12-chapter 6

SUMMARY AND CONCLUSIONS
CHAPTER6
SUMMARY AND CONCLUSIONS
The LEAFY (LFY)IFLORICAULA (FLO) gene isolated originally from Arabidopsis
and Antirrhinum was earlier shown to be necessary for normal flower development in
these species respectively. These genes encode for plant specific transcription factors
required for the transition of plants from vegetative phase to the reproductive phase.
They have dual roles in flowering - first in flower initiation and later in activation of
floral homeotic genes (Parcy et al., 1998). The LEAFY gene has been cloned and
characterized from a few species viz. tobacco (Kelly et al., 1995), Eucalyptus
(Southerton et al., 1998), Pinus (Mouradov et al., 1998), tomato (Molinero-Rosales et
al., 1999), poplar (Rottmann et al., 2000), apple (Wada et al., 2002), rubber
(Dornealas and Rodrigues, 2005), papaya (Yu et al., 2005) and cedar (Dornealas and
Rodrigues, 2005).
Despite its sequence conservation in many species, significant differences in
the expression pattern have been reported which point towards the functional
divergence between them. In Antirrhinum majus, the expression is limited to the
reproductive phase (Coen et al., 1990) while low levels have been detected also
during vegetative phase in Arabidopsis, N. tabacum, Impatiens, P. sativum, Petunia
and L. esculentum (Kelly et al., 1995; Blazquez et al., 1997; Bradley et al., 1997;
Hofer et al.,
199~;
Pouteau et al., 1997; Souer et al., 1998; Molinero-Rosales et al.,
1999). The loss-of-function mutants of tomato and pea indicate its role in leaf
development also (Hofer et al., 1997; Molinero-Rosales et al., 1999). The expression
pattern in monocots is more divergent. The expression of the LFY homolog in rice
(RFL) is restricted to young panicles (Kyozuka et al., 1998). Unlike LFYIFLO gene
expression which precedes AP1 expression, LtLFY from Lolium temulentum is
expressed later than the AP1 ortholog, LtMADS2 (Gocal et al., 2001). In maize,
zjl1zjl2 double mutants shows dramatically reduced number of tassel branches,
indicating that these genes play a role in promoting branch establishment in addition
to its role in flower development (Bomblies et al., 2003). The WFL expression pattern
indicated that WFL is associated with spikelet formation rather than floral meristem
identity in wheat (Shitsukawa et al., 2006).
Constitutive expression of the LEAFY gene promotes flower initiation and
development from shoot apical and axillary meristems in Arabidopsis and other dicot
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and monocot species (Weigel and Nilsson, 1995; He et al., 2000; Pena et al., 2001),
suggesting that its role during transition from the vegetative to the reproductive phase
is conserved between widely related species within Angiosperms. The ability of the
LFY gene to accelerate flowering has generated considerable interest to study its role
in transition from vegetative to the reproductive phase and in the potential use of this
gene for modulating flowering time in agriculturally important crops.
The major objective of the present investigation was to overexpress the
LEAFY gene from Arabidopsis thaliana in Brassica juncea and to clone and study the
expression pattern of its homolog from B. juncea. The highlights of the present
investigation are as follows:
•
The LEAFY eDNA from Arabidopsis thaliana was cloned under the control of
a constitutive CaMV 35S promoter in a binary vector, pCAMBIA 1304,
containing gfp:gus reporter gene, hygromycin phosphotransferase II (hpt II)
gene as the plant selectable marker gene and neomycin phosphotransferase II
(npt II) gene as bacterial selection gene. The constructs harbouring the LFY
gene in the sense and antisense orientation were named as pSL and pASL
respectively.
•
Brassica juncea
cv.
Varuna was
transformed
using Agrobacterium
tumefaciens strain GV3101 harbouring pSL and pASL
•
The TO and Tl transgenic plants were characterized at the molecular level by
Southern and northern blot analysis. All the antisense plants analyzed had a
single copy T-DNA insertion while some of the sense lines had multiple
copies of the LFY gene of A. thaliana inserted in the B. juncea genome. A
fusion transcript was observed in all the transgenic plants but not in the wild
type untransformed control plant.
•
Transgenic plants of B. juncea overexpressing the LFY gene from A. thaliana
in sense orientation flowered about a week earlier as compared to the wild
type untransformed control plant and the antisense transgenic plants. The early
flowering in transgenic plants was not accompanied with any yield loss. The
transgenic plants overexpressing the LFY gene had similar morphology and
growth pattern as the wild type untransformed control plants.
•
The expression of endogenous LFY gene in different vegetative and
reproductive organs was analyzed in B. juncea. The LFY transcript was more
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abundant in sepals and pistils compared to the other floral organs. The
expression of this gene was also detected in stem, mature leaves and bracts.
•
The expression of LFY gene in leaves during different stages of plant growth
was also investigated. The expression was more in the cotyledonary leaves,
decreased in the developing leaves during the vegetative phase and again
increased in these leaves during the reproductive phase.
•
The expression pattern of LFY gene was studied in the shoot apices of
different cultivars of B. juncea with varying flowering time. The level of the
transcript was more in the early flowering variety, TN1, compared to the late
flowering ones, PNMB and RNBL, during the initial stages (1 0 to 24-days ).
The LFY gene expression could also be detected in the vegetative apices and
the expression increased during the transition phase from the vegetative to the
reproductive phase.
•
The effect of sucrose on in vitro flowering of B. juncea was investigated and
the consequent effect on LFY gene expression studied. Sucrose promotes
flowering in B. juncea probably by acting as an inducer of the LFY gene.
•
The effect of different cytokinins and gibberellic acid on LFY transcript level
was also studied. While cytokinin had a promotive effect on LFY expression
gibberellin inhibited LFY gene expression.
•
A LEAFY eDNA (BjLEAFY-P) of 1261 bp was cloned from eDNA library of
B. juncea cv. Pusa Bold. This clone, however, had a single base deletion at
762 bp and therefore encoded for a truncated protein of 284 amino acids with
a molecular weight of 31 kDa. This truncated protein showed significant
homology with the other LFY sequences present in the database.
•
A eDNA (1290 bp) encoding for a full length LFY protein (420 amino acids)
was also cloned from B. juncea cv. Varuna. This was 99% similar to
A. thaliana LFY protein and the truncated protein from B. juncea cv. Pusa
Bold. The encoded protein had a pi 6.48 and molecular weight 46 kDa.
•
Dendrogram analysis of the LFY proteins from different cultivars of Brassica
juncea showed that it is closer to the A. thaliana (LFY) and B. oleracea
(BOFH) proteins.
•
Analysis of the deduced amino acid sequence of the LFY proteins from
B. juncea showed the presence of typical FLOILFY motifs, characteristic of
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the transcription factors. These were proline rich regions at theN-terminus and
the acidic domain.
•
The CO gene expression in B. juncea cv. Varuna was also studied. It is
circadian regulated with a peak in expression after 20 hr and then a decrease
after 40 hr of transfer to continuous darkness. The CO mRNA levels remained
high till 16 hr when a night break (red light irradiation for 5 min) was given
following which its level decreased. The maximum expression was observed
in the photoperiod 8 hr dark and 6 hr light.
•
A eDNA (1033 bp) encoding CONST ANS-like 1 protein (337 amino acids)
was cloned from B. juncea cv. Varuna. It has a pi 6.1 and molecular weight 39
kDa. It has the typical B-box type zinc finger motifs and CCT motif as found
in CO and CO-like proteins.
•
Dendrogram analysis of COL proteins showed that the B. juncea, B. napus and
B. nigra are very close to each other.
Conclusions
In this study, transgenic plants of B. juncea constitutively expressing the LFY gene
were developed which flowered one week earlier as compared to the untransformed
control plant. No yield penalty accompanied this early flowering. The LFY homologs
from two cultivars of B. juncea (Pusa Bold and Varuna) were isolated. While the LFY
eDNA from Varuna encoded for a full-length protein, the one isolated from Pusa Bold
encoded for a truncated protein. The expression pattern of the LFY gene in B. juncea
was also analysed during the vegetative and the reproductive development. The
expression increased during the transition from the vegetative to the reproductive
phase and the abundance of the transcript was more in the early flowering varieties.
The CO gene expression pattern in B. juncea cotyledonary leaves was observed to
follow a circadian rhythm. A COLI eDNA from B. juncea cv. Varuna has also been
isolated.
Future Perspectives
Isolation and characterization of the cis elements present within the promoter of the
BjLFY eDNA isolated in the present investigation could provide an insight into the
expression pattern of the gene. A study of the phosphorylation pattern of BjLFY
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protein would help in elucidating its effect on LFY function. The downstream targets
of LFY gene can be identified and the effect of overexpression of LFY on those could
be analyzed in the B. juncea transgenic plants overexpressing the LFY gene. If the
overexpression of the BjLFY eDNA in a heterologous system also leads to early
flowering, it can be of use in horticulture. The COLI eDNA isolated in the present
investigation needs further characterization and elucidation of its role in flowering
time. Thus, a lot of basic research needs to be continued in this complex area to solve
many intriguing questions.
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