Postharvest Biology and Technology 58 (2010) 48–56

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Postharvest Biology and Technology

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The gladiolus GgEXPA1 is a GA-responsive alpha-expansin gene expressed

ubiquitously during expansion of all floral tissues and leaves but repressed
during organ senescence
Abdul Azeez a , Aniruddha P. Sane a,∗ , Siddharth Kaushal Tripathi a,1 , D. Bhatnagar b , Pravendra Nath a
a
Plant Gene Expression Lab, National Botanical Research Institute, Lucknow 226001, India
b
School of Biochemistry, Devi Ahilya Vishwavidyalaya, Indore 452001, India

a r t i c l e i n f o a b s t r a c t

Article history: The final shape and size of the flower is genetically and developmentally controlled by tight regula-
Received 29 March 2010 tion of cell number and cell size with cell expansion playing an important role The gladiolus expansin
Accepted 16 May 2010 gene, GgEXPA1, was expressed prominently during phases of active tepal expansion and cell elongation
in stamen filaments, gynoecium styles and expanding leaves but not in tissues where expansion had
Keywords: ceased and senescence had been initiated. Within tepals, differential expression between the proximal
Gladiolus
and distal portions that differ in cell elongation was observed. The expression of the gene was responsive
Expansin
to GA and inhibited by the GA biosynthesis inhibitor, paclobutrazol. The promoter of GgEXPA1 showed
Cell expansion
Gibberellin
strong expansion-responsive GUS expression in young agro-infiltrated gladiolus tepals and in etiolated
Promoter hypocotyls and light grown expanding cotyledonary leaves of transgenic Arabidopsis seedlings. Inhibi-
GUS tion of hypocotyl elongation by paclobutrazol blocked the expression of the promoter-driven reporter
gene indicating GA responsiveness of the promoter. GgEXPA1 provides an interesting example of a single
expansin gene being involved in expansion processes in different plant tissues such as tepals, stamens,
pistils and leaves that are both spatially as well as temporally distinct in their development. The studies
provide a basis for GA mediated expansion of floral organs via expansins prior to anthesis.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction cytoskeleton and the matrix of the cellulose/hemicellulose/pectin

The development of a flower is a complex process that begins
with initiation of floral primordia, differentiation of the floral
organs, increase in floral size, bloom and finally senescence. The
shape and size of the floral organs is genetically and developmen-
tally controlled through regulation of cell number (by division), cell
size (by expansion) and influenced by growth rate, anisotropy and
direction (Meyerowitz, 1997; Mizukami, 2001; Rolland-Lagan et
al., 2003; Weiss et al., 2005; Anastasiou and Lenhard, 2007). While
early development in most flowers is characterized by cell division,
cell wall expansion plays an important role in determining the final
shape of flower (Kotilainen et al., 1999; Reale et al., 2002; Yamada
et al., 2009a). Stamens and pistils expand longitudinally while
petals expand in all directions. Cell wall expansion requires not
only cellulose biosynthesis and deposition but also changes in the
∗ Corresponding author. Tel.: +91 522 2297959; fax: +91 522 2205836/839.
E-mail address: saneanil@rediffmail.com (A.P. Sane).
1
Current address: National Center for Natural Products Research, Thad Cochran
Research Center, School of Pharmacy, University of Mississippi, Oxford, MS 38677,
USA.
network which allow flexibility for wall expansion (Fagard et al.,
2000; Martin et al., 2001; Pagant et al., 2002; Smith, 2003). Promi-
nent amongst the wall proteins that provide flexibility for cell
expansion are expansins that are believed to cause wall loos-
ening in a non-hydrolytic, turgor driven manner by disrupting
hydrogen bonds that link cellulose and hemicellulose microfibrils.
Their action causes slippage between the cellulose–hemicellulose
polymers followed by water absorption and expansion of the cell
wall (McQueen-Mason and Cosgrove, 1995). Expansins belong to a
multi-gene family in all plants studied so far and are conserved in
structure but display diversity in function (Sampedro and Cosgrove,
2005). Thus expansins have been shown to play an important role
in several plant processes that require wall modification such as
cell expansion, hypoxic stem elongation in rice, leaf and root ini-
tiation and other non-expansion processes such as wall softening
in fruit ripening and organ abscission (Rose et al., 1997; Fleming et
al., 1997; Brummell et al., 1999; Cho and Cosgrove, 2002; Choi et
al., 2003; Belfield et al., 2005; Sane et al., 2007; Asha et al., 2007).
Individual members of the large expansin family in each plant are
believed to provide specificity of function in each tissue and may
be governed differentially by hormones especially gibberellins. The
expansion of petals has previously been reported to be due to accu-

0925-5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2010.05.006
 A. Azeez et al. / Postharvest Biology and Technology 58 (2010) 48–56
mulation of GA in the petals (Murakami, 1973, 1975; Koning, 1984).
GA is also known to affect stamen filament length and pistil length
in flowers (Murakami, 1973, 1975; Pharis and King, 1985). We are
interested in studying genes that are actively involved in floral
organ growth and expansion and the role of hormones like GA
in their regulation during expansion. In this paper, we describe
the characterization of a new expansin gene from gladiolus that
is closely associated not only with tepal growth and expansion
but also with stamen, pistil and leaf elongation and functions in
a GA-responsive manner thus providing a basis for GA mediated
expansion of floral organs via expansins prior to anthesis.
2. Materials and methods
2.1. Plant material
Flowers of gladiolus (Gladiolus grandiflorus cv Snow Princess)
grown in the field were chosen for study. The spikes, containing
about 12–14 flowers in different stages of growth and senescence,
were cut with a sharp blade and immediately placed in water. Flow-
ers on the spike were marked as previously described (Azeez et al.,
2007). The fully open flowers were designated as 0 stage. Flowers
above this stage in different stages of opening were marked as −1,
−2 and −3 in an ascending order with −3 indicating the bud stage.
Flowers below the 0 stage were marked progressively as +1, +2 and
+3 in a descending order and represented flowers in different stages
of senescence with +3 indicating completely wilted flowers.
2.2. Estimation of tepal area and stamen/pistil length
For estimation of tepal area, at least five complete flowers of
each stage were chosen. Individual tepals were spread on a graph
paper and area of each tepal was measured and summed up to
obtain the entire tepal area. Stamens and pistils of the same flow-
ers were chosen for study of expansion in length. For stamens, the
length of the filament was measured while for pistils, the length of
the style was measured. All data were expressed as mean length
(in cm) ± SD for stamen filaments and pistil styles or mean area (in
mm2 ) ± SD for tepal.
2.3. Treatment of flowers with GA and paclobutrazol
To study hormonal regulation of expansin expression, flowers
from three stages viz. −2, 0 and +2 were cut, and placed individ-
ually in beakers containing either water (as control), GA3 (10 ␮M,
Duchefa, Netherlands) or the GA biosynthesis inhibitor, paclobu-
trazol (20 ␮M, Duchefa, Netherlands) for 24 h. Flower tepals were
ground in liquid nitrogen and RNA extracted for reverse transcrip-
tion and semi-quantitative RT-PCR. To study the effect of GA3 and
paclobutrazol on flower growth, buds at −3 stage were placed in
a beaker containing either water, GA or paclobutrazol for 4 days.
Whole tepal area was measured after 4 days when flowers opened.
Studies were carried out on three independent flowers for each
treatment.
2.4. Isolation of RNA
Floral parts (tepals, stamens and pistils) from flowers belonging
to different stages of growth and senescence were ground in liq-
uid nitrogen to a fine powder. RNA was isolated from these tissues
as described by Asif et al. (2000). RNA was also isolated from the
basal and distal portions of the tepals of fully open flower (stage
0) and from five different stages of leaves starting with the very
young (Y ∼ 12 cm), growing (G1 ∼ 25 cm and G2 ∼ 35 cm), mature
(M ∼50 cm) to senescing (S ∼ 58 cm) leaves.
49
2.5. Cloning of expansin cDNA
DNA free RNA from 0 stage flower was reverse tran-
scribed using MuMLV Revertaid reverse transcriptase (MBI
Fermentas) and primed with the 3 RACE adapter primer (5 -
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3 ) from Invitro-
gen. This was used as cDNA template for further amplifica-
tions of expansin. To amplify the expansin gene fragment,
degenerate primers for expansin (forward primer ExpF1 5 -
GCHTCDGGMACHATGGDTGGDGCDTGTGG-3 and reverse primers,
ExpR1 5 -CCRTTKAYBGTRAAYCKDATHCCTCC-3 and ExpR2 5 -
AAGTGNKKNHGDGGDGGRTTRCACC-3 ) were designed from con-
served regions as described by Sane et al. (2005). The F1R1 pair
was used to amplify a product of 374 nt while the F1AP pair was
used to amplify a 979 nucleotide fragment which was subsequently
cloned in pBluescript IISK (Stratagene, USA). Amplified fragments
were sequenced on an automated DNA sequencer (ABI 373A from
Applied Biosystems Inc., USA) using the thermosequenase dye ter-
minator cycle sequencing kit from Amersham-Pharmacia. The 5
end of the gene was obtained from a Gladiolus genome walker
library created using the genome walker kit (Clontech, Palo Alto,
USA). Gene specific reverse primers Gex-R1 (5 -CAG TAA CGA CAA
TTG AAC CCG AAA GAC-3 ) and Gex-R2 (5 -GGT CGT CGT TGC ACT
TCA TCT CAT AG-3 ) were used in combination with the genome
walker adapter primers GWAP1 (5 -GTA ATA CGA CTC ACT ATA GGG
C-3 ) and GWAP2 (5 -ACT ATA GGG CAC GCG TGG T-3 ) as described
in the genome walker manual. A fragment of approximately 1.8 kb,
containing 474 nucleotides of the gene (inclusive of a 200 nt intron)
towards the 5 end and 1.3 kb sequence upstream of the initiation
codon, was cloned and sequenced. Based on the sequence, a for-
ward primer containing the initiation codon, GExpOEF 5 -GGA TCC
ATG TAT TCC CTT TCC AAA ATC-3 was synthesized and used in
combination with the 3 AP primer to obtain the cDNA sequence
containing the complete open reading frame of 762 nt besides a 3
UTR of 329 nt (Accession number FJ042664).
2.6. Northern analyses
Total RNA (30 ␮g) was resolved on a 1.2% denaturing
formaldehyde-agarose gel as described (Sambrook et al., 1989)
and modified in the Qiagen Oligotex handbook, 2002. RNA was
transferred to nylon membranes (Hybond N, Amersham-Pharmacia
Biotech, Uppsala, Sweden) by vacuum transfer using a vacu-
gene apparatus (Pharmacia) and crosslinked by baking for 2 h.
Radio-labeling of probes for northern blots was performed by
random priming using ␣-32 PdCTP and the 3 variable region
(213–762 nt + the 329 nt 3 UTR) of the expansin gene. Hybridiza-
tion and washings of blots were performed as described by
Sambrook et al. (1989). Signals obtained on the blots were quanti-
fied on a phosphorimager (Molecular Imager FX, BioRad) using the
software QuantityOne-4.2.3 version.
2.7. Semi-quantitative RT-PCR
Equal amounts of DNA free RNA (5 ␮g) from the basal and distal
portions of the tepals were reverse transcribed using the MuMLV
Revertaid reverse transcriptase (MBI Fermentas). The primer GEx-
pOEF was used in combination with the reverse primer GExpSM-R
5 -TGC TGC AGT GTT GGT TCC ATA CCC TT-3 to amplify a fragment
of 210 nt. Actin was used an internal control using the primers F1
5 -ATG ACA TGG AGA AGA TCT GGC ATCA-3 and R1 5 -AGC CTG GAT
GGC AAC ATA CAT AGC-3 to amplify a fragment of 179 nt. Reactions
were run on a PerkinElmer 9700 PCR machine at an initial denatura-
tion of 94 ◦ C (2 min) followed by 32 cycles of 94 ◦ C (10 s), 55 ◦ C (10 s)
and 72 ◦ C (20 s) and a final extension at 72 ◦ C for 5 min. The glad-
iolus GgDAD1 (DEFENDER AGAINST APOPTOTIC DEATH1), a marker
50 A. Azeez et al. / Postharvest Biology and Technology 58 (2010) 48–56
gene for senescence in gladiolus tepals (Yamada et al., 2004), was
also amplified using the primers GgDAD1OEF – 5 -GGA TCC ATG
GCA AAA TCA ACT GCT AAT G-3 and GgDAD1OR – 5 -GGA TCC TCC
AAG GAA GTT CAT GAT CAC A-3 to obtain a fragment of 357 nt.
Semi-quantitative RT-PCR was also performed on cDNA from differ-
ent stages of developing leaves and from GA/paclobutrazol treated
flowers using the same primers as described above with actin as an
internal control. At least three independent reactions were carried
out.
2.8. Isolation of the expansin promoter and preparation of the
promoter-GUS fusion construct
Based on the sequence of the expansin gene, a reverse primer
GEproBam 5 -GGA TCC ATT GAT GGT ACT TCC ATT G-3 that
included the initiation codon (underlined) was designed with a
BamHI site introduced at the 3 end of the initiation codon. This
primer was used in combination with the GWAP1/GWAP2 primers
to amplify the 1.3 kb region upstream of the initiation codon from a
genome walker library of gladiolus (Accesion No. HM208309). The
amplified fragment was cloned in pBI101.2 in translational fusion
with the GUS gene.
2.9. Promoter analysis in gladiolus tepals by transient GUS
expression using agroinfiltration
Transient expression of the promoter-GUS fusion was tested by
agroinfiltration of the construct into tepals at three different stages
viz. −3, −1 and +1. The agroinfiltration was performed as described
by Tripathi et al. (2009). Flower tepals were stained for GUS expres-
sion when they reached the −2, 0 and +2 stages respectively using
X-gluc (Biosynth AG, USA). Plasmids pBI121 and pBI101 were used
as positive and negative controls respectively.
2.10. Transformation of Arabidopsis and histochemical GUS
staining of Arabidopsis seedlings
The GgEXPA1promoter-GUS construct was first introduced in
Agrobacterium (strain GV3101) and then transformed into Ara-
bidopsis thaliana by the floral dip method (Clough and Bent, 1998).
Transformants were selected on kanamycin plates and then trans-
ferred to soil. Seeds from the T1 and T2 populations of A. thaliana
containing GgEXPA1pro::GUS fusion constructs were harvested and
used for analysis of GUS activity. They were germinated in ger-
mination medium containing 1/2 MS (Murashige and Skoog basal
medium, 0.8% agar) and grown for 10–12 days at 20–22 ◦ C under
fluorescent lights with cycles of 16 h light:8 h dark. For etiolation,
plates were kept in the dark for 5 days under the same conditions.
For paclobutrazol treatment, dark grown seedlings were grown
in the presence of paclobutrazol (20 ␮M). The etiolated seedlings
were then immersed individually in 1.5 mL microfuge tubes con-
taining the GUS reagent and stained as previously described
(Gattolin et al., 2006). The tissue was destained in 70% ethanol at
37 ◦ C until examination. Light microscopy was performed on a Leica
Wild M3Z microscope (Leica, Germany).
2.11. Estimation of cell number in the proximal and distal
portions of the tepal
Tissue sections of proximal and distal portions of the tepals from
the bud stage (−2), fully open flower (0) and the senescent stage
(+2) were scanned under a confocal microcope (LSM 510 META
from Zeiss, Germany). Cells in an area of 225 ␮m × 225 ␮m were
counted in three different flowers and results expressed as mean
number ± SD.
3. Results
3.1. Isolation of expansin gene
The expansin gene was isolated using a combination of 3 RACE
and genome walking using cDNA from fully open flowers (stage
0). The complete cDNA had a sequence of 1091 bp that contained
an open reading frame of 762 bp and a 3 UTR of 329 bp. The gene
was designated as GgEXPA1 (Accession No. FJ042664) and encoded a
putative protein of 253 amino acids. Essential features of expansins
such as eight conserved cysteine residues overall, four conserved
tryptophan residues at the C-terminus and a conserved HFD sig-
nature, related to the cellulase catalytic domain were conserved in
GgEXPA1 (Fig. 1A). The predicted protein had an N-terminal signal
peptide of 26 amino acids. Phylogenetic analyses of the predicted
protein by CLUSTAL W showed that it was closest to MaEXP1 from
the monocot fruit banana. Similarity to other expansins that are
unrelated in their site of action was also obvious. It appeared to
be closer to the alpha-expansins from petals of Mirabilis jalapa,
Rosa bourboniana and PhEXPA3 from Petunia hybrida but was dis-
tinct from the expansion related expansins of rice and other petal
expansins of P. hybrida such as PhEXP1 and PhEXP2 (Fig. 1B).
3.2. Expression analyses during organ expansion and senescence
In order to study the expression pattern of the gene during
flower growth and senescence in gladiolus, a northern blot of RNA
isolated from flowers from different stages on the spike was probed
with the 3 region of GgEXPA1, inclusive of the 3 UTR, which is vari-
able when compared to other expansin genes. As shown in Fig. 2A,
the steady state level of transcripts of GgEXPA1 was high in the
early stages of tepal growth during the expansion phase of the
flower and could be detected only until the +1 stage. The expression
was correlated with the progression of expansion in tepals (mea-
sured as an increase in total tepal area). Tepal area increased by
almost 4-fold between stages −3 and 0 with the most prominent
increase between stages −2 to −1 (1.75-fold with respect to the
previous stage) and from −1 to 0 (1.5-fold with respect to the pre-
vious stage). After the cessation of expansion from stage 0 onwards,
flowers wilted and turned senescent and there was a drastic reduc-
tion in the transcript levels of the gene at stage +1 and no expression
was detectable at stages +2 and +3.
Apart from tepals, rapid expansion is also observed in other flo-
ral tissues such as stamen filaments and pistil styles particularly
from stages −3 to −1. This expansion was associated with cell elon-
gation as observed under a light microscope (data not shown). In
order to test if the expansion was associated with expression of
GgEXPA1 we screened northern blots containing RNA isolated from
stamens and pistils from the same stages of flowers as used for tepal
study. The analysis revealed a rapid increase in transcript levels of
the expansin gene during the early stages of stamen development
at stages −2 and −1 that correlated with an increase of almost 6-
fold in stamen filament length between stages −3 and 0 (Fig. 2B).
Once the stamen filament had reached its maximum length at stage
0, no expression of GgEXPA1 was observed from stages +1 to +3
which also corresponded to stages of stamen senescence. In pistils,
the length of the style increased by 3.5-fold between stages −3 and
0 with maximum increase between stages −2 and −1 (1.7-fold).
High levels of transcript could be detected in pistils at this stage
(Fig. 2C). Surprisingly, very low expression was observed in the −2
stage in spite of the 2-fold increase in style length. No expression
was observed in senescent pistils where expansion had ceased.
Apart from testing expression in different stages of the flower,
we also tested for GgEXPA1 expression in the basal and distal parts
of the tepals in the fully open flower (stage 0). Although these flow-
ers appear healthy, the process of senescence has already initiated
 A. Azeez et al. / Postharvest Biology and Technology 58 (2010) 48–56 51

Fig. 1. (A) Deduced amino acid sequence of the GgEXPA1 gene in gladiolus. The putative signal peptide has been underlined. The conserved C and W residues and the putative
cellulose binding site HFD are shown in bold. (B) Phylogenetic tree of the alignment of GgEXPA1 deduced amino acid sequence with other alpha-expansins. Full-length protein
sequences were aligned using CLUSTAL, and a phylogenetic tree was constructed using PHYLIP with the PROTPARS program. Numbers above the branches indicate bootstrap
values. A Phleum pratense pollen allergen (Phlp1), identified as beta-expansin, was used as outgroup. Sequences are GenBank accession numbers: At (Arabidopsis thaliana) EXPB
beta-expansin (AAD20920), EXPA10 (AAF61713), Fa (Fragaria ananassa), EXPA2 (AF159563); Le (Lycopersicon esculentum), EXPA1 (U82123), Ma (Musa acuminata), EXPA1
(AY083168), EXPA2 (AAN16378); Md (Malus domestica), EXPA1 (AY083166); Pa (Prunus armeniaca), EXPA1 (U93167); Phlp1 (Phleum pratense) pollen allergen (X78813);
Mja (Mirabilis jalapa) EXPB2 (AAN86683), EXPA1 (AAL87025), EXPA2 (AAL87021), EXPA3 (AAL87022), EXPA4 (AAL87023), EXPA7 (AAN86682); Ph (Petunia hybrida) EXPA1
(AAR82849), EXPA2 (AAR82850), EXPA3 (AAR82851); Pce (Prunus cerasus) EXPA3 (AAK48847); Sni (Sambucus nigra) EXPA2 (AAP48989), EXPA4 (AAP48991); Gh (Gossypium
hirsutum) EXPA1 (AAY59532); Fa (Fragaria ananassa) EXPA2 (AAF21101); Pa (Prunus armeniaca) EXPA1 (AAC33529); Pco (Pyrus communis) EXPA3(BAC67190) and Rb (Rosa
bourboniana) EXPA1 (DQ320657).

in the distal portions where the petals are thin and expanded. This
results in appearance of the first symptoms of wilting/senescence
at the edges of the fully open tepals in stage 1. We confirmed this by
studying the expression of GgDAD1 (DEFENDER AGAINST APOPTOTIC
DEATH1), a gene known to be repressed during tepal senescence
(Yamada et al., 2004). Expression of GgDAD1 was high in the basal
portion of the tepal (attached to the tube) but low in the distal
expanded portion of the tepals indicating the onset of senescence
in the distal portion (Fig. 3A). Interestingly, within the same tepal,
expression of GgEXPA1 gene was also higher in the basal portion but
lower in the distal portions of the petal. We measured the number
of cells in the basal and distal portions of the tepal in a fixed area
52 A. Azeez et al. / Postharvest Biology and Technology 58 (2010) 48–56

Fig. 2. (A) Correlation of transcript accumulation of GgEXPA1 during tepal growth and senescence with tepal area. Top panel: flowers of gladiolus in different stages of
development and senescence. Numbers indicate the stages of flowers on the spike that were chosen for study. Middle panel: progressive changes in whole tepal area in
different flowers of a spike during growth. Lower panel: transcript accumulation of GgEXPA1 during tepal growth and senescence in the same stages on a northern blot (30 ␮g
total RNA/lane). (B) Correlation of transcript accumulation of GgEXPA1 during stamen growth and senescence with stamen filament length. Top panel: stamens of gladiolus in
different stages of development and senescence. Numbers indicate the stages of flowers on the spike chosen for study. Middle panel: progressive changes in stamen filament
length in different flowers of a spike during growth. Lower panel: transcript accumulation of GgEXPA1 during stamen growth and senescence in the same stages on a northern
blot (30 ␮g total RNA/lane). (C) Correlation of transcript accumulation of GgEXPA1 during pistil growth and senescence with pistil length. Top panel: pistils of gladiolus in
different stages of development and senescence. Numbers indicate the stages of flowers on the spike chosen for study. Middle panel: progressive changes in pistil length
in different flowers of a spike during growth. Lower panel: transcript accumulation of GgEXPA1 during pistil growth and senescence in the same stages on a northern blot
(30 ␮g total RNA/lane).

(225 ␮m × 225 ␮m) in three different stages (−2, 0 and +2) to get an Table 1
Number of cells in the tepal per unit area (225 ␮m × 225 ␮m) (number of cells in
indication of the cell area and observed that the cell number/area
three tepals of each stage were counted).
differed between the basal and distal portions (Table 1). As evident
from the table, the number of cells in the basal portion of the flower Stage of flower Basal portion Distal end (periphery)
decreased from 48 ± 3 in the bud stage to 26 in stage 0 and further Bud (−2) 48 ± 3 43 ± 5
to 18 ± 1 in the +2 stage. This corresponded to an increase in area of Fully open (0) 26 ± 0 26 ± 2
1.85-fold from the bud stage to the fully open stage and further by Senescent (+2) 18 ± 1 23 ± 1

1.44-fold from fully open stage to the senescent stage. On the other
hand, cell number in the distal portion decreased from 43 ± 5 at the
bud stage to 26 ± 2 in stage 0 and further to 23 ± 1 in stage +2. This
corresponded to a 1.65-fold increase in cell size from the bud stage
to the fully open stage but only 1.1-fold from stage 0 to stage 3.
Thus, there was a total increase of 2.66-fold in cell size in the basal
portion but only 1.87-fold in the distal portion of the cell from the
bud stage to the senescent stage. Importantly, the increase in cell
size in the basal portion continued substantially beyond the fully
open stage indicating that it was still in a growing phase unlike the
distal portion.
3.3. Expression in leaves
Considering that GgEXPA1 was expressed in all expanding tis-
sues of the flower, expression was also tested in five different
stages of leaves starting with the young (Y), expanding (G1 and
G2), mature (M) to senescing (S) leaves. Semi-quantitative RT-PCR
of RNA from these tissues revealed maximum expression in the
growing phase of the leaf. As leaves turned mature there was a
decrease in expression and no expression could be detected after
the onset of developmental senescence in leaves (Fig. 3B).
3.4. GA induced expression of GgEXPA1
Since the increase in GA levels overlapped with the phase of
tepal expansion, we tested if GgEXPA1 expression was responsive to
GA mediated expansion. Flowers from three stages viz. −2, 0 and +2
were treated independently with water (control), GA and paclobu-
trazol for 24 h. Semi-quantitative RT-PCR of GgEXPA1 using tepal
cDNA from these samples revealed no change in expression at the
−2 stage when treated with either GA or paclobutrazol. However,
samples of the 0 stage showed a substantial increase in expression
after GA treatment when compared to water-treated samples and
a marked decrease in expression after treatment with paclobutra-
zol (Fig. 4A). In samples at the senescent stage (+2), transcripts of
GgEXPA1 were barely detectable and no change in transcript lev-
els was observed in GA or paclobutrazol treated samples (data not
shown).
3.5. Effect of GA and paclobutrazol on flower growth
Since paclobutrazol exhibited no effect at stage −2 we tested
the effect of GA and paclobutrazol on flower expansion at stage −3.
Buds at −3 stage were placed in beakers containing GA or paclobu-
trazol for 4 days. After 4 days, flowers placed in GA showed a 10%
increase in total tepal area as compared to the control. On the other
hand, flowers treated with paclobutrazol were about 10% smaller
than water-treated flowers (Fig. 4B).
3.6. Isolation of the expansin promoter and study of expansion
related activity
In view of the specific expansion related expression of
the gene, we isolated a 1.3 kb region upstream of the trans-
lational initiation codon, containing the putative promoter
of the expansin gene, by genome walking. Sequence and
analysis of the promoter by PlantCARE (http://bioinformatics.
psb.ugent.be/webtools/plantcare/html) revealed the rice P-box ele-
ment CCTTTTG which is a GA-responsive element at position −293
with respect to the start codon. The promoter was introduced
as a translational promoter-GUS fusion construct (that included
the first ATG codon of GgEXPA1 in frame with the GUS gene)
into gladiolus (for transient expression) and Arabidopsis (for sta-
ble expression) so as to study its ability to drive GUS expression.
In gladiolus, agroinfiltration of the promoter at different stages
 A. Azeez et al. / Postharvest Biology and Technology 58 (2010) 48–56
Fig. 3. (A) Semi-quantitative RT-PCR analysis of GgEXPA1 and GgDAD1 expression in
the basal and distal portions of the tepals of the fully open flower (stage 0). Actin was
used as a loading control. Top panel: figure showing the regions chosen as basal and
distal parts of the tepal. (B) GgEXPA1 expression during leaf growth and senescence
as determined by semi-quantitative RT-PCR. Actin was used as a loading control. Y
– young leaf; G1 – growing leaf (stage 1); G2 – growing leaf (stage 2); M – mature
leaf; S – senescent leaf.
revealed high expression in young −2 stage flowers especially in
the basal portion of the tepals (Fig. 5A). As flowers moved into 0
and +2 stages there was a decrease in GUS expression. The decrease
was most prominent in the periphery of the tepals which began to
show senescence symptoms. Expression was also tested in stable
transgenic arabidopsis plants during early seedling development in
the dark and light grown conditions. As shown in Fig. 5B, develop-
ment in dark caused etiolated seedlings to elongate. This elongation
was associated with activation of the expansin promoter as evident
from the histochemical GUS stain seen in hypocotyls. No colour was
observed in the unexpanded cotyledonary leaves. When seedlings
were grown in light, expansion of the cotyledonary leaves could
be observed. This expansion was associated with increased GUS
activity in the expanding cotyledonary leaves and hypocotyls but
not in the young unexpanded leaves. These results indicated that
the 1.3 kb DNA sequence upstream of the gene possessed cis ele-
ments that could drive the expression of the gene in response
53
Fig. 4. (A) Response of GgEXPA1 transcription to GA treatment and inhibition by
paclobutrazol. Flowers from −2 and 0 stages were treated with water (control), GA
(10 ␮M) or paclobutrazol (20 ␮M) for 24 h and cDNA prepared. This was used for
semi-quantitative RT-PCR analysis using actin as a loading control. (B) Effect of GA
and paclobutrazol on flower size. Buds from stage −3 were excised and placed in
beaker containing water (control), GA3 or paclobutrazol. After 4 days, tepal area
(from at least three flowers) was measured and plotted on a graph. Data were
analyzed and expressed as mean ± standard deviation. Letters (a–c) over the bars
indicate significant differences at P < 0.05.
to expansion related growth even in a heterologous system as
distantly related to gladiolus as Arabidopsis and that it was not
active in non-expanding tissues. To further study the hormonal
regulation of the expansion related expression of the promoter,
we tested the expression of the promoter in etiolated seedlings
after treatments that inhibited hypocotyl growth. When seedlings
were grown in the presence of paclobutrazol, hypocotyl elonga-
tion was inhibited and radial thickening could be observed. Under
these conditions, no GUS expression could be detected along the
length of the hypocotyls. GUS activity could only be seen at the
base of the shoot, which is the primary elongation zone in etio-
lated seedlings (Gendreau et al., 1997). These results showed that
inhibition of elongation/expansion inhibited the expression of the
promoter. Importantly, inhibition by paclobutrazol demonstrated
that the promoter functioned in a GA-responsive manner. In addi-
tion, GUS expression was also detected in Arabidopsis petals and
stamens as in the case of gladiolus.
4. Discussion
Floral organ development is characterized by rapid growth of
floral organs (sepals, petals, stamens and pistils) just prior to and
during anthesis with cell elongation playing a major role in deter-
mining final shape. In petunia, up to 60% of the flower length is
attained by cell elongation after cell division has ceased (Martin
and Gerats, 1993; Reale et al., 2002). In rose also much of the ear-
lier development occurs primarily through cell division while the
later development after flower opening occurs through cell elonga-
tion (Yamada et al., 2009a). Expansins are believed to be important
in cell wall extension and modification and their action is likely
to play a role in determining the final shape of flowers. However,
there have only been a few reports on the study of expansin in rela-
tion to flower development and size (Gookin et al., 2003; Zenoni et
54 A. Azeez et al. / Postharvest Biology and Technology 58 (2010) 48–56
Fig. 5. Histochemical analysis of ß-glucuronidase driven by the 1.3 kb GgEXPA1 promoter. (A) Transient expression in agro-infiltrated gladiolus tepals from three stages viz.
−2, 0 and +2. (B) Transgenic Arabidopsis seedlings. Expression is shown in elongated etiolated hypocotyls, in light grown expanded cotyledonary leaves, after treatment with
paclobutrazol (20 ␮M) and in Arabidopsis flowers and stamens.
al., 2004; Yamada et al., 2009b) and none in monocot flowers that
include a large number of ornamentals.
In this paper, we show that expansion of petal lobes that occurs
primarily from the stages −3 to 0 is strongly correlated with expres-
sion of an alpha-expansin gene, GgEXPA1, in these tissues. This is
similar to the increase in expression observed in expansin genes
MjEXP1, MjEXP3 and MjEXP4 of M. jalapa during the increase in
length of the flower tube (Gookin et al., 2003). However, unlike
in Mirabilis, expression of GgEXPA1 is also seen during expansion of
the limb (stages −1 and 0) as has also been reported for PhEXPA1
in mesophyll cells of Petunia petal limbs (Zenoni et al., 2004) and
for RhEXPA1 in the rose petal expansion (Yamada et al., 2009b). We
have extended our studies to include other elongating floral tissues
such as stamen filaments and pistil styles as well, and shown that
GgEXPA1 expresses during those stages that mark the maximum
increase in length of these organs. The increase in length in both
stamen filaments and pistil styles was a consequence of increased
cell expansion as determined by light microscopy (data not shown)
like in the case of tepals (Table 1). Surprisingly, very low expression
of GgEXPA1 was observed at the −2 stage of pistils in spite of the 2-
fold increase in its length. Since expansins belong to a multi-gene
family, it is possible that expansion at this stage may be brought
about by some other as yet unidentified expansin. No expression
was observed in any of the flower parts in the later stages of +2
and +3 once expansion had ceased and senescence was in progress.
In this respect, GgEXPA1 differs from MjEXP2 and MjEXP6 which
are expressed prominently during senescence of petals (Gookin
et al., 2003). This indicates that GgEXPA1 is not involved in cell
wall modification that occurs during senescence unlike in Mirabilis.
Interestingly, within tepals, GgEXPA1 expression was higher in the
developmentally ‘younger’ growing basal cells than in the senesc-
ing distal cells. The expression of GgDAD1, which is down-regulated
upon the onset of senescence in tepals (Yamada et al., 2004) was
used to confirm the earlier occurrence of senescence in the distal
portion of the tepal as against the basal portion (Fig. 3A).
GgEXPA1 was observed not only in floral organs but also in
young growing/expanding leaves but not in mature leaves where
senescence had started indicating general expansion associated
expression.
Previous studies have reported GA accumulation in the petals
during expansion (Murakami, 1973, 1975; Koning, 1984). GA also
affects expansion in stamen filaments and pistils (Murakami, 1973,
1975; Pharis and King, 1985) and the GA mutant gai-1 shows abnor-
malities in filament length due to reduced cell elongation (Cheng
et al., 2004). When we estimated GA levels in flowers during the
expansion phase we observed an increase in its levels at the −2
stage followed by a decrease to basal levels by stage 0 (data not
shown). This was substantiated experimentally by treatment of
flowers with exogenous GA3 and the GA biosynthesis inhibitor,
paclobutrazol. In the 0 stage flowers, when endogenous GA lev-
els were at their lowest, the addition of exogenous GA was able
to stimulate GgEXPA1 expression considerably. Treatment with
paclobutrazol at this stage inhibited GA biosynthesis bringing down
levels of already limiting GA and causing a marked decrease in
expansin expression. When endogenous GA levels were at their
highest in stage −2, very little effect of additional exogenous GA
was seen since GgEXPA1 was already at its peak expression. Because
of these high GA levels, paclobutrazol was also not able to exert
its inhibitory effect. At the +2 stage, flowers are at an advanced
stage of senescence with barely detectable expansin expression. At
this stage, neither GA nor paclobutrazol have any effect on expres-
sion, most likely because the transcription factor(s) responsible for
GgEXPA1 expression are probably lacking. These results show that
GgEXPA1 is responsive to GA during tepal growth and probably
mediates its action on expansion through GA. Indeed, when GA and
paclobutrazol were applied earlier at the −3 stage when expansin
expression was very low, significant changes in flower size were
observed.
Sequence analysis of the 1.3 kb region of the GgEXPA1 promoter
revealed the presence of the GA-responsive P element CCTTTTG
 A. Azeez et al. / Postharvest Biology and Technology 58 (2010) 48–56
that may respond to gibberellins. In fact, the 1.3 kb promoter of the
GgEXPA1 gene was able to drive the expression of the GUS gene
in growing gladiolus tepals (particularly at the base) but not in
senescent tepals (especially in the periphery) indicating expansion
mediated expression. This was further substantiated in transgenic
arabidopsis where GUS expression was observed in etiolated and
expanding hypocotyls of arabidopsis (which have saturating con-
centrations of GA, Cowling and Harberd, 1999) as well as light
grown expanding cotyledonary leaves but not in the etiolated or
light grown, non-expanding leaves. These patterns showed that
the promoter of GgEXPA1 was responsive to expansion related cues
even in a heterologous system such as Arabidopsis and in different
tissues such as hypocotyls, leaves, petals and stamens. The process
of elongation in etiolated hypocotyls is mediated by GA and the
gai-1 mutant shows reduced hypocotyl elongation while paclobu-
trazol inhibits dark elongation (Cowling and Harberd, 1999). When
dark induced hypocotyl elongation of transgenic pGgEXPA1:GUS
expressing Arabidopsis plants was inhibited with paclobutrazol,
no GUS expression was observed in the hypocotyls indicating that
the 1.3 kb region of GgEXPA1 promoter responds to endogenous
GA during expansion (as observed in gladiolus). Our results col-
lectively suggest a mechanism whereby GA accumulation may
activate the expression of GgEXPA1 leading to organ expansion in
gladiolus. Such GA mediated up-regulation of expansin expression
has been observed for OsEXPA4 in submerged rice within 30 min
of GA treatment (Cho and Kende, 1997) as well as three tomato
expansins, LeEXP4, LeEXP8 and LeEXP10 in endosperm cap cells
(Chen and Bradford, 2000; Chen et al., 2001). Recently, a several fold
increase in transcript accumulation of the cotton expansin gene was
observed within 3 h of GA application in cultured ovules (Aleman
et al., 2008).
In conclusion, GgEXPA1 provides an interesting example of a
single expansin gene being involved in expansion processes in dif-
ferent plant tissues such as tepals, stamens, pistils and leaves that
are both spatially as well as temporally distinct in their develop-
ment. This shows that even though expansins belong to multi-gene
families with distinct roles for different members, certain mem-
bers may be ubiquitously expressed during organ development.
The study also provides a basis for GA mediated expansion of floral
organs via expansins prior to anthesis.
Acknowledgements
We thank Dr SK Datta and Dr RK Roy, NBRI for their help in
providing gladioli corms and Mr Ram Awadh for taking care of the
plants. This work was supported by the Indian National Science
Academy and Department of Biotechnology, India (APS, PN) and
the Council for Scientific and Industrial Research, New Delhi, India
(AA, SKT).
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