Ectopic expression of AtICE1and OsICE1 transcription factor delays stress-induced senescence and
improves tolerance to abiotic stresses in tobacco
Journal name: Journal of Plant Biochemistry and Biotechnology
Nagaveni Budhagatapalli1, Rama Narasimhan1, Jeyaraman Rajaraman1, Chinnusamy Viswanathan2 and
Karaba N Nataraja1*
1
Department of Crop Physiology, University of Agricultural Sciences, Gandhi Krishi Vignana Kendra,
Bangalore, 560 065, Karnataka, India.
2
Division of Plant Physiology, Indian Agricultural Research Institute, Pusa Campus, New Delhi, 110 012, Delhi,
India.
*Corresponding author:
Karaba N Nataraja
Department of Crop Physiology,
University of Agricultural Sciences,
Gandhi Krishi Vignana Kendra,
Bangalore, 560 065, Karnataka, India
Telephone: 918023636713 Telefax: 918023636713
nataraja_karaba@yahoo.com; nnkaraba@uasbagalore.edu.in
Supplementary table: List of primers used in the study
Primers
Sequences (5’-3’)
a. Used for cloning the gene (with attB flanking sequence underlined)
OsICE1
F-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGGACTCCTCTTCTCTTCTC
R-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCAGTTACATCATCATCA
AtICE1
F-GGGGACAAGTTTGTACAAAAAAGCAGGCTCATGAAATCCTCGTGATTCTAC
R-GGGGACCACTTTGTACAAGAAAGCTGGGTGTATCCAACAACAACAACCT
b. Used for expression studies
OsICE1
F-TTGAGGGCTCTCGACAGCCTT
R-TCTGAAGGCCAGCAGAGTGCAG
AtICE1
F-GACCGGGTCTGTTGCTCGCTACCA
R-ACCAGCATACCCTGCTGTATCGA
c. ICE1 downstream gene primers with their respective tobacco locus ID in brackets
HSF1 (TC5803)
COR47 (TC4052)
ERD10 (TC5349)
WRKY6 (TC3961)
HVA22 (TC5661)
EREBP3 (TC9256)
Actin
F-CAGCTGGTGGAAAGTCCGTGG
R-AGCTTCTCGTTTTCGTCCGACAA
F-ATGAGAAGGCAGAGGACACAGC
R-CAGTCTTTGAGTGGTATCGTGGT
F-ACACCACTGGCATACTTGGTGG
R-ATCGTCTTTGTGACCTCCTGGCAA
F-TTGTAGTTCAAACCACAAGCGAC
R-TCATGAGAAGCTCGTTCCACG
F-ACTAGAGGGCTTGTGCTGGTCTTT
R-GTCTCATGCCTTAACACTACGGGT
F-TCACCGACGGAGAATCAGAGCCCA
R-CCAACTTCCCCAGAACGGCGA
F- TCCATAATGAAGTGTGATGT
R- GGCCCTGACTCGTCATACTC
Supplementary Fig. 1 Development and molecular characterization of ICE1 tobacco transgenics. a T-DNA
region of p7WG2.0-ICE1. RB: right border; LB: left border: CaMV35S –promoter; TCaMV35S – terminator,
Npt II- neomycin phosphotransferase; AtICE1- Arabidopsis thaliana ICE1; OsICE1 – Oryza sativa ICE1. b RTPCR of ICE1 over-expressing tobacco lines along with actin gene (M-1kb marker, W-wild-type, 1-3 AtICE1
lines and 4-6 OsICE1 lines). c Quantitative RT-PCR analysis of ICE1 over-expressing tobacco lines represented
as relative transcript levels and normalized with housekeeping gene actin.
Supplementary Fig. 2 Response of transgenic tobacco plants expressing AtICE1 and OsICE1 to different
stresses. Leaf discs were incubated in ethrel (1200ppm) for 42 h, chlorophyll content was estimated and used to
calculate chlorophyll stability index (CSI, a). Similarly, leaf discs were incubated for 42 h in NaCl ( b), PEG
8000 (c) and MV (d) and cell membrane stability (CMS) was estimated. Data shown are mean of three
replicates, error bars represents standard error and asterisks indicate the significance between WT and ICE1
over-expressed tobacco plants, as described by Student’s t test: *P<0.05, **P<0.005 and ***P<0.0001.
Supplementary Fig. 3 RT-PCR of ICE1 target genes in WT and transgenic tobacco plants under drought stress.
Expression analysis was carried out using leaf tissues collected from 30 d old plants exposed to drought stress by
controlled irrigation for 20 d. M-1kb marker, W-wild-type, 1-3 AtICE1 lines and 4-6 OsICE1 lines.