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Posidonia oceanica seedling root structure and development
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Aquatic Botany 88 (2008) 203–210 www.elsevier.com/locate/aquabot
Posidonia oceanica seedling root structure and development Miriam Belzunce a,*, Rafael M. Navarro b, Hava F. Rapoport a a
Instituto de Agricultura Sostenible, CSIC, Apdo. 4084, 14080 Co´rdoba, Spain Dpto. Ingenierı´a Forestal, E.T.S.I.A.M., Apdo. 3048, 14080 Co´rdoba, Spain
b
Received 20 October 2006; received in revised form 26 June 2007; accepted 10 October 2007 Available online 4 November 2007
Abstract The seedling root system of the seagrass Posidonia oceanica consists of a primary root and up to four adventitious roots. Under culture, germination and early growth began with the emergence of the primary root in the first week. Then the two adventitious root primordia originally present in the seed emerged at 3 and 5 weeks respectively, followed successively by further adventitious roots. Primary roots reached 17 mm at 4 weeks, but then their growth decreased markedly. In contrast the adventitious roots showed a pattern of continued elongation. Anatomical observations of both primary and adventitious roots revealed a multilayered hypodermis of thick-walled cells enclosing a wide cortex (99% of the root transverse area) and narrow stele. A well-distinguished endodermis was only observed in the primary roots, while differentiated xylem elements were found solely in the adventitious roots, but it is unclear to what degree differences between the two root types are due to different root maturity or to their role in water and nutrient uptake. Overall, the P. oceanica seedling root system is composed of multiple, rapidly formed roots which are strong yet flexible due to a large proportion of cortical tissue and further strengthened by a multilayered hypodermis, characteristics which could potentially facilitate initial anchorage and establishment. # 2007 Elsevier B.V. All rights reserved. Keywords: Seagrass; Seedling; Root system; Germination; Hypodermis
1. Introduction Posidonia oceanica meadows are of recognised ecological importance in the Mediterranean Sea (Procaccini et al., 2003). In recent years, however, an alarming decline of many populations of P. oceanica has been observed, attributed to both natural and human disturbances (Duarte, 2002). P. oceanica meadows are maintained largely by vegetative propagation (Marba´ and Duarte, 1998), with sexual reproduction showing great variability both in frequency and intensity (Buia and Mazzella, 1991; Balestri and Cinelli, 2003). Flowering occurs throughout the Mediterranean Sea from September to December, and mature buoyant fruits are released from March to June (Buia and Mazzella, 1991). Although drifting fruits contain a germinating seed which is well-adapted for survival (Belzunce et al., 2005), the success of sexual reproduction in P. oceanica is considered low, with large losses occurring during both seed and seedling stages (Piazzi et al., 1999; Balestri and Cinelli, 2003). * Corresponding author. Tel.: +34 957 499216; fax: +34 957 499252. E-mail addresses: [email protected], [email protected] (M. Belzunce). 0304-3770/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2007.10.008
Considerable pre-dispersal seed losses due to abortion and predation, as suggested by Balestri and Cinelli (2003), may reduce reproductive success in P. oceanica. Furthermore, even during years of high fruit production, hundreds of fruits may be lost during drifting and seeds get washed ashore (Balestri et al., 2005). P. oceanica seed germination and seedling development studies in the laboratory (Balestri et al., 1998a; Balestri and Bertini, 2003) and in field transplantation experiments (Balestri et al., 1998b; Piazzi et al., 2000) suggest high seed vigor for this species. Despite the high germinability of seeds, however, seedling establishment in natural conditions is rare (Balestri et al., 1998a; Piazzi et al., 1999). In seagrasses, seedling establishment strongly depends on water movement, depth and sediment stability (Clarke and Kirkman, 1989). In P. oceanica high losses of seedlings were reported during the first months after planting (Balestri et al., 1998a). According to Balestri and Bertini (2003), a major reason was the insufficient root system of the young seedling to cope successfully with physical disturbance and to exploit resources. In adult seagrasses, the relative contribution of root production to net primary production was reported to be similar to the range described for land plants (10–40% of net
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primary production) (Duarte et al., 1998). P. oceanica root production estimates, however, have been low (2.8% of total production) compared to other seagrass species (Duarte et al., 1998). Roots in the Helobiales, the order of aquatic higher plants to which Posidonia species belong, show anatomical adaptations related to an aquatic environment. Amongst the anatomical features associated with aquatic habitats are: specialized root hairs, a narrow exodermis with thickened and suberized walls, an aerenchymatous cortex, and a narrow stele with xylem elements frequently poorly differentiated (Tomlinson, 1982). Thickened and histochemically differentiated cell walls of the exodermis and endodermis are characteristic of adult seagrass roots. These tissues appear to function in the absorption and transport of water and solutes (Tomlinson, 1969; Cambridge and Kuo, 1982; Barnabas, 1994b), and in the mechanical processes of anchorage and plant support (Barnabas, 1991, 1994b). The anatomical study of the seed and early plantlet indicated the presence of a potentially efficient root anchorage system in P. oceanica (Belzunce et al., 2005). These seeds have a conspicuous, emerging primary embryonic root and two adventitious root primordia, all of which are well linked by vascular tissues to the nutritional supplies of the carbohydraterich hypocotyl (Belzunce et al., 2005). However, knowledge is lacking about the growth of those primordia and their subsequent behavior in the young seedling. In this study, initiation of root development and growth was observed in P. oceanica mature seeds collected from drifting fruits, and the structure of the young seedling roots described. The aim of this study was to contribute to the understanding of the P. oceanica seedling establishment process and their physiological response to the environment.
record emergence and growth. Growth measurements were: maximum length of primary root, total number of adventitious roots and maximum length of adventitious roots. Roots were counted as emerged and length measurements started once the root reached a minimum length of 2 mm. Statistical analysis was by t-test and ANOVA, Tukey test. 2.2. Anatomical and histochemical studies For anatomical studies, primary and adventitious roots of 7week old P. oceanica seedlings were used. Apical and subapical portions of 2–3 mm were obtained, fixed in FAE (formalin– acetic acid–ethyl alcohol) dehydrated in a tertiary butyl alcohol series and processed in paraffin as described by Belzunce et al. (2005). Apical samples were sectioned longitudinally and transverse sections were obtained from the subapical samples, all at 12 mm thickness. Sections were stained with 0.05% (w/v) toluidine blue O (Tol. Bl.) (Sakai, 1973) for general cell and tissue organization; tannic acid–ferric chloride–safranin–fast green (TA–FC–SAF–FG) (Jensen, 1962), was used for meristematic tissues and cell walls, and periodic acid-Schiff’s (PAS)-amido black (Ruzin, 1999), was used for total carbohydrates and proteins. Observations were made with a Nikon Labophot TK microscope and images were captured by means of a color Leica JVC camera and image analysis system (Leica QWIN 5001, Leica Imaging Systems Ltd., Cambridge, U.K.) connected to the microscope. Fluorescence observations were made of unstained and toluidine blue stained sections
2. Material and methods Fruits of P. oceanica were collected along 1500 m of coastline at Las Salinas beach, Almerı´a, south Mediterranean coast of Spain (368470 , 26 N, 0028350 ,40 W) during March 2004. The collected material was all drift material, recently deposited on the beach. Undehisced fruits, with the peduncle present and no signs of predation or other damage, were selected, transported to the laboratory and maintained in natural seawater at 5 8C for a maximum of 48 h. seeds were extracted carefully from dissected fruits, washed in sterilized natural sea water, and incubated for germination. The pre-selected seeds had a mean weight of 0.47 0.10 g. 2.1. Seed culture Four replicates of 25 seeds were placed in folded filter paper inside semitransparent covered plastic containers, saturated with sterilized natural seawater (38 psu). Seeds were then placed in a germinating chamber (Frisol, TR-610, Cordoba, Spain) at 17 8C, with continuous light and a photon irradiance of 30 mmol m2 s1 (400–700 nm). The seeds were checked weekly to be sure seawater saturation was maintained and to
Fig. 1. Posidonia oceanica seeds showing initiation, emergence and initial growth of root system. (a) Longitudinal fresh section in the apical part of the mature P. oceanica seed showing the two spherically shaped adventitious root primordia (arrows). The seedling shoot apex (SA) is visible above the hypocotyl node where the adventitious root primordia are formed. Scale bar = 1 mm. (b) Two-week seedling; only primary root (PR) has emerged. (c) Three-week seedling. Primary root has emerged, and first adventitious root (AR) has begun emergence. (d) Seven-week seedling. Primary root remains relatively unchanged and various adventitious roots have emerged and grown. For (b– d) scale bar = 10 mm.
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Table 1 Percentages of seedlings with total numbers of one (1AR), two (2AR), three (3AR) and four (4AR) adventitious roots at successive weeks of culture Weeks of culture 1 1AR 2AR 3AR 4AR
– – – –
2 – – – –
3 21.0 2.0 – –
4
5 a
53.6 10.4a – –
29.1 40.2 14.0 0.0
6 ab a ab b
11.9 51.9 17.6 2.1
7 b a b b
8.3 26.5 48.2 7.9
b ab a b
AR minimum size = 2 mm; n = 4 25. Different letters indicate significant differences between weeks at P 0.05 (ANOVA, Tukey test). a Statistically significant difference between means at the 95.0% confidence level by t-Student.
(Peterson, 1992; Ruzin, 1999) using a Leica DMR microscope with a barrier filter LP 425 and an ultra-violet (UV) excitation filter 340–380 nm under a 100 W mercury lamp. Cross-sectional area of root and stele were measured with the above-mentioned image analysis system connected to a stereomicroscope (root) or an optical microscope (stele). Cortex area, including the hypodermis, was obtained by calculating the difference between the root and stele areas for
Fig. 2. Root growth of primary root (PR) and first adventitious root (1AR) on successive weeks following emergence in culture. Roots were counted as emerged and length measurements started once the root reached a minimum length of 2 mm. All PR emerged 1 week after culture. 1ARa, 2ARb and 1ARc represent first adventitious roots which emerged 3–5 weeks after culture, successively. Each point represents mean root length for all roots of each type which emerged at the same time. Bars indicate S.E. n = 83 (PR); 23 (1ARa); 31 (1ARb); 16 (1ARc).
Fig. 3. Apical and subapical zones of primary (PR) and adventitious root (AR). Scale bars = 100 mm. Stained with Tol. Bl. (a and b) Longitudinal section of PR and AR root apex showing apical zone (AZ) and root cap (RC). (c and d) Transverse section of PR and AR approximately 3 mm from the apex showing the predominant cortex (C).
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each section. A representative five to six non-consecutive sections were measured for each sample, and the means of those measurements used to calculate the area values of that sample. The percentage of areas of different structures, root, cortex and stele were calculated for all samples. 3. Results 3.1. Emergence and growth After 7 weeks under culture conditions 83% of the seeds had germinated, grown and survived. Emergence and growth of the root system during seed germination and early seedling growth is shown in Fig. 1. The primary root (PR), located at the basal end of the seed, was the first root to emerge and develop (Fig. 1b). After 3 weeks of culture, one of the two adventitious root (AR) primordia, present in the seed at maturity (Fig. 1a), emerged and started to grow (Fig. 1c), followed after approximately 1–2 weeks by the second AR. Subsequently, further adventitious roots which were not present as primordia in the mature seed started developing (Fig. 1d). By week 4 of culture, 54% of the plantlets had developed one adventitious root, and the percentage of seedlings with adventitious roots were significantly different (Table 1). During weeks 5 and 6 the second adventitious root was formed in the majority of seeds, and was the most significantly different group from the seeds that had 1AR, 3AR and 4AR. At week 7, seedlings with three adventitious roots were the most significant group. Although variability in root lengths was high, partially due to different dates of emergence, primary and adventitious root showed very different patterns of growth. The primary roots elongated rapidly in the first 3–4 weeks after emergence and then ceased growth or grew extremely slowly (Fig. 2). The first adventitious root (1AR), which started emergence at week 3 or later of culture, grew more rapidly and was longer than the primary root, despite its later emergence (Fig. 2). First adventitious roots (1ARa, 1ARb, 1ARc) which emerged on different dates followed the same pattern of rapid and continuous elongation. Following their emergence (Table 1), the successive adventitious roots (2AR, 3AR and A4R), also elongated in a similar manner to that of the first adventitious root. 3.2. Structure and anatomy The root apex of both primary and adventitious roots had a well-defined root cap covering the apical meristem, but which differed between the two root types. The root cap of adventitious roots consisted of a prominent cone-shaped mass of cells that extended approximately 200–400 mm below the apical meristem. In contrast, primary roots had a less conspicuous root cap extending 110–170 mm from the meristematic region (Fig. 3a and b). In general, root cap cells had large nuclei and thick walls but contained no starch grains as commonly noted for mature root cap cells of adult Posidonia australis (Kuo and Cambridge, 1978). In adventitious roots the meristematic region, located just behind the root cap, was
Fig. 4. Cell form and tissue organization of exterior zones of primary (PR) and adventitious (AR) roots. Scale bars = 50 mm. Stained with Tol. Bl. root hairs (RH) of epidermal cells, epidermis (Ep), hypodermis (Hp), cortex parenchyma cells (PC) and parenchyma cells containing unidentified substance (stars). (a) Primary root (PR). Note the densely staining cell walls of hypodermis. (b) Adventitious root (AR) with thinner hypodermis.
larger, composed of a broader zone with many more layers of apical initials (Fig. 3a and b), and contained more densely staining nuclei than in primary roots. The root exterior of both types, as observed in transverse sections, was composed of an epidermis consisting of a single layer of thin-walled tangentially elongated and relatively large cells. Root hairs with maximum lengths of approximately 300 mm formed from epidermal cells at scattered, separated positions around the root circumference (Figs. 3 and 4). Both root types showed a pronounced predominance of the cortex in their structure (Fig. 3c and d). Cortex area of both primary and adventitious roots represented 99% of total root cross-section area, although the total cross-sectional area of adventitious roots was slightly higher (1.1 0.4 mm2) than that of primary roots (0.75 0.38 mm2). For both root types the predominant cortical area was formed by thin-walled parenchymatous cells which were roughly isodiametric (Fig. 3c and d). No air spaces or lacunae appeared in this tissue, nor were starch grains observed in the cortical cells (Fig. 4a and b). The outermost cell layers of the cortex constituted a hypodermis, composed of compacted layers of hexagonalshaped cells smaller than the other cortical cells (Fig. 4a and b). In primary roots the hypodermis was thicker, usually composed of more cell layers than in the adventitious roots. The hypodermal cell walls were notably thicker, particularly in primary roots, and histochemically different from the general cortical cells. Those differences were confirmed by their intense UV autofluorescence (Fig. 5a, c and d). The endodermis of the primary roots was initially apparent approximately 150 mm from the apical meristem as a welldefined row of small cells with radial wall thickenings
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Fig. 5. Autofluorescence of cell walls of different root tissues observed in transverse sections with UV light. Scale bars = 20 mm, except (c) scale bar = 100 mm. Epidermis (Ep), root hair (RH), hypodermis (Hp), cortex (C), endodermis (En) and stele (St). (a) Primary root showing hypodermis with autofluorescence. (b) Primary root showing autofluorescence of endodermis surrounding the stele. (c) Adventitious root showing faint autofluorescence in hypodermis (arrows). Note the absence of fluorescence in the endodermal region. (d) Detail of hypodermis autofluorescence of adventitious root.
surrounding the stele (Fig. 6a). The characteristically thickened radial walls of this tissue were apparent with Tol. Bl. staining (Fig. 6a and b) and vivid UV autofluorescence (Fig. 5b). In adventitious roots, however, the endodermis was hardly distinguishable (Figs. 5c and 6c), and no endodermal autofluorescence was observed (Fig. 5c). The stele represented 1% of the total root cross-sectional area in all roots. Within the stele, anatomical differences between primary and adventitious roots were observed, particularly in the degree of differentiation of vascular tissue. In the adventitious roots, xylem elements in polyarch arrangement with differentiated secondary walls were clearly distinguishable (Fig. 6c and d). In contrast, vascular tissue was not very differentiated in primary roots (Fig. 6a). 4. Discussion As in other Posidonia species (Kuo and Kirkman, 1996), P. oceanica seeds have high potential for germination and survival
due to their nutrient reserves and embryo structure, which includes both a primary root and two prominent lateral adventitious root primordia (Belzunce et al., 2005). The present study, in agreement with previous work (Balestri et al., 1998b; Piazzi et al., 1999), indicates that both root types emerge and grow rapidly once the seed germinates. The multiple roots which emerge rapidly could increase the capacity of seedlings to become established and anchored. In culture conditions, the primary root emerged and initially grew rapidly, followed by successive emergence of up to four adventitious roots. The sequence of root initiation we observed is similar to that reported by Balestri et al. (1998b) and Balestri and Bertini (2003) for P. oceanica grown in culture. However, Balestri et al. (1998b) reported longer primary and adventitious roots (3.0 7 and 35 7.1 mm respectively) after 2 months of culture, similar to lengths they measured in naturally occurring seedlings. The pattern of root emergence would suggest that during the settlement process, the primary root emerges, growing rapidly to facilitate initial anchorage,
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Fig. 6. Endodermis and stele of primary (PR) and adventitious (AR) roots in transverse sections. Scale bars = 10 mm, except (c) scale bar = 20 mm. (a and c) stained with Tol.Bl. (b and d) stained with TAC–FC–SAF–FG. (a) Primary root endodermis showing slightly thickened radial walls (arrows) and stele with vascular tissue hardly distinguished. (b) Detail of primary root thickened endodermal cell walls (arrows). (c) Stele of adventitious root with xylem elements present in polyarch arrangement (arrows). No differentiated endodermis is distinguished surrounding the stele. (d) Detail of portion of stele containing xylem elements (X).
followed by successive adventitious roots assuring final settlement. Structural evidence obtained in studies of different seagrasses indicates that the roots are effective in water and nutrient absorption (Kuo and Cambridge, 1978; Hemminga et al., 1994; Duarte et al., 1998). Seagrass root structure appears to be speciesspecific, with some degree of intraspecific variability (Barnabas, 1994b), and show multiple functional adaptations to the environment (Barnabas, 1991; Pe´rez et al., 1994). A hypodermis was observed in both primary and adventitious roots of the young P. oceanica seedling, although it showed differences in the number of component cell layers. Other studies have described a clearly differentiated hypodermis in the adult roots of different seagrass genera: Thalassia (Tomlinson, 1969), Posidonia (Kuo and Cambridge, 1978) Zostera (Barnabas and Arnott, 1987), Thalassodendron (Barnabas, 1991), Halodule (Barnabas, 1994a) and Phyllospadix (Barnabas, 1994b). The hypodermis may prevent salt entering the cortex and penetrating into other tissues of the root, representing an apoplastic barrier essential for osmotic
adjustment in the marine environment. The multilayer thickened cell walls of the hypodermis we observed in older primary roots of young P. oceanica seedlings may also provide strength, similar to the cellulose mechanical layer present in seagrasses in surf-exposed habitats (Barnabas, 1994a). In our study, the primary roots of the young P. oceanica seedling had a notable endodermis, in contrast to the adventitious roots. The presence of an endodermis has been confirmed in the roots of adult seagrass species (Tomlinson, 1969; Kuo and Cambridge, 1978; Cambridge and Kuo, 1982; Barnabas and Arnott, 1987; Barnabas, 1991, 1994a,b). According to Perumalla and Peterson (1986), the absolute and relative positions along the root axis at which the endodermis and exodermis differentiate depend on root age and growth rate. The differences we observed in endodermal tissue structure, particularly cell wall composition as indicated by UV autofluorescence, between primary and adventitious roots in the young P. oceanica seedlings could be explained by root differentiation patterns, by response to culture condition stress factors or by different root function at this age.
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Even though the cortex of the young P. oceanica seedling roots occupied a substantial proportion of the root transactional area, it did not have aerenchyma. In rooted aquatic macrophytes the survival of roots and rhizomes inserted in anoxic sediments depends on their aeration by internal transport of oxygen from leaves and stems (Rascio, 2002). Seagrasses are no exception, and in those cases where adult roots have been studied ample aerenchyma tissues have been observed (Kuo, 1983; Roberts et al., 1985; Connell et al., 1999). For example the aerenchyma of P. australis has been described as a wide lacunose area of the root cortex, composed of a series of longitudinal air spaces separated by groups of collapsed, elongated cells (Kuo and Cambridge, 1978). In P. oceanica the large cortex observed in seedlings, regardless of whether aerenchyma will form in the adult plant, aids in ensuring a supply of oxygen by increasing porosity (Jackson and Amstrong, 1999; Striker et al., 2007). Additionally, a substantial cortex provides a large, metabolically inexpensive structure for support. The narrow stele area we observed in young P. oceanica seedling roots has also been reported in adult seagrass roots (Barnabas and Arnott, 1987; Barnabas, 1991, 1994b; Connell et al., 1999), although without detailed measurements. In contrast, Wahl and Ryser (2000) found that the stele occupied 11.7–21% of the root transverse area of 19 terrestrial grass species, ten to twenty times greater than the 0.75–1% of stele area we observed. Possibly the small stele area of P. oceanica is related to reduced vascular development which is typical for aquatic plants (Peterson, 1992). Within the stele, the young adventitious roots contained differentiated xylem elements, while none were observed in the primary roots. In seagrass adult roots studied by different authors the position and degree of vascular tissue differentiation varied greatly. For instance, in Thalassia testudinum tracheids occurred only at the basal zones of mature roots (Tomlinson, 1969) and in P. australis the xylem had tracheids with poorly lignified walls (Kuo and Cambridge, 1978). Roberts et al. (1985) found xylem maturation in Halophila ovalis in the same region as root hairs and endodermal Casparian strip formation, suggesting the functional significance of this synchrony (Roberts et al., 1985). The differences in vascular tissue differentiation between the primary and adventitious roots in young P. oceanica seedlings, as in the case of different endodermal development, may be due to varied root tissue maturity or different root function at this age. P. oceanica seedling root anatomy was similar to that of roots of terrestrial plants, but differed in the amounts of cells and tissues formed or their degree of differentiation. Those differences represent structural adaptations to a marine environment which, with the exception of the absence of aerenchyma, are similar to those described previously for adult sea grasses and other aquatic plants. Initial anchorage and further establishment on the sea floor are facilitated by the sequential development of numerous roots, nourished by substantial hypocotyl reserves. Structurally the roots are strengthened by a multilayered hypodermis of thick-walled cells surrounding a wide cortex, increasing their capability for anchorage, a critical activity for survival on the sea floor.
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Aquatic Botany 88 (2008) 203–210 www.elsevier.com/locate/aquabot
Posidonia oceanica seedling root structure and development Miriam Belzunce a,*, Rafael M. Navarro b, Hava F. Rapoport a a
Instituto de Agricultura Sostenible, CSIC, Apdo. 4084, 14080 Co´rdoba, Spain Dpto. Ingenierı´a Forestal, E.T.S.I.A.M., Apdo. 3048, 14080 Co´rdoba, Spain
b
Received 20 October 2006; received in revised form 26 June 2007; accepted 10 October 2007 Available online 4 November 2007
Abstract The seedling root system of the seagrass Posidonia oceanica consists of a primary root and up to four adventitious roots. Under culture, germination and early growth began with the emergence of the primary root in the first week. Then the two adventitious root primordia originally present in the seed emerged at 3 and 5 weeks respectively, followed successively by further adventitious roots. Primary roots reached 17 mm at 4 weeks, but then their growth decreased markedly. In contrast the adventitious roots showed a pattern of continued elongation. Anatomical observations of both primary and adventitious roots revealed a multilayered hypodermis of thick-walled cells enclosing a wide cortex (99% of the root transverse area) and narrow stele. A well-distinguished endodermis was only observed in the primary roots, while differentiated xylem elements were found solely in the adventitious roots, but it is unclear to what degree differences between the two root types are due to different root maturity or to their role in water and nutrient uptake. Overall, the P. oceanica seedling root system is composed of multiple, rapidly formed roots which are strong yet flexible due to a large proportion of cortical tissue and further strengthened by a multilayered hypodermis, characteristics which could potentially facilitate initial anchorage and establishment. # 2007 Elsevier B.V. All rights reserved. Keywords: Seagrass; Seedling; Root system; Germination; Hypodermis
1. Introduction Posidonia oceanica meadows are of recognised ecological importance in the Mediterranean Sea (Procaccini et al., 2003). In recent years, however, an alarming decline of many populations of P. oceanica has been observed, attributed to both natural and human disturbances (Duarte, 2002). P. oceanica meadows are maintained largely by vegetative propagation (Marba´ and Duarte, 1998), with sexual reproduction showing great variability both in frequency and intensity (Buia and Mazzella, 1991; Balestri and Cinelli, 2003). Flowering occurs throughout the Mediterranean Sea from September to December, and mature buoyant fruits are released from March to June (Buia and Mazzella, 1991). Although drifting fruits contain a germinating seed which is well-adapted for survival (Belzunce et al., 2005), the success of sexual reproduction in P. oceanica is considered low, with large losses occurring during both seed and seedling stages (Piazzi et al., 1999; Balestri and Cinelli, 2003). * Corresponding author. Tel.: +34 957 499216; fax: +34 957 499252. E-mail addresses: [email protected], [email protected] (M. Belzunce). 0304-3770/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2007.10.008
Considerable pre-dispersal seed losses due to abortion and predation, as suggested by Balestri and Cinelli (2003), may reduce reproductive success in P. oceanica. Furthermore, even during years of high fruit production, hundreds of fruits may be lost during drifting and seeds get washed ashore (Balestri et al., 2005). P. oceanica seed germination and seedling development studies in the laboratory (Balestri et al., 1998a; Balestri and Bertini, 2003) and in field transplantation experiments (Balestri et al., 1998b; Piazzi et al., 2000) suggest high seed vigor for this species. Despite the high germinability of seeds, however, seedling establishment in natural conditions is rare (Balestri et al., 1998a; Piazzi et al., 1999). In seagrasses, seedling establishment strongly depends on water movement, depth and sediment stability (Clarke and Kirkman, 1989). In P. oceanica high losses of seedlings were reported during the first months after planting (Balestri et al., 1998a). According to Balestri and Bertini (2003), a major reason was the insufficient root system of the young seedling to cope successfully with physical disturbance and to exploit resources. In adult seagrasses, the relative contribution of root production to net primary production was reported to be similar to the range described for land plants (10–40% of net
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primary production) (Duarte et al., 1998). P. oceanica root production estimates, however, have been low (2.8% of total production) compared to other seagrass species (Duarte et al., 1998). Roots in the Helobiales, the order of aquatic higher plants to which Posidonia species belong, show anatomical adaptations related to an aquatic environment. Amongst the anatomical features associated with aquatic habitats are: specialized root hairs, a narrow exodermis with thickened and suberized walls, an aerenchymatous cortex, and a narrow stele with xylem elements frequently poorly differentiated (Tomlinson, 1982). Thickened and histochemically differentiated cell walls of the exodermis and endodermis are characteristic of adult seagrass roots. These tissues appear to function in the absorption and transport of water and solutes (Tomlinson, 1969; Cambridge and Kuo, 1982; Barnabas, 1994b), and in the mechanical processes of anchorage and plant support (Barnabas, 1991, 1994b). The anatomical study of the seed and early plantlet indicated the presence of a potentially efficient root anchorage system in P. oceanica (Belzunce et al., 2005). These seeds have a conspicuous, emerging primary embryonic root and two adventitious root primordia, all of which are well linked by vascular tissues to the nutritional supplies of the carbohydraterich hypocotyl (Belzunce et al., 2005). However, knowledge is lacking about the growth of those primordia and their subsequent behavior in the young seedling. In this study, initiation of root development and growth was observed in P. oceanica mature seeds collected from drifting fruits, and the structure of the young seedling roots described. The aim of this study was to contribute to the understanding of the P. oceanica seedling establishment process and their physiological response to the environment.
record emergence and growth. Growth measurements were: maximum length of primary root, total number of adventitious roots and maximum length of adventitious roots. Roots were counted as emerged and length measurements started once the root reached a minimum length of 2 mm. Statistical analysis was by t-test and ANOVA, Tukey test. 2.2. Anatomical and histochemical studies For anatomical studies, primary and adventitious roots of 7week old P. oceanica seedlings were used. Apical and subapical portions of 2–3 mm were obtained, fixed in FAE (formalin– acetic acid–ethyl alcohol) dehydrated in a tertiary butyl alcohol series and processed in paraffin as described by Belzunce et al. (2005). Apical samples were sectioned longitudinally and transverse sections were obtained from the subapical samples, all at 12 mm thickness. Sections were stained with 0.05% (w/v) toluidine blue O (Tol. Bl.) (Sakai, 1973) for general cell and tissue organization; tannic acid–ferric chloride–safranin–fast green (TA–FC–SAF–FG) (Jensen, 1962), was used for meristematic tissues and cell walls, and periodic acid-Schiff’s (PAS)-amido black (Ruzin, 1999), was used for total carbohydrates and proteins. Observations were made with a Nikon Labophot TK microscope and images were captured by means of a color Leica JVC camera and image analysis system (Leica QWIN 5001, Leica Imaging Systems Ltd., Cambridge, U.K.) connected to the microscope. Fluorescence observations were made of unstained and toluidine blue stained sections
2. Material and methods Fruits of P. oceanica were collected along 1500 m of coastline at Las Salinas beach, Almerı´a, south Mediterranean coast of Spain (368470 , 26 N, 0028350 ,40 W) during March 2004. The collected material was all drift material, recently deposited on the beach. Undehisced fruits, with the peduncle present and no signs of predation or other damage, were selected, transported to the laboratory and maintained in natural seawater at 5 8C for a maximum of 48 h. seeds were extracted carefully from dissected fruits, washed in sterilized natural sea water, and incubated for germination. The pre-selected seeds had a mean weight of 0.47 0.10 g. 2.1. Seed culture Four replicates of 25 seeds were placed in folded filter paper inside semitransparent covered plastic containers, saturated with sterilized natural seawater (38 psu). Seeds were then placed in a germinating chamber (Frisol, TR-610, Cordoba, Spain) at 17 8C, with continuous light and a photon irradiance of 30 mmol m2 s1 (400–700 nm). The seeds were checked weekly to be sure seawater saturation was maintained and to
Fig. 1. Posidonia oceanica seeds showing initiation, emergence and initial growth of root system. (a) Longitudinal fresh section in the apical part of the mature P. oceanica seed showing the two spherically shaped adventitious root primordia (arrows). The seedling shoot apex (SA) is visible above the hypocotyl node where the adventitious root primordia are formed. Scale bar = 1 mm. (b) Two-week seedling; only primary root (PR) has emerged. (c) Three-week seedling. Primary root has emerged, and first adventitious root (AR) has begun emergence. (d) Seven-week seedling. Primary root remains relatively unchanged and various adventitious roots have emerged and grown. For (b– d) scale bar = 10 mm.
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Table 1 Percentages of seedlings with total numbers of one (1AR), two (2AR), three (3AR) and four (4AR) adventitious roots at successive weeks of culture Weeks of culture 1 1AR 2AR 3AR 4AR
– – – –
2 – – – –
3 21.0 2.0 – –
4
5 a
53.6 10.4a – –
29.1 40.2 14.0 0.0
6 ab a ab b
11.9 51.9 17.6 2.1
7 b a b b
8.3 26.5 48.2 7.9
b ab a b
AR minimum size = 2 mm; n = 4 25. Different letters indicate significant differences between weeks at P 0.05 (ANOVA, Tukey test). a Statistically significant difference between means at the 95.0% confidence level by t-Student.
(Peterson, 1992; Ruzin, 1999) using a Leica DMR microscope with a barrier filter LP 425 and an ultra-violet (UV) excitation filter 340–380 nm under a 100 W mercury lamp. Cross-sectional area of root and stele were measured with the above-mentioned image analysis system connected to a stereomicroscope (root) or an optical microscope (stele). Cortex area, including the hypodermis, was obtained by calculating the difference between the root and stele areas for
Fig. 2. Root growth of primary root (PR) and first adventitious root (1AR) on successive weeks following emergence in culture. Roots were counted as emerged and length measurements started once the root reached a minimum length of 2 mm. All PR emerged 1 week after culture. 1ARa, 2ARb and 1ARc represent first adventitious roots which emerged 3–5 weeks after culture, successively. Each point represents mean root length for all roots of each type which emerged at the same time. Bars indicate S.E. n = 83 (PR); 23 (1ARa); 31 (1ARb); 16 (1ARc).
Fig. 3. Apical and subapical zones of primary (PR) and adventitious root (AR). Scale bars = 100 mm. Stained with Tol. Bl. (a and b) Longitudinal section of PR and AR root apex showing apical zone (AZ) and root cap (RC). (c and d) Transverse section of PR and AR approximately 3 mm from the apex showing the predominant cortex (C).
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each section. A representative five to six non-consecutive sections were measured for each sample, and the means of those measurements used to calculate the area values of that sample. The percentage of areas of different structures, root, cortex and stele were calculated for all samples. 3. Results 3.1. Emergence and growth After 7 weeks under culture conditions 83% of the seeds had germinated, grown and survived. Emergence and growth of the root system during seed germination and early seedling growth is shown in Fig. 1. The primary root (PR), located at the basal end of the seed, was the first root to emerge and develop (Fig. 1b). After 3 weeks of culture, one of the two adventitious root (AR) primordia, present in the seed at maturity (Fig. 1a), emerged and started to grow (Fig. 1c), followed after approximately 1–2 weeks by the second AR. Subsequently, further adventitious roots which were not present as primordia in the mature seed started developing (Fig. 1d). By week 4 of culture, 54% of the plantlets had developed one adventitious root, and the percentage of seedlings with adventitious roots were significantly different (Table 1). During weeks 5 and 6 the second adventitious root was formed in the majority of seeds, and was the most significantly different group from the seeds that had 1AR, 3AR and 4AR. At week 7, seedlings with three adventitious roots were the most significant group. Although variability in root lengths was high, partially due to different dates of emergence, primary and adventitious root showed very different patterns of growth. The primary roots elongated rapidly in the first 3–4 weeks after emergence and then ceased growth or grew extremely slowly (Fig. 2). The first adventitious root (1AR), which started emergence at week 3 or later of culture, grew more rapidly and was longer than the primary root, despite its later emergence (Fig. 2). First adventitious roots (1ARa, 1ARb, 1ARc) which emerged on different dates followed the same pattern of rapid and continuous elongation. Following their emergence (Table 1), the successive adventitious roots (2AR, 3AR and A4R), also elongated in a similar manner to that of the first adventitious root. 3.2. Structure and anatomy The root apex of both primary and adventitious roots had a well-defined root cap covering the apical meristem, but which differed between the two root types. The root cap of adventitious roots consisted of a prominent cone-shaped mass of cells that extended approximately 200–400 mm below the apical meristem. In contrast, primary roots had a less conspicuous root cap extending 110–170 mm from the meristematic region (Fig. 3a and b). In general, root cap cells had large nuclei and thick walls but contained no starch grains as commonly noted for mature root cap cells of adult Posidonia australis (Kuo and Cambridge, 1978). In adventitious roots the meristematic region, located just behind the root cap, was
Fig. 4. Cell form and tissue organization of exterior zones of primary (PR) and adventitious (AR) roots. Scale bars = 50 mm. Stained with Tol. Bl. root hairs (RH) of epidermal cells, epidermis (Ep), hypodermis (Hp), cortex parenchyma cells (PC) and parenchyma cells containing unidentified substance (stars). (a) Primary root (PR). Note the densely staining cell walls of hypodermis. (b) Adventitious root (AR) with thinner hypodermis.
larger, composed of a broader zone with many more layers of apical initials (Fig. 3a and b), and contained more densely staining nuclei than in primary roots. The root exterior of both types, as observed in transverse sections, was composed of an epidermis consisting of a single layer of thin-walled tangentially elongated and relatively large cells. Root hairs with maximum lengths of approximately 300 mm formed from epidermal cells at scattered, separated positions around the root circumference (Figs. 3 and 4). Both root types showed a pronounced predominance of the cortex in their structure (Fig. 3c and d). Cortex area of both primary and adventitious roots represented 99% of total root cross-section area, although the total cross-sectional area of adventitious roots was slightly higher (1.1 0.4 mm2) than that of primary roots (0.75 0.38 mm2). For both root types the predominant cortical area was formed by thin-walled parenchymatous cells which were roughly isodiametric (Fig. 3c and d). No air spaces or lacunae appeared in this tissue, nor were starch grains observed in the cortical cells (Fig. 4a and b). The outermost cell layers of the cortex constituted a hypodermis, composed of compacted layers of hexagonalshaped cells smaller than the other cortical cells (Fig. 4a and b). In primary roots the hypodermis was thicker, usually composed of more cell layers than in the adventitious roots. The hypodermal cell walls were notably thicker, particularly in primary roots, and histochemically different from the general cortical cells. Those differences were confirmed by their intense UV autofluorescence (Fig. 5a, c and d). The endodermis of the primary roots was initially apparent approximately 150 mm from the apical meristem as a welldefined row of small cells with radial wall thickenings
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Fig. 5. Autofluorescence of cell walls of different root tissues observed in transverse sections with UV light. Scale bars = 20 mm, except (c) scale bar = 100 mm. Epidermis (Ep), root hair (RH), hypodermis (Hp), cortex (C), endodermis (En) and stele (St). (a) Primary root showing hypodermis with autofluorescence. (b) Primary root showing autofluorescence of endodermis surrounding the stele. (c) Adventitious root showing faint autofluorescence in hypodermis (arrows). Note the absence of fluorescence in the endodermal region. (d) Detail of hypodermis autofluorescence of adventitious root.
surrounding the stele (Fig. 6a). The characteristically thickened radial walls of this tissue were apparent with Tol. Bl. staining (Fig. 6a and b) and vivid UV autofluorescence (Fig. 5b). In adventitious roots, however, the endodermis was hardly distinguishable (Figs. 5c and 6c), and no endodermal autofluorescence was observed (Fig. 5c). The stele represented 1% of the total root cross-sectional area in all roots. Within the stele, anatomical differences between primary and adventitious roots were observed, particularly in the degree of differentiation of vascular tissue. In the adventitious roots, xylem elements in polyarch arrangement with differentiated secondary walls were clearly distinguishable (Fig. 6c and d). In contrast, vascular tissue was not very differentiated in primary roots (Fig. 6a). 4. Discussion As in other Posidonia species (Kuo and Kirkman, 1996), P. oceanica seeds have high potential for germination and survival
due to their nutrient reserves and embryo structure, which includes both a primary root and two prominent lateral adventitious root primordia (Belzunce et al., 2005). The present study, in agreement with previous work (Balestri et al., 1998b; Piazzi et al., 1999), indicates that both root types emerge and grow rapidly once the seed germinates. The multiple roots which emerge rapidly could increase the capacity of seedlings to become established and anchored. In culture conditions, the primary root emerged and initially grew rapidly, followed by successive emergence of up to four adventitious roots. The sequence of root initiation we observed is similar to that reported by Balestri et al. (1998b) and Balestri and Bertini (2003) for P. oceanica grown in culture. However, Balestri et al. (1998b) reported longer primary and adventitious roots (3.0 7 and 35 7.1 mm respectively) after 2 months of culture, similar to lengths they measured in naturally occurring seedlings. The pattern of root emergence would suggest that during the settlement process, the primary root emerges, growing rapidly to facilitate initial anchorage,
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Fig. 6. Endodermis and stele of primary (PR) and adventitious (AR) roots in transverse sections. Scale bars = 10 mm, except (c) scale bar = 20 mm. (a and c) stained with Tol.Bl. (b and d) stained with TAC–FC–SAF–FG. (a) Primary root endodermis showing slightly thickened radial walls (arrows) and stele with vascular tissue hardly distinguished. (b) Detail of primary root thickened endodermal cell walls (arrows). (c) Stele of adventitious root with xylem elements present in polyarch arrangement (arrows). No differentiated endodermis is distinguished surrounding the stele. (d) Detail of portion of stele containing xylem elements (X).
followed by successive adventitious roots assuring final settlement. Structural evidence obtained in studies of different seagrasses indicates that the roots are effective in water and nutrient absorption (Kuo and Cambridge, 1978; Hemminga et al., 1994; Duarte et al., 1998). Seagrass root structure appears to be speciesspecific, with some degree of intraspecific variability (Barnabas, 1994b), and show multiple functional adaptations to the environment (Barnabas, 1991; Pe´rez et al., 1994). A hypodermis was observed in both primary and adventitious roots of the young P. oceanica seedling, although it showed differences in the number of component cell layers. Other studies have described a clearly differentiated hypodermis in the adult roots of different seagrass genera: Thalassia (Tomlinson, 1969), Posidonia (Kuo and Cambridge, 1978) Zostera (Barnabas and Arnott, 1987), Thalassodendron (Barnabas, 1991), Halodule (Barnabas, 1994a) and Phyllospadix (Barnabas, 1994b). The hypodermis may prevent salt entering the cortex and penetrating into other tissues of the root, representing an apoplastic barrier essential for osmotic
adjustment in the marine environment. The multilayer thickened cell walls of the hypodermis we observed in older primary roots of young P. oceanica seedlings may also provide strength, similar to the cellulose mechanical layer present in seagrasses in surf-exposed habitats (Barnabas, 1994a). In our study, the primary roots of the young P. oceanica seedling had a notable endodermis, in contrast to the adventitious roots. The presence of an endodermis has been confirmed in the roots of adult seagrass species (Tomlinson, 1969; Kuo and Cambridge, 1978; Cambridge and Kuo, 1982; Barnabas and Arnott, 1987; Barnabas, 1991, 1994a,b). According to Perumalla and Peterson (1986), the absolute and relative positions along the root axis at which the endodermis and exodermis differentiate depend on root age and growth rate. The differences we observed in endodermal tissue structure, particularly cell wall composition as indicated by UV autofluorescence, between primary and adventitious roots in the young P. oceanica seedlings could be explained by root differentiation patterns, by response to culture condition stress factors or by different root function at this age.
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Even though the cortex of the young P. oceanica seedling roots occupied a substantial proportion of the root transactional area, it did not have aerenchyma. In rooted aquatic macrophytes the survival of roots and rhizomes inserted in anoxic sediments depends on their aeration by internal transport of oxygen from leaves and stems (Rascio, 2002). Seagrasses are no exception, and in those cases where adult roots have been studied ample aerenchyma tissues have been observed (Kuo, 1983; Roberts et al., 1985; Connell et al., 1999). For example the aerenchyma of P. australis has been described as a wide lacunose area of the root cortex, composed of a series of longitudinal air spaces separated by groups of collapsed, elongated cells (Kuo and Cambridge, 1978). In P. oceanica the large cortex observed in seedlings, regardless of whether aerenchyma will form in the adult plant, aids in ensuring a supply of oxygen by increasing porosity (Jackson and Amstrong, 1999; Striker et al., 2007). Additionally, a substantial cortex provides a large, metabolically inexpensive structure for support. The narrow stele area we observed in young P. oceanica seedling roots has also been reported in adult seagrass roots (Barnabas and Arnott, 1987; Barnabas, 1991, 1994b; Connell et al., 1999), although without detailed measurements. In contrast, Wahl and Ryser (2000) found that the stele occupied 11.7–21% of the root transverse area of 19 terrestrial grass species, ten to twenty times greater than the 0.75–1% of stele area we observed. Possibly the small stele area of P. oceanica is related to reduced vascular development which is typical for aquatic plants (Peterson, 1992). Within the stele, the young adventitious roots contained differentiated xylem elements, while none were observed in the primary roots. In seagrass adult roots studied by different authors the position and degree of vascular tissue differentiation varied greatly. For instance, in Thalassia testudinum tracheids occurred only at the basal zones of mature roots (Tomlinson, 1969) and in P. australis the xylem had tracheids with poorly lignified walls (Kuo and Cambridge, 1978). Roberts et al. (1985) found xylem maturation in Halophila ovalis in the same region as root hairs and endodermal Casparian strip formation, suggesting the functional significance of this synchrony (Roberts et al., 1985). The differences in vascular tissue differentiation between the primary and adventitious roots in young P. oceanica seedlings, as in the case of different endodermal development, may be due to varied root tissue maturity or different root function at this age. P. oceanica seedling root anatomy was similar to that of roots of terrestrial plants, but differed in the amounts of cells and tissues formed or their degree of differentiation. Those differences represent structural adaptations to a marine environment which, with the exception of the absence of aerenchyma, are similar to those described previously for adult sea grasses and other aquatic plants. Initial anchorage and further establishment on the sea floor are facilitated by the sequential development of numerous roots, nourished by substantial hypocotyl reserves. Structurally the roots are strengthened by a multilayered hypodermis of thick-walled cells surrounding a wide cortex, increasing their capability for anchorage, a critical activity for survival on the sea floor.
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