International Research Journal of Biotechnology (ISSN: 2141-5153) Vol. 2(3) pp. 072-077, March, 2011
Available online http://www.interesjournals.org/IRJOB
Copyright © 2011 International Research Journals
Full Length Research Paper
Molecular analysis of lower germinating rate in TLP
18.3 mutant of Arabidopsis thaliana
Mohammad Israil Ansari* and Tsan-Piao Lin
Institute of Plant Biology, National Taiwan University, Taipei, Taiwan
Amity Institute of Biotechnology,Amity University Uttar Pradesh, Lucknow Campus, Lucknow-226 010. India
Accepted 11 March 2011
Gibberellin (GA) play important role in Arabidopsis thaliana seed germination. GA play important role to
control the plant growth and seed germination process. Arabidopsis thaliana thylakoid lumen 18.3 kDa
protein (TLP18.3) gene has a domain of unknown function. To examine the germination developmental
regulation of this gene, Arabidopsis thaliana TLP18.3 T-DNA insertion mutant (SALK_109618),
transformed and wild type plant were observed for the effect of GA3 in the germination of seeds and
seedlings. The transformation was done with Agrobacterium mediated transformation of Arabidopsis
thaliana TLP18.3 T-DNA insertion mutant with pPZP200GB-TLP18.3 construct. The examination of
expression pattern of TLP 18.3 gene in germinating seeds of mutant plants as well as with exogenous
application of GA3 was examined. GA3 improved the germination rate 97% after treatment while before
treatment was only 63% in 48 hours in mutant plants. The transformed plant recued the germination as
well as transcriptional level. Our data suggest that GA3 is required for the up-regulation of Arabidopsis
thaliana TLP18.3 gene and Arabidopsis thaliana TLP18.3 gene regulate the germination process by
modulating the level of GA3 enzymes in GA3 biosynthesis.
Keywords: Gibberellin, Arabidopsis thaliana, thylakoid lumen, germination, transcript.
INTRODUCTION
Arabidopsis thaliana thylakoid lumen 18.3 kDa protein
(TLP18.3) gene (At1g54780) has a domain of unknown
function, which is a family of uncharacterized protein.
Arabidopsis thaliana is a plant which is having separated
vegetative and reproductive phases. In this plant the
change of vegetative shoot meristem into a reproductive
inflorescence meristem very apparent by changes in the
structure and in the pattern of mitotic activity of the shoot
apex (Besnard-Wibaut 1977). Plant hormones play an
important role in the regulation of plant life. Gibberellin
(GA) required for the germination and successive
elongation growth. GA is an essential phytohormone that
controls several aspects of plant development, including
seed germination, leaf expansion, stem elongation,
flowering, maturation, and seed development (Davies,
1995).
Seed germination is a complex physiological process
*Corresponding
author
Email:
ansari_mi@hotmail.com,
miansari@amity.edu. Phone: ++91-983-954-1698; Fax: ++91522-2721-934
that is controlled by a range of developmental and
external factors (Koornneef et al., 2002). Germination
starts with water uptake by seeds and terminates with the
initial elongation of the embryonic axis (Bewley, 1997;
Razem et al., 2006; Weiss and Ori, 2007). Completion of
germination is visible by the emergence of the radicle. It
is well known that GA promotes germination and ABA
inhibits germination. GA-deficient mutant that require
exogenous GA for the completion of germination (Groot
and Karssen, 1987; Ogawa et al., 2003 Toh et al., 2008)
while, ABA-deficient seeds fail to enter dormancy and
germinate precociously (Groot and Karssen, 1992). It has
been reported that signal transduction pathways are
regulators of this event, ie. RGA–Like2 (RGL2) is a
component of GA signal transduction (Lee et al., 2002;
Bassel et al., 2004). This protein is a member of the
DELLA family in Arabidopsis, transcription factors that
function to inhibit GA responses (Lee et al., 2002).
We have already reported that Arabidopsis thaliana
TLP18.3 gene is up-regulated during dehydration,
localizes into chloroplast and has important role in
flowering (Ansari et al., 2011). In this study we have
Ansari and Lin 073
observed that germination rate in Arabidopsis thaliana
ecotype Columbia wild type and Arabidopsis thaliana
TLP18.3 homozygous T-DNA insertion mutant plants
(SALK_109618) and transformed plants. We have
investigated the temporal abundance of TLP18.3
transcripts in Arabidopsis seeds in relation to germination
to determine whether expression of this gene is
correlated with the completion of germination. Our data
provide evidence suggesting that TLP18.3 plays a
significant important role in a GA3 signaling pathway that
controls seed germination in Arabidopsis thaliana.
MATERIALS AND METHODS
Plant material
Arabidopsis thaliana ecotype Columbia wild type, Arabidopsis
thaliana TLP18.3 homozygous T-DNA insertion mutant plants
(SALK_109618 obtained from ABRC, Ohio State University) and
transformed plants were used in this study. The homozygous
Arabidopsis thaliana TLP18.3 T-DNA insertion mutant plants were
find out using PCR with primer from left and right border of T-DNA
and primer from flanking region. Plants were grown at 22o C for
long day condition (16 h light / 8 h dark cycle) aseptically or on soil.
For soil growth, seeds were sown in Bio-Mix Potting Substratum
(Tref group, Netherlands) and placed at 4o C for 4 days in dark to
break residual dormancy and later transferred to normal growth
conditions. For aseptic growth condition, seeds were treated with
70% ethanol for 5 min and then with 30% household bleach for 15
min, washed 10 times with sterile double distilled water. Seeds
were germinated on filter paper in 90 mm diameter with MS medium
(Murashige and Skoog,1962) in presence of 100 µM GA3 (SigmaAldrich Ltd., St Louis, MO) and control seeds were germinated with
MS media only.
Transformation of
mutant
Arabidopsis thaliana T-DNA insertion
Coding region of Arabidopsis thaliana TLP18.3 gene (GeneBank
Accession No. NM_104353) was cloned into binary vector
pPZP200GB using XbaI and BamHI restriction enzymes. This
pPZP200GB with β -glucuronidase and BAR (BASTA resistance
gene) cassettes was derived from pBI221 (Clontech Laboratories,
Palo Alto, CA) and pSK-35S-BAR (Chu et al. 2005). The obtained
plasmid construct was named pPZP200GB-TLP18.3. This binary
vector has spectinomycin resistance for E. coli and glufosinate
resistance for plant. The pPZP200GB-TLP18.3 construct was
transformed into Agrobacterium tumefaciens strain C-58 by
electroporation. Arabidopsis thaliana TLP18.3 T-DNA insertion
mutant (SALK_109618) plants were transformed through
Agrobacterium mediated transformation by floral dipping method
(Clough and Bent 1998). Transformed plants were selected by
spraying seedlings at 7, 9 and 11 days after germination with a
solution of 0.4% of BASTA herbicide (McDowell et al. 1998). T2
generations were selected for isolating homozygous lines. Plants
were grown in growth chamber at 22o C for long day condition (16 h
light / 8 h dark cycle) on soil and placed at 4o C for 4 days in dark to
break residual dormancy and later transferred to normal growth
conditions. Transformed plants were selected from non transformed
by spraying 0.4% of BASTA herbicide.
RNA isolation and Northern blot analysis
Total RNA from Arabidopsis thaliana leaf sample was isolated by
REzol reagents kit (PROtech Technology, Taiwan) according to the
manufacturer’s instructions. RNA from Arabidopsis thaliana seeds
at different germinating stages was isolated using the method of
Vicient and Delseny (1999). 100 mg seeds at germinating stages
were ground in liquid nitrogen and placed in a tube with 1 ml of
extraction buffer (8 M LiCl, 2% v/v b-mercaptoethanol). Centrifuged
at 13 000 rpm for 30 min and the supernatant removed. The pellet
was washed with 70% (v/v) ethanol and air dried, and then
dissolved in 0.5 ml of buffer (0.5% w/v SDS, 100 mM NaCl, 25 mM
EDTA, 10 mM Tris-HCl, pH 7.6, 2% v/v b-mercaptoethanol) before
extraction twice with phenol, twice with phenol:chloroform:isoamyl
alcohol (25:24:1) and twice with choloform:isoamyl alcohol (24:1).
The final aqueous phase was taken to a new tube and 0.1 volume
of 3 M sodium acetate and 1.5 volumes ethanol were added and
the tube stored at -20o C for overnight. The next day the tube was
centrifuged at 13 000 rpm for 20 min, the supernatant removed and
the pellet washed with 70% ethanol and the pellet air-dried. The
pellet was suspended in di-ethyle-pyrocarbonate (DEPC) treated
water.
For RNA gel blot analysis RNA was analyzed on 1.2%
formaldehyde agarose gel. After electrophoresis, the RNA was
transferred from agarose gel to a positively charged nylon
membrane (Boehringer Mannheim GmbH, Mannheim, Germany).
Hybridization was performed at 65o C in the FastHyb-hybridization
solution (BioChain Institute, USA) with DIG labeled Arabidopsis
thaliana TLP18.3 full length cDNA using DIG Luminescent
Detection Kit (Roche, Germany). Signals were captured on a
LXA3000 Image System (Fiji) after 2 h of exposure.
DNA sequencing and computational analysis
DNA sequencing was performed by the Applied Biosystems 3730 xl
DNA Analyzer. Homology search against the sequence database
was performed using the BLAST program at the National Center for
the Biotechnology Information, Bethesda, MD. Amino acid and
nucleotide sequence were analyzed with Vector-NTI Suit 5.5
(Informax Inc., Bethesda, MD).
RESULTS
Effect of GA3 on germination of Arabidopsis thaliana
TLP18.3 T-DNA insertion mutant, transformed and
wild type plants
To investigate the role of Arabidopsis thaliana TLP18.3
gene in germination process, we have homozygous
Arabidopsis thaliana TLP18.3 mutant (SALK_109618),
caused by T-DNA insertion in the second exon,
transformed and wild type plants. The mutant plants at
control condition have shown 51%, 62%, 63% (24h, 36h,
48h after germination) while after treatment with 100 µM
of GA3 this germination rate significantly rises up 18%,
19%, 34% (24h, 36h, 48h after germination) (Figure 1A).
In transformed plants at control condition have shown
67%, 82%, 91% (24h, 36h, 48h after germination) while
after treatment with 100 µM of GA3 this germination
increase was marginally significant, it was 2%, 3%, 6%
only (24h, 36h, 48h after germination) (Figure 1B). In wild
type plants, germination rate was same just like
transformed plants, at control condition germination rate
was 67%, 81%, 33% (24h, 36h, 48h after germination)
while after treatment with 100 µM of GA3 this germination
rate was 80%, 93%, 98% (24h, 36h, 48h after
074 Int.Res.J.Biotechnol.
100
% Germination
A
80
60
40
C ontrol
20
T re a te d
0
12
18
24
30
36
42
48
Time after treatment (h)
100
% Germination
B
80
60
40
C ontrol
20
T re a te d
0
12
18
24
30
36
42
48
Time after treatment (h)
% Germination
100
C
80
60
40
C ontrol
20
T re a te d
0
12
18
24
30
36
42
48
Time after treatment (h)
Figure 1. Effect of GA3 on seed germination of Arabidopsis thaliana TLP18.3 T-DNA insertion mutant
(SALK_109618), transformed and wilt type plants. Seeds were stratified at 4o C in the dark for 48h and
incubated at 22o C with GA3 (treated with 100 µM GA3) or mock solution (control containing only MS media)
for seed germination. Germination percentage was counted with time course. A- Mutant; B- Transformed;
C- Wild.
germination) (Figure 1C). As GA3 induced the
germination rate in mutant plants, probably Arabidopsis
thaliana TLP18.3 gene play important role in GA
metabolism that control seed germination. GA deficient
mutant of tomato gib-1 and Arabidopsis thaliana ga1-3
mutant after treatment with 100 µM GA3 gives better
germination (Bassel et al., 2006). Increase in germination
rate have been reported with GA3 in other plants as well
as in mutant studies (Weiss et al., 2007; Toh et al., 2008).
Ansari and Lin 075
Figure 2. Changes in Arabidopsis thaliana TLP18.3 mRNA level at seed germination stage response to
GA3 treated and without treated seeds of TLP18.3 T-DNA insertion mutant (SALK_109618), transformed
and wilt type plants. Sample was taken after 12h, 24h and 48h after treatment. RNA gel blot was
hybridized with the Arabidopsis thaliana TLP18.3 cDNA. Ethidium bromide (EtBr) staining is shown as
loading control. A-Mutant; B- Transformed; C-Wild type. Control: only MS media, Treated: MS media
containing 100 µM GA3.
Arabidopsis thaliana TLP18.3 gene expression during
seed germination in Arabidopsis thaliana mutant,
transformed and wild type plants
To study the role of Arabidopsis thaliana TLP18.3 gene in
seed germination. The mutant seed at control condition,
northern blot analysis with TLP18.3 full length cDNA the
transcript level did not increase in 12-18h duration, while
after treating with 100 µM GA3 the transcript level upregulated (Figure 2A). In transformed seeds as well as in
wild type seeds at control condition the transcript level
with the time course did not increase at 12h, 24h and 48h
after germination (Figure 2B, 2C). The up-regulation of
TLP18.3 transcript in mutant after GA3 treatment gives us
a kind of message that GA3 must have important role in
seed germination. Bassel et al., (2006) up-regulation of
gene LeAB13 in tomato mutant gib-1(GA deficient) have
reported after treating with GA3 in germinating seeds.
hormone was made to the two weeks old growing
seedlings. It was observed that the seedling growth was
increased significantly in a medium containing the 100
µM of GA3 compare to the control seedlings which were
not treated with GA3. The expression of Arabidopsis
thaliana TLP18.3 gene was analyzed before and after
treatment with GA3 in mutant seedlings. The expression
of TLP 18.3 gene in two week old seedlings of mutant
plants, after exogenous application of GA3 just after the
day one of treatment the transcript level goes-up and day
three and day five of the treatment abundance of
TLP18.3 gene expression (Figure 3A). In control mutant
seedlings the transcript level was very low even after the
day five of sampling (Figure 3B). This demonstrated that
GA3 is required for the up-regulation of Arabidopsis
thaliana TLP18.3 gene. Exogenous application of GA3 in
mutant plants which induced the gene expression have
been reported by several scientist (Groot and Karssen,
1987; Nonogaki et al., 2001; Yamaguchi and Kamiya,
2002).
Exogenous application of GA3 reverses stunting and
up-regulate the Arabidopsis thaliana TLP18.3 gene in
TLP18.3 mutant plants
DISCUSSION
Compare to the wild type plant Arabidopsis thaliana
TLP18.3 T-DNA insertion mutant have stunted growth. To
investigate whether the reduced growth in the TLP18.3
mutant was due to GA, exogenous application of GA3
It is well documented that GA play important roles in seed
germination (Finkelstein et al., 2002; Razem et al., 2006;
Weiss et al., 2007; Toh et al., 2008; Liu et al., 2010). The
studies on numerous mutants have demonstrated that
076 Int.Res.J.Biotechnol.
Figure 3. RNA gel blot analysis investigating the TLP18.3 expression pattern in TLP18.3 T-DNA
insertion mutant in 2 weeks old plant after treatment with 100 µM of GA3, leaves sample were
collected at day 1 (D1), day 3 (D3) and day 5 (D5). RNA gel blot was hybridized with the Arabidopsis
thaliana TLP18.3 cDNA. Ethidium bromide (EtBr) staining is shown as loading control. A-Mutant
treated with GA3 (treated), B-Mutant without GA3 treatment (control).
GA biosynthesis is required for seed germination and
dormancy (Groot and Karssen, 1987; Ogawa et al., 2003;
Kushiro et al., 2004; Okamoto et al., 2006). We have
examined the effect of GA3 on the germination of seeds
for the developmental regulation of TLP18.3 gene. 100
µM GA3 treatment in Arabidopsis thaliana TLP18.3 TDNA insertion mutant significantly enhanced the
germination rate 18%, 19%, 34%, while in wild type and
transformed TLP18.3 T-DNA insertion mutant plant the
germination rate was same (Figure 1). As GA3 induced
the germination rate in mutant plants, probably
Arabidopsis thaliana TLP18.3 gene play important role in
GA3 metabolism that control seed germination. GAdeficient mutant that require exogenous GA for the
completion of germination have been reported by other
groups also (Groot and Karssen, 1987; Weiss et al., 2007;
Toh et al., 2008; Yano et al., 2009). It has been reported
that GA deficient mutant of tomato gib-1 and Arabidopsis
thaliana ga1-3 mutant after treatment with 100 µM GA3
gives better germination (Bassel et al., 2006).
The temporal profile of TLP18.3 transcript accumulation
in seed germination was evaluated. The mutant seed at
control condition the transcript level did not change with
time course, while after treating with 100 µM GA3 the
transcript level up-regulated. In transformed seeds as
well as in wild type seeds at control condition and after
treating with 100 µM GA3 the transcript level did not
change at 12h, 24h and 48h after germination (Figure 2).
The up-regulation of TLP18.3 transcript with GA3
treatment, probably this gene has GA biosynthesis
metabolism. GA3 induced transcript level during seed
germination in mutant plants have been reported by
others also (Steber and McCourt, 2001; Holdsworth, et al.,
2008; Carrera, et al., 2008). Bassel et al., (2006) upregulation of gene LeAB13 in tomato mutant gib-1(GA
deficient) have reported after treating with GA3 in
germinating seeds.
Exogenous application of GA3 in mutant seedlings have
been observed by Groot and Karssen, 1987; Nonogaki et
al., 2001; Yamaguchi and Kamiya, 2002, they have
reported the increased growth and up-regulated transcript
in different plants as well as in Arabidopsis thaliana with
different T-DNA insertion mutants. Application of GA3
hormone on the 2 weeks old growing seedlings on
Arabidopsis thaliana TLP18.3 T-DNA insertion mutant,
the expression of TLP 18.3 transcript in TLP18.3 mutant
plants after GA3 treatment was up-regulated (Figure 3A).
In control condition the mutant seedlings transcript level
was very low even after the day five of sampling (Figure
3B). Liu et al., (2010) reported that ABA negatively
regulates GA biosynthesis in germinating seeds of
Arabidopsis thaliana. Further the suppression of GA
biosynthesis gene by exogenous application of ABA also
been reported (Liu et al., 2010).This demonstrated that
GA3 is required for the up-regulation of Arabidopsis
thaliana TLP18.3 gene. Our data provide the evidence
suggesting that Arabidopsis thaliana TLP18.3 gene
regulate the germination process by modulating the level
of GA3 enzymes in GA3 biosynthesis that control the
germination process.
ACKNOWLEDGEMENTS
This research work was supported by National Science
Council of Taiwan Government grant (Grant No. NSC952811-B-002-021). I am thankful to Mr. Vineet Kumar
Srivastava, Assistant System Administrator, Amity
University, Lucknow Campus, Lucknow, India for his
support for the preparation of this manuscript.
Ansari and Lin 077
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