Effect of GeO<sub>2</sub> on embryo development and photosynthesis in <italic>Fucus vesiculosus</italic> (Phaeophyceae)
Effect of GeO2 on embryo development and photosynthesis in Fucus vesiculosus (Phaeophyceae)
ALGAE. 2012. Jun, 27(2): 125-134
Copyright ©2012, The Korean Society of Phycology
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://cre-ativecommons. org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : December 12, 2011
  • Accepted : May 05, 2012
  • Published : June 15, 2012
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About the Authors
Elena R. Tarakhovskaya
Department of Plant Physiology and Biochemistry, St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg 199034, Russia
Eun Ju Kang
Department of Oceanography, Chonnam National University, Gwangju 500-757, Korea
Kwang Young Kim
Department of Oceanography, Chonnam National University, Gwangju 500-757, Korea
David J. Garbary
Department of Biology, St. Francis Xavier University, Antigonish, Nova Scotia, B2G 2W5, Canada
Germanium dioxide (GeO 2 ) has been used for many years in the cultivation of red and green algae as a means of controlling the growth of diatoms. Brown algae are sensitive to GeO 2 , however, the basis of this sensitivity has not been characterized. Here we use embryos of Fucus vesiculosus to investigate morphological and physiological impacts of GeO 2 toxicity. Morphometric features of embryos were measured microscopically, and physiological features were determined using pulse amplitude modulated (PAM) fluorometry. At 5 mg L -1 GeO 2 , embryos grew slower than controls and developed growth abnormalities. After 24 h, initial zygote divisions were often oblique rather than transverse. Rhizoids had inflated tips in GeO 2 and were less branched, and apical hairs were deformed, with irregularly aligned, spheroidal cells. Minimum fluorescence ( F 0 ) showed minor differences over the 10 days experiment, and pigment levels (chlorophylls a , c and total carotenoids) showed no difference after 10 days. Optimum quantum yield increased from ca. 0.52 at 24 h to 0.67 at 5 days, and GeO 2 -treated embryos had higher mean values (significant at 3 and 5 days). Optimum quantum yield of photosystem II (Φ PSII ) was stable in control thalli after 5 days, but declined significantly in GeO 2 . Addition of silica (as SiO 2 ) did not reverse the effects of GeO 2 . These results suggest that GeO 2 toxicity in brown algae is associated with negative impacts at the cytological level rather than metabolic impacts associated with photosynthesis.
Lewin (1966) reported that germanium (Ge) was toxic to diatoms. Subsequently, this element (as GeO 2 ) has had an important role in the laboratory culture of marine algae. Accordingly, researchers will typically use 5-10 mg L -1 of GeO 2 in their medium to prevent growth of diatoms during initial stages of algal isolation (e.g., McLachlan 1973, Garbary et al. 2004). The basis for this use is that Ge and silica (Si) are chemically very similar, and Ge inhibits deposition of Si into the frustule of the diatoms (Chiappino et al. 1977). This application of GeO 2 has proven effective in the culture of red and green algae (e.g., Garbary and Tam 1989, Scrosati et al. 1994), and only in very high concentrations (20-30 mg L -1 GeO 2 ) is there any inhibition of red algal growth (Tatewaki and Mizuno 1979). It has long been known, however, that brown algae have negative responses to GeO 2 . West (unpublished, see Parker 1969) first noticed the inhibition of brown algae in the presence of Ge, and McLachlan et al. (1971) later reported growth asabnormalities in Fucus embryos when grown in the presence of GeO 2 . McLachlan (1977), Tatewaki and Mizuno (1979) and Yang (1993) later showed that this was a more general phenomenon for Phaeophyceae, with inhibition in most species occurring at concentrations as low as 1 mg L -1 . In addition, Markham and Hagmeier (1982) and Hubbard et al. (2004) reported inhibition of growth of blade discs of Saccharina and kelp gametophytes when grown with medium containing GeO 2 .
The mechanism of Ge toxicity for brown algae is unknown. While species of Phaeophyceae may contain Si (Parker 1969), this is no more than the concentration in seawater. In addition, the omission of Si from the growth media for brown algae has no apparent deleterious effects (McLachlan 1977). Thus the physiological basis for Ge toxicity in brown algae is unlikely to be similar to that in diatoms, and Shea and Chopin (2007) even reported positive growth responses of low concentrations of GeO 2 on S. latissima (L.) Lane et al. (as Laminaria ). More recently Mizuta and Yasui (2011) showed extensive silicon localization in sporophyte tissue of S. japonica (Areschoug) Lane et al. There have been several studies on the direct effects of Ge on algal growth and morphology (McLachlan 1977, Yang 1993, Shea and Chopin 2007). In this paper we examine the effects of GeO 2 on dei veloping embryos of F. vesiculosus L. during the first 10 days following fertilization from the perspective of both morphological changes and photosynthetic physiology. Thus, are the inhibitory effects of Ge primarily associated with developmental phenomena, and are these impacts associated with negative effects of GeO 2 on photosynthetic processes?
- Experimental material and culturing
Fucus vesiculosus was collected at Tor Bay Provincial Park (45.19° N, 61.34° W) on the Atlantic coast of Nova Scotia, Canada from mid-June to mid-July 2007. Mature receptacles were collected in the mid-intertidal zone during low tide, washed with seawater, dried with filter paper, and stored in the dark at 4-10℃ for up to 10 days. We used the standard technique (Jaffe and Neuscheler 1969, Quatrano 1974) to obtain gametes and to carry out fertilization. Immediately after fertilization (AF) the zygotes were divided into several groups, and each group was placed into 60-mm plastic Petri dishes in Tyndalized seawater at 15℃. Later, the developing zygotes and embryos were kept in Petri dishes with seawater under continuous light provided by cool white fluorescent bulbs at an irradiance of 20-25 μmol photons m -2 s -1 . Embryos were grown in seawater alone or in seawater supplemented with 5 mg L -1 GeO 2 (electronic grade, lot A019218501; Acros Organics, Geel, Belgium). Embryos exposed to GeO 2 are referred to as Ge-treated. Samples for fluorescence measurement and morphometry were taken at 1, 3, 5, 8, and 10 days AF.
- Morphometry
Embryos were measured using a calibrated Olympus BH2 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) at 200× or 400× magnification. Ten or 15 embryos from each condition were measured for embryo length (top of embryo, excluding hairs, to tip of rhizoid), embryo diameter, and rhizoid tip diameter. In addition, the number of apical hairs was counted. The apical hairs usually appeared in 6-7 days old embryos and were counted at 7-8 days AF.
- FM4-64-staining of membranes
Membrane aggregations were stained in embryos (22-24 h AF) with 5 μM of FM4-64 [N-(3-triethylammonium- propyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide] (Invitrogen, Burlington, ON, Canada) for 30 min (Belanger and Quatrano 2000, Bolte et al. 2004, Hadley et al. 2006). The stain was prepared from the stock solution (10 mM in dimethyl sulfoxide [DMSO]) and diluted in seawater. The embryos were observed by Nikon Microphot FXA microscope (Nikon, Tokyo, Japan) as described by Belanger and Quatrano (2000).
Images from both transmitted light and epifluorescence microscopy were captured using the CCD SPOT2 camera system (Diagnostic Instruments, Sterling Heights, MI, USA) mounted on the Nikon Microphot FXA microscope.
- Pulse amplitude modulated (PAM) fluorometry
Fluorescence measurements of F. vesiculosus embryos were performed using a microscopy-PAM apparatus (Walz GmbH, Effeltrich, Germany) as described previously (Garbary and Kim 2005, Kim et al. 2006). This system uses a blue (MC-L470) actinic and saturating-pulse light source. The active field diameter was set at 65 μm. The maximum ( F m ) and the minimum ( F 0 ) fluorescence, and the intrinsic potential quantum efficiency of PSII ( F v / F m ) were determined for all embryos after being acclimated to darkness for 10 min. Microscopy-PAM was used to assess the effective quantum yield of charge separation in PSII, Φ PSII = ( F m ′ - F )/ F m ′ (Genty et al. 1989); where F m is the maximum fluorescence yield for a light-acclimated embryo exposed to the actinic light, and F is the steady-state fluorescence yield of a light-acclimated embryo.
From the rapid light curves (RLC) of photosynthesis we calculated relative electron transport rate (rETR max ), photosynthetic efficiency (α ETR ) and light saturation ( I k = rETR max ETR ). Electron transport rate was calculated as rETR = 0.84 × Φ PSII × PFD × 0.5, where PFD is photon flux density of photosynthetically active radiation (PAR 400-700 nm). RLCs were fitted to a double exponential decay curve (Platt et al. 1980). In cases where photoinhibition was not observed, an asymptotic function was applied (Harrison and Platt 1986).
The photosynthesis experiment was repeated using a different collection from the same site. Based on the similarity of results in the two runs, we only report on the one for which the morphometric data was collected.
- Pigment analysis
Ten-days old attached embryos were resuspended and concentrated by centrifugation (500 g , 5 min). The supernatant was discarded and the pellet was ground using a manual tissue grinder in 100% acetone with small quantities of Na 2 SO 4 and NaHCO 3 . Several rounds of extraction were made with additional acetone until the extract was colourless. The concentration of acetone was adjusted to 90% (in distilled water), and quantities of chlorophylls a and c ( c 1 + c 2 ) as well as total carotenoids were calculated from data obtained with a Unicam UV2 spectrophotometer (Unicam, Cambridge, UK) using published equations (Jeffrey and Humphrey 1975, Henley and Dunton 1995).
- Data analysis
Photosynthetic data were analyzed using a general linear model (GLM) procedure of SYSTAT version 9 (SPSS Inc., Chicago, IL, USA) to determine if significant differences occurred among treatments and times for F 0 , F v / F m , rETR max , α ETR , and I k . Data were tested for normality and homogeneity of variance using the Kolmogorov-Smirnov and Cochran’s tests, respectively. Where a significant difference occurred, a Tukey’s HSD post hoc comparison was used to determine which times were different. Values are all expressed as mean and standard error (i.e., mean ± SE) or as mean and 95% confidence intervals.
- Changes in embryo morphogenesis
GeO 2 at 5 mg L -1 altered the growth and morphogenesis of F. vesiculosus embryos. Differences between embryos in seawater without ( Fig. 1 A & B) and with Ge ( Fig. 1 C-E) were often noticeable after the first zygote division, 22-24 h AF. Within 24 h AF nearly all control embryos became polarized, the rhizoid developed and at least one cell division occurred. The first division of the zygote was perpendicular to the polar axis of the embryo. The cell division pattern of Ge-treated embryos was much less regular, and up to 30-35% of the embryos had irregularities. Some zygotes failed to polarize forming apolar embryos. More commonly, while the rhizoid protuberance developed, the division plane of the first division was not perpendicular to the polarity axis of the embryo but oblique ( Fig. 1 C-E).
By 3-4 days AF conspicuous morphological differences developed. Ge-treated embryos were smaller, and had wider diameter rhizoid tips ( Table 1 ), which looked inflated ( Fig. 1 G & H). Differences in rhizoid morphology were particularly apparent, as control embryos had attenuated tips ( Fig. 1 F). Many of the Ge-treated embryos also had curved rhizoids ( Fig. 1 H). Whole 5 days AF embryos were significantly longer than those in Ge ( Table 1 ). This size difference was largely a function of rhizoid elongation as there was no difference in embryo ‘body’ length (i.e.,
Characteristics of control and GeO2treated embryos of Fucus vesiculosusNote: hair numbers for 8 days from different experiment. Values indicate mean ± SE (n = 10).
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Characteristics of control and GeO2 treated embryos of Fucus vesiculosus Note: hair numbers for 8 days from different experiment. Values indicate mean ± SE (n = 10).
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Developing embryos of Fucus vesiculosus. (A & B) 24 h control embryos with one (A) or two (B) whole or partial cell divisions stained with FM4-64 and showing bright membrane concentrations at rhizoid tip and along cell plates. (C) GeO2 treated 24 h embryo stained with FM4-64 showing oblique division. (D) GeO2 treated 24 h embryo showing oblique first division. (E) GeO2 treated 24 h embryo showing oblique first division (short arrow) and subsequent division of thallus cell perpendicular to the first (long arrow). (F) Control embryo at 4 days showing normal development of rhizoid and thallus. (G) GeO2 treated embryo at 4 days showing rhizoid with inflated tip. (H) GeO2 treated embryo at 4 days showing hook-shaped rhizoid with inflated tip. (I) Apex of control embryo at 8 days with three normal apical hairs. (J) Base of 8 days control embryo with typical rhizoid development. (K) Whole 8 days GeO2 treated embryo with irregular shape, inflated rhizoid tip, poor development of apical hairs and secondary rhizoid development from thallus base; arrows indicate common point of breakage of GeO2 treated embryos. (L & M) Apex of 8 days GeO2 treated embryo showing the abnormal development of apical hairs. Note the cell disaggregation. (N) Primary and secondary rhizoids of 8 days GeO2 treated embryo showing the inflated tips. Scale bars represent: A-K, 50 μm; L-N, 20 μm.
the non-rhizoidal portion). All of these metrics were significantly different between control and treatment conditions (Student’s t-test, p < 0.01).
At 7-8 days AF most control embryos had developed conspicuous up to 3 apical hairs ( Fig. 1 I). The hairs were up to six cells long, and cells were regularly cylindrical. The rhizoids were often highly branched and often developed a rhizoidal pad ( Fig. 1 J). The Ge-treated embryos were often misshapen with irregular outlines ( Fig. 1 K). These embryos were often fragile, and tended to break near the base of the ‘body’ part of the embryo ( Fig. 1 K). The apical hairs in Ge-treated embryos were sometimes absent, but always highly deformed. The cells in a given filament were spheroidal, and poorly aligned ( Fig. 1 L & M), sometimes appearing as a cell cluster at the embryo apex. The rhizoids showed the same inflated structures as previously ( Fig. 1 K & N). The tips of rhizoids were fragile and sometimes burst following resuspension (by gentle pipetting), which was never observed in control embryos. The number of rhizoid branches was reduced in Ge-treated embryos; however, some embryos did have a more usual number of, albeit deformed, rhizoid tips ( Fig. 1 N).
- Photosynthetic responses
Photosynthetic characteristics in embryos developing in seawater alone and in seawater supplement with 5 mg L -1 GeO 2 were measured over the first 10 days AF. The controls differed significantly from Ge-treated embryos
Summary of ANOVA results for two-way analyses of photosynthetic data shown inFigs 2-6
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Summary of ANOVA results for two-way analyses of photosynthetic data shown in Figs 2-6
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Dynamics of minimum fluorescence (F0) in control and GeO2 treated embryos of Fucus vesiculosus. AF, after fertilization.
in their minimum fluorescence ( Table 2 ) and generally showed higher levels of F 0 . There were several reversal over time, and at the end of the experiment, the two conditions were not significantly different ( Fig. 2 ). This corresponds well with the pigments data, that saw no differences in chlorophyll and carotenoid contents between control and Ge-treated embryos after 10 days ( Table 3 ).
While the ANOVA suggested no significant overall difference between treatments for maximum quantum yield
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Dynamics of maximum quantum yield (Fv/Fm) in control and GeO2 treated embryos of Fucus vesiculosus. AF, after fertilization.
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Dynamics of maximum electron transport rate (rETRmax) in control and GeO2 treated embryos of Fucus vesiculosus. AF, after fertilization.
Photosynthetic pigment content in 10 days after fertilization and GeO2treated embryos of Fucus vesiculosusMeans given (± conf. int., α = 0.05; n = 3); none of the differences are significant (Student’s t-test).
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Photosynthetic pigment content in 10 days after fertilization and GeO2 treated embryos of Fucus vesiculosus Means given (± conf. int., α = 0.05; n = 3); none of the differences are significant (Student’s t-test).
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Dynamics of photosynthetic efficiency (αETR) in control and GeO2 treated embryos of Fucus vesiculosus. AF, after fertilization.
( Table 2 ), this is confounded by the differences attributable to time and the highly significant time × treatment interation. The F v / F m in 1 day Fucus embryos was about 0.53 ( Fig. 3 ). During the first 5 days AF it gradually increased, and then stabilized at about 0.65. In Ge-treated embryos, quantum yield did not differ from controls after 24 h AF ( Fig. 3 ). F v / F m then increased sharply, and over the next 4 days of embryogenesis it remained significantly higher than in control embryos. The maximum difference between control and experiment conditions was about 6% at 4 days AF. After 5 days when F v / F m in control embryos reached a steady-state level, it gradually decreased in Ge-treated embryos. Thus at the end of the experiments (10 days AF) maximum quantum yield of photosystem II was significantly lower in Fucus embryos growing in the presence of Ge. Despite the decline, the mean of about 0.6 still indicates normal functioning (Garbary and Kim 2005, Kim et al. 2006).
Time, treatment and the time × treatment interaction were all significant for rETR max ( Table 2 , Fig. 4 ). The maximum value of rETR max was about 13.5 in 8 days control embryos. Higher values for rETR max were evident at the end of the experiment when controls were over 30% higher than Ge-treated embryos. Photosynthetic efficiency (α ETR ) in both control and experimental embryos remained relatively stable during the whole experiment, fluctuating slightly between 0.18-0.22 ( Fig. 5 ). While time and treatment × time interaction were significant factors, there was no overall difference between Ge and control embryos ( Table 2 ).
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Dynamics of optimum light intensity (Ik) in control and GeO2 treated embryos of Fucus vesiculosus. AF, after fertilization.
After 24 h, control and Ge-treated embryos had different optimal light intensities ( I k ). Control embryos saturated at an irradiance of about 25 μmol photons m -2 s -1 while the I k of Ge-treated embryos was ~28% higher ( Fig. 6 ). This value of optimum light intensity in Ge-treated embryos remained stable throughout the experiment, while the I k of control embryos increased gradually in the first five days AF, and then dramatically at eight days AF up to ~60 μmol photons m -2 s -1 . Thus at the end of the experiments (10 days AF) the I k in control embryos was significantly higher than in Ge-treated ones.
The rhizoid cells of control and Ge-treated two-celled embryos were significantly differed in minimum fluorescence: F 0 of rhizoid cells in Ge-treated embryos was almost 30% higher than in controls ( Table 4 ). At the same time, there were no differences in other photosynthetic parameters between these cells.
Comparison of photosynthetic parameters in rhizoid cells of control and GeO2treated 2-celled embryos of Fucus vesiculosusMeans given (± conf. int., α =0.05), significant differences marked with an asterisk (Student’s t-test, p <0.01).
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Comparison of photosynthetic parameters in rhizoid cells of control and GeO2 treated 2-celled embryos of Fucus vesiculosus Means given (± conf. int., α =0.05), significant differences marked with an asterisk (Student’s t-test, p <0.01).
- Morphological responses
Many aspects of germanium toxicity were manifest as abnormalities of Fucus embryo morphogenesis, including zygote polarization, the pattern of rhizoid growth and apical hair formation. The induction of axis polarity in fucoid zygotes has been extensively investigated (e.g., Quatrano 1974, Kropf 1997, Homblé and Léonetti 2007). Fucoid eggs have spherical shape and are initially apolar. Polarity can be observed within 14-16 h AF when the rhizoid protuberance appears on the zygote surface. The rhizoid appearance is preceded by a series of processes, including asymmetric ion fluxes (Homblé and Léonetti 2007), redistribution of cytoskeletal elements (e.g., Bisgrove and Kropf 2001 a , Bisgrove et al. 2003) and targeted vesicular secretion (Kropf 1997). Subsequently, the zygote has a single axis of symmetry that defines the embryo growth axis, and normally the first division of the zygote is perpendicular to this axis and positioned by telophase nuclei (Bisgrove and Kropf 2004). In the presence of Ge, many zygotes failed to polarize before the first division, and many 2-celled embryos had a cell plate at an oblique angle to the polarity axis. The relatively high level of minimum fluorescence ( F 0 ) in rhizoid cells of Ge-treated embryos ( Table 3 ) also may be the consequence of disturbed polarization. Generally, high values of this parameter reflect either a stressed state, or relatively high chlorophyll a content. The former is less likely, since there was no decrease in F v / F m or ETR ( Table 3 ). Thus, the high chlorophyll content (i.e., numerous plastids) in rhizoid cells could result from incorrect positioning of cell plates during the first division. This division is normally highly asymmetric, with most mitochondria in the rhizoid cell and most chloroplasts in the non-rhizoidal one (Quatrano 1974). In brown algae, cytokinesis includes participation of microfilaments in cell plate formation (Bisgrove and Kropf 2004). The delay of polarization and abnormal position of cell plates in Ge-treated zygotes suggests that Ge affects (possibly indirectly) the localization of microfilaments in Fucus cells.
Yang (1993) observed morphological changes in Ge-treated cells of brown algae. He showed that Ge altered cell wall structure in fucoid embryos, particularly in the rhizoid region; this is consistent with our data. The wall looseness and disorganized arrangement of fibrils in the rhizoid tips might be a direct effect of Ge on the cell wall (Yang 1993), but these symptoms also point to cytoskeletal malfunction. In fucoid algae the cell wall organization and function is regulated by F-actin (Bisgrove and Kropf 2001 b ).
Another hypothesis for how GeO 2 affects cell walls of brown algae is the substitution for other elements with similar chemical properties to Ge. In diatoms, Ge toxicity can be reduced by the addition of Si to the growth medium (Lewin 1966), although McLachlan et al. (1971) pointed out that this was not the case with fucoids. In our case, Ge was also not less toxic even when Si (as SiO 2 ) was added to the medium in double the concentration of added Ge (data not shown). Although this does not rule out the possibility that Si-dependent processes have no role in Ge toxicity, inhibitory effects of GeO 2 may not always be based on Si substitution.
A possible mechanism for Ge toxicity comes from higher plants where Ge leads to increased root branching and leaf necrosis (Skok 1957, McIlrath and Skok 1966). Ge may partially substitute for boron (B), where it affects cell permeability, structural integrity of cell membranes and cell wall formation (McIlrath and Skok 1966, Cakmak et al. 1995). Boron resembles Si in physical and chemical properties, although unlike Si, which is not essential for most plants, B is almost universally required. Boron is normally absorbed by plants as undissociated boric acid, and it accumulates in both the cytoplasm and cell walls (Dembitsky et al. 2002, Bassil et al. 2004). Free and complex forms of accumulated borate occur in different seaweeds, including green, red and brown algae. Unlike other algae, brown algae contain considerable B in diverse B-containing compounds, including complexes with mannitol, laminarin, and alginic acid (Chuda et al. 1997, Dembitsky et al. 2002). If Ge can also substitute for B in brown algae, that might explain their increased sensitivity to GeO 2 compared with green or red algae (Tatewaki and Mizuno 1979). Both B and Ge can form complex compounds with other membrane and cell wall components such as phenols (Cakmak et al. 1995), and brown algae have high contents of phenolic compounds (Schoenwaelder 2002, 2008). Phenolics play a critical role in early development of fucoids participating in cell wall formation, zygote adhesion to the substratum and polar axis formation (Schoenwaelder and Wiencke 2000, Bisgrove and Kropf 2001 b ). Most Ge-treated embryos had abnormal rhizoid growth. Algal rhizoid elongation is mediated via tip growth. The requirements for this process were extensively investigated in pollen tubes and root hairs (e.g., Holdaway-Clarke and Hepler 2003, Galway 2006). Elimination of boric acid from culture media often leads to pollen tubes bursting at their tips (Cheng and McComb 1992, Holdaway-Clarke and Hepler 2003); we observed this symptom in rhizoids of Ge-treated Fucus embryos.
- Photosynthetic responses
Our observations on the development of the photosynthetic apparatus in fucoid embryos are consistent with the previous investigations of this process carried out with similar (Lamote et al. 2003, Kim et al. 2006, Kim and Garbary 2009) or other methods (McLachlan and Bidwell 1978, Major and Davison 1998, Tarakhovskaya and Maslov 2005). Kim et al. (2006) had similar results with another fucoid, Ascophyllum nodosum . The major difference in photosynthetic development during embryogenesis between Fucus and Ascophyllum is that the latter did not show a significant increase in maximum quantum yield, whereas in Fucus we observed a 20% increase during the first 5 days AF.
There were no conspicuous changes in F 0 in Ge-treated embryos. Since F 0 provides a relative measure of chlorophyll a content, we conclude that Ge apparently had no effect on synthesis or degradation of this pigment. The direct measurements of photosynthetic pigment content in Fucus embryos support this conclusion, for even after 10 days there was no difference between control and experimental conditions in chlorophylls a and c and total carotenoids.
While maximum quantum yield and rETR max significantly increased in Ge-treated embryos during the first 5 days AF, at the same time, total growth was reduced and highly abnormal. This seems counterintuitive; however, Lewin (1966) showed that GeO 2 , though extremely deleterious for diatoms, had no negative influence on energy metabolism of the cells, and even at 400 mg L -1 GeO 2 , cell respiration did not decrease. Shea and Chopin (2007) also reported that low concentrations of GeO 2 (up to 0.5 mg L -1 ) might sometimes have a slightly positive effect on growth of brown algae. After 5 days, F v / F m and rETR max significantly decreased in Ge-treated embryos, relative to control. This effect may reflect the morphological observations of embryos. Normally, apical hairs form at this stage of embryo development. We showed that Ge-treated embryos failed to form normal hairs, though they previously formed primary and secondary rhizoids. Thus Fucus embryos can develop (though highly abnormally) in the presence of Ge, up to about the 5-6 days AF. Subsequently, development nearly ceases, embryos never form normal apical hairs, and embryo disintegration occurs. McLachlan et al. (1971) observed apical necrosis in older Fucus embryos treated with Ge. The decline of photosynthetic processes may also reflect this general collapse of embryo development. At 5 days AF, F v / F m in control embryos reached a steady-state, suggesting that electron transport processes and coupled biochemical reactions in the Calvin cycle were equilibrated (Roháček 2002). This was not the case in Ge-treated embryos where it was not the structural components of the photochemical part of the assimilation apparatus (e.g., pigments) that were affected, but the processes maintaining the balance between electron transport and carbon reduction.
The developmental abnormalities of Fucus that result from exposure to GeO 2 support the notion that Ge is interfering with essential cell processes including growth rate, morphogenesis, and to a lesser extent, photosynthesis. The affects of Ge in Phaeophyceae as shown here are more complex than previously considered and warrant further study.
We thank Taizo Motomura for providing key literature, and Denise Wallbank, Moira Galway, Lori Graham, William Marshall and Judith Pink for providing reagents and laboratory facilities. Katelyn White assisted with preparation of the plate. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada to DJG. Partial support was provided by the project titled “Long-term change of structure and function in marine ecosystems of Korea” funded by the Ministry of Land, Transport and Maritime Affairs, Korea.
Bassil E. , Hu H. N. , Brown P. H. 2004 Use of phenylboronic acids to investigate boron function in plants: possible role of boron in transvacuolar cytoplasmic strands and cell-to-wall adhesion. Plant Physiol. 136 3383 - 3395
Belanger K. D. , Quatrano R. S. 2000 Membrane recycling occurs during asymmetric tip growth and cell plate formation in Fucus distichus zygotes. Protoplasma 212 24 - 37
Bisgrove S. R. , Henderson D. C. , Kropf D. L. 2003 Asymmetric division in fucoid zygotes is positioned by telophase nuclei. Plant Cell 15 854 - 862
Bisgrove S. R. , Kropf D. L. 2001a Asymmetric cell division in fucoid algae: a role for cortical adhesions in alignment of the mitotic apparatus. J. Cell Sci. 114 4319 - 4328
Bisgrove S. R. , Kropf D. L. 2001b Cell wall deposition during morphogenesis in fucoid algae. Planta 212 648 - 658
Bisgrove S. R. , Kropf D. L. 2004 Cytokinesis in brown algae: studies of asymmetric division in fucoid zygotes. Protoplasma 223 163 - 173
Bolte S. , Talbot C. , Boutte Y. , Catrice O. , Read N. D. , Satiat-Jeunemaitre B. 2004 FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J. Microsc. 214 159 - 173
Cakmak I. , Kurz H. , Marschner H. 1995 Short-term effects of boron, germanium and high light intensity on membrane permeability in boron deficient leaves of sunflower. Physiol. Plant. 95 11 - 18
Cheng C. , McComb J. A. 1992 In vitro germination of wheat pollen on raffinose medium. New Phytol. 120 459 - 462
Chiappino M. L. , Azam F. , Volcani B. E. 1977 Effect of germanic acid on developing cell walls of diatoms. Protoplasma 93 191 - 204
Chuda Y. , Ohnishi-Kameyama M. , Nagata T. 1997 Identification of the forms of boron in seaweed by11B-NMR. Phytochemistry 46 209 - 213
Dembitsky V. M. , Smoum R. , Al-Quntar A. A. , Abu Ali H. , Pergament I. , Srebnik M. 2002 Natural occurrence of boron-containing compounds in plants, algae and microorganisms. Plant Sci. 163 931 - 942
Galway M. E. 2006 Root hair cell walls: filling in the framework. Can. J. Bot. 84 613 - 621
Garbary D. J. , Kim K. Y. 2005 Anatomical differentiation and photosynthetic adaptation in brown algae. Algae 20 233 - 238
Garbary D. J. , Kim K. Y. , Hoffman J. 2004 Cytological damage to the red alga Griffithsia pacifica from ultraviolet radiation. Hydrobiologia 512 165 - 170
Garbary D. , Tam C. 1989 Blidingia minima var. stolonifera var. nov. (Ulvales, Chlorophyta) from British Columbia: systematics, life history and morphogenesis. Nord. J. Bot. 9 321 - 328
Genty B. , Briantais J. -M. , Baker N. R. 1989 The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990 87 - 92
Hadley R. , Hable W. E. , Kropf D. L. 2006 Polarization of the endomembrane system is an early event in fucoid zygote development. BMC Plant Biol. 6 5 -
Harrison W. G. , Platt T. 1986 Photosynthesis-irradiance relationships in polar and temperate phytoplankton populations. Polar Biol. 5 153 - 164
Henley W. J. , Dunton K. H. 1995 A seasonal comparison of carbon, nitrogen and pigment content in Laminaria solidungula and L. saccharina (Phaeophyta) in the Alaskan Arctic. J. Phycol. 31 325 - 331
Holdaway-Clarke T. L. , Hepler P. K. 2003 Control of pollen tube growth: role of ion gradients and fluxes. New Phytol. 159 539 - 563
Homblé F. , Léonetti M. 2007 Emergence of symmetry breaking in fucoid zygotes. Trends Plant Sci. 12 253 - 259
Hubbard C. B. , Garbary D. J. , Kim K. Y. , Chiasson D. M. 2004 Host specificity and growth of kelp gametophytes symbiotic with filamentous red algae (Ceramiales, Rhodophyta). Helgol. Mar. Res. 58 18 - 25
Jaffe L. F. , Neuscheler W. 1969 On the mutual polarization of nearby pairs of fucaceous eggs. Dev. Biol. 19 549 - 565
Jeffrey S. W. , Humphrey G. F. 1975 New spectrophotometric equations for determining chlorophylls “a”, “b”, “c1” and “c2” in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz. 167 191 - 194
Kim K. Y. , Garbary D. J. 2009 Form, function and longevity in fucoid thalli: chlorophyll a fluorescence differentiation of Ascophyllum nodosum, Fucus vesiculosus and F. distichus (Phaeophyceae). Algae 24 93 - 104
Kim K. Y. , Jeong H. J. , Main H. P. , Garbary D. J. 2006 Fluorescence and photosynthetic competency in single eggs and embryos of Ascophyllum nodosum (Phaeophyceae). Phycologia 45 331 - 336
Kropf D. L. 1997 Induction of polarity in fucoid zygotes. Plant Cell 9 1011 - 1020
Lamote M. , Darko E. , Schoefs B. , Lemoine Y. 2003 Assembly of the photosynthetic apparatus in embryos from Fucus serratus L. Photosynth. Res. 77 45 - 52
Lewin J. 1966 Silicon metabolism in diatoms. V. Germanium dioxide, a specific inhibitor of diatom growth. Phycologia 6 1 - 12
Major K. M. , Davison I. R. 1998 Influence of temperature and light on growth and photosynthetic physiology of Fucus evanescens (Phaeophyta) embryos. Eur. J. Phycol. 33 129 - 138
Markham J. W. , Hagmeier E. 1982 Observations on the effects of germanium dioxide on the growth of macro-algae and diatoms. Phycologia 21 125 - 130
McIlrath W. J. , Skok J. 1966 Substitution of germanium for boron in plant growth. Plant Physiol. 41 1209 - 1212
McLachlan J. 1973 Growth media: marine. In Stein, J. R. (Ed.) Handbook of Phycological Methods: Culture Methods and Growth Measurements. Cambridge University Press Cambridge 25 - 51
McLachlan J. 1977 Effects of nutrients on growth and development of embryos of Fucus edentatus Pyl. (Phaeophyceae, Fucales). Phycologia 16 329 - 338
McLachlan J. , Bidwell R. G. S. 1978 Photosynthesis of eggs, sperm, zygotes, and embryos of Fucus serratus. Can. J. Bot. 56 371 - 373
McLachlan J. , Chen L. C. -M. , Edelstein T. 1971 The culture of four species of Fucus under laboratory conditions. Can. J. Bot. 49 1463 - 1469
Mizuta H. , Yasui H. 2011 Protective function of silicon deposition in Saccharina japonica sporophytes (Phaeophyceae). J. Appl. Phycol.
Parker B. C. 1969 Occurrence of silica in brown and green algae. Can. J. Bot. 47 537 - 540
Platt T. , Gallegos C. L. , Harrison W. G. 1980 Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J. Mar. Res. 38 687 - 701
Quatrano R. S. 1974 Developmental biology: development in marine organisms. In Mariscal, R. N. (Ed.) Experimental Marine Biology. Academic Press New York 303 - 346
Roháček K. 2002 Chlorophyll fluorescence parameters: the definitions, photosynthetic meaning, and mutual relationships. Photosynthetica 40 13 - 29
Schoenwaelder M. E. A. 2002 The occurrence and cellular significance of physodes in brown algae. Phycologia 41 125 - 139
Schoenwaelder M. E. A. 2008 The biology of phenolic containing vesicles. Algae 23 163 - 175
Schoenwaelder M. E. A. , Wiencke C. 2000 Phenolic compounds in the embryo development of several northern hemisphere fucoids. Plant Biol. 2 24 - 33
Scrosati R. , Garbary D. J. , McLachlan J. 1994 Reproductive ecology of Chondrus crispus (Rhodophyta, Gigartinales) from Nova Scotia, Canada. Bot. Mar. 37 293 - 300
Shea R. , Chopin T. 2007 Effects of germanium dioxide, an inhibitor of diatom growth, on the microscopic laboratory stage of the kelp, Laminaria saccharina. J. Appl. Phycol. 19 27 - 32
Skok J. 1957 The substitution of complexing substances for boron in plant growth. Plant Physiol. 32 308 - 312
Tarakhovskaya E. R. , Maslov Y. I. 2005 Description of the photosynthetic apparatus of Fucus vesiculosus L. in early embryogenesis. Biol. Bull. (in Russian). 32 456 - 460
Tatewaki M. , Mizuno M. 1979 Growth inhibition by germanium dioxide in various algae, especially in brown algae. Jpn. J. Phycol. 27 205 - 212
Yang W. X. 1993 Morphological study on the inhibitory effect of germanium dioxide on growth and development of brown algae. Sci. Pap. Inst. Algol. Res. Fac. Sci. Hokkaido Univ. 9 33 - 64