2.3.2 clock–controlled genes, promoting photoperiod mediated flowering (Putterill et

2.3.2 Sex determination

The
floral development of Jatropha has been divided into 12 phases starting from
vegetative to reproductive transition resulting the formation of inflorescence
meristem. During the first five phases no sexual differentiation occurs and the
females are present as bisexual tissue. Further development causes the abortion
of male tissue allowing the development of female flower. No traces of female
tissues were found in the male flowers. Thus, there are two modes of
development in Jatropha: formation of female flowers after the abortion of male
tissues and the other is the formation of male flowers with early adolescence
and no occurrence of female primordia (Wu et al 2012). As male can occupy
female flowering site, causing the decreased ratio of female to male flowers. Thus,
to increase the number of female flowers, the most effective approach is either
to transform male type inflorescences to middle of intermediate type or by
increasing the male abortion rate, allowing female flowers to develop. This
would only be possible by having the knowledge of genetic switches causing the
transition towards female flowers. This is one of the method to increase seed
yield, as fruits are formed from the female flowers only.

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2.3.2 Molecular cue for flowering of
Jatropha

Floral
initiation, a process in which shoot apical meristem develops into the
inflorescence meristem which forms reproductive organs. The process is
controlled at environmental and genetic level, regulating various genes
associated with triggering floral pathways. Factors contribute to flowering initiation are photoperiod, vernalization, hormones,
autonomous and age related pathways. These signals initiate the
reproductive phase by inducing meristem identity genes which directs the certain groups of cells of SAM to
differentiate into floral meristems in an irreversible manner.

 

The floral induction signals through various signaling pathways
are transmitted via floral integrator genes FLOWERING LOCUS T (FT), FLOWERING LOCUS
C (FLC)  and SUPPRESSOR OF OVEREXPRESSION OF
CONSTANS1 (SOC1) 
to the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) at the apical meristem. FT is a florigen i.e. a mobile flowering signal which
plays a crucial role in the transition from vegetative to reproductive phase. Activity
of FT is induced by CO and GI  which are circadian clock–controlled genes, promoting
photoperiod mediated flowering (Putterill et al.,
1995, Fowler et al., 1999(Li et al 2013).). FT
is expressed in leaf phloem when activated and moves to the shoot apex. AT the apex it forms a complex
with FD, a basic domain/leucine
zipper protein. This FT/FD heterodimer then activates a floral meristem
identity gene AP1and a floral promoter SOC1 (Michaels et al., 2005). AP1, FRUITFULL (FUL) and CAULIFLOWER (CAL) genes act redundantly to control the flower
meristem identity and inflorescence architecture by regulating the expression
of  LFY and TFL1  (Ferrándiz et al., 2000). FT & LFY genes have been isolated and
characterized in Jatropha. JcFT was expressed mainly in the reproductive
organs. Overexpression of JcFT & JcLFY induced early flowering, showing that they act as flowering promoters (Ye et al 2014; Tang et al 2016).

 

 

 

2.3.3 Molecular basis of sex determination

In plants, sex determination is the process
through which unisexual flowers are formed. There are two dominant ways of
unisexual flower development: One is the emergence of only one type of sex
organ without formation of any bisexual tissue at any stage floral development.
Whereas in other; there is initiation of a bisexual floral meristem with both
stamens and pistils followed by a developmental arrest or abortion of one sex
with only the stamens or the carpels attaining functional maturity. The step
impeding the development of floral sex organs is at an immature stage well
prior to reaching sexual maturity (reviewed by Kinney, Columbus, & Friar,
2008). Many monoecious species progresses through an early hermaphroditic stage
to differentiated (unisexual) stages later in floral development, by aborting
or arresting either of the sexual organs (Ainsworth, 2000). Jatropha being a monoecious
plant, sexual differentiation occurs by abortion of stamens, allowing the
female flowers to develop. No female tissues were found in fully developed male
flowers however remains of male tissues (aborted stamens) were found in a fully
developed female flowers. Transcriptome analysis of Jatropha floral buds identifies
Tasselseed 2 (TS2), a sex
determination gene which is required for stamen development and its reduced
expression promotes the carpel development by aborting male tissues. Recently,
transcriptome analysis of male and female floral buds at different
developmental stages of Jatropha identifies CRABS CLAW gene for sex
differentiation. They have also identified an ATP-binding protein promotes
stamen degeneration in female flower at later stage of development. Chlorophyll A/B-binding protein, ubiquitin
carboxyl-terminal hydrolase and inorganic phosphate transporter contribute to
the female organ development whereas cytochrome C oxidase subunit 1 contributes
to the development of the stamen. Gibberellin-regulated protein 4-like protein
and AMP-activated protein kinase  genes were
found to be associated with stamen differentiation, whereas auxin response
factor 6-like protein, AGL-20, CLV1, auxin-induced protein 22D, RING-H2 finger
protein ATL3J, and r2r3-myb transcription factor contribute to embryo sac
development in female flowers. COX, ARP1, GID1 and auxin-induced protein X10A
are expressed in both male and female flowers (Xu et al 2016). Functional study of JcFT, a floreign and a key
regulator of flowering pathway showed highest expression level in female
flowers (Li et al 2014).

Transcriptome analysis of other monoecious
plants have been performed to identify genes associated with sex determination.
In Quercus sober POLYGALACTURONASE-1, CYTOCHOME
P450 and ENDO-BETA-1,3-1,4 GLUCANASE
genes were identified for female flowering and CHALCONE SYNTHASE A, DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) and
4-COURAMATE–CoA
LIGASE-LIKE 1 were
found to be associated with pollen development (Rocheta et al 2014). ENDO-BETA-1,3-1,4
GLUCANASE gene possibly inhibits the development of male structure in females
and defect in DAD1 showed defects in anther dehiscence, pollen maturation, and
flower opening (Ishiguro et al., 2001). In Ricinus communis PDC related genes (cysteine protease) identified for
female development and its expression level was increased at the peak of anther
abortion (Lorrain et al., 2003; Wei et al., 2013). 

In cucumber sex differentiation has been
studied extensively and is genetically controlled by F locus (for females) and
M locus (for male). AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASEs
(ACS1 & ACS2), ETR2 and ERS genes associated with ethylene biosynthesis and signaling
pathways were found to be involved in sex determination.  ACS1 & ACS2 promotes gynoecia development by inhibiting male
reproductive organs (Saito et al 2007; Boualem et al., 20089).  ETR2 and ERSI, an ethylene receptors accumulated in
gynoecia, thus promoting female development (Yamasaki et al 2001).  A MADS-box protein ERAF-17 in cucumber induces female flowering. CTR1-like
kinase protein (CTR1 and CTR2), negative regulators of ethylene signaling promotes
male flower development by lowering ethylene accumulation, as males are
sensitive for ethylene. CmWIP1
gene in cucumber also
promotes male flowers.  Suppression of LESS ADHERENT POLLEN 3 (LAP3) and Nodulin MtN3 resulted in sterile pollen and their abortion
in female flowers in Vitis vinifera I and rice 60. In Medicago
truncatula Nodulin, MtN3 (Xa1) when suppressed resulted in
small anthers and reduced fertility due to abortive pollen 61.  In Pea (Pisum sativum L.), carpel senescence is associated with high levels of
lipoxygenase gene
expression 62.  Pentatricopeptide repeat-containing genes can
restores cytoplasmic male sterility in rice and petunia 63, 65. These
proteins have also been reported in Jatropha where they are involved in
differentiation of stamen and carpel and in later stages they are active in
embryo sac of females. Still there is a limited information on genetic cues for
female flower transition and sex determination in Jatropha. Also, the expression
status of genes at three floral buds (male, female and hermaphrodite) is still
unknown and is important to identify the possible targets for increasing the
female flower ratio.

 

ABCDE model of sex determination

ABCDE
model elucidate the role of floral homeotic genes in sex determination and
floral development.  Expression of the A-class genes specifies sepal
formation in whorl 1. A- and B-class genes combinedly specifies the development
of petals in whorl 2. In whorl 3, the combination of B- and C-class genes form stamens.
The expression of the C-class genes in whorl 4 determines the development of
carpels.  D-type activity involved in the
specification of ovules and an E function necessary for the determination of
the corolla, androecium and gynoecium (Angenent and Colombo, 1996; Pelaz et al., 2000). B and C class genes belong to MADS-box
family which are highly important transcription factors with its role in floral
organogenesis. These transcription
factors have highly conserved DNA binding domain known as the MADS box and moderately conserved domain known as K
box. MADS box genes have been isolated and
characterized in both dioecious species (white campion and sorrel) and in
monoecious species maize and  cucumber (Kater et al., 1998; Perl-Treves et al., 1998Hardenack et al., 1994) ,Schmidt et al., 1993; Mena et al., 1995, Ainsworth et al., 1995). Previous studies identified AGAMOUS,
APETALA3 and PISTILLATA
genes of B and C classes were differentially expressed in the male flowers in Thalictrum
dioicum and Spinacia
oleracea (Di
Stilio, Pfent C, Sather DN). In Elaeis guineensis mutation
in AP3 and PI male tissues,
allowing females to develop. In cucumber FLORAL BINDING PROTEIN 11 (FBP11),
a D-class gene determines ovule. AG2 has mixed C/D function gene as it
is expressed only in ovule primordia
and carpel in A. thaliana and Elaeis
guineensis (Favaro et al., 2003;Pinyopich et al., 2003). In monoecious
species Liquidambar styraciflua L and Rumex acetosa L, the expression of the C gene has been associated
with the arresting of the sexual organs. 
However, in Liquidambar styraciflua the arresting could be the
result of cell death. In Populus
trichocarpa, a genetic switch at a sex locus controls
expression of B and C-class genes, thereby controlling the development of male
or female flowers.

 

 

 

2.3.4 Transcription factors in female flowering
and sex determination

Apart from homeotic and floral organ identity
genes, transcription factors also plays an important role in sex deteremination
in plants. CUC2, a NAC transcription factor
was
expressed at boundaries between meristems and organ primordia and plays an
important role in organ separation and female reproductive organs in Arabidopsis thaliana
and Silene latifolia 32.  bZIP  (homolog of PERIANTHIA (PAN)) was involved in flower development by altering floral
organ number and initiation pattern by activation of the C-class MADS box
protein AG in A.
thaliana  (Running and Meyerowitz, 1996; Wynn et al., 2014; Das et al., 2009; Maier et al.,2009). SUP a C2H2 type zinc finger protein is a negative regulator of B class of genes. CEN1, TFL1 and FT belongs to the TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) family of
transcription factors. TFL1 and FT have
antagonist effects where FT promotes flowering and TFL1 is a suppressor of
flowering.