Structural organization of the gynoecium and pollen tube path in Himalayan sea buckthorn, Hippophae rhamnoides (Elaeagnaceae)

Gynoecium of Hippophae rhamnoides (Elaeagnaceae) is comprised of a single carpel and develops by enfolding of the carpel margins. The enfolding results in a vertical slit over the ventral surface of ovary. The pollen tube path is initially sub-stigmatic and is subsequently along the epidermal surface of slit. The tube accesses the solitary ovule through a pore positioned in the slit. These findings would be useful in understanding the evolution of transmitting tract in general and knowledge of pollen-pistil interaction of the species in particular.


Introduction
A conspecific pollen-pistil interaction encompassing pollen germination on a stigma, pollen tube growth and guidance through the style followed by successful fertilization constitutes the progamic phase in angiosperms (Heslop-Harrison 1975a, b;Linskens 1986). The functional attributes of the phase, such as duration, male gametophyte competition and regulation of the number of pollen tubes (selection/screening), are controlled by structural modifications that have accompanied the closure of carpels (angiospermy) in flowering plants (Bell 1995;Herrero and Hormaza 1996;Sage et al. 2009;Williams et al. 2010). Among these modifications, specialization of an elaborate receptive surface into a terminal stigma and the internalized pollen tube transmitting tract (internal compitum) are two of the conspicuous developmental events (Endress and Igersheim 2000;Williams 2009;Endress 2011). These processes have apparently followed different evolutionary lines (Endress 1982;Scutt et al. 2006) and may significantly reflect the habitat conditions or the pollination syndromes (biotic and/or abiotic) which proved effective in maximizing fitness in a species (Wang et al. 2011).
The pollen tube path in a majority of plants has evolved from an extragynoecial compitum to an internalized one through syncarpy, which has offered greater advantages by providing favourable conditions and the necessary nutrition for a prolonged, lengthier and even distribution of tube growth inside the pistil (Labarca and Loewus 1973;Herrero and Dickinson 1979;Herrero and Hormaza 1996). In spite of these advantages, there have been evolutionary reversals to apocarpy and/or unicarpellate condition (Endress 2011;Prychid et al. 2011;Wang et al. 2011). Nearly 10 % of the angiosperms have apocarpous gynoecia and 10 % are unicarpellate (Endress 1994(Endress , 2001. Apocarpy has greater preponderance in basal eudicots (Endress and Igersheim 2000;Endress and Doyle 2009). In higher eudicots and monocots, apocarpy is thought to be secondarily derived (Endress et al. 1983). As the mechanism of angiospermy has largely evolved independently from syncarpy (carpel fusion) (Williams et al. 2010), studies on core eudicots with unicarpellate or apocarpous gynoecia may provide fresh insights into the evolution and modularity in the structure and function of the progamic phase.
Elaeagnaceae, a core eudicot family nested in Rosales, exhibits apocarpous/unicarpellate condition and shares this feature with its sister family Barbeyaceae (Dickinson and Sweitzer 1970;Thulin et al. 1998). In its related families Rhamnaceae and Dirachmaceae, syncarpy is a prevalent condition. In Colletia spinosissima (Rhamnaceae), an extragynoecial compitum is present in the form of stigmatic exudate (Medan and Basilio 2001). Elaeagnaceae is represented by three genera: Hippophae, Elaeagnus and Shepherdia. The structural details of the gynoecia and information on the progamic phase in these species are not available. Among these, Hippophae rhamnoides (Himalayan sea buckthorn) is an Old World species distributed from Europe to China across the Himalaya (Mabberley 2008). It is a small to medium-size dioecious shrub and is largely exploited for its berries, known as Leh berry in India, from the wild. There are attempts to bring the species under cultivation and breeding programmes for sustainable utilization. Knowledge of the progamic phase including pollen-pistil interaction would be useful for these endeavours. In the present work on H. rhamnoides, we provide a detailed account on the structural and functional aspects of its gynoecium, and demonstrate that the species exhibits several plesiomorphic features in the gynoecium and a partly internalized pollen tube path.

Study species
In India, H. rhamnoides grows naturally between 3000 and 5000 m above sea level in the State of Jammu and Kashmir, Himachal Pradesh, Uttarakhand and Sikkim (Raina et al. 2012 The female flowers of sea buckthorn are borne in condensed axillary and terminal racemes. Each female flower is pedicellate and the gynoecium is enveloped in two partly fused perianth lobes. Anthesis in female flowers is marked by the emergence of a yellowish-green stigma from the perianth lobes, between 09:00 and 12:00 h [see Supporting Information].

Gynoecium architecture
Freshly opened female flowers (n ¼ 100) were dissected from randomly collected inflorescences and the morphological details of flowers were studied with the aid of a stereo-zoom microscope (Magnü s MS 24, Olympus, India). The morphometric details were recorded with the help of a calibrated ocular micrometer (Erma, Tokyo).
For anatomical details, the pollinated and unpollinated gynoecia were fixed in Karnovsky's fixative for 4 h, rinsed with freshly prepared sodium cacodylate buffer (0.2 M) twice, dehydrated in an ethanol series, and then infiltrated and embedded in a glycol methacrylate monomer mixture (Merck). The sections (2, 4 and 5 mm) were obtained using glass knives on a rotary microtome (AO Spencer, USA), stained with toluidine blue O ′ (Sigma, pH 4.4; Feder and O'Brien 1968) and viewed under a photomicroscope (Primo Star, Carl Zeiss, Germany). Additionally, some sections were stained with oil red (EX1-359 or EX2-518) to localize the lipids (Lillie and Fuller 1976)

The pollen tube path and progamic phase
The pollen tube path was established by localizing the tubes from manually pollinated and open-pollinated gynoecia. For manual pollinations, the most receptive stage of stigma was ascertained through the peroxidases test (Shivanna and Rangaswamy 1992). The pollinated gynoecia (n ¼ 450) were fixed in a lactic acid -ethanol (70 %) solution (1 : 3 v/v) at intervals of 12,20,24,30,36,42,48,56,60,70 and 72 h after pollination (hap), for 4 h at 4 8C, rinsed with 70 % ethanol and then stored in the same solution. The temporal details of tube growth in the pollinated gynoecia were established by the decolourized aniline blue staining method (Martin 1959). Observations were made under an epifluorescence photomicroscope (Eclipse 80i Nikon, Japan). The mean tube length + standard deviation, at each time interval, was computed and plotted against the duration at which the gynoecia were chemically fixed. The average growth rate of the pollen tube was computed by dividing the mean length of pollen tubes (n ¼ 20 pollinated gynoecia) by 48 h (i.e. 72 h minus 24 h), because the grains took nearly 24 h to show significant germination. Pollen grains with a tube nearly equal to the diameter of pollen grains were considered germinated.
The ultrastructural details of the cells lining the pollen tube path were studied by transmission electron microscopy. For this, the gynoecia were fixed in Karnovsky's fixative for 4 h at 4 8C, and washed twice with sodium phosphate buffer (0.2 M). Post-fixation was done in osmium tetroxide (1 %) prepared in the buffer, for 2 h at 4 8C, dehydrated through ascending cold acetone series and embedding was done in a CY212 aralditeresin mixture. Ultrathin sections (60 -90 nm) were obtained using an ultracut rotary microtome (E-Reichert, Germany), collected on carbon-coated copper grids and stained with alcoholic uranyl acetate for 10 min followed by staining with lead acetate for 8 min (Reynolds 1963). Observations were made under a transmission electron microscope (Morgagni 268D, FEI Electron Optics).

Structural organization of the gynoecium
The gynoecium of sea buckthorn is unicarpellate and the carpel exhibits a distinct dorsiventral symmetry. It is shortly stipitate and measures 3.4 + 0.4 mm in length (n ¼ 30) (Fig. 1A). The ventral side is marked by an apical and prominently fanned out stigma. There is a continuous groove (ventral slit) that runs downwards, half-way from the stigma to the sub-basal region of a dorsally protruded ovary (Figs 1A and 2A).
A freshly emerged yellowish-green stigma is the most conspicuous part of the gynoecium (2.0 + 0.1 mm in length, n ¼ 30), which becomes exposed to air, like a baseball glove. It is dry and of non-papillate type (Heslop-Harrison and Shivanna 1977) with an undulated (scorbiculate) surface (Figs 1B, 2C and D).
The stigma is subtended by a short constricted region (0.3 + 0.1 mm in length), termed here as the pseudostyle (Figs 1A and 2A). The internalized transmitting tissue (compitum)-like organization is absent in this zone (Figs 2F and G). The margins of the stigma taper and merge downwards in this region to form a stigma -pseudostyle junction (Figs 1A, C and 2E). The corresponding dorsal surface of the pseudostyle is covered with dendroid trichomes (Figs 1A and 2E). The ventral slit becomes conspicuous in the pseudostyle region due to involution of the carpel margins (Fig 1A  and C).
The ovary (0.8 + 0.1 mm in length) is superior, unilocular with a single bitegmic, anatropous and crassinucellate ovule ( Fig. 2A and I). The ovarian region is distinctly conduplicated as is evident by a ventral slit, which eventually leads to an ovarian pore near the base of the ovary (Figs 1A, F, 2B and 3B). The ovarian pore makes a continuum between the pollen tube path and the locule of the ovary (Figs 2H-J and 4B). The ovule develops from the submarginal position on the ventral carpellar wall of the locule (Fig. 2J). The micropyle invariably faces away from the ovarian pore (Fig. 2I).

The progamic phase and pollen tube path
The pollen grains measured 26.47 + 3.27 mm in diameter (n ¼ 100 pollen grains, 10 flowers) and are shed from anthers at the two-celled stage [see Supporting Information]; the exine is smooth (Fig. 1D). On average, the pollen grains took 20 h to produce a small protuberance from the germ pore. After 24 h of pollination, 81.88 + 14 % of the pollen showed tube emergence nearly equal to the mean diameter of pollen grain (27.05 + 3.01 mm, n ¼ 18 gynoecia). Thirty AoB PLANTS www.aobplants.oxfordjournals.org hours after pollination, the tubes attained a length of 51.0 + 5.41 mm (n ¼ 30 gynoecia) and penetrated into the subdermal tissue (Figs 1E and 5).
The stigma attains receptivity soon after its emergence from the perianth and reaches its peak once it outgrows the subtending bract. Before pollination, the stigma is covered with a continuous cuticle, which is thicker over the median dorsal and non-receptive surface. After pollination, the cuticle lining the ventral surface becomes discontinuous (Figs 6A and 7A). Five or six layers of the subdermal cells of stigma are large, vacuolated and possess intercellular spaces. These interstitial spaces accumulate a predominantly polysaccharidic and proteinaceous extracellular matrix (ECM) (Figs 6B, 7B and C). The secretory nature of these cells was evident from the presence of numerous mitochondria and endoplasmic reticulae along the plasma membrane (Fig. 6B). The marginal cells of stigma showed accumulation of oils and phenolics (Fig. 7B, D and E).
Tubes grew through the intercellular spaces of the substigmatic tissue, before reaching the stigma -pseudostyle junction, 48 hap (Figs 3A, B and 5). Interestingly, after a subdermal phase of growth, the tube(s) emerge near the junction and grow over the epidermal surface of the slit before making their entry into the locule through the ovarian pore (Figs 3, 4A and B).
Pollen tube growth along the ventral slit is supported by ECM secreted from the secretory activity of juxtaposed cells. In the pseudostyle region, the cells lining the slit are smaller, globular and compactly arranged whereas the hypodermal region possess interstitial spaces. These spaces accumulate osmiophilic and protein-rich ECM (Figs 6C, 7D and E); carbohydrates are sparsely distributed.
The pollen tube reaches the pore (Figs 1F, 3C and 4B) by 60 hap (n ¼ 20 gynoecia) and enters the micropyle (Fig. 4C) by 72 hap (n ¼ 15 gynoecia). The ventral slit in the ovarian region and the ovarian pore is lined by a continuous layer of compactly arranged and thickwalled epidermis (Fig. 2H-J). A sparse osmiophilic ECM could be localized all along the ventral slit of the ovarian region and in the intervening space of the ovarian pore (Fig. 6D). In nearly 90 % of the instances (n ¼ 30), only one pollen tube traversed through the pseudostyle, and in the remaining 10 %, two pollen tubes were observed (Fig. 3A). In the latter case, however, invariably only one pollen tube could make its entry through the ovarian pore (Fig. 3C) and reached the micropyle. The mean pollen tube length in the pollinated gynoecia after 72 h was 908.13 + 180.43 mm (range: 600-1300 mm, Fig. 5). Thus, the pollen tube growth rate from the time of pollen germination to ovule penetration was 18.75 mm h 21 .

Discussion
This is the first comprehensive account of the structural and functional aspects of the progamic phase  in any taxa of Elaeagnaceae. Many character states in H. rhamnoides are plesiomorphic, as they have preponderance in basal angiosperms and the basal eudicots, and provide new insight into the pollen tube path.

Structural organization of the gynoecium
The gynoecial features in sea buckthorn, such as conduplication, sparse exudation of ECM along the site of angiospermy and incomplete fusion of carpel margins, together strongly suggest that angiospermy in sea  buckthorn is contributed mainly by secretion and to some extent by the closely appressed carpel margins (Lloyd and Wells 1992;Endress and Igersheim 2000). The condition is close to type 2 angiospermy, where carpel closure is achieved by secretion and postgenital fusion (Endress and Igersheim 2000). The intervening constricted region between the stigma and ovary lacks the usual specialized transmitting tissue (compitum), and hence is termed a pseudostyle rather than a style (Bailey and Swamy 1951).
More than half of the ventral (adaxial) surface is receptive (stigma). This is in contrast to some extant basal angiosperms with style-less, conduplicated carpels where the receptive secretory/stigmatic hairs extend to the inner surface of the carpel and pollen grains are received on two independent papillate margins known as stigmatic crests (Bailey and Swamy 1951;Endress 2005). During the evolutionary modification of the carpel, deformation of either the abaxial or adaxial surface is instrumental in displacing the stigma from a stigmatic crest to the terminal region of the style (Bailey and Swamy 1951). In H. rhamnoides, adaxial deformation seems to have developmentally organized the stigma. An incomplete connation of carpel margins in sea buckthorn is also evident from the continuation of their epidermis into the ovarian locule, which leaves a ventral pore in the lower end of the slit. In taxa such as Kadsura, Schisandra (Schisandraceae) and Sagittaria (Alismataceae), the ventral pore may serve as the exit as well the entry point of pollen tubes, in a continuum of extragynoecial compitum (Lyew et al. 2007). In H. rhamnoides, the ventral pore is the only route taken by the pollen tubes to gain access to the ovule.
The formation of a hunchback-like and dorsally protruded ovary in sea buckthorn is largely a consequence of the syntropous orientation of anatropous ovule, which is most likely accompanied by conduplication to accommodate the more or less basally positioned ovule. This causes the micropyle of the ovule to face away from the ventral pore. A similar condition is seen where an ovule borne on the submarginal position in free carpels is curved away from the margins in the direction of involution (Endress 1994). The dorsal bulge in the ovary around the ovule is considered an evolutionary specialization during carpel development, as in some taxa of Monimiaceae with a weakly peltate carpel and a dorsal bulge (Endress 2011).

The progamic phase and pollen tube path
Sea buckthorn is a wind-pollinated plant and inhabits ecological conditions suitable for anemophily. The floral contrivances such as dry stigma, copious production of dry, starchy pollen with smooth exine and their morphometric range are similar to a majority of anemophilous taxa (Culley et al. 2002). Further, a large stigma in H. rhamnoides is also an obvious adaptive attribute to capture the airborne pollen grains.
During the functional phase of a dry stigma, the cuticle-pellicle layer is usually complete (Heslop-Harrison and Shivanna 1977). The cuticle layer develops discontinuities after pollination, which is accompanied by accumulation of secretion products beneath the cuticle-pellicle layer. This build-up of stigmatic ECM results in the formation of irregular bulges over the stigmatic surface, as seen in other systems with dry stigma (Shivanna and Johri 1985). The stigmatic ECM available through the cuticular discontinuities possibly facilitates adhesion and hydration of airborne pollen grains of species with dry stigma (Elleman et al. 1992).
Except for the fact that the species lacks an extragynoecial compitum by virtue of having a solitary and unicarpellate gynoecium, the partly hidden pollen tube growth pattern is similar to that reported in Winteraceae (Frame 2003), Schisandraceae (Lyew et al. 2007) and the other basal angiosperms (Endress and Igersheim 2000;Koehl 2002;Bernhardt et al. 2003;Thien et al. 2003;Lora et al. 2010). Enfolding of the carpel margins in the pseudostyle offers protection to the growing pollen tube (Lyew et al. 2007) and to a minor amount of ECM present over the secretory slit. The role of the pseudostyle is more or less similar to that of the usual compitum in confining the pollen tube growth along the secretory path and regulating the number of pollen tubes (Erbar 2003). In Annona cherimola and Amborella trichopoda, the stigma-style interface has been described as the site where male gametophyte selection and reduction in the number of pollen tubes occur; and only one or two pollen tubes enter the stylar canal (Williams 2009;Lora et al. 2010). In H. rhamnoides, male gametophyte competition is possibly confined to the stigmatic region and those with greater vigour nearly reach the pseudostyle. However, further down, the most vigorous one is able to grow beyond the stigma -pseudostyle interface due to spatial constraints (Erbar 2003) and thus in this context the interface appears to serve as the site of regulation of tube number.
The pollen tube growth rate is species specific and may be influenced by the length of the pollen tube path and ambient temperature regimen (Williams 2008(Williams , 2009Steinacher and Wagner 2012). The average growth rate of the pollen tube in H. rhamnoides (18.75 mm h 21 ) is comparable to that of gymnosperms such as Ephedra distachya (14 mm h 21 ) and E. trifurca (6 -19 mm h 21 ) (Williams 2009). In H. rhamnoides, the slow metabolic growth due to sub-zero temperature could be the cause of a slower tube growth rate.

Conclusions
In the evolutionary history of flowering plants, several ancestral reproductive features in the relatively advanced taxa are believed to be a consequence of reversals (Doyle and Endress 2000;Endress and Doyle 2009;Endress 2011). The presence of many plesiomorphic traits in the gynoecia of derived lineages suggests homoplasic apomorphy either by reversion or secondary derivation (Endress 1982), which is most likely influenced by the fitness requirements under the prevailing ecological conditions of a plant species (Armbruster et al. 2002). Additionally, many features such as open habitats, dioecy, simple and small flowers, uniovulate condition and wind pollination have been shown to exhibit correlated evolution (Friedman and Barrett 2008). The occurrence of conduplication, an incomplete connation of the carpellar margins resulting in a ventral pore and a partly internal progamic phase represent a peculiar combination of gynoecial features in H. rhamnoides, a core eudicot nested within Rosales (APG III 2009). It is likely that, together, these features are the derived ones that have AoB PLANTS www.aobplants.oxfordjournals.org accompanied modification of the gynoecium of sexually diverging individuals in a dioecious system.

Contributions by the Authors
Y.M. and R.T. made equal contributions in conducting the research. R.T. and S.G. were involved in planning the research and in writing the manuscript. S.N.R. was involved in the planning and supervision of the research work.