Chapter 6
FLORAL BIOLOGY OF SUNFLOWER:
A HISTOLOGICAL AND PHYSIOLOGICAL ANALYSIS
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla*
Laboratory of Plant Physiology and Biochemistry, Department of Botany,
University of Delhi, Delhi 10007, India
ABSTRACT
The development of sunflower inflorescence can be considered under three phases,
namely inflorescence initiation, floret development and anther formation. Floret
primordia appear at the rim of the receptacle where ray or disc florets are generated. Disc
florets are arranged in Fibonacci series whereby a spiral pattern emerges as new florets
arise in rows of bumps consisting of a bract and a floret. Floral morphogenesis in
sunflower occurs according to the ABC model, whereby genes of the MADS box are
activated. Anthesis of disc florets is a phytochrome-mediated response and is also
modulated by plant hormones, such as auxins. The disc florets are hermaphrodite and
protandrous in nature, whereas the ray florets are sterile, incomplete and have an
attractive, fused and flag-like corolla. Stigma in sunflower is semi-dry in nature,
producing lipid rich exudates in the crevices of the adjacent papillae. Stigma undergoes
physiological maturity with the passage of development from bud, staminate and, finally
to the pistillate stage. The production of extracellular lipid rich secretions is initiated at
the staminate stage of stigma development and increases at the receptive stage through
the availability of elaioplasts and endoplasmic reticulum network in the basal regions of
the papillae. Transfer cells, earlier identified only in the wet type of stigma, are also
present in the transmitting tissue of sunflower stigma. Neutral esters and triacylglycerols
(TAGs) are the major lipidic constituents in pollen grains and stigma, respectively.
Lignoceric acid (24:0) and cis-11-eicosenoic acid (20:1) are specifically expressed only
in the pollen coat. Similar long-chain fatty acids have earlier been demonstrated to play a
significant role during the initial signalling mechanism leading to hydration of pollen
grains on the stigma surface. Lipase activity is expressed both in the pollen grains and
stigma papillae. Stigma exhibits a better expression of acyl-ester hydrolase activity the
pollen grains. Specific expression of lignoceric acid (24:0) in the pollen coat and
*
Corresponding author: (Professor S.C.Bhatla), E mail: [email protected].
2
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
localization of lipase in pollen and stigma are likely to have possible roles during pollenstigma interaction. During the course of stigma development in sunflower, a correlation is
evident in the accumulation of reactive oxygen species (ROS), nitric oxide (NO) and the
activities of ROS scavenging enzymes [superoxide dismutase (SOD) and peroxidase
(POD)]. Mn-SOD (mitochondria localized) and Cu/Zn-SOD (cytoplasmic) exhibit
differential expression during the staminate stage of stigma development. An increase in
total SOD activity at the staminate stage is followed by a peak of POD activity during the
pistillate stage of stigma development, indicating the sequential action of the two
enzymes in scavenging ROS in maturing stigma. The number of POD isoforms increases
with the passage of stigma development and two POD isoforms are unique to pistillate
stage. This highlights their role in ROS scavenging mechanism. ROS and NO
accumulation exhibit reverse trends during pollen-stigma interaction. All these recent
findings indicate the modulation of floral development in sunflower by an array of
biomolecular signalling components which influence development through a series of
cross-talk mechanisms.
Keywords: Lipids, Nitric oxide, Non-specific esterase, Pollen, Peroxidase, Reactive oxygen
species, Superoxide dismutase, Stigma, Pollen-stigma interaction.
INTRODUCTION
A large diversity of floral structures of varying complexities are evident in plants for the
attraction of pollinators. Cross-pollinated plants exhibit a kind of synchrony among
themselves and also with their pollinators in order to bring about optimal seed set. It is
necessary that plants must flower at the correct time of the year for optimal reproductive
fitness. Such a strong correlation with the environment for the onset of flowering poses many
questions about the mechanisms of sensing of the environmental signals by the plants and
also about the sequence of biochemical events which ultimately bring about flowering in
response to the environmental signals. A vegetative shoot bud exhibits noteworthy differences
when compared with a floral bud, in terms of the constituent forms and types of cells. A
change in the fate of cells at the shoot apex is governed by the expression of a set of genes,
leading to various biochemical events in the shoot apex which bring about floral evocation,
i.e the ability of the apical meristem to produce flowers. Floral evocation is regulated by
endogenous factors, such as circadian rhythms and hormones, and exogenous factors such as
photoperiod and temperature. The initiation of four types of floral organs from the floral
meristem is observed in whorls around the flanks of the meristem. The floral primordial start
as small bumps of cells and their further development into reproductive structures is governed
by the environmental signals, various metabolic events and also by the activation of specific
genetic programs. Broadly, an attempt has been made in the following chapter to understand
the histological, physiological and biochemical changes taking place in the shoot apex of
sunflower during this process of phase change, i.e, transition from adult vegetative phase to
adult reproductive phase. The initiation of whorls of disc florets in the inner core and
development of peripheral ray florets during capitulum development in sunflower is a gradual
process, showing various stages of development of florets in a capitulum. The present chapter
provides detailed information on the ultrastructural changes associated with stigma
development in sunflower. These features have been analyzed in relation with the
Floral Biology of Sunflower: A Histological and Physiological Analysis
3
biochemical events accompanying stigma maturation. Likewise, a detailed structural analysis
of pollen (intact and germinating) has been discussed. Finally, the mechanism of pollenstigma interaction has been analyzed under natural and experimental conditions.
PATTERN OF FLORAL DEVELOPMENT AND ITS MODULATION BY
LIGHT, HORMONES AND GENETIC FACTORS
Members of Asteraceae maximize their reproductive output by condensing inflorescence
and forming a capitulum. Vegetative apex is indeterminate, domed and densely meristematic
whereas reproductive apex broadens, flattens and becomes determinate (Teeri et al., 2006).
Sunflower inflorescence is a disc-shaped capitulum located at the shoot tip and its shape and
size vary according to the cultivar, season and agricultural conditions (Weiss, 2000). The
capitulum is surrounded by three rows of ovate to ovate-laceolate involucral bracts or
phyllaries which function as sepals and protect the capitulum during its development (Figure
1). Various phases of reproductive development in sunflower have earlier been categorized
under nine stages (Schneiter and Miller, 1981). Beginning from the initiation of floral bud
(R1) to the attainment of physiological maturity (R9), R5 marks the beginning of flowering.
This stage (R5) is further subdivided from R5.1 to R5.2 and so on, representing the percent of
disc florets which have completed or are flowering. The florets in the capitulum are arranged
in a spiral and geometric pattern (Hernández and Green, 1993). Floret primodia appear at the
rim of the receptacle where ray or disc florets are generated. Disc florets are arranged in
Fibonacci series leading to the emergence of a spiral pattern as new florets arise in rows of
bumps consisting of a bract and a floret (Hernández, 1997). Ray florets (outer) are sterile,
incomplete and have an attractive, fused and flag-like corolla whereas disc florets (inner) are
complete and exhibit centripetal maturation pattern. Each disc floret consists of an inferior
ovary, two pappus scales (modified sepals) and a tubular corolla, which is fused, except at the
tip (Figure 2). Flowering begins with the unfolding of the ray florets in the capitulum. Disc
florets gradually open in whorls towards the centre of the head, as a consequence exhibiting
different stages of floret maturation in a single capitulum. Such a pattern of development also
increases flowering time of a capitulum, thereby attracting insects for pollination. The
maturation stages of disc florets are referred as bud, staminate, transitional and pistillate
(Figure 2). The disc florets are protandrus and are cross-pollinated by insects, particularly
bees. At the bud stage, disc florets exhibit the development of corolla, androecium and
gynoecium. Stigma is clasped and the pollen grains inside the anther lobes have a well
developed exine. Anthesis begins in the morning at the staminate stage when staminal
filaments elongate and black syngenesious stamens are exposed through the tubular corolla.
Protandry in sunflower is induced by photoperiod and corolla has been suggested to be the
site of light perception, stimulating the growth of anther filament (Lobello et al., 2000).
Elongation of the antheridial filaments is initiated after the dark period and occurs for about
2-6h. The pollen grains are then released inside the anther tube as three-celled structures. At
the transitional stage, the stigma elongates through the anther lobe and hairy pseudopapaillae
are observed at the apex of the anther tube. Members of Asteraceae exhibit the phenomenon
of secondary pollen presentation whereby pollen grains are relocated from the anther to
another floral organ, which then presents pollen for pollination. The pollen grains left in the
4
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
anther tube adhere to the sweeping hair (pseudopapillae) of the stigma and they are
mechanically forced out and exposed to the pollinators (Hong et al., 2008). Stigma exhibits
biphasic growth kinetics at the pistillate stage during which it first elongates, detaches along
the median, and the tip curls outward (Sammataro et al., 1985).
Figure 1. Stages of the development of capitulum in sunflower.
Figure 2. Various stages of disc floret development in sunflower. A: Young bud, B: Mature bud, C:
Staminate stage, D: Transitional stage, E: Pistillate stage.
Floral Biology of Sunflower: A Histological and Physiological Analysis
5
Floret maturation is reported to be under the control of phytochrome and plant hormones
(Baroncelli et al., 1990; Koning, 1983). The development of capitulum from R2 to R4 is
affected by photoperiod (Rezadoust et al., 2010). Although sunflower is considered to be a
day neutral plant, the development of floral buds and their maturation is known to be affected
by daylength. Light leads to an enhancement of photosynthesis, causing growth of the tissue
for the formation of floral bud. Short days are known to modulate anthesis by promoting postinitiation development of the floral buds (Marc and Palmer, 1981). Depending on the
influence of photoperiod in green house plantations (from the period of emergence to floral
bud development), different sunflower genotypes have been classified as long-day, short-day
or day-neutral plants. In addition, some genotypes have been observed to be
ambiphotoperiodic, a condition in which the floral buds can develop in long or short day
conditions but their further development is delayed in intermediate day length (Goyne and
Schneiter, 1987).
The young capitula (heads) exhibit heliotropism which is marked by the eastward
movement of head in the morning and its westward turning along the direction of sun. As the
capitulum matures, the opened heads are locked in the eastward direction (Weiss, 2000). An
eastwardly direction of the capitulum dries the night dew in the morning hours and decreases
the possibility of fungal attack. It also prevents overheating of the developing stigmas and
preserves pollen viability, consequently enhancing the efficiency of fertilization. It has been
proposed that the heliotropic movement of the young capitulum is related to auxin
distribution in the actively growing parts of the plants (Weiss, 2000). Growing regions of
plants contain relatively higher concentration of IAA than as compared to the fully developed
plant parts resulting in the accumulation of assimilated substances (Duca, 2006). The content
of gibberellic acid also increases, particularly in the cytoplasmic male sterile lines.
Gibberellic acid is involved in floral induction and the process of sexual differentiation (Duca
et al., 2003; Duca, 2006). The role of phytochrome in favouring protandry, and hence cross
pollination, has also been established (Baroncelli et al., 1990; Lobello et al., 2000). Variations
in photoperiod and relatively higher concentrations of gibberellic acid are known to cause a
deviation in floral development (Blackman et al., 2011). The elongation of antheridial
filaments is stimulated by auxins (IAA and NAA) or light. Auxins are known to be involved
in the light-regulated expansion of cells. In vitro experiments have confirmed that auxins can
reduce the inhibitory action of red or dichromatic treatment (far red + red light) on the
elongation of antheridal filaments. Filament elongation caused by light and dark cycles or
auxins, is also known to be dependent on the critical phase of growth of the florets (Lobello et
al., 2000). High concentrations of gibberellic acid (GA3) are known to inhibit filament and
style elongation in favourable photoperiodic conditions (Lobello et al., 2000). Light, thus,
plays an important role in altering the availability of gibberellic acid which is essential for
cell expansion.
The development of floral organs in each whorl is regulated by the differential activities
of various genes encoding MADS-box transcription factors (Dezar et al., 2003). The
identification of the genes responsible for sunflower morphogenesis has highlighted two types
of floral differentiation. Reproductive meristem follows the ABC model of flower
development which refers to the class of genes that are required for the development of
whorls of sepals, petals, stamens or carpels. The genes corresponding to ABC encode the
MADS-box transcription factors which are conserved motifs controlling transition from
vegetative to reproductive growth, thus determining the identity of floral meristem and
6
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
organs. Floral homeotic mutants of Arabidopsis thaliana, Antirrhinum majus and Petunia
hybrida have extensively been used to understand the controlling factors for the initiation of
floral buds by the ABC model (Coen and Meyerowitz, 1991; Angenent et al., 1995). Seven
full length cDNAs of HAM genes (Helianthus annuus MADS) have been isolated and their
control in the development of pistil, stamen and petals has been established in sunflower
(Shulga et al., 2008). Blot hybridization from different parts of sunflower has further revealed
that HAM 75 and HAM 92 genes are expressed in petals, seed coat HAM 45 is expressed in
the ovule and HAM 59 is expressed in the ovules, stamens and pistil. HAM 59 is expressed
essentially in the disc florets and is absent in ray florets, causing sterility in the ray florets.
HAM 59 expression during ray floret initiation seems to be important for the structural and
functional differences in the developing inflorescence. A correlation between the structure
and function of these proteins is evident. Since HAM genes code for proteins that belong to
different subfamilies of MADS-box, a duplication of the sunflower genome during evolution
has been proposed. Antimicrobial proteins are known to be produced by plants which
contribute to resistance. Among the antimicrobial peptides, cysteine-rich thionine, lipidtransfer proteins, defensins and snakin have been described. In sunflower, defensins have
been reported to accumulate as florets mature. The defensins are known to be localized
mainly in cell wall or vacuoles (Urdangarín et al., 2000).
Figure 3. Structural analysis of receptive stigma and pollen in sunflower. A: Scanning electron
micrograph of receptive stigma surface (65X); B: Transverse section of mature stigma showing the
presence of papillae (P), vascular strand (VS) and secretory canal (SC) (400X); C: Localization of
proteins in the papillae and transmitting tissue (TT) after staining with mercuric bromophenol solution
(400X); D: Electron micrograph from the basal region of the papillae showing the accumulation of
Floral Biology of Sunflower: A Histological and Physiological Analysis
7
extracellular secretions (1,150X); E: Transmission electron micrograph showing cluster of
mitochondria at the base of papillae at the staminate stage of stigma development (8000X); F:
Transmission electron micrograph showing transfer cells in the transmitting tissue below the papillae in
the receptive stigma; G: Transmission electron micrograph of cells of transmitting tissue showing
plasmodesmatal connections (1,150X); H and I: Transmission electron micrograph of the pollen wall
showing spinular region (4,600X) and the inter-spinular region (8,400X). Abbreviations: P, Papillae;
PP, Pseudopapillae; E, Extracellular secretions; PD, Plasmodesmata; N, Nucleus; V,Vacuole; M,
Mitochondria; WI, Wall Ingrowths; TT, Transmitting tissue; VS, Vascular strand.
STRUCTURAL ANALYSIS OF DEVELOPING STIGMA
AND TRANSMITTING TISSUE
Mature stigma in sunflower is forked, bifid and consists of two parts- the peripheral
brush-like pseudopapillae and the inner, thin, finger-like papillae, which are raised and
densely arranged outgrowths of the peripheral cells (Figure 3A). The papillate surface of
stigma increases the pollen capturing area and ensures the proper interaction of pollen with
stigma surface (Heslop-Harrison and Shivanna, 1977). A transverse section of stigma shows
the presence of a four-layered transmitting tissue immediately below the papillae where the
cells are surrounded by an intercellular matrix. Beneath the transmitting tissue is the ground
tissue, in the centre of which is a vascular canal and a large secretory canal (Figure 3B).
Papillae and the transmitting tissue are abundant in proteins (Figure 3C). In order to attain
receptivity, stigma undergoes many structural and physiological changes which allows it to
become competent for the directional growth of pollen tubes (Kandasamy et al., 1994; Yi et
al., 2006). Transmission electron microscopic analysis has revealed that the papillae in the
bud stage of developing stigma are densely cytoplasmic as compared to the ones in mature
stigma, in which they are elongated and vacuolated. The papillae have a large nucleus with a
prominent nucleolus and abundant mitochondria. Nature of stigma surface in sunflower has
remained controversial over the years. It has earlier been described by some investigators as
dry and lacking secretions on the surface (Heslop-Harrison and Shivanna, 1977; Vithanage
and Knox, 1977; Gotelli et al., 2010). Recently, it has also been described as semi-dry in
sunflower (Shakya and Bhatla, 2010), and in some other members of Asteraceae, namely
Senecio squalidus (Hiscock et al., 2002a), Lessingianthus grandiflorus and Lucilia
lycopodioides (Teixeira et al., 2011). Semi-dry stigma possesses a surface cuticle on the
papillae similar to the dry stigma which, however, is not continuous at the base of the
papillae. Like the wet stigma, mature semi-dry stigma possesses a small amount of
extracellular secretion at the base of the papillae (Allen et al., 2010). The initiation of
secretory activity in Helianthus annuus is observed at the staminate stage which leads to an
accumulation of lipid-rich extracellular secretion at the base of the papillae during the
pistillate stage of stigma development (Figure 3D; Sharma, 2012). The secretory activity
coincides with the presence of endoplasmic reticulum and elaioplasts in the basal region of
the papillae. These secretions accumulate in the intercellular and subcuticular gaps, causing a
disruption of the cuticle and release of exudates on the stigma surface. Dry stigma in Brassica
rapa, Arabidopsis thaliana and Raphanus sp. do not show extracellular secretions and the
cuticle extends to the base of the papillae (Hiscock et al., 2002a). Lipids are known to be the
major components of the exudates in some wet stigmas and are responsible for pollen
8
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
hydration (Cresti et al., 1986). During the course of evolution, the function of hydration has
been taken over by the pollen coat in dry stigmas (Sage et al., 2009). Probably, the pollen coat
and lipids on the stigma surface of semi-dry stigma aid during the processes of adhesion and
hydration.
The cells of transmitting tissue are loosely arranged below the papillae. The secretory
products of the transmitting tissue in sunflower are rich in pectins and other polysaccharides,
as has also been observed in Tibouchina sp. (Ciampolini et al., 1995), Vitis vinifera
(Ciampolini et al., 1996), Passiflora edulis (Souza et al., 2006). The extracelluar secretions of
the transmitting tissue increase as stigma attains maturity, showing that it acts as a source of
nutrition for the growing pollen tube. Cells of the transmitting tissue are polyhedral to
spherical, with a prominent nucleus, plastids and endoplasmic reticulum. Cells of the
transmitting tissue can be differentiated into three types. Type I cells are vacuolated with
parietal cytoplasm and few mitochondria. Type II cells have a large nucleus, several
mitochondria and a vacuole smaller than that in Type I cells. Type III cells have a dense
cytoplasm (Figure 3G). Some cells of the transmitting tissue show internal ramification
(Figure 3F). The finger-like projections into the cytoplasm formed by secondary wall
ingrowths increase the surface-volume ratio for enhanced metabolic activities across the cells.
The involvement of these cells in secretory activity has also been reported in the members of
Rosaceae (Heslop-Harrison and Shivanna, 1977) and watermelon (Sedgley, 1981). Cells of
the transmitting tissue possess numerous plasmodesmic connections which are involved in the
transfer of metabolic signals from the ovary (Figure 3G). Some plasmodesmic connections
are also present between the basal region of papillae and cells of the transmitting tissue,
indicating their involvement in the symplastic pathway for the transport of metabolites from
the transmitting tissue to the papillae (Figure 3F).
ACCUMULATION OF INTRACELLULAR AND
EXTRACELLULAR LIPIDS AND ASSOCIATED ENZYMES IN
RELATION WITH STIGMA MATURATION
During pollen-stigma interaction, lipids are known to play a role in pollen hydration,
germination and pollen tube penetration into the style (Wolters-Arts et al., 1998). Lipids
prevent evaporation of stigmatic tissue in dry stigma and prevent desiccation of exudates in
wet stigma (Shivanna, 2003). Some lipids in the exudates serve as attractants and are of
nutritional value for the pollinators (Lord and Webster, 1979). In wet stigmas, lipids are
present in the stigmatic exudates, as observed in Phaseolus vulgaris (Lord and Webster,
1979), Nicotiana tabacum (Cresti et al., 1986), Olea europaea (Serrano et al., 2008), while in
the dry stigmas, lipids are present as a continuous layer of cuticle beneath the pellicle, as
demonstrated in Zephyranthus sp. (Ghosh and Shivanna, 1984). In semi-dry stigmas, such as
those in Helianthus annuus and Senecio squalidus, a small amount of lipid-rich extracellular
secretion is evident in the crevices of the papillae, and cuticle is not continuous (Shakya and
Bhatla, 2010; Allen et al., 2010; Sharma, 2012). Lipid content in sunflower stigma increases
with the attainment of stigma receptivity, as observed in wet stigmas of Forsythia intermedia
and Nicotiana tabacum (Matsuzaki et al., 1985; Dumas, 1977). Triacylglycerol (TAGs)
content decreases from bud to staminate stage in sunflower and shows an increase at the
Floral Biology of Sunflower: A Histological and Physiological Analysis
9
pistillate stage. It is probable that TAGs at the bud stage are degraded during the growth of
the stigma and are synthesized during the pistillate stage of stigma development. In addition,
terpenes have also been detected at the pistillate stage of stigma development. Cis-unsaturated
fatty acids have been reported to be essential components of stigma secretions among wet
stigmas and are required for restoring stigma fertility (Wolters-Arts et al., 2002). Fatty acid
composition in Helianthus annuus shows the abundance of some saturated and unsaturated
fatty acids, as has also been observed in Nicotiana tabacum and Forstythia intermedia
(Shakya and Bhatla, 2010; Matsuzaki et al., 1983; Dumas, 1977). Palmitic acid (16:0) is the
major saturated fatty acid at all the stages of stigma development and its content increases as
the stigma attains maturity (Figure 4A; Shakya and Bhatla, 2010; Sharma, 2012). Stearic acid
(18:0) content, however, decreases with stigma maturation. Linoleic acid is the major
unsaturated fatty acid in the lipids of sunflower stigma (Figure 4B). Its content decreases on
maturity, indicating its role in stigma maturation. In contrast, linolenic acid increases as the
stigma attains maturity. Linolenic acid is required as a substrate for octadecanoid pathway
which results in the production of signalling molecules, such as jasmonic acid (McConn and
Browse, 1996).
Figure 4. Relative content of major saturated (A) and unsaturated (B) free fatty acids from stigma at
different maturation stages as resolved by gas liquid chromatography. Each value is a mean of three
independent values (±standard error). Zymographic detection of fatty acyl esterase isoforms in different
fractions of pollen (C) and developmental stages of stigma (D) following treatment with α-napthyl
acetate and fast blue B.
10
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
The cuticle on the surface of stigmatic papillae in sunflower has an outer proteinaceous
pellicle (Figure 3C). Cytochemical studies have indicated the presence of glycoproteins and
some enzymes, predominantly esterases, peroxidases and acid phosphatase, as the major
components of the pellicle (Vithanage and Knox, 1977; Shakya and Bhatla, 2010). Nonspecific esterase activity has earlier been implicated in the attainment of stigma receptivity
and has a role to play in the metabolism of fatty acids and in host-pathogen interaction
(Bilková et al., 2009; Shivanna and Rangaswamy, 1992). Non-specific esterases include
carboxylesterase (EC 3.1.1.1), arylesterase (EC 2.1.1.2) and acetyl esterase (EC 3.1.1.6). The
activity of these enzymes has been detected in dry stigmas [Linum grandiflorum (Ghosh and
Shivanna, 1980), Pennisetum americanum (Reger, 1989) and Brassica (Mattsson et al.,
1974)], wet stigmas [Impatiens sp. (Kulloli et al., 2010), Moringa oleifera (Bhattacharya and
Mandal, 2004) and Nicotiana sylvestris (Kandasamy and Kristen, 1987)] and semi-dry
stigmas, as in Helianthus annuus and Senecio squalidus (Shakya and Bhatla, 2010; Hiscock et
al., 2002a). Non-specific esterase activity is evident at all stages of stigma development in
sunflower and it increases as stigma attains maturity. As in Helianthus annuus, esterase
activity has also been detected in the early stages of stigma development in Nicotiana
sylvestris (Kandasamy and Kristen, 1987) and Linum grandiflorum (Ghosh and Shivanna,
1980), which can be correlated with the initiation of cell differentiation (Bílková et al., 1999).
Qualitative analysis of stigma proteins has revealed a change in the isoform pattern of
esterases as stigma approaches maturity (Figure 4D). An increase in the number of isoforms
indicates their correlation with stigma maturation (Bhattacharya and Mandal, 2004). Nonspecific esterases present on the stigma surface have been reported to be involved in forming
a cutinase complex (Knox et al., 1976; Hiscock et al., 2002b). The cutinase complex interacts
with esterases from pollen grain wall and pellicle which allows the breakdown of cuticle,
facilitating the penetration of pollen tube into the stigmatic tissue. Removal of stigma surface
components by chemical treatment or using an inhibitor of serine esterases, reduces the ability
of pollen tubes to penetrate the stigma (Knox et al., 1976). This suggests that serine esterases
are associated with cutinase complex formation needed for pollen tube penetration in dry
stigmas (Hiscock et al., 2002b). Non-specific esterases have the ability to hydrolyze crossbonds of cell wall polysaccharides and are, therefore, important in the establishment and
reorganization of cell wall. Lipase (Triacylglycerol acyl hydrolase; EC 3.1.1.3)- like proteins
have also been reported in mature stigma papillae of sunflower (Shakya and Bhatla, 2010),
Petunia and Nicotiana (Beisson et al., 2003). Lipases act on the triacylglycerols (TAG) and
might be involved in altering the lipidic composition of the stigmatic surface and have been
located on the stigmatic papillae and pseudopapillae of the receptive stigma (Shakya and
Bhatla, 2010).
REACTIVE OXYGEN SPECIES ACCUMULATION DURING STIGMA
DEVELOPMENT AND ASSOCIATED SCAVENGING MECHANISMS
Recent reports have suggested that during stigma receptivity, angiosperms exhibit an
accumulation of high levels of reactive oxygen species (ROS), principally H2O2 (McInnis et
al., 2006a,b; Allen et al., 2011; Losada and Herrero, 2012). ROS are the byproducts of
aerobic metabolism which are removed by enzymes and antioxidants. In recent years ROS
Floral Biology of Sunflower: A Histological and Physiological Analysis
11
have been shown to act as signalling molecules in various phases of growth and development,
response to environmental stress and during pollen germination and tube growth (McInnis et
al., 2006a; Hiscock et al., 2007; Zafra et al., 2010). ROS-mediated signalling is controlled by
a fine balance between the production and scavenging of ROS. The main sites of ROS
production are located in the plasma membrane, NADPH-oxidases, plastids and peroxisomes
(Karuppananpandian et al., 2011; Apel and Hirt, 2004). An increase in ROS accumulation is
observed in sunflower stigma accompanying the attainment of stigma receptivity (Figure 5 I;
Sharma and Bhatla, 2013). Stigmas are a source of nutrients for the pollen grains and may be
prone to microbial attack. It is suggested that ROS play a role in defence mechanisms since
high levels of ROS have been detected in the floral nectar which never experience microbial
attack (Carter and Thornburg, 2004). ROS may directly be toxic to the pathogen or may
trigger hypersensitive reaction and programmed cell death at the site of pathogen attack (De
Rafael et al., 2001). It has also been demonstrated that ROS is required for the polarized
growth of the pollen tube (Potocký et al., 2007).
Superoxide dismutases (SOD; EC 1.5.1.1) form the first line of defence against the
accumulation of superoxide anion (O2-) and lead to its conversion into H2O2 and dioxygen.
SOD activity is associated with redox cycle in the nectar in Nicotiana (Carter and Thonburg,
2004; Carter et al., 2007). The SOD in stigma activity is mainly due to Mn-SOD
(mitochondria localized) which may be associated with enhanced nectar secretion at the base
of florets during pollen producing stage and also when stigma is receptive (Figure 5 III;
Tripathi and Singh, 2008; Sharma and Bhatla, 2013). Coinciding with the increased SOD
activity, a cluster of mitochondria is evident at the basal region of papillae cell cytoplasm,
showing the increased metabolic activity accompanying stigma elongation and dehiscence of
anther (Figure 3E; Sharma, 2012). A slight reduction in SOD activity at the pistillate stage of
stigma development leads to a reduction in O2- form of ROS and an increase in H2O2 in the
stigmatic papillae. Peroxidases (POD; EC 1.11.7) are heme-containing glycoproteins and
their activity has been reported both in wet and dry types of stigmas (McInnis et al., 2005).
Peroxidase activity in the stigmatic tissue increases as the floret development reaches its
maximum at receptivity. In Arabidopsis thaliana, Petunia hybrida and Senecio squalidus,
POD activity increases as stigma attain receptivity (Dafni and Maués, 1998; McInnis et al.,
2006b). Younger stages of developing stigma show reduced POD activity in Peduclaris
canadensis, Clintonia borealis and Helianthus annuus which has been correlated with poor
pollen adhesion and germination (Figure 5 IV; Galen and Plowright, 1987; Sharma and
Bhatla, 2013). Recent investigations have reported peroxidase isoforms specific to stigma.
Expression of stigma-specific peroxidases is developmentally regulated, their activity being
maximally observed during stigma receptivity (McInnis et al., 2005). Among the stigmaspecific peroxidases, three isoforms have earlier been identified in Arabidopsis, one in
Senecio squalidus, and one in hazelnut (Beltramo et al., 2012). Stigma-specific expression of
POD indicates the key role of peroxidases in the loosening of cell wall components of stigma
to allow pollen tube growth into the stigma. They might be involved (through H2O2
metabolism) in signalling network, mediating species-specific pollen recognition (McInnis et
al., 2005). Some peroxidases are also known to be induced/upregulated in association with
hypersensitive response or stress, thereby indicating their role in defense mechanism of
stigma (McInnis et al., 2005; Beltramo et al., 2012).
Nitric oxide (NO) is a gaseous signalling molecule known to be involved in different
plant processes related to growth and development and in responses to stress. Recent
12
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
investigations have also revealed that NO is likely to be involved in plant reproductive
processes (Hiscock et al., 2007; Seligman et al., 2008; Yadav et al., 2013). In sunflower, NO
accumulation increases as stigma attains maturity (Figure 5 II; Sharma and Bhatla, 2013).
Similar increase in NO has been reported in the developing stigma of olive and Arabidopsis
(Zafra et al., 2010; Seligman et al., 2008). NO is important in imparting immunity to the
receptive stigma and it also increases thermotolerance by activating ROS scavenging
enzymes, thereby playing a role in ROS-mediated signalling processes (Piterková et al. 2013;
Sharma and Bhatla, 2013). Upon pollination, a crosstalk between pollen-localized NO and
stigmatic ROS has been proposed which may have a role in pollen recognition and signalling
between the stigmatic papillae and pollen grains (Hiscock et al., 2007; Sharma and Bhatla.,
2013).
Figure 5. Accumulation of reactive oxygen species (ROS) and expression of associated scavenging
enzymes during stigma development. I: Localization of ROS on the surface of developing stigma after
treatment with fluorescent probe-dichlorodihydrofluorescein diacetate (DCFH-DA). Magnification:
100X. Inset shows intense ROS accumulation in the papillae. II: Localization of nitric oxide (NO) on
surface of developing stigma after treatment with MNIP-Cu {Copper derivative of (4-methoxy-2-(1Hnapthol [2,3-d] imidazol-2-yl) phenol)}. Magnification: 200X. III: Zymographic detection of
Floral Biology of Sunflower: A Histological and Physiological Analysis
13
superoxide dismutase (SOD) isoforms in developing stigma, after treatment with nitro blue tetrazolium.
IV: Zymographic detection of peroxidase (POD) isoforms in developing stigma following benzidine
treatment.
LIPIDS IN POLLEN COAT AND THEIR ROLE IN FOOT FORMATION
Mature pollen grains are released in a highly desiccated condition and are metabolically
inactive. Pollen grains are suboblate, echinate, tectate and tricolporate (Gotelli et al., 2008).
The innermost layer of pollen wall (intine) is thin as compared to the outer layer (exine)
which is spinulate and has pollen coat substances embedded on it. The spines of pollen are
conical and have spinular microperforations, indicating their entomophilous nature (Figure
3H; Harry et al., 1978; Shakya, 2005; Coutinho and Dinis, 2007). The exine is differentiated
into ektexine and endexine, both of which are separated by a space designated as cavus 2.
Ektexine is formed of spinules, tectum, internal foramina (openings), columella, large internal
spaces (cavus 1) and foot layer (Figure 3I). Proteins originating from tapetal cytoplasm attach
to the cavae and internal foramina of exine, which are known to act as allergens and
recognition substances for interspecific compatibility (Horner and Pearson, 1978). The exine
pattern is of ‘Helianthoid type’, referring to abundant internal foramina in the columella and
tectum, equal length of columella having basally fused regions and presence of cavae and a
thin foot layer (Skvarla and Turner, 1966). Connected basal region of columella, internal
formina in columella and enlarged cavae in sunflower (as in other members of Asteraceae,
such as Pallenis maritiama, Jasonia tuberose and Astericus aquaticus) allows easy
communication between pollen surface and cavae, facilitating the exchange of water and
physiologically active substances between them (Coutinho and Dinis, 2007). Pollen coat is
rich in lipids which originate from the tapetal cytoplasm (Horner and Pearson, 1978). Pollen
capture is exine-dependent and at a later stage it involves the formation of “attachment foot”
at the point of its contact with the stigmatic papillae. Pollen coat contains essential
components required for adhesion and cell to cell interaction between the stigmatic cells and
pollen. The pollen coat material flows out from between the columellae of exine to form an
adhesive foot at the surface of the papillae (Wheeler et al., 2001). Lipidic constituents in the
pollen grains of sunflower belong to two different domains- the external tryphine (pollen
coat) and the internal cytoplasmic (internal pollen). The lipidic content of pollen coat is more
than that of the internal pollen (Shakya and Bhatla, 2010). Among the total neutral lipids,
neutral esters (wax esters) and free fatty acids are the major components of the two pollen
fractions. Gas chromatographic profile of free fatty acids has revealed an abundance of
saturated and unsaturated free fatty acids in the internal pollen and the pollen coat. As in
stigma, the major saturated fatty acids are palmitic (16:0) and stearic (18:0) acids both in
pollen coat and internal pollen, thus indicating a functional similarity in the lipidic
constituents of stigmatic exudates and pollen (Piffanelli et al., 1997; Shakya and Bhatla,
2010). Lignoceric acid (24:0), which is expressed more in the pollen coat than in the internal
pollen fraction, is specifically expressed only in pollen grains, pointing to its role in the
involvement of long fatty acids in the signalling mechanism for hydration of pollen on the
stigma surface. Among the unsaturated fatty acids, oleic (18:1), linoleic (18:2), linolenic
(18:3) and cis–eicosenoic (20:1) acids have been detected in the pollen grains. Linolenic acid
(18:3) is the major unsaturated fatty acid in the pollen coat and internal pollen in sunflower,
14
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
and Brassica napus (Evans et al., 1987; Shakya and Bhatla, 2010). Cis-eicosenoic acid (20:1)
is the major component of intact pollen grains in sunflower (Schulz et al., 2000). Pollen coat
in sunflower exhibits lipase activity, as has also been reported in Arabidopsis thaliana
(Mayfield et al., 2001; Shakya and Bhatla, 2010). Lipid profile of the pollen fractions shows
an absence of triacylglycerides in the pollen coat fraction. It is likely that lipases in pollen
coat are activated when pollen coat makes a contact with the stigmatic tissue. Pollen coat
lipases may be involved in the degradation of the lipids, such as cuticle present in the
stigmatic tissue, and they may also participate in various signalling activities (Murphy, 2006).
Several esterase isoforms have been detected in the two fractions of pollen (internal
pollen and pollen coat). Four and three isoforms have been detected in the pollen coat and
internal pollen, respectively (Figure 4C; Shakya and Bhatla, 2010). Esterase activity has
earlier been localized in pollen intine suggesting its role in cuticle degradation during the
entry of pollen tube (Vithanage and Knox, 1979). It has been proposed that esterases from the
pollen and stigma cause a species-specific recognition event resulting in the formation of a
‘cutinase complex’ which digests the cuticle for the successful penetration of pollen tube into
the stigma (Knox et al., 1976; Hiscock and Allen, 2008). Only one peroxidase isoforms is
present in the intact pollen of sunflower (Shakya, 2008). Three isoforms have been detected
in the internal pollen whereas pollen coat does not show peroxidase activity. Pollen
peroxidases are known to degrade phenolic compounds (such as chlorogenic acid, caffeic acid
and cinnamic acid) present in the stigmatic exudates (Bredemeijer, 1984, 1982). Phenolics
present on the stigmatic exudates or stigma surface seem to be involved in the stimulation or
inhibition of IAA oxidase activity which influences growth activity in stigma (Shakya, 2008;
Bredemeijer and Blaas, 1975).
OTHER BIOCHEMICAL FEATURES OF POLLEN GRAINS
AND RECEPTIVE STIGMA
Glycoproteins believed to have important role/s in pollen-stigma interaction, are known
to be present in the pollen grains (Suraez-Carvera et al., 2005; Kimura et al., 2002; Aelst and
Went, 1992; Shakya, 2008). In the pollen grains of sunflower, four glycoproteins have been
detected. As in the pollen grain of Elaeis guineensis, an isoforms of 31kDa has been detected
in sunflower as well. The other three glycoproteins correspond to the presence of xylanases
which help in the hydrolysis of xylan in the cell wall of stigma (Shakya, 2008). Some
glycoproteins in stigma are known to be correlated with the expression of S gene proteins,
namely SLG (S locus glycoprotein) and SLR-1 (S locus related glycoprotein-1; Luu et al.,
1997b). SLG is a polymorphic protein known to have a role in self-incompatibility and it is
secreted into the cell wall of the stigmatic papillae (Kandasamy et al., 1989; Umbach et al.,
1990; Doughty et al., 1993). The cytosolic fraction of receptive stigma of sunflower contains
a single glycoprotein of 31 kDa. The expression of SLR1 gene is reduced at the later stages of
pollen-stigma interaction, showing its involvement in pollen-stigma cross-linking (Luu et al.,
1997b). These glycoproteins on the papillae surface interact with pollen coat proteins (PCP)
and facilitate in the process of adhesion. SLG has been demonstrated to bind to PCP-A1 and
about ten PCP-like proteins, pointing towards the involvement of SLG in many other
processes in addition to pollen-stigma adhesion (Swanson et al., 2004). Various enzymes,
Floral Biology of Sunflower: A Histological and Physiological Analysis
15
including proteases, are required for the proteolytic digestion of proteinaceous pellicle during
the initial stages of pollen-stigma interaction (Luu et al., 1997a; Graff de et al., 2001;
Swanson et al., 2004). Protease activity may also result in the damage of proteins and
enzymes on the stigma surface, which leads to the activation of pollen cutinase necessary for
the degradation of cuticle (Radlowski, 2005). A protease 54kDa protease has been reported in
the cytosolic fraction of sunflower at the receptive stage, in contrast with Cynara cardunculus
where two proteases have been reported in the storage vacuoles of stigmatic papillae and
transmitting tissue of mature stigma (Verissimo et al., 1996; Shakya, 2008). Calcium is an
important constituent of in vitro germinating pollen and serves as a chemoattractant for
guiding the growth of pollen tube. Membrane-bound calcium appears to be generally
distributed in the papillae at all the stages of development. Young buds, however, show lesser
accumulation of bound calcium in the stigmatic papillae. At the staminate stage, calcium
content increases, reaching a maximum at the pistillate stage of stigma development. Bound
calcium has been detected in the pellicle and upper region corresponding to the cytoplasmic
organelles in the papillae at the pistillate stage of stigma development. The tip region of
stigma also shows an increase in bound calcium content. Investigations using 45Ca2+ have
revealed calcium uptake by the germinating pollen from the stigma tissue (Bednarska and
Butowt, 1995). De-esterified pectins in the apoplast are capable of binding with calcium ions.
Upon pollination, bound calcium is liberated due to the enzymatic lysis of de-esterified
pectin, leading to an increase in the levels of free calcium (Bednarska, 1989; Lenartowska et
al., 2001). In the growing pollen tubes, callose synthesis is also a calcium-dependent process
(Bednarska, 1989).
THE PROCESS OF POLLEN ADHESION, HYDRATION AND
GERMINATION ON THE RECEPTIVE STIGMA
Pollination involves the transfer of viable pollen onto the receptive stigmatic surface.
Adhesion is initiated due to non-specific van der Waal forces between the rough surface of
stigma bearing papillae and the spikes of pollen grains (Ferrari et al., 1985; Thio et al., 2009).
The process of pollen adhesion is rapid, maximal at the receptive stage of stigma and initiates
in a similar manner both in self- and cross-pollinated conditions. As has also been reported in
Brassica oleracea, the papillae undergo physiological changes with the attainment of stigma
maturity thereby affecting their ability to interact with pollen grains (Heizmann et al., 2000;
Sharma, 2012). The process of adhesion involves the interaction of proteins present in the
pellicle (arabinogalactan proteins) and the pollen wall (Hiscock and Allen, 2008; Losada and
Herrero, 2012). As in Brassica sp., the initial stages of adhesion are not dependent on Sterlity
locus (S). Therefore, recognition or rejection of compatible or incompatible pollen does not
occur at this stage. At a later stage, however, with the involvement of S locus glycoproteins,
adhesion between incompatible pollen grains does not increase (Heizmann et al., 2000). The
pollen coat and the stigmatic pellicle of the papillae have an important role in the process of
adhesion. Upon removal of the pollen coat, a reduction in the degree of adhesion between the
internal pollen and stigmatic papillae is evident. Adhesion of few decoated pollen and
stigmatic papillae has, however, revealed that pollen coat proteins and carbohydrate-based
molecules associated with exine are involved in the process of adhesion (Zinkl et al., 1999;
16
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
Sage et al., 2009). The importance of pellicle in the process of adhesion is further evident by
the treatment of pellicle with acetone which leads to a significant decrease in pollen adhesion
suggesting that glycolipids and lipids located at the base of the papillae play a role in the
process of adhesion. The lipids of the pollen coat and cuticle and extracellular lipids in the
basal region of papillae are likely to form hydrophobic bonds, leading to adhesion between
the pollen grains and the stigmatic papillae. After adhering to the stigmatic papillae, the
pollen grains hydrate. The exact mechanism by which water, nutrients and other essential
molecules are taken up by the pollen grains from the stigmatic papillae, is not yet fully
understood (Frion et al., 2012). Various proteins from pollen coat, including glycine-rich
proteins, calcium-binding proteins, lipases and cutinases aid in the process of hydration by
causing the breakdown of lipidic constituents at the pollen-stigma interface (Mayfield and
Preuss, 2000; Hiscock et al., 2002b; Updegraff et al., 2009; Shakya and Bhatla, 2010). Pollen
grains hydrate leading to an increase in their volume, both in self- and cross-pollinated
conditions (Vithanage and Knox, 1977). After making a contact with stigma, the pollen coat
material flows out from the columella of the exine to the surface of papillae (Ellemen et al.,
1992; Allen et al., 2011). This leads to the formation of ‘attachment foot’ where an interaction
between pollen and stigma-derived biomolecules takes place. Lipids create conditions and
govern the movement of water for guidance cue for the development of the pollen tube which
emerges from the pore in the middle of colpus area to grow through the attachment foot
towards the basal region of the papillae (Ellemen et al., 1992; Hiscocok et al., 2002a). The
acceptance or rejection of pollen grains on the stigmatic surface seems to be dependent on the
interaction between the pollen and stigma-derived attachment foot (Hiscock, 2000; Sharma,
2012). Some of the incompatible pollen grains are not able to germinate while others pass
perpendicularly towards the basal region of the papillae. Some pollen grains grow parallel to
the papillate surface of stigma thereby showing some incompatible rejection reactions
(Figure 6).
Figure. 6. Transverse section through germinating pollen grain and the associated stigma following
cross (A) and self (B) pollination. Magnification: 400X.
Floral Biology of Sunflower: A Histological and Physiological Analysis
17
In sunflower, cross pollination is favoured due to the presence of self-incompatibility, as
also observed in the members of Brassicaceae. Members of both the families (Asteraceae and
Brassicaceae) have some similarities in pollen-stigma interaction. The common features
include the presence of dry stigma surface (Vithanage and Knox, 1977), three-celled pollen
grains (Hiscock and Allen, 2008), release of pollen coat on contact with papillae (Elleman et
al., 1992) and arrest of incompatible pollen soon after germination accompanying the
deposition of callose on the pollen tube and papillae (Allen et al., 2011; Vithanage and Knox,
1977). Habura (1957) was first to report sporophytic self-incompatibility in sunflower and it
was later confirmed by various other plant scientists (Luciano et al., 1965; Asthana, 1973).
Recent reports have, however, pointed out flexibility in sporophytic self incompatibility in
other members of Asteraceae (Hiscock, 2000; Ortiz et al., 2006). As in Senecio squalidus,
sunflower shows germination of self-incompatible pollen. Some of the incompatible pollen
grains are not able to germinate, while others germinate and grow between the papillae
(Vithanage and Knox, 1977). It has been suggested that the degree of self-incompatibility and
self-fertility depends on genetic control, environmental factors and the morphology of the
inflorescence (Miller and Fick, 1997). Self-incompatibility is compensated in situations when
the reproductive opportunity of the pistil is affected. Such cases exhibit pseudo selfcompatibility which involves a delayed acceptance of the otherwise incompatible pollen
(Brennan et al., 2011). The modification of self-incompatibility may also be brought about by
G locus, which is a second gametophytic ancestral locus, remnant of ancestral gametophytic
self incompatibility in Brassicaceae, which permits compatible cross between the individuals
with the same S alleles, which are otherwise incompatible (Lewis et al., 1988; Hiscock, 2000;
Brennan et al., 2011). Recent molecular analysis has also pointed out that sporophytic selfincompatibility in Asteraceae operates by a mechanism different from the SRK/SCR
molecular mechanism operative in Brassicaceae (Hiscock et al., 2003; Tabah et al., 2004;
Allen et al., 2011). Further investigations related to seed set and the molecular mechanisms in
sunflower are required to understand the physiological nature of self-incompatibility.
To sum up, the present work highlights the complex interaction between floral
development and environmental factors, which seem to operate through a critical balance of
auxin and gibberellic acid distribution at the bud primordium. Subsequently, in order to
facilitate an effective pollen and stigma interaction, both the reproductive structure undergo
biomolecular changes required for ‘foot’ formation on the surface of receptive stigma
papillae. The knowledge gained so far in this direction highlights the role of reactive oxygen
species and associated scavenging enzymes, glycoproteins, calcium and many more
biomolecules. Alterations in the availability of these biomolecules correlate with distinct
subcellular structural changes both in pollen and stigma. Although a lot is known by now
about these structural and biochemical events accompanying floral development in sunflower,
further investigations on their physiological, biochemical and genetic aspects will provide
further information on the crosstalk mechanisms regulating floret development and pollenstigma interaction.
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