Chapter causes the vegetative shoot apical meristem to form

Chapter 5


Overall increase in oil production is the
long-term objective for plant-based biodiesel production. J. curcas due to its various merits is considered as a potential
source of biodiesel. However, some major constraints like variation in oil
content among genotypes, low female flower ratio, low productivity in terms of
yield, susceptibility to various biotic stresses and non-availability of
sufficient feedstock have been limiting this plant as a feasible alternative
for biodiesel production. As Jatropha has lower female to male flower ratio,
which in turn reduces seed yield and overall seed oil, identification of
molecular insights to comprehend female flowering and its transition was
crucial. Also, there was a requirement to identify molecular components
associated with yield in response to cytokinin treatment in J. curcas since after BA application
there was no significant increase in yield. Therefore, present study was
carried out with an aim of elucidating molecular basis of female flowering and
its transition, oil content variation among genotypes and understanding
molecular mechanisms and components underlying carbon capture and flux in
response to cytokinin application in J.
curcas to identify the factors affecting the overall yield. This research
work has provided leads which can be taken forward to carry out genetic
improvement for enhancement of seed yield in J. curcas.

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results obtained are discussed as under:














5.1 Transition from vegetative to reproductive

Meristem behavior
largely influences the plant floral cycle producing two types of meristems: first
is the inflorescence meristem consisting of organ primordia and floral organs
and the second meristem which does not bear any reproductive organs. Signal for
flowering causes the vegetative shoot apical meristem to form reproductive
organs.  When flowering signal is
triggered, FT/FD heterodimer moves to the shoot apex and activates AP1 which in turn induces flowering by
activating floral organ genes. AP1, a
floral meristem identity gene belongs to MADS-box family transcription factor, initiates the
floral development by integrating growth, patterning, and hormonal pathways
25.  In Jatropha, AP1 showed ~426 folds increase in RSI stage (SAM with emerging
floral buds) showing its strong association with transition to inflorescence
meristem. AP1 acts as a phase
activator or switch for transition to reproductive phase has been reported in wheat,
maize, tomato etc (Pena et al. 2001; Ellul et al. 2004, Murai et al 2003, Alter
et al 2016).  The promoter of AP1 gene is a flower-specific promoter which
directs the expression of SUP gene. SUP, a cadastral
gene and C2H2
type zinc finger protein family transcription factor, defines the boundary of floral organs and the
expression of organ identity genes (Yun et al. 2002). TFL1,
a TCP transcription factors showed ~ 65 folds higher expression in RSI suggesting
its role in floral transition and maintaining inflorescence meristem. Li et al
also reported that JcTFL1b was highly
expressed in reproductive phase in Jatropha.  It maintains the inflorescence meristem, floral buds and
reproductive growth in Arabidopsis, Populus and Bambusa oldhamii (Conti and Bradley 2007; Zeng et al.,
2015; 23). Apart from floral homeotic genes, circadian rythyms also plays an
important rale in phase transition. CRY2
encodes a cryptochrome proteins which was expressed ~3 folds higher in
reproductive phase. It interacts with circadian clock and promotes flowering by
inducing the expression of CO gene which then activates
floreign. This activates the floral meristem identity genes resulting in floral
transition (Guo el al 1988). Along
with floral homeotic transcription factors and circadian rhythms, hormones
plays an important role in reproductive phase transition. Auxins              Thus, the expression xpression pattern of TFL1, SUP, AP1, CRY2, CUC2, PIN1 and TAA1 indicated their involvement in
vegetative to reproductive phase transition.






5.2 Formation of floral organs

After commitment
to reproductive phase, undifferentiated buds from and in which plant hormonal
signaling plays a major role. Genes RGL,
IPT2 and EIN2 showed increased transcript abundance in initial buds stage
and then expressed at similar levels in intermediate, male and female floral
buds, thereby, suggesting their involvement in floral organ development but not
in organ differentiation. EIN2 has
been implicated in ethylene signaling which is involved in vegetative to
reproductive phase transition whereas RGL
protein is involved in modulating floral development in Arabidopsis 26-27.
Genes IPT2, CYP735A, AHK2, CRE1, CUC2 are associated with cytokinin biosynthesis and signaling
whereas genes TAA1 and PIN1 with auxin signaling and might be
playing role in floral organ formation.


buds further differentiate into male, female and intermediate types. We
observed that genes TFL1, CRY2 and SUP might be involved in floral transitions. Genes AP1 and TypA1 are possibly contributing to male flower development while
genes CKX1, CUC2 and TAA1 towards
female flower development. In present study, expression of TFL1 gene was in the order of intermediate>male>female
suggesting its involvement in floral transition and contributing to male flower
buds development. From our study we inferred that SUP and CRY2 genes might
be contributing towards female flower transition as they were expressed in
intermediate>female>male. Expression of CUC2, TAA1 and CKX1 increased progressively from
vegetative to reproductive stage, then in initial bud and finally highest in
female flower buds indicating their role in floral organ formation and further
in female flower development. It
has been reported
that CUC2, a NAC transcription factor
was expressed at boundaries between
meristems and organ primordia indicating its role in organ separation in Arabidopsis 28. CUC2 develops female reproductive organ by controlling meristematic
activity in Arabidopsis 11 and Silene latifolia 29. TAA1 involved in local auxin production,
tissue-specific ethylene effects, and organ development and its involvement in
female flower development was reported by Stepanova et al 8. CKX1 gene
causes oxidative cytokinin degradation and its accumulation in reproductive
tissues of transgenic maize resulting in male-sterile plants 30. Expression
pattern of PIN1 in J. curcas reflected
its role in initial buds formation and then in female flowering. CUC1 and CUC2 regulate PIN1
activity which further regulates ovule primordial development in Arabidopsis 31. Genes AP1 and TypA1 showed highest expression at initial bud stage, confirming
their role in floral organ development. AP1
is reported to be involved in patterning of floral organs in Arabidopsis 32. In cucumber, CsTypA1 is expressed differentially
during male and female flower development with higher expression in ovary 33.
Whereas in our study, expression of TypA1
was ~5.5 folds higher in male floral buds compared to female flower buds
suggesting its role in male flower development in J. curcas.

Expression pattern
of genes involved in endogenous cytokinin signaling indicates its strong role
in female flowering. Till now IPT1 is
the only isopentenyltransferase–encoding gene that is expressed in ovules of Arabidopsis
thaliana 34. From our
expression data we observed that genes IPT3
and IPT9 are also involved in female
flowering as their expression was higher in female floral buds in J. curcas.
Cytokinin signal transduction is mediated by receptors histidine kinases AHK2 and AHK4/CRE1. AHK2 signaling in floral development is
mediated by the effector gene CUC2
35. When expression of AHK2 and CUC2 was compared between RSIII, RSIV
and RSV, both expressed higher in RSV indicating that they are active in female
flowering. CUC1 and CUC2 may be involved in the increase of
CKs which regulates PIN1 expression
needed for primordia formation 36. CKI1
and PIN1 follow the same expression
pattern indicating correlation between endogenous cytokinin signalling and
auxin flux. Therefore, CKI1 might be
regulating directly or indirectly auxin flux and thus female flowering.
Expression pattern of CRE1 and BEL1 is similar in all stages and is in
agreement with the previous study. Thus, BEL1
is regulated by cytokinin signaling mediated through CRE1 receptor. BEL1 along
with SPL is involved in ovule
development by modulating auxin fluxes through controlling PIN1 expression 37.

Flowering transition is mediated through SUP which regulates the process by
cytokinin signaling. Previous studies have shown that SUP blocks the expression of B class floral identity genes in Arabidopsis
and develops gynoecia by aborting stamen development. SUP, itself is under the control of floral meristem identity gene LFY,
which activates it through AP3/PI-dependent and -independent
pathways 38. Also, exogenous cytokinin (6-benzyladenine) treatment
upregulated AP3 and repressed class B
floral homeotic genes and TS2
homolog, which increased the female to male flower ratio by arresting pistil
primordia in male flowers in J. curcas 15. These studies indicate
involvement of SUP gene in female
flowering and further our observations suggest its role in flower induction and
female flower transition. CYR2 and FT genes were expressed higher in female
floral buds as compared to male indicating that circadian rhythms also
influence female flowering. Also, FT
and TFL1 act antagonistically and in
our study FT was found to be
associated with female flowering while TFL1
with the development of male flowers. Hence it is inferred that auxin and
cytokinin signaling pathways and circadian rhythms control feminism in J.
curcas and are involved in sex determination.

genes identified through expression analysis at floral developmental stages may
have been associated to a particular stage due to developmental pattern rather
influenced by the inherent genetic differences, which could only be inferred by
testing expression in genotypes differing for female to male flower ratio.
Difference in female to male flower ratio among different genotypes could be
due to environmental or molecular effects. Since genotypes were grown at the
same location this nullifies the role of environmental factors for variation in
ratio of female flowers. These
high and low female flower accessions differ with respect to the presence of
female flowers at the apical position of each sub branches of the inflorescence
in high accession whereas in low accession it is occupied by only intermediate
buds. To identify the role of molecular cues, we selected male, female
and intermediate type buds and relative expression status of genes SUP, CRY2,
PIN1, CKX1, TAA1, CUC2, TypA1 and AP1 was
compared in high female to male flower ratio genotypes. After analyzing
expression at different developmental stages we observed that the difference in
female to male flower ratio occurs mainly in intermediate buds depending upon
the molecular signals it receives. We observed when the rate of stamen abortion
is high; more females are formed indicating various internal cues are causing
the arrest of stamens thus allowing female flowers to develop otherwise
resulting in development of males with fused stamens. Through expression data
it was observed that SUP along with TAA1, CRY2, and CKX1 genes play
an important role in female flower transition as its expression was correlated
with the ratio of female flower. The expression of SUP was increased upto 7 folds in intermediate buds when compared
with low genotypes and was significantly decreased in males indicating that
this might have increased the stamen abortion resulting in more female flower
number. This is also indicated by dissection of buds and the position of female
flowers where higher number of female flowers is achieved at the position
occupied by intermediate types with fused stamens in low ratio genotypes. This
strongly suggests that SUP might be
responsible for transition towards female flowers. Even CUC2 showed 10 folds upregulation in female flower buds when
compared to low female flower ratio genotype, thereby suggesting its
association with female flower development. It is interesting to note that both
SUP and CUC2 genes are involved in differentiating floral organs by forming
a boundary between them. Functions of both SUP
and CUC2 genes have been validated in
different plant systems for female flower development by creating mutants but
not for transition towards female flower development. We are suggesting that
endogenous auxin and cytokinin signaling plays an important role in this
transition. Previous studies on exogenous cytokine treatment also indicated the
stamen arrest with downregualtion of TS2 15.
Through these data we are suggesting that cytokinin treatment did increase the
total flower number but it might have enhanced the female to male flower ratio
by increasing the rate of male abortion in intermediate buds. This can be an
important factor to enhance the overall female flower number either by inducing
more females to develop at the intermediate or increasing the arrest of stamens
thus allowing female tissues to develop. SUP
gene can be a strong candidate to enhance overall female flower number in Jatropha curcas through genetic
modification, however its function needs to be validated.

Higher expression of genes involved in
reproductive phase transition as well as in female flowering might be governed
by common regulatory elements ARR1AT, BIHD1OS,
MYB1AT, POLLEN1LELAT52, and WRKY71OS in promoter regions of genes FLT, SUP, AP1, CRY2, CUC2, CKX1, TAA1 and
PIN1. ARR1AT, a cis-regulatory cytokinin response motif is reported in Arabidopsis 39. ARR1 element is
present in promoters of both WUS and STM genes involved in meristem formation
40. BIHD1OS, a BELL homeodomain transcription factor encodes a protein involved in
patterning of ovule primordia in the Arabidopsis
41.  POLLEN1LELAT52 element is required for pollen specific expression in
tomato 42. WRKY71OS is a binding site of rice WRKY71, a transcriptional repressor of the
gibberellin signaling pathway 43. Female flower formation
seems to be regulated by the presence of elements UP2ATMSD, GAREAT and MYB1AT
in genes CUC2, TAA1, CKX1, SUP,
CRY2 and PIN1. UP2ATMSD, a cis-regulatory
element was reported to regulate gene expression during initiation of axillary
bud outgrowth in Arabidopsis 44. GAREAT regulates gibberellin biosynthesis and
signalling in Arabidopsis 45
whereas MYB1AT functions as transcriptional activators in abscisic acid
signaling 46. GARE2OSREP1 and CARGATCONSENSUS were identified as unique
elements in the promoter region of CRY2 gene, possibly regulating reproductive
and female flowering transition. CARGATCONSENSUS element is reported to
regulate the flowering time genes and floral homeotic genes in Arabidopsis 47-48. Also, GARE2OSREP1
element is involved in gibberellin regulation in rice (Sutoh and Yamauchi 2003).