two selected genes are not beyond from transgenic lines expressing either CsDFR and CsANR alone as published earlier in terms of flavan-3-ols accumulation. However, the unique flower color and better seed germination rate are some interestingly comparable differences that were reported in pyramided lines in relation to individual transgenic plants. In conclusion, the present results reveal an interesting dynamic between CsDFR and CsANR in modulating flavan-3-ol/anthocyanin levels and functional analysis of stacked CsDFR and CsANR transgenic tobacco lines.

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Abbreviations

4CL:

4-coumarate: CoA ligase

ABTS:

2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid)

AN2:

Anthocyanin 2

ANR1:

Anthocyanidin reductase 1

ANR2:

Anthocyanidin reductase 2

ANS:

Anthocyanidin synthase

C4H:

Chalcone-4-hydrolase

Cat:

Catechin

cDNA:

Complementary deoxyribonucleic acid

CHI:

Chalcone isomerase

CHS:

Chalcone synthase

DFR:

Dihydroflavonol 4-reductase

DPPH:

2,2-diphenyl-1-picryl-hydrazyl

EC:

Epicatechin

ECG:

Epicatechin gallate

EGC:

Epigallocatechin

F3H:

Flavanone 3-hydroxylase

FLS:

Flavonol synthase

MS:

Murashige and Skoog medium

Nt :

Nicotiana tabacum

PAL:

Phenylalanine lyase

PCR:

Polymerase chain reaction

RT:

Reverse transcriptase

ROS:

Reactive oxygen species

TT1:

Transparent testa 1

TT2:

Transparent testa 2

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Acknowledgements

The authors are thankful to the Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur for providing the necessary facility to conduct the research and valuable suggestions during course of work. Thanks are due to Dr. Gireesh Nadda for expert technical assistances in anti-feeding experiment. The Council of Scientific and Industrial Research (CSIR), GOI sponsored this work under NMITLI program (TLP003). VK is also thankful to CSIR for award of SRF.

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Authors and Affiliations

  1. Centre for Plant Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, 151001, India

    Vinay Kumar

  2. Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur, HP, 176061, India

    Vinay Kumar & Sudesh Kumar Yadav

  3. Center of Innovative and Applied Bioprocessing (CIAB), Knowledge City, Sector-81, Mohali, Punjab, India

    Sudesh Kumar Yadav

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  1. Vinay Kumar
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Contributions

VK and SKY conceived and designed present research. VK conducted experiments. VK and SKY analyzed data. VK wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Vinay Kumar.

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13205_2017_819_MOESM1_ESM.pptx

Table S1 Sequences of primers used in the present study. Figure S1 HPLC spectra details of standard (Catechin, Cat; Epicatechin, EC; Epicatechin gallate, ECG; Epigallocatechin, EGC) and identification of similar peaks in pyramided transgenic tobacco lines as compared to control tobacco plant in left and right side, respectively (PPTX 150 kb)

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Kumar, V., Yadav, S.K. Pyramiding of tea Dihydroflavonol reductase and Anthocyanidin reductase increases flavan-3-ols and improves protective ability under stress conditions in tobacco. 3 Biotech 7, 177 (2017). https://doi.org/10.1007/s13205-017-0819-1

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