УДК 581.1 Influence of Phytohormones on Morphology and Chlorophyll a Fluorescence Parameters in Embryos of Fucus vesiculosus (Phaeophyceae) 1 © 2013 E. R. Tarakhovskaya*, E. J. Kang**, K. Y. Kim**, D. J. Garbary*** * Department of Plant Physiology and Biochemistry, St. Petersburg State University, St. Petersburg ** Department of Oceanography, Chonnam National University, Gwangju, Korea *** Department of Biology, St. Francis Xavier University, Antigonish, Nova Scotia, Canada Received March 11, 2012 While a variety of plant hormones from brown algae were described, there were few studies that examined the combined effects of these hormones on morphogenesis and photosynthetic physiology in developing fucoid embryos. We evaluated the effects of phytohormones to determine the extent, to which responses were similar to those of terrestrial plants. Kinetin, IAA, ABA, GA3, and kinetin + IAA were added to seawater at a physiological concentration (1 mg/L), and embryos of Fucus vesiculosus L. were grown for 10 days. Photosynthetic activity of single embryos or embryo cells were characterized using the following fluorescence parameters: minimum fluorescence yield (F0), maximum quantum yield (Fv/Fm), relative maximum rate of electron transfer to photosystem II under saturation irradiances (rETRmax), photosynthetic efficiency under non-saturating irradiances (ETR) and saturation irradiance (Ek). In addition, embryo length and diameter and apical hair length and number were determined. Morphological changes associated with hormone treatments included an increase in the embryo length in the presence of IAA, an increase in the embryo diameter in the presence of IAA, kinetin, and kinetin + IAA, an increase in the maximum hair length and number in the presence of kinetin + IAA, and a decrease in the hair length and number in the presence of ABA. With respect to fluorescence parameters, significant effects of phytohormones included an increase in the F0 and Fv/Fm at kinetin treatment, a synergistic effect of kinetin + IAA on Fv/Fm, rETRmax, and ETR, a promotion of Fv/Fm by GA, and a decrease of the parameters by ABA. These results are consistent with responses of land plants to the same hormones and suggest that brown algae have evolved regulatory mechanisms for morphogenesis and photosynthetic regulation similar to plants. ------------------------- 1 This text was submitted by the authors in English. ------------------------- Abbreviations: AF  after fertilization; ETR  photosynthetic efficiency under non-saturating irradiances; Ek  saturation irradiance; F0  minimum fluorescence yield of PSII photochemistry; Fv/Fm  maximum quantum yield of PSII photochemistry; PAM  pulse amplitude modulation; PS  photosystem; rETRmax  relative maximum rate of electron transfer to PSII under saturation irradiances. Corresponding author: E. R. Tarakhovskaya. Department of Plant Physiology and Biochemistry, St. Petersburg State University. Universitetskaya nab., 7/9, St. Petersburg, 199034 Russia. E-mail: elena.tarakhovskaya@gmail.com Keywords: Fucus vesiculosus  photosynthetic activity  chlorophyll a fluorescence  phytohormones  morphogenesis INTRODUCTION Phytohormones contribute to the regulation of most aspects of the plant life cycle, including the development and functioning of the photosynthetic apparatus. Although hormone function and mechanism of action have been extensively studied (for review see [1, 2]), the role of most hormones in the regulation of photosynthetic processes is still poorly understood. Cytokinins and ABA were most active and most thoroughly investigated. The hormones produce a variety of responses and influence photosynthetic processes through different mechanisms (e.g., [35]). The study of other hormones is even more preliminary and they produced ambiguous results. This may be a consequence of the narrow spectrum of model systems used in such studies. Thus, most investigations used representatives of higher plants or microalgae, and algal groups with their diversity of photosynthetic systems were poorly investigated. Seaweeds possess almost all known phytohormones [6]. Furthermore, auxins, cytokinins, and ABA occur in brown seaweeds (e.g., Fucus, Ascophyllum, Laminaria) at concentrations similar to those in land plants [68]. Numerous elaborate physiological and biochemical processes, including thallus growth and morphogenesis, formation of reproductive structures, regeneration, and stress adaptation were reported as being regulated by phytohormones in some algal species [6]. Exogenous application of natural and synthetic auxins and cytokinins affected the induction of polarity, germination and growth of fucoid zygotes [8, 9]. Zygotes and embryos of these algae develop independently of maternal tissues and are photosynthetically competent [10, 11]. Thus, the energy and structural elements necessary for embryo development must be, at least partially, produced by photosynthesis. Here we address the question: can phytohormones regulate photosynthetic parameters characterized by chlorophyll a fluorescence measurements in developing zygotes and embryos of fucoids? Fertilization of fucoid eggs occurs externally in seawater. Consequently, the development of zygotes and embryos is independent of maternal tissues. This feature makes fucoid embryos an excellent model system, especially since they are easily cultivated in laboratory conditions [12, 13]. After the first asymmetric division (within 24 h of fertilization), fucoid zygotes form two cells that differ in morphological and physiological characteristics. The upper or "thallus" cell subsequently gives rise to the frond, thus producing all the organs and structures of the erect plant. The lower cell initially forms a rhizoid, and this eventually develops into the holdfast. This anatomical differentiation defines a physiological differentiation that has implications for the photosynthetic characteristics of the embryo as a whole. Even as two-celled, the thallus and rhizoidal cells are obviously different because most chloroplasts after the first division are in the thallus cell [12]. Thus, from the perspective of photosynthetic processes, these two cells and their division products should be considered separately. Pulse amplitude modulation (PAM) fluorometry uses fluorescence characteristics of chlorophyll a to characterize the photosynthetic processes in plant cells in vivo [1416]. Microscopy-PAM (“Walz”, Germany) was developed to allow for the characterization of fluorescence parameters that reflect photosynthetic activity on very small spatial scales, and also in single cells. This technology provides an excellent approach for investigating the development of the photosynthetic apparatus during fucoid embryogenesis. PAM fluorometry has been used previously for studies of fucoid photosynthesis [17, 18]. Kim et al. [11] used Microscopy-PAM to study photosynthetic parameters in unfertilized eggs and developing embryos of Ascophyllum nodosum. The objective of this paper was to examine the influence of several key phytohormones (IAA, kinetin, GA, and ABA) on the photosynthetic activity of developing embryos of Fucus vesiculosus. In addition, we examined differentiation of photosynthetic parameters in rhizoid and thallus cells of two-celled embryos. MATERIALS AND METHODS Experimental material and culturing. Fucus vesiculosus L. was collected at Tor Bay Provincial Park (45.19° N, 61.34° W) on the Atlantic coast of Nova Scotia, Canada in June and 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°C for up to ten days. We followed Quatrano [12] to obtain gametes and fertilization. Immediately after fertilization (AF) the zygotes were divided into several groups and each group was placed in Petri dishes in seawater previously heated to 90°C on two successive days. Developing zygotes and embryos were maintained in Petri dishes with seawater at 15°C under continuous light provided by cool white fluorescent tubes at an irradiance of 2025 µmol photons/(m2 s). Embryos were grown in seawater alone (control) or in seawater supplemented with 1 mg/L IAA (I-3750), kinetin (K3378), GA3 (G7645), and ABA (862169) (all from “Sigma-Aldrich”) as well as with the mixture of IAA and kinetin (each at 1 mg/L). Phytohormones were added to the experimental cultures from the stock solutions just AF. Stock solutions of the hormones IAA, ABA, and GA were prepared in 96% alcohol, whereas kinetin was dissolved in 0.05 M HCl. The final concentrations of alcohol and HCl in seawater were not higher than 0.05%. Preliminary experiments showed that this had no significant effect on pH in the medium or embryo development (data not shown). To minimize breakdown of the phytohormones, medium was replenished daily. Samples for fluorescence measurement and analysis of morphology were harvested 2 h after egg release from oogonia and then 1, 2, 3, 6, 8, and 10 days AF. Morphometric analysis. Eight-day-old embryos were measured using a calibrated Olympus BH2 microscope at 200 magnification. We measured embryo length and maximal diameter of the thallus part of embryos (excluding apical hairs). In addition, the number and maximal length of apical hairs were determined. Chlorophyll fluorescence measurement. Variable chlorophyll a fluorescence of F. vesiculosus embryos was measured using a Microscopy-PAM apparatus comprising a computer-operated PAM-Control Unit (“Walz”) as described previously [11, 19]. This instrument employs a blue light emitting diode with a peak emission at 470 nm, which not only serves as sources of measuring light, but of actinic light and saturation pulses as well. Light was measured using a special pin-hole Micro Quantum Sensor (“Walz”) that had been calibrated against a Li-Cor quantum sensor (“Li-Cor”, United States). During photosynthetic measurements the active fluorescence field was set at 65µm diameter. Fluorescence parameters were determined for 1015 separate embryos in each phytohormone treatment at each time interval. The maximum (Fm) and minimum (F0) fluorescence, and the maximum quantum yield of PSII (Fv/Fm = (Fm  F0)/Fm) were determined for all embryos after being acclimated to darkness for 10 min in the object plane. Minimum fluorescence provides a proxy of chlorophyll biomass in an unstressed photosynthetic system [20], and the maximum quantum yield provides a measure of the charge separation in the reaction centre of PSII [14]. The higher value for Fv/Fm, the greater the proportion of photons that is resulting in charge separation within the reaction centre. For healthy leaves of flowering plants, values of 0.790.82 are typical [14], and in fucoids these values are usually lower, i.e., values of 0.5 to 0.7 [11, 19]. The effective quantum yield was determined as PSII = F/Fm' = (Fm'  F)/Fm' following Genty et al. [21]; where F is the steady-state fluorescence, Fm' is the maximum fluorescence and F is variable fluorescence of a light-acclimated samples. Relative electron transport rates (rETR) were calculated as the product of the quantum efficiency of PSII, the absorbed photon flux density, and absorbance factor; rETR = PSII × PPFD × 0.84 × 0.5, where PPFD is photosynthetic photon flux density of photosynthetically active radiation (PAR, 400700 nm). This equation assumes that PSII absorbs half the quanta of available light and an average photosynthetic unit absorbs about 84% of incident PAR [14]. The rapid light curves were generated automatically with a Microscopy-PAM using an incremental sequence of actinic illumination periods, with light intensities increasing in nine steps from 0 to 130 µmol photons/(m2 s). Each illumination period lasted 10 s, at the end of which time, F, and following a saturation pulse, Fm', were measured. To determine the photosynthetic parameters ETR, Ek, and rETRmax, where ETR is photosynthetic efficiency under non-saturating irradiances, Ek is saturation irradiance, and rETRmax is the relative maximum rate of electron transfer to PSII under saturation irradiances, empirical data from the RLC were mathematically fitted to an asymptotic function. For data analysis ANOVA was carried out using STATISTICA 5.5 (“StatSoft”, United States). Prior to testing, data were evaluated for homogeneity of variances using Cochran’s C-test. Fv/Fm data were arcsine square-root transformed and rETRmax and Ek data were log transformed prior to analysis to meet assumptions of normality. Values are expressed as means and standard errors. RESULTS Dynamics of fluorescence parameters reflecting the photosynthetic activity in early embryogenesis Minimum fluorescence (F0) in zygotes and developing embryos of F. vesiculosus was stable throughout the study period with values of 280310; these values did not differ from F0 in unfertilized eggs (data not shown). Maximum quantum yield (Fv/Fm) was about 0.54 in Fucus eggs (fig. 1a). After fertilization the Fv/Fm increased, and at two days the Fv/Fm significantly exceeded that of eggs. During the first six days of embryo development, it gradually increased and stabilized at approximately 0.65. The maximum electron transport rate (rETRmax) also changed during the first ten days of embryogenesis (fig. 1b). The rETRmax in eggs was ~2.89, and this value increased significantly by one day AF. The rETRmax increased gradually over five days and then sharply to ~13.44 at eight days AF. During the following two days, this value decreased slightly. Photosynthetic efficiency (ETR) was ~0.11 in Fucus eggs, then almost doubled by day one and remained stable until the end of the experiment (data not shown). Consequently, the saturation irradiance (Ek) was largely a reflection of rETRmax (fig. 1c). The same fluorescence parameters were measured separately in thallus and rhizoid cells of two-celled embryos (table 1). These two cells showed significantly different values of F0 (by ~45% higher in the thallus cell). There was also a difference in Fv/Fm and Ek between the cells, and these parameters were higher in rhizoid cells. Though not large, these differences were still statistically significant. There were no significant differences in rETRmax and ETR between the two types of cells of the embryo, although rETRmax in the rhizoid cell was slightly higher than in the thallus cell. Phytohormone influence on chlorophyll fluorescence parameters We determined the fluorescence parameters in embryos after one and six days of exposure to various hormone treatments. Kinetin increased minimum fluorescence (F0) and maximum quantum yield (Fv/Fm) after six days (figs. 2a, 2b), without significantly affecting other photosynthetic parameters. The combination of kinetin and IAA produced even greater stimulation of Fv/Fm, rETRmax, and ETR (figs. 2b2d), and increased the optimal light intensity (Ek) for the embryos (fig. 2e). With this treatment the stimulatory effect on photosynthetic processes was already apparent by day one (fig. 2b). When applied without cytokinin, IAA decreased Fv/Fm, rETRmax, and ETR (figs. 2b2d). In addition, the inhibitory effect of ABA was even stronger both in one- and six-day embryos (figs. 2b2e). Gibberellic acid had no effect after one day, but increased Fv/Fm in six-day embryos (fig. 2b). Morphological responses to phytohormones Auxin, kinetin, and their combination significantly affected the growth of F. vesiculosus during the first eight days of development (table 2). In the presence of IAA, the length and diameter of the “thallus part” of eight-day embryos were about 20% greater than in control conditions. When applied together with kinetin, IAA instead of inducing the uniform increase of embryo size stimulated only widening of the embryos. Kinetin alone had the same effect. The majority of control embryos had apical hairs after eight days. Up to four hairs were present, but most embryos had only a single hair. In the presence of IAA + kinetin, hair number increased to at least two well developed hairs (table 2). Embryos in ABA often had no hairs at this stage of embryogenesis, and they never had more than one hair. The length of the largest apical hair was also measured, and the combination of IAA + kinetin apparently stimulated apical hair growth, whereas ABA inhibited both hair formation and growth (table 2). GA had no significant effect on any measured size characteristics of F. vesiculosus embryos. DISCUSSION Dynamics of fluorescence parameters during Fucus embryogenesis McLachlan and Bidwell [10] initially demonstrated that the eggs, sperm, and zygotes of F. serratus were photosynthetically competent and that photosynthetic activity of the embryos increased rapidly during the first days of embryogenesis. The dynamics of the photosynthetic apparatus development in F. vesiculosus embryos we observed is consistent with previous studies based on 14C incorporation, oxygen evolution, and fluorescence measurement [10, 11, 22]. Thus, our results confirmed that chlorophyll a fluorescence data is a proxy for photosynthetic processes. We demonstrated for the first time that thallus and rhizoid cells of two-celled Fucus embryos had significantly different photosynthetic characteristics (table 1). These two cells are the products of the highly asymmetric division of the Fucus zygote, which usually occurs about 2022 h AF. These cells differ fundamentally in their morphology, physiology, and subsequent fate [12]. The most evident morphological differences are the following. The thallus cell is larger, approximately spherical, and does not undergo conspicuous growth, whereas the rhizoid cell is conical and elongates actively via tip growth. Organelles are not symmetrically distributed: chloroplasts are concentrated in the thallus cell, whereas mitochondria and Golgi structures are more abundant in the rhizoid cell [12, 23]. The different values of F0, we observed in these cells (table 1), most likely reflect the uneven chloroplast numbers and chlorophyll a content [20]. Tip growth is highly energy-dependent based on the constant production and redistribution of numerous membrane and cell wall components. We suggest that intense growth of rhizoids is more dependent on local energy production than on import from the thallus part of the embryo. This is supported by the high quantum yield shown by these cells. However, high quantum yield in rhizoid cells is not maintained for a long time, as chloroplasts in rhizoids tend to degrade, beginning from the four-celled stage of embryogenesis [23]. Responses to phytohormones Application of phytohormones to embryos of F. vesiculosus affects chlorophyll fluorescence characteristics that reflect their photosynthetic activity. Kinetin increased both F0 and Fv/Fm (figs. 2a, 2b). In the absence of photoinhibition the level of F0 apparently provides a measure of relative chlorophyll a content. Thus, we concluded that kinetin increased pigment quantity. This is a common response of terrestrial plant cells where cytokinins contribute to the regulation of many physiological processes connected with photosynthetic function. This includes leaf senescence, de-etiolation, and plastid formation (e.g., [4]). Cytokinin treatment also inhibits the degradation of chlorophyll and proteins of light-harvesting complexes (LHC) [3]. Cytokinins induce the biosynthesis of various proteins encoded both in the nuclear and plastid genomes, including Rubisco small subunit and LHC chlorophyll-binding proteins [3, 24, 25]. A number of cytokinins have been found in various algae, including fucoids [6]. Accordingly, we concluded that these hormones may function as activators of photosynthetic processes in fucoids similar to land plants. It is interesting that kinetin increased Fv/Fm, but not rETRmax. The latter parameter is calculated based on effective quantum yield; hence, it shows the photosynthetic efficiency that is actually achieved. The maximum quantum yield is only a potential efficiency (the quantum efficiency if all PSII centers are open). We suggested that the improvement of Fv/Fm without significant changes in rETR may be interpreted as a result of the increase of the structural elements of photosynthetic machinery (e.g., chlorophyll a including the PSII reaction centres) without subsequent functional integration of components of the system. Our results suggest that IAA has an inhibitory effect on photosynthetic processes in Fucus (figs. 2b2d). The role and importance of this hormone in photosynthetic regulation is still poorly understood. Natural and synthetic auxins have both positive and negative influences on pigment content, intensity of CO2 fixation, etc., depending upon the model system and experimental conditions (e.g., [26]). Our experiments suggest that when treated with auxin, Fucus embryos use cell resources to accelerate growth and differentiation instead of developing the photosynthetic potential. It was shown here (table 2) and elsewhere [8, 9] that exogenous IAA stimulated growth and development of fucoid zygotes and embryos, as demonstrated by accelerated polarization and rhizoid and apical hair formation. The most pronounced IAA effect is the transient decrease in the initial slope of the light curve (ETR) resulting in the increase of the saturation irradiance (figs. 2d, 2e). The possible explanation is the increasing demand of ATP in the period of zygote polarization, cell wall formation, and first division (first 24 h AF). The extra ATP may be produced via cyclic phosphorylation reactions, which require PSI activation. This may lead to the transient decrease of PSII photosynthetic efficiency. Phytohormones interact, and these interactions form the part of a complex regulatory system. There are both metabolic (one hormone influencing the synthesis or degradation of the other) and functional (joint regulation of physiological processes) interactions among plant hormones. Characteristic examples of this phenomenon are the interactions between GA and ABA (joint regulation of gene expression coding hydrolytic enzymes in the cereal aleurone layer), and especially between IAA and cytokinins (cell cycle regulation, morphogenetic processes) [27, 28]. The joint action of phytohormones in algae has been virtually unstudied, thus highlighting the importance of our results. Having opposite effects on photosynthetic processes when applied separately, IAA and cytokinin worked synergistically in combination. This combination stimulated photosynthesis of Fucus embryos even more than kinetin alone (figs. 2b2d). In these conditions, cell growth and the development of assimilatory systems may be stimulated simultaneously. We may suggest that this effect may be similar to the synergistic action of these hormones contributing to the cell cycle regulation in higher plants [28]. Unlike kinetin alone, the IAA + kinetin combination increased not only potential PSII quantum yield (Fv/Fm), but also the parameters characterising actually achieved photosynthetic efficiency (rETRmax and ETR). The ETR improvement may be of special interest due to its environmental implications. This parameter shows photosynthetic efficiency under low irradiances. The typical habitat of the fucoid embryos is under the canopy of the adult macroalgal thalli; thus, the effective use of low irradiances can be crucial for their development. ABA regulates a diverse range of physiological processes in higher plants, including seed formation and dormancy, adaptation to environmental impacts, and senescence [1]. This hormone accumulates in aging tissues, and this leads to protein and chlorophyll degradation, inhibition of photosystem activity, CO2 consumption and Rubisco biosynthesis [3, 5, 29]. Thus, decreases in chlorophyll a fluorescence parameters we observed in embryos of F. vesiculosus (figs. 2b2d) are entirely consistent with ABA effects on tissues of terrestrial plants. It remains to be determined whether cytokinins and ABA contribute to photosynthetic regulation in seaweeds via the same mechanisms as in higher plants. One of the key functions of ABA both in land plants and in algae is participation in a systemic response to stress conditions. ABA content increases in plant tissues during environmental stresses, such as high temperature and irradiance, and this is especially important for developing fucoid embryos [5, 6, 17]. Thus, by artificially increasing ABA concentration we modeled a stress situation, and reductions in rETRmax and ETR may reflect the first stage of stress adaptation  transient inhibition of anabolic processes. Maximum quantum yield was significantly improved in GA-treated embryos after six days (fig. 2b). In general, photosynthetic regulation is not considered among the primary functions of gibberellins in higher plants [1]. However, using tobacco plants with modified gibberellin biosynthesis, Biemelt et al. [30] showed an increase in photosynthetic activity caused by overproduction of the hormone. Our data support the hypothesis that GA stimulates at least some elements of the photosynthetic machinery, such as maximum quantum yield of PSII in plants, including seaweeds (fig. 2b). It might seem counterintuitive that there was no influence of GA on embryo growth (table 2) because growth stimulation is usually the most evident effect of this hormone both in terrestrial plants [1] and in algae [6]. This apparent contradiction is explained by Major and Davison [17] observation that growth is not always a direct function of the photosynthetic rate in early embryogenesis of fucoids. There was a strong congruence between the responses of embryos of F. vesiculosus that we observed and what would be predicted based on the more fully explored responses of land plants. This is in spite of the huge phylogenetic gap of at least one billion years that separate these lineages. Indeed, the evolutionary separation between Phaeophyceae and land plants is deep in the eukaryotic domain. Furthermore, these two multicellular lineages, that independently evolved tissues and meristems, are separated by numerous classes and phyla of unicellular lineages. 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Phytohormone influence on minimum fluorescence (F0) (a), maximum quantum yield (Fv/Fm) (b), maximum electron transport rate (rETRmax) (c), photosynthetic efficiency (ETR) (d) and saturation irradiance (Ek) (e) in F. vesiculosus embryos. Values indicate means ± SE (n = 10). Asterisks indicate values significantly differing from control (ANOVA, p < 0.01). 1  control; 2  IAA; 3  kinetin; 4  IAA + kinetin; 5  GA; 6  ABA.