Central Cell Degeneration Leads to Three-Celled Female Gametophyte in Zeylanidium Lichenoides Engl

South African Journal of Botany 91 (2014) 99–106

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South African Journal of Botany

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Central cell degeneration leads to three-celled female gametophyte in Zeylanidium lichenoides Engl. (Podostemaceae)

A. Chaudhary a, P. Khanduri a,R.Tandona,⁎, P.L. Uniyal a, H.Y. Mohan Ram b a Department of Botany, University of Delhi, Delhi 110007, India b Shriram Institute for Industrial Research, 19 University Road, Delhi 110007, India article info abstract

Article history: The absence of double fertilization and lack of endosperm formation are key embryological features of Received 13 June 2013 Podostemaceae, which is unique among the angiosperms. Double fertilization fails in spite of the fact that Received in revised form 8 January 2014 two male gametes are carried in the pollen tube and the incipient embryo sac is equipped with an egg Accepted 8 January 2014 cellandacentralcell.Littleisknownabouttheproximatecausesofthefailureofdoublefertilization.The Available online 27 January 2014 female gametophyte of Zeylanidium lichenoides, (subfamily — Podostemoideae) has a monosporic mode Edited by GV Goodman-Cron of development resulting in an initial four-celled/four-nucleate condition. However, the central cell degenerates before the arrival of the pollen tube in the vicinity of micropyle, thus the mature and functional Keywords: female gametophyte of Z. lichenoides is ontogenically a three-celled/three-nucleate structure. It is inferred Female gametophyte that the formation of a highly reduced female gametophyte, presumably is a result of degeneration of the Podostemaceae central cell. Podostemads © 2014 SAAB. Published by Elsevier B.V. All rights reserved. Single fertilization Three-celled/three-nucleate embryo sac

1. Introduction rocks or boulders. The plant body of podostemads is thalloid. The conventional demarcation into root, shoot and leaf, is lacking, leading Double fertilization leading to endosperm formation is believed to to a ‘fuzzy morphology’ (Cook and Rutishauser, 2007; Mohan Ram be a universal feature and a key developmental innovation in the diver- and Sehgal, 2007). The occurrence of high developmental plasticity sification of angiosperms (Williams and Friedman, 2004). Molecular and perplexing polymorphism has made taxonomic delimitation of cross-talk between the endosperm and the embryo at various stages Podostemaceae a daunting task. Position of the family in the phylogeny of development is considered crucial in forming a successful sporophyt- of angiosperms was also unresolved, till recently it was placed at differ- ic phase in the sexually reproducing flowering plants (Ungru et al., ent positions in the phylogeny of angiosperms. Ruhfel et al. (2011) 2008; Nowack et al., 2010). Yet some plants successfully defy the defin- provide evidences that Podostemaceae belong to Clusioid clade of the ing second fusion of double fertilization. Among these, all the species order Malpighiales with Bonnetiaceae, Calophyllaceae, Clusiaceae and of Podostemaceae invariably lack double fertilization despite the forma- Hypericaceae as its sister families. tion of two male gametes and differentiation of an egg cell and a central Podostemaceae has been cited as an embryological family (Kapil, cell in the female gametophyte (Sehgal et al., 2011). 1970). Besides the absence of double fertilization, occurrence of a With 50 genera and 280 species, Podostemaceae represent the larg- four-celled/four-nucleate embryo sac, absence of antipodals, presence est natural assemblage of fresh water flowering plants with several of a pseudo-embryo sac (Nucellar plasmodium) and lack of plumule unusual vegetative and reproductive features (Cook and Rutishauser, and radicle in the mature embryo are believed to be among the other 2007; Mohan Ram and Sehgal, 2007). The family is pan-tropical in defining embryological features of the family (Kapil, 1970; Mohan occurrence, found in Africa, Asia, Australia and the Americas. Africa Ram and Sehgal, 2007). (including Madagascar) has 16 genera and ca. 77 species (Engler, Absence of double fertilization in an angiospermic family is an in- 1930; Cusset, 1987), 21 genera (ca. 135 species) are documented triguing feature, since the presence of double fertilization has historical- from the Americas (Tippery et al., 2011) and 18 genera (ca. 84 spp.) ly defined reproduction in the angiosperms. The absence of double occur in Asia and Australia (Cusset and Cusset, 1988; Cusset, 1992). fertilization in Podostemaceae was attributed to the inability of the sec- All taxa of this family begin their life in flowing water attached to ond male gamete to unite with the central cell (Mukkada and Chopra, 1973). However, a recent report in Dalzellia zeylanica Wight (subfamily ⁎ Corresponding author. Tel.: +91 9810760111. Tristichoideae), has shown that early degeneration of central cell just E-mail address: [email protected] (R. Tandon). before the release of the male gametes is the cause of this condition in

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D. zeylanica (Sehgal et al., 2011). Whether or not the degeneration of the Podostemoideae. Zeylanidium (Tul.) Engl., with its four species central cell is a prerequisite condition in the other taxa of this family has viz. Zeylanidium olivaceum (Gardn.) Engl., Zeylanidium johnsonii not been clearly established. (Wight) Engl., Z. lichenoides Engl., Zeylanidium maheshwarii Mathew The occurrence of a single fertilization has been documented in and Satheesh, is conﬁned to peninsular India, the southern Indian sub- Spiranthes sinensis (Pers.) Ames. (Orchidaceae) and some natural hy- continent and Myanmar (Mathew and Satheesh, 1997). The earlier brids of maize (Terasaka et al., 1979; Kato, 1997, 1999). However, all embryological investigations in Z. olivaceum and Z. johnsonii have these three instances are distinct. S. sinensis represents an abnormality shown that the two species exhibit a four-celled/four-nucleate condi- of microgametogenesis as only one male gamete is formed due to the tion of embryo sac corresponding to the Apinagia (monosporic) and failure of the generative cell division (Terasaka et al., 1979). In maize Polypleurum (bisporic) type of embryo sac development, respectively hybrids single fertilization is a rare case (Kato, 1997, 1999). There is a (Arekal and Nagendran, 1977). We report that the female gametophyte rare example of Commiphora wightii (Arn.) Bhandari in which the egg development in Z. lichenoides is monosporic, Apinagia ‘b’ type and apparatus and the antipodal cells degenerate and the two polar nuclei its mature female gametophyte is similar to that described for fuse to form an autonomous diploid endosperm (Gupta et al., 1996). D. zeylanica. We also demonstrate that syngamy is the only fertiliza- The present work is the ﬁrst report on the development of female tion event, as the central cell degenerates before the arrival of the gametophyte in Zeylanidium lichenoides Engl., of the subfamily pollen tube in the vicinity of the micropyle.

Fig. 1. (A) Site of collection of plant material; (B) Habitat showing a patch of Zeylanidium lichenoides growing attached to partly submerged rocks; (C–H) Stages of ﬂower bud (bd) to fruit development; (C) Flower bud (bd) (Stage I), (bract br; spathella, sp); (D) Flower at anthesis (Stage II); (E) Anthers (an) in contact with an interlocking biﬁd stigma (st) effecting self- pollination (Stage III) (ovary, ov); (F) The anthers (an) have shed all pollen and the andropodium (ap) has moved away from the interlocking stage (Stage IV); (G) The andropodium (ap) has elongated 2–3 times and the pedicel (pd) ~3–4 times andtheandropodium (ap) is far above the stigma (st) (Stage V); (H) A fully developed fruit (capsule) (Stage VI), bar = 0.5 mm. A. Chaudhary et al. / South African Journal of Botany 91 (2014) 99–106 101

2. Material and methods axile placenta. Ovules are anatropous, bitegmic and tenuinucellate (Fig. 2A). The two integuments of the ovule arise from the base of the The study material (Z. lichenoides) was collected in December nucellus; the outer integument arises first and contributes to the forma- 2009–2010 from a tributary of Periyar river near Campanywadi tion of the micropyle. The inner integument is shorter and is restricted (10°45′N and 77°9′S) in Idukki district, Kerala state in Southern India to the base of embryo sac. (Fig. 1A). In this season, the plant is available at various stages of repro- Although flowering is initiated under water, anthesis and pollination duction, spanning from initiation of floral primordia to completely invariably occurred above water. As the flowers are naked, the timing of mature but undehisced fruits (capsules). A patch of about 200 plants rupture of the spathella was considered as the time of anthesis (Fig. 1D, was selected to observe anther dehiscence, transfer of pollen grains Table 1). Pollination occurs soon after anthesis (Fig. 1F, Table 1); the and their deposition on the stigmas. stigmatic lobes become stretched and the anthers that lie entangled be- Flowers at several stages of development were fixed in Karnovsky's tween the stigmatic lobes, release the pollen grains thereby facilitating fixative (8% paraformaldehyde + 12% glutaraldehyde in 0.2 M sodium self-pollination (Fig. 1E) (Autopollination). After pollination, there is a cacodylate buffer, pH 7) (Karnovsky, 1965) for 6 h at 28 °C and then considerable increase in the length of the andropodium and the pedicel. washed in sodium cacodylate buffer. The material was dehydrated These changes help in recognizing developmental stages of the flower through ethanol series and infiltrated and embedded with freshly pre- (Fig. 1G, H, Table 1). pared glycol methacrylate monomer mixture (O' Brien and McCully, 1981). Semi-thin (3 and 4 μm) sections were cut using glass knives on 3.2. Megasporogenesis and megagametogenesis a rotary microtome (820, AO Spencer, USA). Sections were stained with either 0.1% toluidine blue or with decolourized aniline blue A densely protoplasmic hypodermal archesporial cell of the nucellus, (Martin, 1959). Observations were made using photomicroscope (Carl which differentiates early in a developing ovule directly functions as the Zeiss, Axio Scope A1). megaspore mother cell/megasporocyte (Fig. 2A). The megasporocyte Clearing of ovules was done by two methods. For differential inter- undergoes Meiosis-I to give rise to two vertically arranged nuclei ference contrast (DIC) microscopy, ovules fixed in FAA {formaldehyde: followed by an asymmetric division to result in two dyad cells of un- acetic acid:70% ethanol; 1:1:18} were gently rinsed in 50% acetone equal sizes (Fig. 2B). Out of these two, the micropylar dyad cell is and subsequently passed through an ascending dehydration series of smaller and degenerates and may persist as a dark pyknotic body until acetone (70%, 80%, 90%, and 100% for 45–50 min each). Clearing was fertilization (Fig. 2C). The surviving larger chalazal dyad cell, enclosing done with methyl benzoate for 120 min and with methyl benzoate: a large nucleus undergoes the second meiotic division and produces Spurr's resin (7:1) for 12 h (Siddiqi et al., 2000). Mounting was done two vertically arranged nuclei in configuration similar to those that ap- in the same mixture. For fluorescence microscopy, the ovules were pear after Meiosis I (Fig. 2D). However, nuclear division is not followed cleared with 1N NaOH for 24 h, followed by washing with distilled by cell wall formation. Out of the two nuclei, the one located at the water. RNase treatment was given for 30 min before staining with chalazal end degenerates and forms a crescent shaped structure 4′,6-diamino-2-phenylidole (DAPI) (10 μg/ml). The path of the pollen (Fig. 2E), whereas the micropylar nucleus further divides mitotically tube was localized from pollinated pistils at various stages employing (Mitosis-I) to form two nuclei, positioned one above the other along decolourized aniline blue (Shivanna and Rangaswamy, 1992). the micropylar and chalazal axis. The cell wall is not formed after Mitosis-I and both the nuclei undergo a second mitotic division (Mito- sis-II). During the second mitotic division, the nucleus at the micropylar 3. Results end divides vertically and the cells differentiate into two synergids. The chalazal nucleus undergoes a transverse division and the resulting cells 3.1. Plant morphology and structure differentiate into an egg cell (near the synergids) and a central cell (at the chalazal end). This sequence of divisions in the nuclei and Z. lichenoides has a green, crustaceous, irregularly branched, dorsi- arrangement of cells results in a T-shaped configuration of a four- ventral and thalloid plant body. It is attached to rocks by means of glan- celled/four-nucleate embryo sac (Figs. 2F, 3A). dular hairs in fast flowing streams (Fig. 1B). Flowering shoots arise on In a majority of the ovules examined (n = 2500), the organization of the thallus with the fall of water level in the streams. Each flowering embryo sac was T-shaped. However, in nearly 5% of the ovules, the shoot bears three or four pairs of bracts, and terminates in a solitary polarity was reversed into an inverted-T-shape, with synergids located flower. The flower bud originates on the margin of the dorsal surface at the chalazal end and the central cell at the micropylar end, with the of the thallus and is enclosed by a protective covering spathella egg cell in the center (Fig. 2H). (Fig. 1C, Table 1). The flower is highly reduced and is represented only by a pistil and two stamens (Fig. 1F). The anthers are tetrasporangiate and dorsifixed. Pollen grains are produced as dyads. The average num- 3.3. Nucellar plasmodium ber of pollen grains produced in a flower was 4571 ± 53 (n = 50 flowers). The gynoecium is bicarpellary and syncarpous. The ovary is Like the other podostemads, Z. lichenoides is characterized by the superior and bilocular with 47 ± 6.9 (n = 50 flowers) ovules on an presence of a nucellar plasmodium, a unique nutritive tissue. It begins

Table 1 Morphological and embryological details of various stages of ﬂower of Zeylanidium lichenoides, recognized for the present study.

Stage of ﬂower Time before or after pollination Characteristic feature(s) Stage of embryo sac

I – Flower enveloped inside the Megasporogenesis and green spathella megagametogenesis II 30 min-1 HBPa Anthesis; the spathella ruptures, Four celled/four nucleate ﬂower is exposed III 0 Pollination Four celled/four nucleate IV 6 HAPa Post pollination I Four celled/four nucleate V12HAPa Post pollination II Three celled/three nucleate VI 24 HAPa Post pollination III Fertilized egg

a HBP and HAP: Hours before and after pollination, respectively. 102 A. Chaudhary et al. / South African Journal of Botany 91 (2014) 99–106 A B C

np ii oi

D E s F

np np np

G H I

c pt np ii oi e

s L J K pt

np np A. Chaudhary et al. / South African Journal of Botany 91 (2014) 99–106 103

A B s s

e e

Fig. 3. Cleared ovule as observed by DIC. (A) A completely developed four-celled/four-nucleate embryo sac (central cell, c; egg cell, e; synergid, s), bar = 2.5 μm; (B) A mature three-celled/ three-nucleate embryo sac, bar = 5 μm.

to organize at the time of differentiation of the megaspore mother cell 4. Discussion (Fig. 2A) and becomes completely organized at the four-celled/four- nucleate stage of the embryo sac. The process of its formation initiates The present study provides clear evidence that Z. lichenoides exhibits from the central row of nucellar cells. The walls of nucellar cells located monosporic type of megagametophyte development. After meiosis II, below the embryo sac disintegrate first followed by those located the chalazal nucleus disintegrates and soon disappears. The nucleus at towards the chalazal end. Subsequently, the protoplasts of all the cells the micropylar end alone contributes to the formation of the embryo merge to form a coenocyte and the inner integument forms the periph- sac. Thus, according to the classification proposed by Battaglia (1987), ery of the nucellar plasmodium. The formation of a nucellar plasmodi- the monosporic type of development observed in Z. lichenoides is um in Z. lichenoides is completed prior to syngamy (Fig. 2G). ‘Apinagia type b’ as the degenerated chalazal megaspore nucleus is ephemeral (Fig. 5). The monosporic type of female gametophyte development found in Podostemaceae is, however, different from that 3.4. Path of pollen tube and fate of the central cell observed in other angiosperms. A linear tetrad of megaspores is absent in Podostemaceae and degeneration of the non-contributing cells is a Pollen grains germinate soon after pollination. Numerous pollen successive type instead of being simultaneous as observed in other tubes traverse through the stigma, a short style and enter the placental angiosperms. Degeneration of the first megaspore takes place immedi- tissue (Stage IV, Table 1). At this stage the female gametophyte is four- ately after the first division of meiosis. After the second division during celled/four-nucleate. The central cell begins to degenerate when the meiosis the chalazal megaspore nucleus degenerates before the wall is pollen tube reaches the tip of ovule (Stage V–VI, Table 1)(Fig. 4A, B). laid. Thus, the embryo sac is monosporic in origin, as it is formed from By the time the pollen tube enters the micropyle, the central cell only one nucleus, positioned at the micropylar end of the lower dyad would have completely degenerated shrinking into a crescent-shaped cell. structure (Fig. 4B, C). Hence, the embryo sac in Z. lichenoides at maturity Before the organization of a functional embryo sac, the is a three-celled/three-nucleate structure, represented only by the egg megametophyte of Z. lichenoides has four nuclei. Three of these apparatus (Figs. 3B, 4D). nuclei constitute the egg apparatus towards the micropyle and the fourth occupies the chalazal end. Nagendran et al. (1977) iden- tified this nucleus as a polar nucleus because it is a sister of the egg 3.5. Fertilization nucleus. This configuration of megametophyte is also found in Nymphaeales and Austrobaileyales, two of the basal angiosperm lineages Pollen tubes release the sperm cells into the receptive synergid at (Friedman and Williams, 2003, 2004). A recent phylogenetic and com- Stage VI (Fig. 2J); the receptive synergid degenerates after the entry of parative analysis of the female gametophyte ontogenies, have shown pollen tube (Fig. 2I, J). One of the male gametes fuses with the egg that the four-celled/four-nucleate female gametophyte is plesiomorphic cell, resulting in the formation of a zygote (Fig. 2K, L). The second and seven-celled/eight-nucleate condition is apomorphic (Friedman and male gamete is rendered partner-less. Williams, 2003, 2004). According to ‘modular duplication hypothesis’,

Fig. 2. Longitudinal sections of ovules showing key stages of megasporogenesis and megagametogenesis (A–H) and the earlier stages of syngamy (I–L) in Zeylanidium lichenoides. (A) Bitegmic and tenuinucellate condition along with nucellar plasmodium (np) and megaspore mother cell (arrow) (inner integument, ii; outer integument, oi), bar = 30 μm; (B) A two-celled megagametophyte after cytokinesis with a smaller micropylar dyad cell (upper arrow) and a larger chalazal dyad cell (lower arrow), bar = 30 μm; (C) A degenerated micropylar dyad as a dark pyknotic body (arrow), bar = 40 μm; (D) Binucleate (arrows) stage of megagametophyte after meiosis II in the chalazal dyad, bar = 10 μm; (E) Two-nucleate megagametophyte after mitosis I with a degenerated chalazal nucleus (arrow), bar = 15 μm; (F) A completely developed four-celled/four-nucleate embryo sac (central cell, c; egg cell, e; synergid, s), bar = 25 μm; (G) Completely organized nucellar plasmodium in an embryo sac before syngamy, bar = 40 μm; (H) Micropylar region of the ovule showing an inverted embryo sac with synergids (s) located towards the chalazal end, bar = 20 μm; (I) An embryo sac with darkly stained synergid which has received the tube, note that the central cell has degenerated (arrow) bar = 30 μm; (J) An obliquely cut ovule showing the pollen tube penetrating into one of the synergids and the other synergid is intact with a nucleus positioned towards its micropylar end, bar = 40 μm; (K) Embryo sac with the male (arrow) and female gametes (arrow), bar = 40 μm; (L) A thick-walled zygote (z) (arrow), the pollen tube (pt) is noticeable above the embryo sac, bar = 40 μm. 104 A. Chaudhary et al. / South African Journal of Botany 91 (2014) 99–106

A B

C D

Fig. 4. (A–C) Fluorescence photomicrographs of pollinated pistils stained with decolourized aniline blue to localize the pollen tube path. (A) A pollen tube (arrow) approaching the micropylar end of the embryo sac bar = 2.5 μm; (B) A pollen tube at the tip of ovule and the degenerated central cell appears as a crescent shaped structure with callose deposition at the chalazal end (Stage VI, arrow), bar = 2.5 μm; (C) A part of the ovule showing pollen tube and a degenerated central cell, bar = 2.5 μm; (D) A portion of the cleared ovule stained with DAPI to localize the nuclei and depict three-celled/three-nucleate embryo sac,(egg cell, e; synergid, s), bar = 5 μm. Note: All the pictures were captured by using a monochromatic CCD camera; green ﬂuorescence is due to the selected pseudocolour.

the four-celled/four-nucleate gametophyte is considered as the basic Although the second fusion has never been documented in the fam- modular unit of development, and the seven-celled/eight-nucleate ily, the reasons for the absence of double fertilization have remained is the byproduct of its duplication (Friedman and Williams, 2003; elusive. Evidence from the study of Z. lichenoides lends support to ‘cen- Friedman and Ryerson, 2009). In this context, the Podostemaceae pos- tral cell degeneration’ as the cause. The collapse of central cell from the sesses only one of the modules. Such a unimodular female gametophyte embryo sac before the arrival of pollen tube reiterates that it is the has also been reported from D. zeylanica (Sehgal et al., 2011) and absence of female counterpart and not the male. Podostemum weddellianum (Tul.) Philbrick and Novelo (de Sá-Haiad So far, reduction in the structural components of female gameto- et al., 2010). A unimodular megagametophyte is a characteristic feature phyte due to the degeneration of central cell is known to occur in of the basal angiosperms. As Podostemaceae is nested in Malpighiales, D. zeylanica (Sehgal et al., 2011). In D. zeylanica, the central cell degen- a core eudicot order, the unimodular megagametophyte condition in erates soon after its formation. However, in Z. lichenoides, the central the family appears to be a secondarily derived evolutionary specializa- cell remains intact even after pollination and up to a reasonable time tion (Mohan Ram and Sehgal, 2007). The other reproductive features of pollen tube growth in the pistil. Nevertheless, a comparison of polli- such as tenuinucellate ovule, bisporic embryo sac and the formation of nation events in D. zeylanica and Z. lichenoides shows that the time of micropyle exclusively by the outer integument also corroborate position central cell degeneration varies but it happens before syngamy. Similar of Podostemaceae as a derived group. observations of central cell degeneration have also been obtained from A. Chaudhary et al. / South African Journal of Botany 91 (2014) 99–106 105

Megasporogenesis Megagametogenesis Three-celled megagametophyte

M1 M2 MT1 MT2

Stage I Stage II-IV Stage V-VI

Fig. 5. A ﬂow-diagram of megasporogenesis and megagametogenesis events in Zeylanidium lichenoides. The diagram is oriented to represent micropyle at the top and chalazal end towards the bottom (meiosis, M; mitosis MT; stages used for the present study, SI–SVI).

Tristicha trifaria, Inversodicraea bifurcata and Inversodicraea keniensis Authors 3 & 4 planned the research and wrote the paper. (Sikolia and Ochora, 2008; Sikolia and Onyango, 2009), but whether Author 5 supervised the overall research work. or not it occurs before the release of sperm cells needs to be ascertained. Z. lichenoides and D. zeylanica are the representatives of two of the three Acknowledgments subfamilies of Podostemaceae; i.e. Podostemoideae and Tristichoideae, respectively. Degeneration of the central cell before fertilization in PK is thankful to the Council of Scientific & Industrial Research for these species suggests that it might be a shared familial trait. the grant of Senior Research Fellowship. Financial assistance from the A female gametophyte with an inverted polarity (Polypleurum type) DST-Purse and DU-Research and Development Grant to RT is gratefully has been noted in many other species of the family (Nagendran et al., acknowledged. HYM thanks the Indian National Science Academy for 1977). However, its occurrence in only a few ovules of Z. lichenoides, the Srinivasa Ramanujan Research Professorship awarded to him. We fi suggests the event to be insigni cant. Similarly, the occurrence of two are thankful to the two anonymous reviewers for their constructive types of embryo sac within the same species has also been reported suggestions. from Hydrobryopsis sessilis (Sehgal et al., 2009). The case of two different arrangements of cells in an ovule is perplexing because gamete specification in embryo sacs is strictly under the regulation of asymmetric dis- References tribution of auxins (Pagnussat et al., 2009; Sundaresan and Alandete- Arekal, G.D., Nagendran, C.R., 1975. Is there a Podostemum type of embryo sac in the Saez, 2010). genus Farmeria? Caryologia 28, 229–235. In the absence of endosperm, nucellar plasmodium is believed to Arekal, G.D., Nagendran, C.R., 1977. 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