Chapter 2
Female gametophyte development in
selected species of Podostemoideae;
Podostemaceae
Introduction
The discovery of the unique process of double fertilization in flowering plants by
Nawaschin (1898, 1899), underlined the essential role of a second fusion event in the
formation of endosperm in an angiospermic seed (Raghavan, 2003). He demonstrated
that in ovules of Lilium martagon and Fritillaria tenella (Liliaceae), both the male
gametes from a pollen tube entered the embryo sac; one of them fused with the nucleus
of the egg cell and the other with the polar nuclei (at that time known as the definitive
nucleus) ensuing a second fertilization event. Later, due to the sustained efforts of
succeeding workers, the occurrence of double fertilization became a defining feature of
the reproductive development of evolutionarily the most successful group of plants, the
angiosperms.
Podostemaceae is the only family of angiosperms where double fertilization is
not known to occur. However, a nucellar plasmodium, also known as the ‘pseudo-
embryo sac’, is formed which provides nutrition to the developing embryo.
Podostemaceae has several notable embryological features and is often cited as an
‘embryological family’ (Kapil, 1970). The defining embryological features of the
family are - (a) diverse developmental patterns of female gametophyte, (b) 4-celled/ 4nucleate condition of the embryo sac, (c) occurrence of single fertilization (only
syngamy) and no endosperm formation, (d) absence of antipodals, (e) presence of a
pseudo embryo sac, (f) pollen grains in dyads (except in Tristichoideae), (g) presence of
suspensor haustoria, (h) lack of a plumule and a radicle in a mature embryo (Razi, 1949,
1955; Maheshwari, 1955; Mukkada, 1962, 1969; Chopra and Mukkada, 1966; Kapil,
1970; Battaglia, 1971; Mukkada and Chopra, 1973; Nagendran, 1975; Nagendran and
Arekal, 1976; Arekal and Nagendran, 1974, 1975a, 1975b, 1977a, 1977b; Nagendran et
al., 1981; Mohan Ram and Sehgal, 1992, 2001, 2007; Sehgal et al., 2010).
Development of female gametophyte is the most extensively studied feature of
the Indian Podostemaceae. Typically, it has been deduced to be a 4-celled/4-nucleate
structure, represented by a large egg cell and two small synergids constituting the egg
apparatus, and a central cell harbouring a solitary polar nucleus (Razi, 1949; Mukkada,
48
1969; Battaglia, 1971; Nagendran et al., 1977, 1980). Four types of four-celled embryo
sac are documented in the family: the bisporic Podostemum and Polypleurum type, and
the monosporic Apinagia type ‘a’ and Apinagia type ‘b’ (Battaglia, 1971, 1987) (Fig. 22).
Figure 22. Diagrammatic representation of the four types of embryo sac development in
Podostemaceae (modified from Nagendran et al., 1977).
There are two views regarding the absence of double fertilization in
Podostemaceae. The first view considers the failure of pollen tube to discharge the
second sperm in the vicinity of embryo sac (Chopra and Mukkada, 1966; Mukkada,
1969) and the second considers the degeneration of central cell as the cause of failure of
double fertilization (Battaglia, 1971; Nagendran et al., 1976, 1980). The latter view
implies that the absence of a true polar nucleus in the embryo sac precludes fusion of
the second male gamete. However, none of these views was supported by evidences,
only till recently. Sehgal et al. (2010) studied the incidence of single fertilization in
Dalzellia zeylanica (Gardner) Wight (subfamily: Tristichoideae) and observed that its
female gametophyte undergoes a transition from 4-celled/4-nucleate to 3-celled/3nucleate condition because the central cell degenerates just before fertilization. Whether
49
or not, this reduction in the structural components of female gametophyte is of universal
occurrence in the family, still needs to be ascertained. Therefore, with the aim of
understanding the developmental constraints associated with the elimination of double
fertilization in Podostemaceae, and verifying reduction and simplification in female
gametophyte in the other species, the presented work was taken up.
To elucidate it, development of female gametophyte was studied for first time in
Willisia arekaliana Shivam. and Sadanand and reinvestigated in three species namely
Zeylanidium olivaceum (Gardn.), Engler, Polypleurum munnarense Nagendran and
Arekal and Podostemum subulatum Gardn. In the latter three species, earlier authors
have only described the type of embryo sac development but the occurrence of any
modifications till fertilization was not the focus of their studies. Therefore, with the
intention to study the occurrence of any ontogenic variability, the embryo sac
development was reinvestigated. The mode of development of female gametophyte in
Z. olivaceum and P. subulatum was first reported as Podostemum type by Magnus and
Werner (1913). After much contradictions and reinvestigations by various authors
(Razi, 1955; Walia, 1965; Battaglia, 1971), embryo sac development in Z. olivaceum
and P. subulatum was confirmed to be Apinagia type (Arekal and Nagendran, 1977a;
Nagendran et al., 1980). The ontogeny of female gametophyte of P. munnarense was
described as Polypleurum type, where the central cell lies towards the micropylar end of
ovule and the synergids towards the chalazal end (Nagendran et al., 1977).
As double fertilization requires coordinated action of the component cells of the
female gametophyte in concert with the male gametes, the female gametophyte was
observed for any changes till fertilization occurred. I have shown that the female
gametophyte of W. arekaliana is a highly reduced 3-celled/3-nucleate structure
represented only by an egg apparatus and lacks the central cell. A key modification i.e.,
the degeneration of central cell before fertilization occurs in the development of female
gametophyte in the other studied podostemads which might be responsible for the
obscure second fertilization event. Finally, the study also highlights the evolutionary
trend in megagametophyte development in the family.
50
Materials and Methods
Species selected
Ontogeny of embryo sac was examined histologically for the first time in Willisia
arekaliana and reinvestigated in Zeylanidium olivaceum, Podostemum subulatum and
Podostemum munnarense. Although the ontogeny of female gametophyte in the latter
three species has been described in the previous investigations (Magnus and Werner,
1913; Razi, 1955; Walia, 1965; Battaglia, 1971; Arekal and Nagendran, 1977a;
Nagendran et al., 1977, 1980), the details of female gametophyte development between
pollination and pre-fertilization relevant to the aim of the present study have been
investigated for the first time.
Methodology
The pistils at appropriate stages of development (different stages of flower bud, freshly
anthesized flowers and pistils at different times after pollination) were carefully excised
from the flowers and fixed in Karnowsky’s fixative (Karnowsky, 1965) for 6 h at room
temperature (20°C), washed in sodium cacodylate buffer and dehydrated through pure
2-Methoxy ethanol, ethanol, n-propanol, and butanol series for one day each, in
separate screw cap glass vials (10 ml). Infiltration (for 2 days at 40 ºC in oven) and
embedding (for 1 day at 60ºC) were done by using freshly prepared glycol methacrylate
monomer mixture (Feder and O’ Brien, 1968; O’ Brien and McCully, 1981). Semi-thin
(3 and 4 µm) sections were cut using glass knives on a rotary microtome (AO Spencer,
USA). The sections were expanded in a drop of water on a slide kept on a hot plate set
at 40˚C, dried, stained as per the requirement and mounted in DPX.
To trace the path of pollen tubes from stigma to megagametophyte, the naturally
pollinated flowers were fixed in acetic alcohol (ethanol:acetic acid, 3:1) at different
times beginning from pollination (0 HAP) to 25 h after pollination (25 HAP). Pistils of
each species were cleared in 1 N NaOH for 3-4 h at 40○C and later thoroughly washed
with water. The cleared pistils were left overnight in 0.05% decolorized aniline blue at
4○C in dark, mounted in aniline blue and observed under an epifluorescence microscope
51
(Zeiss axioscope A1, Germany) using UV excitation (Martin, 1959; Shivanna and
Rangaswamy, 1992). Photographs were captured using Zeiss Axiocam digital camera.
Images were processed with Adobe Photoshop CS5 (Adobe Systems, San Jose, CA).
Image manipulations were applied to the entire image.
52
Results
Female gametophyte development
In all the investigated species, the ovary is bilocular and contains anatropous, bitegmic
and tenuinucellate ovules on a massive axile placenta (Fig. 23A, 24A, 25A, 26A). The
nucellus is comprised of an inner row of mostly five to seven cells surrounded by a
unicellular layer. The well differentiated outer integument alone organizes the
micropyle whereas the inner integument develops only upto the base of megaspore
mother cell or the developing female gametophyte (embryo sac). The further course of
development varies in different species and is therefore provided separately for each
species.
Podostemum subulatum
A densely protoplasmic hypodermal archesporial cell directly functions as the megaspore
mother cell. Megaspore mother cell attains the size of the future embryo sac and
possesses a centrally placed nucleus (Fig. 23B). After meiosis-I, an asymmetric division
results in two unequal dyad cells (Fig. 23C). The micropylar dyad is smaller, degenerates
soon and persists as a crescent shaped cap (Fig. 23D). The chalazal dyad cell is larger and
alone completes meiosis-II, resulting in two free megaspore nuclei that occupy the two
poles of the embryo sac (Fig. 23E). Soon, the chalazal megaspore nucleus becomes
smaller and degenerates (Fig. 23F). The micropylar megaspore nucleus alone undergoes
mitotic division to produce two nuclei (Fig. 23G) which in turn undergo mitosis II. All
these four nuclei take part in the embryo sac organization and get arranged in a T-shaped
configuration, typical of the Podostemoideae. In a 4-celled/4-nucleate embryo sac, the
two juxtaposed cells towards the micropyle are differentiated into synergids (Fig. 23H).
One largest cell in the center is organized into an egg cell and the cell towards the
chalazal end is differentiated into a central cell but invariably, with only one nucleus (Fig.
23H). Shortly, the polar cell degenerates and the mature female gametophyte becomes 3celled/3-nucleate with two synergids and an egg cell (Fig. 23I).
53
Polypleurum munnarense
Within each ovule an axial row of cells becomes conspicuous, the terminal cell of
which is distinct by virtue of a large nucleus and dense cytoplasm and it directly
functions as the megaspore mother cell (Fig. 24B). The megaspore mother cell
undergoes meiosis I to produce two unequal dyad cells. The upper dyad cell is smaller
and it soon degenerates (Fig. 24C). The nucleus of the larger chalazal dyad divides
further (Meiosis II) to produce two megaspore nuclei of equal size (Fig. 24D). In a great
majority of podostemads, the chalazal megasporial nucleus degenerates and thus the
four-celled megagametophyte is a product of two mitotic divisions of the micropylar
megasporial nucleus alone. However, in the ovules of P. munnarense, the chalazal
nucleus does not degenerate; instead both the micropylar and the chalazal nuclei
undergo one mitotic division each (Fig. 24E). The resultant four nuclei get arranged in
inverted T-shaped configuration. During the mitotic division, the nucleus at the chalazal
end divides vertically and the cells differentiate into two synergids (at the chalazal end).
The nucleus at the micropylar end undergoes a transverse division and the resulting
cells differentiate into an egg cell and a central cell (at the micropylar end). This
sequence of divisions in the nuclei and arrangement of cells results in an inverted Tshaped configuration of an embryo sac with two small synergids at the chalazal end,
centrally positioned large egg cell and a micropylar central cell with one polar nucleus
(Figs. 24F). The synergids are usually juxtaposed and elongated (Fig. 24F); and
occasionally, they are obliquely placed (Fig. 24G). Sometimes, the chalazal cell does
not divide and the synergid is a single bi-nucleate cell (Fig. 24H). Just before
fertilization, the central cell placed at the micropylar end degenerates and at maturity
the female gametophyte becomes 3-celled/3-nucleate (Figs. 24I).
Willisia arekaliana
The megasporocyte is hypodermal in origin; the uppermost cell of the inner nucellar row
of cells functions as the megaspore mother cell (Fig. 25B). The megasporocyte enlarges
to almost the size of a mature embryo sac. It has a centrally placed nucleus and evenly
distributed dense cytoplasm (Fig. 25B). The first meiotic division is asymmetric which
54
results in vertically arranged two daughter cells (dyads), of unequal sizes (Fig. 25C). The
micropylar dyad cell is smaller and ephemeral. The larger chalazal dyad cell undergoes
second meiotic division to produce two vertically arranged megaspore nuclei (Fig. 25D).
The two megaspores migrate to the micropylar and chalazal poles (Fig. 25E). Like P.
munnarense both the micropylar and the chalazal nuclei took part in mitotic division.
Thus, the resultant megagametophyte is a product of two megaspore nuclei and is
bisporic in origin. Mitotic division is not followed by cytokinesis and the resultant four
nuclei lie freely in the coenocyte (Fig. 25F). Out of the four nuclei, one nucleus at the
chalazal pole degenerates followed by the cell wall formation resulting in a 3-celled/3-
nucleate female gametophyte representing an egg apparatus alone (Fig. 25G,H). The
central cell is not formed, which is a novel feature in the family. This reduced female
gametophyte is organized when the flower is still inside the spathella. By the time flower
anthesizes (i.e. comes out of spathella), the development of female gametophyte is
completed.
In nearly 5% of the ovules (n= 31/643 ovules) in W. arekaliana, two
megagametophytes developed in an ovule.
In such ovules, both the dyad cells
underwent meiosis II, hence the two developing embryo sacs in such ovules were the
products of a single megasporocyte (Fig. 25I).
Zeylanidium olivaceum
A densely protoplasmic and hypodermally located archesporial cell of the nucellus,
which differentiates early in a developing ovule, directly functions as the megaspore
mother cell (megasporocyte). Sooner, the megasporocyte enlarges in size and at this
stage it possesses a dense and a conspicuous nucleus; the cytoplasm is devoid of
vacuoles (Fig. 26B). The megasporocyte divides meiotically (Meiosis I) to give rise to
two vertically arranged dyad cells. The division is asymmetric and thus the two
resulting daughter cells (dyad cells) are of unequal sizes (Fig. 26C). The smaller, upper
dyad cell degenerates and the lower dyad cell enlarges (Fig. 26D) and alone undergoes
second division of the meiotic process to produce two vertically arranged nuclei (Fig.
26E). However, nuclear division is not followed by cell wall formation and of the two
55
resulting nuclei; the chalazal megaspore nucleus gradually becomes smaller and
degenerates (Fig. 26F). The micropylar nucleus further divides mitotically (Mitosis I) to
form two nuclei, positioned one above the other along the micropylar-chalazal axis
(Fig. 26G). The cell wall is not laid after mitosis I and both the nuclei undergo a second
mitotic division. During the second mitotic division, the nucleus at the micropylar end
divides vertically and the cells differentiate into two synergids. The nucleus located at
the chalazal end undergoes a transverse division and the resulting cells differentiate into
an egg cell (near the synergids) and a central cell (at the chalazal end). This sequence of
divisions in the nuclei and arrangement of cells results in a T-shaped configuration of
an embryo sac with two synergids vertically organized at the micropylar end and a
centrally placed egg cell between the central cell and the synergids (Fig. 26H). In later
stages, the central cell degenerates and consequently the mature female gametophyte is,
represented by an egg apparatus alone (Fig. 26I).
Temporal details of pollen tube growth and central cell degeneration
In the investigated species, the central cell is either not formed (W. arekaliana) or it
degenerates before fertilization (Z. olivaceum, P. munnarense and P. subulatum). When
the precise time of central cell degeneration was correlated with pollen tube growth it
was found that the time of loss of central cell was different among the species.
Podostemum subulatum (Fig. 27)
At the time of anthesis, megagametophytes in most of the ovules had attained four-
celled/ four-nucleate stage but a few ovules also showed megagametophytes with
degenerated central cell. The pollen tubes reached the middle of placenta by 4 HAP
(Fig. 27A,B), and by this time most of the megagametophytes had attained the final
configuration of 3-celled/3-nucleate (Fig. 27C). Hence, when the pollen tubes reached
the tip of the ovule (12-16 HAP) and brought about fertilization (24 HAP), only two
synergids and an egg cell were present at the micropylar domain of the
megagametophyte, whereas the central cell had degenerated and megagametophyte was
3-celled/3-nucleate.
56
Polypleurum munnarense (Fig. 28)
Pollen grains germinate and reach the middle of placenta by 4 HAP (Fig. 28A,B). At
this time, most of the megagametophytes were 4-celled/4-nucleate. Further growth of
the pollen tube is slow, as it takes 12 h to reach the tip of the ovule from the placenta
(16 HAP). By the time pollen tube enters the micropyle, the central cell located at the
micropylar end of the ovule degenerates reducing the megagametophyte to 3-celled/3nucleate condition (Fig. 28C).
Willisia arekaliana
W. arekaliana represents an exclusive system where the nucleus destined to become the
central cell degenerates before cell walls are laid. Hence, the megagametophyte is threenucleate only. Therefore, at all the stages of pollen tube growth beginning from the time
of pollination (08:00- 10:00 am) till fertilization (24 HAP), the megagametophyte is a
3-celled/3-nucleate structure. Hence no figure is provided for the species.
Zeylanidium olivaceum (Fig. 29)
Pollen grains begin to germinate as soon as they land onto the receptive stigma (Fig.
29A,B). Numerous pollen tubes traverse through the stigma and reach its base. They
grow through the short style and enter the placental tissue (8 HAP). At this stage, the
female gametophyte is 4-celled/4-nucleate. It was observed that during the postpollination stages (12-24 HAP), after travelling through the placenta, when pollen tubes
reach the tip of ovule, the central cell is degenerated (Fig. 29C).
Fertilization
Fertilization took place ca. 24 HAP in all the species examined. The pollen tubes
rupture terminally in one of the receptive synergids in P. subulatum, W. arekaliana and
Z. olivaceum, whereas in P. munnarense, it directly enters the egg cell as the synergids
are towards the chalazal end (Fig. 30A,B,C,D). However, only syngamy took place
(Fig. 30E,F) at 24–26 HAP as the central cell had already degenerated and the usual
second fertilization event, characteristic of double fertilization, was invariably absent in
all the species.
57
Nucellar plasmodium
Like all the other podostemaceous species, W. arekaliana also develops a nucellar
plasmodium (Fig. 25B). As soon as the megaspore mother cell is organized, cell walls
of the nucellar cells below the megaspore mother cell disintegrate and all the nuclei
pool in a common cytoplasm. Disintegration of walls of nucellar cells progresses from
the micropyle towards the chalazal end. The two-layered inner integument remains
intact in the process and delimits the nucellar plasmodium (Fig. 25B). Development of
the nucellar plasmodium in P. subulatum, Z. olivaceum and P. munnarense has been
documented by earlier workers; and no deviation was observed in the developmental
pattern.
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Discussion
Ever since the significant discovery of double fertilization was made in the flowering
plants, it is considered a distinctive feature of angiosperms (Raghavan, 2003). Double
fertilization is a mating of the products of two ontogenies, the female gametophyte and
the male gametophyte. The female gametophyte of the flowering plants produces two
components: an egg cell and a central cell which mate with two sperm cells and
contribute to the process of double fertilization. Podostemaceae is the only family of
flowering plants whose members do not indulge in double fertilization and undergo
only single fertilization i.e., syngamy (Mukkada, 1962; Mukkada and Chopra, 1973).
Other than Podostemaceae, single fertilization has also been reported in a member of
Orchidaceae, Spiranthes sinensis (Terasaka et al., 1979), in which the generative cell
does not divide to form two male gametes.
In spite of the normal development of the female and male gametophytes,
double fertilization fails to occur in Podostemaceae for the reasons unknown. Typically,
an organized embryo sac of Podostemaceae is 4-celled, consisting of two small
synergids, a large egg cell constituting the egg apparatus, and a central cell harboring a
polar nucleus. The 4-celled/4-nucleate female gametophyte like in Podostemaceae is
also known to occur in the earliest divergent angiosperms (Williams and Friedman,
2002, 2004) and the members of Onagraceae except Trapa (Maheshwari, 1937), but
double fertilization does occur in both the latter cases (Baroux et al., 2002; Williams
and Friedman, 2002) leading to the formation of a diploid endosperm, which makes the
absence of double fertilization in Podostemaceae even more perplexing.
However, Sehgal et al. (2010), while demonstrating the incidence of single
fertilization in Dalzellia zeylanica, a member of the subfamily Tristichoideae
(Podostemaceae), showed that early degeneration of central cell leads to the formation
of a novel 3-celled/3-nucleate mature female gametophyte comprising only two
synergids and an egg cell. The polar nucleus of the central cell degenerates prior to the
59
entry of the pollen tube into the synergid. Therefore, out of the two male gametes only
one gets a counterpart for fusion whereas the other eventually degenerates due to the
absence of its partner. In light of this report, I studied female gametophyte development
from differentiation of megaspore mother cell till fertilization in the other species of
Podostemaceae, specifically in the subfamily-Podostemoideae. The findings of the
present investigation are discussed below.
Megagametophyte development in Podostemaceae can be either monosporic or
bisporic
The megagametophyte development in Podostemaceae has been defined in different
ways by different authors. In Podostemaceae, after the first meiotic division of the
megaspore mother cell, the upper dyad cell degenerates and the female gametophyte
develops from the lower dyad cell. After the second meiotic division of the lower dyad
cell, the two megasporial nuclei are vertically arranged. In some species (Vanroyenella
plumosa Novelo and Philbrick, Hydrobryum griffithii (Wall. ex Griff.) Tul.,
Zeylanidium olivaceum, Indotristicha ramosissima), embryo sac develops from the
upper nucleus alone and the lower nucleus degenerates whereas in others (Polypleurum
munnarense, P. dichotomum (Gard.) Hall, Hydrobryopsis sessilis, Willisia selaginoides
and Zeylanidium johnsonii (Wight) Engl.), both the nuclei divide and give rise to an
embryo sac (Mukkada, 1962, 1964; Arekal and Nagendran, 1975b, 1976, 1977a, 1977b;
Nagendran et al., 1976, 1977; Murguìa-Sánchez et al., 2002). Since, the embryo sac
arises from the lower dyad cell; some authors considered it to be of bisporic in origin
(Chiarugi, 1933; Maheshwari, 1947). The authors regarded the number of megaspores
present in the initial cell of embryo sac as the basis of classifying the megagametophyte
development and concluded it to be bisporic in Podostemaceae. While the other widely
accepted classification considers the number of megaspores that actually contribute to
the formation of a mature embryo sac (Arekal and Nagendran, 1975a). It was opined
that the type of embryo sac development should be considered monosporic, if only one
megaspore nucleus participate in the formation of an embryo sac and bisporic, when
both the megaspore nuclei are involved.
60
Based on the latter classification, monosporic development is the most common
type reported from the family. Under monosporic development, the two types of
embryo sacs are Apinagia ‘a’ and Apinagia ‘b’ type. In the ‘Apinagia’ type of embryo
sac, the two synergids are positioned at the micropylar pole, and the egg cell and central
cell are at the chalazal side of the embryo sac. Interestingly, the single polar nucleus has
no apparent function, as double fertilization does not occur and antipodal cells are never
formed (Battaglia, 1987). In true bisporic type development, only one mitotic division
takes place (instead of two in the ‘Apinagia’ type) to form the quartet embryo sac.
Under this type of embryo sac development, ‘Podostemum’ type and ‘Polypleurum’
type are described. In ‘Podostemum’ type the two synergids arise from the upper
megasporial nucleus. The spindle of the mitotic division of the lower megaspore is
vertical and gives rise to an egg cell and a single nucleated central cell. The
arrangement of four cells is thus T-shaped in the embryo sac. The ‘Polypleurum’ type
differs from the ‘Podostemum’ type in the orientation of the spindles of the only mitotic
division of the two megaspores. The upper spindle is vertically oriented, and the lower
is horizontal such that synergids are towards chalazal end and the egg cell is centrally
placed and the central cell is towards micropylar end.
However, the occurrence of bisporic megagametophyte in Podostemaceae has
long been questioned (Battaglia, 1987). Battaglia (1987) challenged the occurrence of
all bisporic embryo sacs in Podostemaceae and strongly encouraged a reinvestigation of
both the ‘Polypleurum’ type (Mukkada, 1962, 1964; Arekal and Nagendran, 1975b,
1976, 1977a; Nagendran et al., 1977) and the ‘Podostemum type’ (Arekal and
Nagendran, 1975b).
Evidences in the present study clearly suggest that the megagametophyte
development in Willisia arerkaliana and Polypleurum munnarense is of bisporic type
where both the megaspores in the chalazal dyad undergo one mitotic division each. The
occurrence of bisporic female gametophyte in P. munnarense confirms the observations
of Nagendran et al. (1977) and together with the observations in W. arerkaliana the
existence of bisporic embryo sac in Podostemaceae is validated. The megagametophyte
development is of monosporic type in Zeylanidium olivaceum and Podostemum
subulatum.
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Evidences in angiosperms suggest that bisporic and tetrasporic embryo sacs are
the derived conditions that have evolved multiple times, especially in relatively derived
groups, but also in some early-divergent angiosperms such as Piperaceae (Williams and
Friedman, 2004). Since monosporic development is more common in Podostemaceae,
bisporic development can be presumed to be derived from it.
Megasporogenesis and Megagametogenesis
After tracing the stages of megasporogenesis and megagametogenesis in Z. olivaceum,
P. subulatum and P. munnarense, it was noticed that the ontogenetic pattern of the
female gametophyte in all the species is same till the differentiation of an egg apparatus
and central cell as described by the earlier embryologists. Megagametogenesis results in
four nuclei after two mitotic divisions in Z. olivaceum and P. subulatum.
Megagametogenesis comprises only one mitotic division in P. munnarense. After
megagametogenesis the resultant two cells differentiate as synergids and the other two
as an egg cell and a central cell. However, the three species differ from each other in the
organization of cells of the embryo sac. The synergids are towards the micropylar end
and central cell towards the chalazal end in Z. olivaceum and P. subulatum. The polarity
of the embryo sac in P. munnarense is reversed i.e., the central cell is towards the
micropyle and synergids are towards chalazal end. Thus, it can be concluded that
irrespective of the pattern of development of embryo sac and polarity to which it may
align, it invariably consists of two synergids, an egg and a central cell in these species.
Unlike the other podostemaceous species, megagametophyte in W. arerkaliana
is a unique 3-celled/3-nucleate structure. In W. arerkaliana, a 4-celled stage of female
gametophyte is never formed. Out of the four mitotic products of megagametogenesis,
one towards the chalazal end degenerates at the free nuclear stage itself, resulting
female gametophyte without a central cell. It is a known fact that the embryo sac first
develops as a syncytium, and the establishment of cell identity coincides with
cellularization (Sundaresan and Alandete-Saez, 2010), and the observations in W.
arerkaliana suggest that the central cell is not even established in the species since the
fourth nucleus degenerates at the syncytial stage itself before it can be cytologically
62
identified as a central cell. Therefore, female gametophyte of W. arekaliana is a highly
reduced structure with only an egg apparatus.
Pollen tube path and central cell degeneration
Observations in Z. olivaceum, P. subulatum and P. munnarense showed that the 4celled/4-nucleate stage of megagametophyte, as described, is transient and it eventually
differentiates into 3-celled/3-nucleate stage before fertilization. It was also observed
that the time of degeneration of central cell/polar nucleus is not constant among the
species. Precisely, it occurs at the time interval between flower anthesis and 4 HAP in
P. subulatum. In Z. olivaceum and P. munnarense, the central cell degenerates around
12-16 HAP. From the results, it can be assumed that degeneration of central cell is a
key step in the ontogeny of female gametophyte of Podostemaceae. Central cell is an
important component of female gametophyte and its absence from the mature female
gametophyte is not only surprising but also provides an insight into the cause of
absence of double fertilization in Podostemaceae.
Fertilization
For long the reason for the absence of double fertilization in Podostemaceae was
unclear and seen as a probable result of failure of the pollen tube to discharge the
second gamete because of which central cell degenerates (Raghavan, 2003; Sikolia and
Ochora, 2008; Sikolia and Onyango, 2009). In the present study it could be observed
that the male gametes are released into the embryo sac. Moreover, failure of discharge
of the second male gamete as a reason may hold true for species where central cell
degenerates just before fertilization but may not be true for W. arerkaliana where the
polar nucleus perishes even before it attains its cellular identity. Absence of central cell
in W. arekaliana and its disorganization before fertilization in other investigated species
(present study) confirms that loss of double fertilization in Podostemaceae is a result of
lack of central cell as rightly conjectured by Sehgal et al. (2010), and not the male
counterpart as proposed by some earlier authors. However, molecular basis for this
early ‘programmed’ degeneration of polar nucleus still need to be explored.
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Evolution of female gametophyte in Podostemaceae
The origin of 8-nucleate/7-celled Polygonum type of embryo sac was probably a major
ontogenic event in angiosperms because it stabilized triple fusion and endosperm
formation (Rudall et al., 2008). Occurrence of a 4-celled/4-nucleate megagametophyte
in some early-divergent angiosperms gave rise to a hypothesis that the 4-nucleate
condition is ancestral, and by duplication it gave rise to the 8-nucleate Polygonum type
of embryo sac, which is present in more than 70% of angiosperms (Friedman and
Williams, 2003). However, the current phylogenetic position of Podostemaceae places
it in the order Malphigiales, and the occurrence of bisporic megagametophyte and other
embryological characteristics like tenuinucellate ovules and the formation of micropyle
only by the outer integument further corroborate its position as a derived group.
Therefore, the occurrence of a 4-celled/4-nucleate megagametophyte in Podostemaceae
appears to be a probable result of the reduction from an 8-nucleate megagametophyte.
This might hold true in aquatic groups like Podostemaceae, because aquatic plants are
frequently associated with strong morphological reduction, high intraspecific variation
and high plasticity (Arber, 1920; Cook, 1999). Further, reduction of the
megagametophyte in the family might have prompted the occurrence of a 3-celled/3-
nucleate megagametophyte. The elimination of central cell at different times and the
formation of mature 3-celled/3-nucleate female gametophyte in the studied genera
represent a novel evolutionary line of megagametophyte development by reduction in
Podostemaceae and megagametophyte of W. arekaliana represents the extreme end of
this paradigm as the nucleus that would have become central cell, degenerates before its
establishment as central cell. Clues like the presence of two megagametophytes in the
same ovule, which develops from a single megasporocyte in W. arekaliana and
frequent observations of embryo sacs with inverted polarities in Z. olivaceum and W.
arekaliana also provide a hint of such a reduction.
Differentiation of megasporocyte is the beginning of transition from the
sporophytic to gametophytic phase in plants. While the development of more than one
megasporocyte from a multicellular archesporium is a common phenomenon in
angiosperms (Maheshwari, 1950; Eames, 1961), development of multiple gametophytes
from a single megasporocyte is unusual and surprising given the fact that organization
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of embryo sacs in angiosperms is under the purview of many developmental
checkpoints (Grossniklaus and Schneitz, 1998). There are very few instances of more
than one megaspore giving rise to multiple embryo sacs like in Pellodendron amurense
(Starshova and Solntseva, 1973; Poddubnaya-Arnoldi, 1976), early-divergent
angiosperms such as Schisandraceae (Friedman et al., 2003; Williams and Friedman,
2004) and Hydatellaceae (Rudall et al., 2008) where development of two gametophytes
in the same ovule are reported. Interestingly, mem mutants in Arabidopsis are known to
have similar double gametophytes wherein mutation affects archespore selection and
megaspore mother cell (mmc) specification, leading to the initiation of two
gametophytes in the same ovule (Schmidt et al., 2011).
In the ovules of W. arekaliana, separate archesporial cells within the same
nucellus were never observed, but megagametophytes developing from both the dyads
could be seen. In about 33 ovules out of 640 ovules (~5%) observed, the megaspores in
both the dyads were observed to be undergoing second meiosis. However, meiosis II
was never followed by cell wall formation in any of the dyads. In ovules with two
gametophytes, the chalazal one is functional because in most of the instances the
micropylar embryo sac usually degenerates or stops dividing after two-nucleate stage.
In many ovules, the micropylar gametophyte contains two prominent nuclei, such that a
four-nucleate embryo sac can sometimes give a wrong impression of a five-nucleate
embryo sac. Observations of two female gametophytes developing in the same ovule in
W. arekaliana are significant because they not only indicate a high degree of
developmental plasticity but may also provide a clue to the specialization of 4-celled/4-
nucleate female gametophyte in Podostemaceae from an ancestral eight-nucleate
gametophyte, probably as a result of reduction.
Friedman and Williams (2003) have regarded the development of female
gametophyte to be modular, with one micropylar and one chalazal module. Such that the
eight-nucleate embryo sac has two mirror image domains. According to this modular
hypothesis, embryo sacs in the early divergent angiosperms have only the micropylar
domain and rest of the angiosperms have two domains which is the result of duplication.
The condition seems to be analogous in Podostemaceae but in reverse direction where it
seems that ancestral condition might have been an eight nucleate embryo sac with both
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the domains but due to reduction, one domain was lost, and only one domain was left.
Frequent observation of embryo sacs with inverted polarities in many genera of
Podostemaceae (Z. lichenoides, W. arekaliana, Hydrobryopsis sessilis) also conforms to
the suggestion. These embryo sacs might be representing the chalazal quartet of an
ancestral eight-nucleate embryo sac. However, this hypothesis will become more testable
if double gametophytes are also observed in other species of Podostemaceae.
Central cell has been shown to play a significant role in pollen tube guidance
and in endosperm formation after fertilization (Chen et al., 2007; Curtis and
Grossniklaus, 2008). Its absence from the female gametophyte of Podostemaceae not
only questions the indispensible role of this cell but also underlines the fact that
endosperm is not required for embryo development in these plants, unlike the other
angiosperms.
Imprinting and parent-of-origin effects in the seed development of Arabidopsis
and Zea mays (Haig and Westoby, 1989, 1991; Grossniklaus and Schneitz, 1998; Scott
et al., 1998; Adams et al., 2000; Leblanc et al., 2002; Danilevskaya et al., 2003) have
emerged as new insights into seed development of angiosperms. It would be interesting
to know whether these imprinted genes are present in Podostemaceae and if present
what is their role in the absence of endosperm.
The loss of endosperm in Podostemaceae appears to be compensated by the
acquisition of a novel structure - the nucellar plasmodium or pseudo embryo sac, which is
an embryo nourishing tissue of sporophytic origin. It is formed as a result of
disintegration of the nucellar cells below the embryo sac (Battaglia, 1980; Davis, 1966;
Arekal and Nagendran, 1975a). It is similar to the perisperm observed in early-divergent
angiosperms where a diploid endosperm formed following double fertilization is often
found to be minute and devoid of reserves (Rudall et al., 2008; Friedman et al., 2012). It
is the maternal sporophyte that is perisperm, which takes over this minute endosperm
when it comes to nutrient storage and resource allocation in these plants (Friedman et al.,
2012). Pseudo embryo-sac and perisperm may be non-homologous by developmental
origin and structure but show similarity of function i.e. to provide nutrition to developing
embryos, in the absence or underdevelopment of endosperm.
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