Abstract
Microsporogenesis and microgametogenesis of Rhododendron ledebourii (semi-deciduous), Rhododendron luteum (deciduous), and Rhododendron catawbiense (evergreen) were studied by light and electron microscopies in order to determine the stages of pollen development in relation to period of winter dormancy and bloom time throughout an annual growth cycle. Development of generative organs starts in June in R. ledebourii and in July in R. luteum and R. catawbiense and reaches completion about 11 months later. R. luteum and R. catawbiense microspores undergo meiosis at the end of the August and spend winter at the vacuolization stage. Mitosis with the formation of bicellular pollen grain occurs shortly before flowering at the beginning of June. R. ledebourii develops two types of flowers which differ in the timing of microgametogenesis. The first type is characterized by early microspore meiosis and mitosis leading to development of bicellular pollen grains by the end of August, and is prone to fall blooming during warm autumn temperatures. Microspores of the second flower type have a more prolonged vacuolization stage with mitosis and subsequent bicellular pollen grains occurring in November. By winter, flower buds in R. ledebourii are more advanced developmentally than in R. catawbiense and R. luteum, and bloom about 1 month earlier. The different strategies of pollen development identified both within and between these three Rhododendron species were recognized which are not associated with leaf drop during winter but appear to be related to the time of spring flowering and the frequency of autumn flowering.
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Introduction
Developmental events through the male gametophyte (or pollen) formation are highly similar between flowering plants indicating their conserved evolutionary character with conserved genetic control (Blackmore et al. 2007; Borg et al. 2009; Ma 2005; McCormick 2004; Wilson and Zhang 2009). However, having mainly the same sequence of structural and cytomorphological events, pollen develophment has very variable duration of each stage from several weeks in herbaceous annuals (Bedinger 1992; Owen and Makaroff 1995; Vinckier et al. 2012; Zhang et al. 2011) to about 1 year in woody and some spring-blooming herbaceous perennials from temperate climate (El-Ghazaly and Grafström 1995; Jacobs and Lersten 1994; Jansson and Douglas 2007; Julian et al. 2011; Khodorova et al. 2010; Miroslavov et al. 2008). The extended flower development is related mostly to adaptation to seasonal climate with period of winter dormancy. Chilling requirement for proper flower development and dormancy release is a well-known phenomenon for many temperate zone perennials (Atkinson et al. 2013; Perry 1971; Samish 1954). Despite extensive research of flower ontogenesis in temperate climate plants, an understanding of the mechanisms involved in temperature-dependent flower development is still limited (Van der Toorn et al. 2000). It has been demonstrated that various factors contribute to proper flowering in response to cold, such as hormonal status (Rietveld et al. 2000), water balance (Van der Toorn et al. 2000; van Kilsdonk et al. 2002), and features of reserve substance transport (Kamenetsky et al. 2003; Khodorova et al. 2010; Lambrechts et al. 1994; Marquat et al. 1999). Pollen development, which is temperature-sensitive (Barton et al. 2014; De Munk 1973; Hak and Russell 2004; Khodorova et al. 2010; Ohnishi et al. 2010), is a good system for studying structural reproductive development in the context of seasonal temperature changes.
The phenology and biology of flowering in different Rhododendron species has been studied extensively (Aleksandrova 1989; Babro et al. 2007; Bell and Burchill 1955; Malciūtė et al. 2011; Schneider 1972; Willingham 1976) with special attention to freezing tolerance (Ishikawa and Sakai 1981; Pellett 1987) or to recent climate changes (Ellwood et al. 2013). In most Rhododendron species originating from temperate climates or high altitudes, inflorescence and flower differentiation occur in the late summer followed by dormancy during the cold season, and it is a well-known chilling requirement to overcome dormancy and proper spring flowering in Rhododendron; however, regulating mechanisms (including the timing of flower initiation and development) are less characterized.
Pollen development in Rhododendron has been studied using light microscopy, showing that meiosis and tetrad formation occur before the onset of winter dormancy, and pollen of some species and hybrids overwinters at the late vacuolization stage of development (Rhododendron canadense, Bell and Burchill 1955; Leach hybrid rhododendrons, Stowe et al. 1989; Rhododendron luteum, Rhododendron schlippenbachii, Shamrov and Babro 2008; R. luteum, Mirgorodskaya et al. 2011). However, it is not known whether this strategy of pollen development is common for all Rhododendron species growing in temperate climate, and how it is related to unproper autumn blooming or to timing of spring bloom. Furthermore, there is little information on the precise sequence of events at the ultrastructural level and the timing of the structural changes involved.
About 25 Rhododendron species have been introduced to Botanical Garden of Komarov Botanical Institute (St. Petersburg, Russia) where the low temperature period lasts for up to 5 months. Most of them originated from the Russian Far East, Caucasus, and Altai. For several years, there has been a steady increase of average autumn temperatures, and as a result, certain Rhododendron species are flowering during early autumn more frequently than they used to do in the past. The taxa used for the present investigation differing in winter leaf drop and frequency of autumn flowering were semi-deciduous Rhododendron ledebourii which blooms at fall, deciduous R. luteum, and evergreen Rhododendron catawbiense. R. ledebourii (combined with Rhododendron dauricum in some systems) is endemic to Altai, Sayan Mountains in Russia (Semenjuk 1976). The area of wild distribution of R. luteum includes eastern part of Middle Europe, Balkan Mountains, Asia, and subalpine zone of the Caucasus. The origin of R. catawbiense is the eastern part of the North America from Virginia to Georgia, Tennessee, and Alabama (Aleksandrova 1989).
The purpose of this study has been to characterize the microsporogenesis and microgametogenesis in three Rhododendron species along with the flower initiation and development, and to compare the advantages of different pollen development strategies to avoid frost damage. In this study, the special attention was devoted to structural features of tapetum function supporting pollen development. Comparative analysis allows to study relationships between winter resting stage of microspore development and the frequency of autumn blooming and the time of spring blooming.
Materials and methods
Flower buds of R. catawbiense Michx. (evergreen), R. luteum L (deciduous), and R. ledebourii Pojark (semi-deciduous) (Ericaceae) were studied in the Botanical Garden of the Komarov Botanical Institute of the Russian Academy of Sciences (St. Petersburg, Russia). The study was done in an over a 3-year period (August 2010 to November 2013); Supplementary Fig. 1A shows the fluctuations of average air temperatures during the winter time in comparison with climatic norm for St. Petersburg region, and Supplementary Fig. 1B represents fluctuations of daily temperatures during autumns under interest. Samples for light (LM) and transmission electron (TEM) microscopy were collected twice per month. Anthers were fixed for 48 h in 2 % (v/v) paraformaldehyde and 2 % (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) followed by 12 h of post-fixation at 4 °C in 2 % OsO4. After dehydration in a 30, 50, 70, and 96 % (v/v) ethanol series followed by 100 % acetone, samples were infiltrated in Epon-Araldit M resin (Fluka, Buchs, Switzerland). Cross sections were made on a Reichert Ultracut E ultramicrotome (Reichert-Jung GmbH, Heidelberg, Germany). Semi-thin sections (1 μm) for LM were stained with 0.1 % (w/v) toluidine blue and observed with an Axio Scope1 equipped with an Axio Cam RMc5 (Zeiss, Germany). Thin sections (70 nm) for TEM were stained with 4 % uranyl acetate and Reynold’s lead citrate and examined using Hitachi-H600 (Tokyo, Japan) transmission electron microscope.
For scanning electron microscopy (SEM), flower buds and anthers were fixed using the above protocol, postfixed in 2 % OsO4, then dehydrated in an ethanol series to 100 % ethanol, critical-point dried, placed onto specimen mounts, sputter-coated with gold, and examined using a JEOL JSM35C (Tokyo, Japan) scanning electron microscope.
For the investigation of pollen germination, the stamens of R. ledebourii flowers were collected near the time of anther opening during the autumn and spring blooming. Pollen germination ability was tested in 1 % sucrose (w/v) at 25 °C during 2–3 h and observed with an Axio Scope1 equipped with an Axio Cam RMc5 (Zeiss, Germany).
Results
Morphology and timing of flower initiation
Early stages of flower initiation and anther development are presented for three Rhododendron species in Fig. 1 . Flower initiation occurs in June in R. ledebourii and in July in R. catawbiense and R. luteum (Table 1), with similar morphogenetic events. R. ledebourii usually has two (up to five) separate flower buds sited close to each other at the apical part of branches (Fig. 1a) while R. catawbiense and R. luteum are characterized by 15–25 flowers initiated per inflorescence (Fig. 1b). Flower initiation in inflorescence occurs in acropetal direction, from the base to the tip, as a result, different stages of flower development can be found in one axis (Fig. 1b).
The sequence of flower development in all three species starts from almost simultaneous initiation of sepals followed by petals and stamens at the periphery of receptacle in alternate pentagonal whorls (Fig. 1a, b). R. luteum forms one whorl of five stamens, while in R. ledebourii and R. catawbiense, two whorls of stamens are formed sequentially (Fig. 1c). Morphologically differentiated stamens are observed in early July for R. ledebourii (Fig. 1c) and in August for R. catawbiense and R. luteum (Table 1). At this stage, anthers contain sporogenous tissue (Fig. 1d).
Sporogenous tissue stage
In all studied species, sporogenous tissue is located within four loculi of anthers (Fig. 1d) and consists of large polygonal cells that are undifferentiated microsporocytes (Fig. 1f). Future tapetum is represented by one cell layer at the periphery of the anther loculus followed by few middle layers, endothecium, and epidermis towards the outside of the anther wall (Fig. 1e). The tapetal cells have rectangular form and are smaller sized than microsporocytes. At the level of ultrastructure microsporocytes and tapetal cells are meristematic cells having a nucleus at the center, abundant cisterns of rough endoplasmatic reticulum (RER), few mitochondria, plastids, dictyosomes, and vacuoles (Fig. 1e, f). Undifferentiated microsporocytes at later stage of development became microspore mother cells and have roundish form. Meiotic stage occurs at the end of July in R. ledebourii and at the end of August-early September in R. catawbiense and R. luteum resulting in tetrad formation (Table 1).
Tetrad stage
The four daughter cells from a meiosis form an aggregation called a pollen tetrad. Those four cells are originally interconnected and surrounded by common callose wall (Fig. 2a, b); tetrads of microspores in genus Rhododendron do not dissociate. At this stage of pollen development, the exine formation is initiated through the primexine deposition as a thin dark layer between the callose wall and plasma membrane in early tetrads (see Fig. 2a, b), followed by development of pro-tectum, pro-columellae, and foot-layer precursors in mid-tetrads (Fig. 2c) with increasing in thickness at the late tetrad (Fig. 2d). During microspore meiosis and the exine formation, tapetal cells show hypersecretory ultrastructure having numerous dictyosomes and cisterns of RER near the plasma membrane, abundant smooth endoplasmic reticulum (SER) in the center of the cell, and polysomes through the cytosol (Fig. 2e). Tapetum of R. ledebourii sometimes contains amyloplasts with starch granules. Tapetum cell wall towards the anther loculus is loose, and orbicules are secreted from the tapetum cells to loculus (Fig. 2c–e).
Stage of vacuolization
This stage starts in early August in R. ledebourii and in early September in R. catawbiense and R. luteum (Table 1). During early vacuolization stage, the nucleus of microspore is located in the center of cell, and only a few vacuoles are in cytoplasm (Fig. 2f). The exine development continues with the endexine formation. Exine now contains ectexine with three layers (tectum, columellae, and foot layer) and endexine (Fig. 2f). Later, the microspore forms an intine which is the inner layer of its wall with very pronounced thickening near the aperture. Tapetal cells contain numerous mitochondria, ribosomes, and vacuoles, and stacking of RER cisterns to parallel arrangement is characteristic for this stage. Density of cytosol in tapetal cell is decreased (Fig. 2g). During the late vacuolization stage, the nucleus and cytoplasm of the microspore are displaced towards to periphery of the cell with large vacuole situated in the center (Fig. 3d, e).
Stage of bicellular pollen grains and the phenomenon of fall bloom in R. ledebourii
This species shows asynchronous microgametogenesis among the developing flowers resulting in flowers at two different stages of development by fall. They differ in the timing of mitosis and subsequent formation of bicellular pollen grains containing generative and vegetative cells (Table 1). In flower buds which are more apically positioned at branch axis (apical buds), mitosis occurs at the end of August. In lower positioned flower buds (subapical buds), microspores remain in the late vacuolization stage until November, with the same structural features in microspores and tapetum cells as R. catawbiense and R. luteum have. In addition, these flowers are smaller sized and have more tightly closed bud scales than the apical buds. In subapical flower buds, mitosis occurs throughout November leading to bicellular pollen grain formation.
R. ledebourii is the only species among the three being studied which is prone to autumn flowering during unusually warm fall temperatures (September–November). In St. Petersburg (Russia), these warm conditions occurred during the 3 years the current study was conducted (Supplementary Fig. 1). Our observations of flowers indicate that the apical flower buds containing bicellular pollen grains are more likely to bloom in the fall than flower buds at the lower position on the stem. Subapical flower buds usually develop bicellular pollen grains later or occasionally pass winter at vacuolated stage and do not flower in fall under favorable temperatures.
Observation of pollen germination during the fall and spring bloom shows the similar percentage of germinated pollens, ~95 %.
Winter resting stage (October–April)
R. catawbiense and R. ledebourii are evergreen with leaves exhibiting thermonasty (curling and drooping) under freezing conditions (Fig. 3a, c) while R. luteum as a deciduous species loses foliage in October (Fig. 3b). Microspores of R. catawbiense and R. luteum overwinter at the stage of late vacuolization until the May (Table 1, Fig. 3d, e; Fig. 4a). Tapetum cells appear to be metabolically inactive in autumn and winter. The vesiculation of tapetal cell cytosol (Fig. 3g), a decrease in number of organelles, and the formation of RER “stacks” are characteristic for this period (Fig. 3h, i), and these are specific features of the cell dormancy. A significant reorganization of tapetal cell ultrastructure is observed in April showing recovery of secretory activity. The main ultrastructural changes are multiple nuclei, disappearance of vesicles, disassembling of RER “stacks,” abundance of Golgi bodies, and accumulation of electron-dense substances with sporopollenin-like material secreted towards the anther loculus (Fig. 4b).
R. ledebourii shows different strategy of pollen development and overwinters with microspores at the pronounced stage of bicellular pollen grains (Fig. 3f). During winter dormancy, the generative cell tends to be located at the periphery of the pollen grain and has smooth plasma membrane towards the inner layer of pollen wall, intine, but has very irregular plasma membrane shape where it joins to the plasma membrane of the vegetative cell. The dense cytosol of both cells and starch granules in amyloplasts of the vegetative cell are characteristic of ultrastructure at this stage (Fig. 3f). Specific position and ultrastructural features remain mainly unchanged during the winter until middle of May with only progressive increasing of sizes of starch granules in vegetative cell in spring. Unlike R. catawbiense and R. luteum, tapetal cells in R. ledebourii during the cold season are not vesiculated and characterized by special concentric arrangement (“whorls”) of RER surrounding the lipid bodies (Fig. 3j). Starch granules are accumulated in the plastids of endothecium of the anther wall (Fig. 3j).
Stage of bicellular pollen grains in R. catawbiense and R. luteum
Pollen grains in R. catawbiense and R. luteum complete development shortly before flowering, which is observed in June (Table 1). Bicellular pollen grains with generative and vegetative cells are distinguished at the second part of May (Fig. 4c, d). The generative cell is located at the center of the pollen grain. Numerous amyloplasts of vegetative cell contain starch grains (Fig. 4c, d). Pollen grain completes cell wall formation after intine deposition. At this time, the tapetum collapses with destruction starting from large vacuole formation and followed by total cytoplasm shrinkage; finally only remains of orbicules are observed (Fig. 4f).
Mature pollen grains
Complete maturation of pollen grains in all three species occurs shortly before flowering. Flower buds which are in process of budburst (Supplementary Fig. 2a, b, c) contain fully mature pollen grains. The pollen grains are shed as tetrads (Supplementary Fig. 2g, h, i) that are typical for Ericaceae. Dehiscent anthers have two pollen sacs which originate from four pollen loculi of the tetrasporangiate anthers during the last stages of pollen maturation. By the time of flowering (May for R. ledebourii (Supplementary Fig. 2d) and June for R. luteum (Supplementary Fig. 2e) and R. catawbiense (Supplementary Fig. 2f)), starch grains in amyloplasts of vegetative cells become very small and rare (compare Fig. 4f, g); in R. ledebourii, the generative cell moves away from the wall to a more central position. Mature pollen grains are bicellular; it was shown for R. luteum that division of generative cell undergoes in the pollen tube (Jakobson 1969). Mature pollen grains in tetrads are 3-colporate with viscin threads at the surface which are easily recognized under the SEM (Supplementary Fig. 2g, h, i) and which hold the pollen grains together when leaving the anther. At maturity, the tapetum is completely destroyed and the remains of orbicules stick to the exine layer of the pollen grain. Plastids of cells in the remaining anther layers contain large grains of starch (Fig. 4g).
Discussion
Winter resting stage of pollen development
The results showed that in the three Rhododendron species studied, pollen development takes about 11 months and includes six stages: I—sporogenous tissue, II—meiosis and tetrads of microsporocytes, III—early vacuolization, IV—late vacuolization, V—mitosis and bicellular pollen grains, VI—mature pollen grains. These stages are common for the most studied plants irrespective of origin, distribution, and features of ontogenesis indicating highly conserved evolutionary character of pollen development among angiosperms (Blackmore et al. 2007; Borg et al. 2009; Ma 2005; McCormick 2004; Wilson and Zhang 2009). In many woody perennial plants from temperate climate, male gametophyte development continues about 10–11 months; however, the time scale of each stage and their durations and winter resting stage vary from species to species (Bell and Burchill 1955; El-Ghazaly and Grafström 1995; Jacobs and Lersten 1994; Jansson and Douglas 2007; Julian et al. 2011).
In this study, the timing of flower initiation and flowering as well as the time scale of stages of pollen development differed in R. ledebourii versus R. catawbiense and R. luteum (Fig. 5). Transition from vegetative to reproductive structures occurred earlier in R. ledebourii (June) than in R. catawbiense and R. luteum (July). Early initiation allows to R. ledebourii to complete pollen differentiation by November, resulting in microspores that overwinter as bicellular pollen grains. Earlier differentiation of pollen in R. ledebourii is associated with earlier spring bloom, at least 1 month earlier than flowering in R. catawbiense and R. luteum. In contrast, the delay in pollen development in R. catawbiense and R. luteum results in prolonged stage of microspore vacuolization occurring during the period of lower temperatures in winter. Maturation of pollen in these two species completes just before blooming in middle of May when the probability of microspore damage from spring frost is lower (Fig. 5).
Among the plant studied, only herbaceous early-spring ephemeral Scilla sibirica (Miroslavov et al. 2008) and woody perennials Epigaea repens and Chamaedaphne calyculata (Bell and Burchill 1955) and Corylus avellana (Frenguelli et al. 1997) have microspores as bicellular pollen grains during the cold season similar to R. ledebourii. Besides R. catawbiense and R. luteum, overwintering at the stage of vacuolization was also shown for herbaceous early-spring ephemeral Corydalis bracteata (Khodorova et al. 2010) and Ericaceae species, Kalmia polifolia and R. canadense (Bell and Burchill 1955), R. schlippenbachii (Shamrov and Babro 2008), and Leach hybrid rhododendrons (Stowe et al. 1989).
Among seed plants, several additional variants were shown which are differing in winter resting stage of pollen development. Microspores of some conifers overwinter at the stage of meiosis (Ekberg et al. 1968; Owens and Molder 1974; Rowley and Walles 1985a; Rowley and Walles 1985b; Walles and Rowley 1982; Zhang et al. 2008) or at the stage of sporogenous tissue (Cecich 1984; Kupila-Ahvenniemi et al. 1978). The latter strategy is also found for angiosperms, Prunus cerasus (Felker et al. 1983), Acer saccharum (Jacobs and Lersten 1994), Cerasus vulgaris and Cerasus tomentosa (Shamrov and Yandovka 2006), Prunus armeniaca (Julian et al. 2011), four Ericaceae species (Bell and Burchill 1955), and Ribes nigrum (Koteyeva et al. 2015). Thus, the most plants studied overwinter at the sporogenous stage, and no one plant found resting at the stage of early vacuolization or completely mature pollen grains. Additionally, only among conifers the prolonged stage of meiosis was found occurring during period of low temperatures. The most Rhododendron species and cultivars studied overwinter at the stage of late vacuolization, with only R. ledebourii having more pronounced stage of pollen development during the winter. The strategy of pollen development is not depending on leaf drop during winter but appear to be associated with the time of spring flowering as R. ledebourii flowers first at the conditions under the current study.
Each species has a fairly definite resting stage of microspore development regardless of year and locality with some rare exceptions (Bell and Burchill 1955). Variations in pollen development reflect different methods that temperate zone plants use to adapt to seasonal climate. It seems that temperature-dependent and/or temperature-sensitive stages of pollen development are different among these strategies. For example, for species overwintering at the stage of sporogenous tissue, the meiosis depends on low temperature applying (Julian et al. 2011; Koteyeva et al. 2015) and does not occur if to avoid chilling during winter dormancy (Koteyeva et al. 2015). In contrast, in the Rhododendron being studied, microspore meiosis does not require low temperatures since meiosis occurs before the first frost. The limited data on pollen development strategies to survive low winter temperatures do not permit us to suggest the most successive between them; more experimental data is needed taking in consideration the pollen viability. It has been shown that overwintering at the meiosis stage of microspores has the largest percent of abnormality, while pre- or postmeiotic stages appear to be more stable (Bazhina et al. 2011; Eriksson 1968; Hak and Russell 2004; Owens and Molder 1974). The sporogenous stage also appears to be freeze tolerant due to its meristematic nature (Julian et al. 2011; Koteyeva et al. 2015). For several annual crops was shown that during anther development the most sensitive stages for chilling injury are meiosis, tetrads, and uni-nucleate microspore (Barton et al. 2014; Ohnishi et al. 2010; Oliver et al. 2005).
Phenomenon of fall bloom in R. ledebourii
R. ledebourii develops initially two different types of flower buds. One type of flower is characterized by early microspore meiosis and mitosis leading to development of bicellular pollen grains by the end of August; they are typically larger and apically positioned on shoot axis. Microspores of the second type have a more prolonged vacuolization stage with mitosis and subsequent bicellular pollen grains occurring in November. All three individuals of R. ledebourii studied were characterized by expanded sparse bloom from end of August to end of October under the warm conditions. Only apical flower buds that have formed bicellular pollen grains very early in August were capable to bloom at fall supposing very short chilling period required for break of dormancy or developmental regulation of this flower blooming independently from dormancy status. To understand the regulating factor provoking the fall bloom, the more plants resting at pronounced stage of microspore development need to be studied, and experiments using controlled conditions should be applied. However, there is very limited number of plants known to have microspores as bicellular pollen grains during the cold season (Bell and Burchill 1955; Frenguelli et al. 1997; Miroslavov et al. 2008) with no data available on their unexpected autumn bloom. According to our observation, Daphne mezereum L. which is natural to St. Petersburg region and overwinters at the stage of bicellular pollen grains is prone to fall and even to early winter bloom with most flower buds enabled resulting often in the whole plant death (Olga Mirgorodskaya, observations during 2010–2012, Botanical Garden of Komarov Botanical Institute, unpublished). In case of R. ledebourii, only part of flowers enables to bloom during the autumn.
The physiological and ecological role of production of two separate populations of flowers in R. ledebourii is not clear. Viability of pollens and percent of germination are not different in autumn and spring blooming flowers; however, no one event of successful pollination was recorded in autumn under conditions studied. Area of origin and recent natural distribution of this species are characterized by seasonal climate with cold winters, and the fall bloom is frequently reported for natural areas from August to November depending on temperature conditions (Miroslava Sakhnevich, annual personal observations in Altaisky State Nature Biosphere Reserve for the more than 14 years). Nevertheless, the existence of floral buds with asynchronous male gametophyte development in R. ledebourii reduces the fraction of flower buds which incorrectly break dormancy due to temperature fluctuations.
Tapetum function during prolonged pollen development
The tapetum, an inner parietal layer of the anther wall adjacent to sporogenous tissue, consists of secretory cells which nourish and regulate the pollen grains during development (Ariizumi and Toriyama 2011; Liu and Fan 2013; Pacini 2010), and which collapse during the last stages of pollen development. Our study shows two different ways of tapetum functioning in flowers associated with two overwintering strategies in R. ledebourii versus R. catawbiense and R. luteum.
Flowers which overwinter with vacuolated microspores (R. catawbiense and R. luteum) have two peaks of secretory activity in tapetal cells interrupted by a winter dormancy period. The first peak of tapetal activity is observed during meiosis and tetrad formation; it is characteristic for this stage of development in all plants studied and was described by Rowley (1993) as a period of metabolic hyperactivity in tapetal cells. The tapetum remains active during late tetrad formation assisting in exine formation, indicated by exocytosis of sporopollenin-like-containing orbicules to the anther loculus and pronounced cytoplasm development. Tapetal cell participation in exine formation has been studied in numerous taxa (Ariizumi and Toriyama 2011; Dickinson and Bell 1976; Gabarayeva et al. 2009; Galati et al. 2007; Liu and Fan 2013; Quilichini et al. 2014; Vinckier and Smets 2005). During the winter period, vesiculation of cytosol, arrangement of RER to “stacks”, and a decrease in mitochondria and Golgi content are indicative of tapetal cell inactivation. It has been shown that “stacks” of RER produced in dormant tissue are a prerequisite configuration for future protein biosynthesis in these organelles (Cecich 1984; Kupila-Ahvenniemi et al. 1978; Muravnik 2008). Reactivation of tapetal cell metabolism and function as secretory structure occurs during further development of pollen grains in the spring, in May–June for R. catawbiense and R. luteum followed by disintegration of the tapetum shortly before flowering.
In contrast, the tapetum of R. ledebourii anthers with bicellular pollen grain formation in autumn actively functions as a secretory structure during the meiosis (July) followed by assistance in exine formation at the tetrad stage (August). During the winter period, the ultrastructure of tapetum cells indicates a dormant state for this tissue, and it finally collapses shortly before flowering in May without reactivation.
In summary, two different strategies of pollen development were shown for three Rhododendron species in this study. The observed differences in the seasonal timing of floral differentiation were not dependent on the foliage behavior (loss/preservation) during the winter but associated with timing of spring bloom. There was a close association between the early pollen development and the probability of fall bloom; floral buds that delayed mitosis and formation of bicellular pollen grains until late fall avoided flowering during warm fall temperatures. Pollen development in more Rhododendron species needs to be studied to understand the advantages of each strategy and their evolution in relation with species origin/distribution, and to predict the species reaction to introduction as ornamental plants or to global climate changes.
References
Aleksandrova МS (1989) Rododendron. Lesnaja promyšlenosť, Мoscow
Ariizumi T, Toriyama K (2011) Genetic regulation of sporopollenin synthesis and pollen exine development. Annu Rev Plant Biol 62:1–24
Atkinson CJ, Brennan RM, Jones HG, Atkinson CJ, Brennan RM, Jones HG (2013) Declining chilling and its impact on temperate perennial crops. Environ Exp Bot 91:48–62. doi:10.1016/j.envexpbot.2013.02.004
Babro AA, Anisimova GM, Shamrov II (2007) Reproductive biology of Rhododendron schlippenbachii and R. luteum (Ericaceae) in the introduction in botanical gardens of St. Petersburg. Rast Res 43:1–13
Barton DA, Cantrill LC, Law AMK, Phillips CG, Sutton BG, Overall RL (2014) Chilling to zero degrees disrupts pollen formation but not meiotic microtubule arrays in Triticum aestivum L. Plant Cell Environ 37:2781–2794. doi:10.1111/pce.12358
Bazhina EV, Kvitko OV, Muratova EN (2011) Specific features of meiosis in the Siberian Fir (Abies sibirica) in the forest Arboretum of the V. N. Sukachev Institute, Russia. Biodivers Conserv 20:415–428. doi:10.1007/s10531-010-9958-y
Bedinger P (1992) The remarkable biology of pollen. Plant Cell 4:879–887
Bell HP, Burchill J (1955) Winter resting stages of certain Ericaceae. Can J Bot 33:547–561
Blackmore S, Wortley AH, Skvarla JJ, Rowley JR (2007) Pollen wall development in flowering plants. New Phytol 174:483–498. doi:10.1111/j.1469-8137.2007.02060.x
Borg M, Brownfield L, Twell D (2009) Male gametophyte development: a molecular perspective. J Exp Bot 60:1465–1478. doi:10.1093/jxb/ern355
Cecich RA (1984) The histochemistry and ultrastructure of jack pine microsporangia during winter. Am J Bot 71:851–864
De Munk VJ (1973) Flower-bud blasting in tulips caused by ethylene. Neth J Plant Pathol 79:41–53
Dickinson HG, Bell PR (1976) The changes in the tapetum of Pinus banksiana accompanying formation and maturation of the pollen. Ann Bot 40:1101–1109
Ekberg I, Eriksson G, Sulikova Z (1968) Meiosis and pollen formation in Larix. Hereditas 59:427–438
El-Ghazaly G, Grafström E (1995) Morphological and histochemical differentiation of the pollen wall of Betula pendula Roth, during dormancy up to anthesis. Protoplasma 187:88–102. doi:10.1007/bf01280236
Ellwood ER, Temple SA, Primack RB, Bradley NL, Davis CC (2013) Record-breaking early flowering in the eastern United States. PLoS ONE 8:e53788. doi:10.1371/journal.pone.0053788
Eriksson G (1968) Temperature response of pollen mother cells in Larix and its importance for pollen formation. Stud For Suec 63:131
Felker FC, Robitaille HA, Hess FD (1983) Morphological and ultrastructural development and stach accumulation during chilling of sour cherry flower buds. Am J Bot 70(3):376–386
Frenguelli G, Feranti F, Tedeschini E, Andreutti R (1997) Volume changes in the pollen grain of Corylus avellana L. (Corylaceae) during development. Grana 36:289–292
Gabarayeva N, Grigorjeva V, Rowley JR, Hemsley AR (2009) Sporoderm development in Trevesia burckii (Araliaceae): II. Post-tetrad period: further evidence for the participation of self-assembly processes. Rev Palaeobot Palynol 156:233–247. doi:10.1016/j.revpalbo.2009.01.004
Galati BG, Monacci F, Gotelli MM, Rosenfeldt S (2007) Pollen, tapetum and orbicule development in Modiolastrum malvifolium (Malvaceae). Ann Bot 99:755–763
Hak O, Russell JH (2004) Environmental effects on yellow-cedar pollen quality. For Genet Council 05:1–9, Extension note
Ishikawa M, Sakai A (1981) Freezing avoidance mechanisms by supercooling in Rhododendron flower buds with reference to water relations. Plant Cell Physiol 22:953–967
Jacobs CA, Lersten NR (1994) Microsporogenesis and endothecial wall patterns in black maple (Acer saccharum subsp. nigrum, Aceraceae). Bull Torrey Bot Club 121:180–187. doi:10.2307/2997170
Jakobson LY (1969) Forming and development sperms in Rhododendron luteum Sweet. Voprosi Biologii 1:41
Jansson S, Douglas CJ (2007) Populus: a model system for plant biology. Annu Rev Plant Biol 58:435–458. doi:10.1146/annurev.arplant.58.032806.103956
Julian C, Rodrigo J, Herrero M (2011) Stamen development and winter dormancy in apricot (Prunus armeniaca). Ann Bot. doi:10.1093/aob/mcr056
Kamenetsky R, Barzilay A, Erez A, Halevy AH (2003) Temperature requirements for floral development of herbaceous peony cv. Sarah Bernhardt. Sci Hortic 97:309–320
Khodorova NV, Miroslavov EA, Shavarda AL, Laberche J-C, Boitel-Conti M (2010) Bud development in corydalis (Corydalis bracteata) requires low temperature: a study of developmental and carbohydrate changes. Ann Bot 105:891–903
Koteyeva NK, Mirgorodskaya OE, Bulisheva MM, Miroslavov EA (2015) Pollen development in Ribes nigrum (Grossulariaceae) in relation to the low temperature period. Bot Zh (under review)
Kupila-Ahvenniemi S, Pihakaski S, Pihakaski K (1978) Wintertime changes in the ultrastructure and metabolism of the microsporangiate strobili of the Scotch pine. Planta 144:19–29
Lambrechts H, Rook F, Kollöfel C (1994) Carbohydrate status of tulip bulbs during cold-induced flower stalk elongation and flowering. Plant Physiol 104:515–520
Liu L, Fan X-d (2013) Tapetum: regulation and role in sporopollenin biosynthesis in Arabidopsis. Plant Mol Biol 83:165–175. doi:10.1007/s11103-013-0085-5
Ma H (2005) Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants. Annu Rev Plant Biol 56:393–434
Malciūtė A, Naujalis JR, Šaulienė I (2011) The seasonal development characteristics of different taxa and cultivars of rhododendrons in Northern Lithuania. 2. Flowering peculiarities. Žemdirbystė 98:81–92
Marquat C, Vandamme M, Gendraud M, Petel G (1999) Dormancy in vegetative buds of peach: relation between carbohydrate absorption potentials and carbohydrate concentration in the bud during dormancy and its release. Sci Hortic 79:151–162
McCormick S (2004) Control of male gametophyte development. Plant Cell 16:S142–S153
Mirgorodskaya OE, Miroslavov EA, Umarov MU (2011) Microsporogenesis and structure of tapetum cells in Rhododendron luteum (Ericaceae) in- and outdoors. Vestnik MANEB 16:89–96
Miroslavov EA, Barmicheva EM, Khodorova NV (2008) Tapetum and pollen grain structure in Scilla sibirica (Liliaceae) growing in- and outdoors. Bot Zh 93:1444–1452
Muravnik LE (2008) Ultrastructure of digestive glands of Dionaea muscipula and Aldrovanda vesiculosa (Droseraceae). Bot Zh 93:288–299
Ohnishi S, Miyoshi T, Shirai S (2010) Low temperature stress at different flower developmental stages affects pollen development, pollination, and pod set in soybean. Environ Exp Bot 69:56–62. doi:10.1016/j.envexpbot.2010.02.007
Oliver SN et al (2005) Cold-induced repression of the rice anther-specific cell wall invertase gene OSINV4 is correlated with sucrose accumulation and pollen sterility. Plant Cell Environ 28:1534–1551. doi:10.1111/j.1365-3040.2005.01390.x
Owen H, Makaroff CA (1995) Ultrastructure of microsporogenesis and microgametogenesis in Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija (Brassicaceae). Protoplasma 185:7–21. doi:10.1007/bf01272749
Owens JN, Molder M (1974) Bud development in western hemlock. II. Initiation and early development of pollen cones and seed cones. Can J Bot 52:283–294
Pacini E (2010) Relationships between tapetum, loculus, and pollen during development. Int J Plant Sci 171:1–11
Pellett H (1987) Tolerance of flower buds of several Rhododendron taxa to a severe fall freeze. J Am Rhododendron Soc 41:196–197, http://scholar.lib.vt.edu/ejournals/JARS/v41n4/v41n4-pellett.htm
Perry T (1971) Dormancy of trees in winter. Science 171(3966):29–36
Quilichini TD, Douglas CJ, Samuels AL (2014) New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana. Ann Bot. doi:10.1093/aob/mcu042
Rietveld PL, Wilkinson C, Franssen HM, Balk PA, van der Plas LHW, Weisbeek P, Douwe de Boer A (2000) Low temperature sensing in tulip (Tulipa gesneriana L.) is mediated through an increased response to auxin. J Exp Bot 51:587–594
Rowley JR (1993) Cycles of hyperactivity in tapetal cells. Plant Syst Evol 7:23–27
Rowley JR, Walles B (1985a) Cell differentiation in microsporangia of Pinus sylvestris. II. Early pachytene. Nord J Bot 5:241–254. doi:10.1111/j.1756-1051.1985.tb01654.x
Rowley JR, Walles B (1985b) Cell differentiation in microsporangia of Pinus sylvestris. III. Late pachytene. Nord J Bot 5:255–271. doi:10.1111/j.1756-1051.1985.tb01655.x
Samish RM (1954) Dormancy in woody plants. Annu Rev Plant Physiol 5:183–204. doi:10.1146/annurev.pp. 05.060154.001151
Schneider EF (1972) The rest period of Rhododendron flower buds: III. Cytological studies on the accumulation and breakdown of protein bodies and amyloplasts during flower development. J Exp Bot 23:1021–1038
Semenjuk NB (1976) About areal and species limits of Rhododendron ledebourii Pojark. Bull Glav Bot Sada 101:51–55
Shamrov II, Babro AA (2008) Development and structure of anther of Rhododendron schlippenbachii and R. luteum (Ericaceae). Bot Zh 93:1219–1239
Shamrov II, Yandovka LF (2006) Pollen fertility in Cerasus vulgaris and C. tomentosa (Rosaceae). Bot Zh 91:208–220
Stowe WC, Fink CVM, Leach DG (1989) Seasonal changes in anther development of cold hardy Rhododendrons. J Am Rhododendron Soc 43:128–132
Van der Toorn A, Zemah H, Van As H, Bende P, Kamenetsky R (2000) Developmental changes and water status in tulip bulbs during storage: visualization by NMR imaging. J Exp Bot 51:1277–1287
van Kilsdonk MG, Nicolay K, Franssen JM, Kolloffel C (2002) Bud abortion in tulip bulbs studied by magnetic resonance imaging. J Exp Bot 53:1603–1611
Vinckier S, Smets E (2005) A histological study of microsporogenesis in Tarenna gracilipes (Rubiaceae). Grana 44:30–44. doi:10.1080/00173130510010530
Vinckier SA, Janssens SB, Huysmans S, Vandevenne A, Smets EF (2012) Pollen ontogeny linked to tapetal cell maturation in Impatiens parviflora (Balsaminaceae). Grana 51:10–24. doi:10.1080/00173134.2011.650194
Walles B, Rowley JR (1982) Cell differentiation in microsporangia of Pinus sylvestris with special attention to the tapetum. I. The pre- and early-meiotic periods. Nord J Bot 2:53–70. doi:10.1111/j.1756-1051.1982.tb01435.x
Willingham FFC (1976) Variation and phenological forms in Rhododendron calendulaceum (Michx.) Torrey (Ericaceae). Castanea 41:215–223
Wilson ZA, Zhang D-B (2009) From Arabidopsis to rice: pathways in pollen development. J Exp Bot 60:1479–1492. doi:10.1093/jxb/erp095
Zhang S-G et al (2008) Development of male gametophyte of Larix leptolepis Gord. with emphasis on diffuse stage of meiosis. Plant Cell Rep 27:1687–1696. doi:10.1007/s00299-008-0579-9
Zhang D, Luo X, Zhu L (2011) Cytological analysis and genetic control of rice anther development. J Genet Genomics 38:379–390. doi:10.1016/j.jgg.2011.08.001
Acknowledgments
This material is based upon work supported by the Research Foundation of American Rhododendron Society, grant # 133 and by the Russian Foundation of Basic Research, grant # 13-04-00876. We thank Irina Descharmes for reviewing the English. We appreciate the Core Center “Cell and Molecular Technology in the Plant Science” at the Komarov Botanical Institute (St. Petersburg) for use of its facilities.
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Mirgorodskaya, O.E., Koteyeva, N.K., Volchanskaya, A.V. et al. Pollen development in Rhododendron in relation to winter dormancy and bloom time. Protoplasma 252, 1313–1323 (2015). https://doi.org/10.1007/s00709-015-0764-y
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DOI: https://doi.org/10.1007/s00709-015-0764-y