Functional & Integrative Genomics

, Volume 17, Issue 4, pp 399–412

Transcriptome response and developmental implications of RNAi-mediated ODC knockdown in tobacco

Original Article

DOI: 10.1007/s10142-016-0539-3

Cite this article as:
Choubey, A. & Rajam, M.V. Funct Integr Genomics (2017) 17: 399. doi:10.1007/s10142-016-0539-3

Abstract

Polyamines (PAs) are ubiquitously present polycationic compounds that play a critical role in various growth and developmental processes including stress responses in plants. Yet, their specific functions and mode of action remain largely unknown. In the present study, we have targeted tobacco ornithine decarboxylase gene (ODC) by RNA interference to modulate cellular PA levels and study the effects at different developmental time points. Down-regulation of ODC resulted in significant physiological and morphological anomalies including reduced leaf size, reduced chlorophyll and carotene content, decreased abiotic stress tolerance, early onset of senescence, delayed flowering, partial male and female sterility, reduced seed setting, delayed seed germination, reduced seed viability, and poor in vitro regeneration response from leaf explants. Also, for the first time, microarray analysis has been attempted to study genome-wide gene expression changes in response to lowered PA titers in an ODC knockdown line. A number of transcription factors, auxin- and ethylene-responsive genes, stress-induced genes, lignin-biosynthesis genes, photosynthesis-related genes, senescence-associated genes, membrane proteins, and protein kinases were found to be affected, suggesting a probable list of PA-responsive genes. Transcriptome analysis has also indicated many genes, which could directly or indirectly be responsible for regulating the PA metabolic pathway. Various phenotypic changes observed upon ODC knockdown along with the identification of a number of gene targets means it is a step forward in envisaging possible mechanisms of PA action and for assigning them with specific roles in various developmental processes they are known to be a part of.

Keywords

Polyamines Ornithine decarboxylase RNA interference Transcriptome analysis Development Nicotiana tabacum 

Supplementary material

10142_2016_539_MOESM1_ESM.jpg (89 kb)
Fig. S1Agrobacterium-mediated tobacco transformations. A) T-DNA map of ODC-RNAi binary vector; B) ODC-hpRNAi transformed tobacco explants on w/o (i) and with 0.5 mM Put (ii) supplemented tobacco shoot regeneration medium (SRM, MS Basal, BAP- 1 mg/L; NAA- 0.1 mg/L; Kan- 100 mg/L; Aug- 300 mg/L); Positive control (iii). (JPEG 89 kb)
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Fig. S2Molecular characterization of transgenics. A) and B) PCR screening of putative tobacco ODC- hpRNAi lines with nptII specific primers. L1–1 kb DNA ladder; Wt- Untransformed wild-type DNA control; ND- No DNA control; +C- Positive control (vector plasmid); Numbers indicate transgenic lines. C) Southern blot analysis of ODC-RNAi using nptII gene probe. (JPEG 81 kb)
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Fig. S3Morphological features of ODC-RNAi lines. A) Lateral root formation in ODC-RNAi lines as seen in culture tubes (ii) and germinating seedlings (iv); i) and iii) are controls; B) Early onset of senescence as seen under culture-room conditions (i) and green house conditions (ii). (JPEG 103 kb)
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Fig. S4Growth pattern of seedlings. Growth after 4 weeks (A), 8 weeks (B) and >12 weeks (C) of inoculation on MS medium, with and without kan (100 mg/L) as indicated. (JPEG 75 kb)
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Fig. S5Abiotic stress response. Representative images showing phenotypic differences between leaf disks of wild-type and ODC-RNAi lines after treatment with 100, 200 and 400 mM NaCl solution for 3 days (A) and 5 days (B). (JPEG 133 kb)
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Table S1Primers used in the cloning of ODC-RNAi construct, screening of transgenics (npt II), and expression analysis by quantitative RT-PCR. (DOC 110 kb)
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Table S2Regeneration frequency in transformation experiments with ODC-RNAi construct (without Put). Regeneration was 100% in case of non-transformed control (DOC 11 kb)
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Table S3Regeneration frequency in transformation experiments with ODC-RNAi construct (with 0.5 mM Put). Regeneration was 100% in case of non-transformed control (DOC 11 kb)

Funding information

Funder NameGrant NumberFunding Note
University of Delhi
  • R & D grant - 2014-15

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of GeneticsUniversity of Delhi South CampusNew DelhiIndia

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