Cytokinins Induce Photomorphogenic Development in Dark-grown Gametophytes of Ceratopteris richardii

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1 Plant Cell Physiol. 45(9): (2004) JSPP 2004 Cytokinins Induce Photomorphogenic Development in Dark-grown Gametophytes of Ceratopteris richardii Mark D. Spiro 1, Behzad Torabi and Catharine N. Cornell Biology Department, Bucknell University, Lewisburg, PA 17837, U.S.A. The cytokinins benzylaminopurine, kinetin and isopentenyladenine induce photomorphogenesis in darkgrown gametophytes of the fern Ceratopteris richardii. At sub-nanomolar concentrations each altered the rate and pattern of cell division, elongation and differentiation, mimicking aspects of the light-mediated transition from filamentous to prothallial growth. Untreated dark-grown gametophytes grow as narrow, elongate, asexual filaments with an apical meristem. Cytokinin treatments as low as M reduced the length-to-width ratio through decreased cell elongation, increased periclinal cell division and induced the formation of rhizoid initials in the cells immediately below the apical meristem. Higher concentrations ( M) induced conversion of the meristem from apical to notch morphology. Cytokinins induced both red- and blue-light-mediated photomorphogenic events, suggesting stimulation of both phytochrome and cryptochrome signaling; however, cytokinin treatment only partially substituted for light in that it did not induce hermaphroditic sexual development or spore germination in the dark. Additionally, cytokinins did not increase chlorophyll synthesis in dark-grown gametophytes, which unlike angiosperms are able to produce mature chloroplasts in the dark. Cytokinin treatment had only slight effects on light-grown gametophytes. These results suggest evolutionary conservation between angiosperms and pteridophytes in the role of cytokinins in regulating photomorphogenesis. Keywords: Ceratopteris richardii Cytokinin Gametophyte Photomorphogenesis. Abbreviations: BAP, benzylaminopurine; ip, N 6 ( 2 -isopentenyl) adenine. Introduction Cytokinins appear to be involved in the regulation of a number of light-mediated responses in higher plants. As early as 1956, 2 years after their discovery, cytokinins were shown to substitute for red light in the expansion of leaf disks and elongation of epicotyl segments in etiolated bean seedlings and in the germination of light-sensitive lettuce seed (Miller 1956). ; Numerous subsequent studies have shown that cytokinins induce a wide range of phytochrome- and cryptochromemediated photomorphogenic events including chloroplast maturation (Stetler and Laetsch 1965), cotyledon expansion (Huff and Ross 1975), inhibition of hypocotyl elongation (Chory et al. 1991) and induction of light-regulated genes (Flores and Tobin 1986, Chory et al. 1994). Dark-grown seedlings transferred to light show a rapid and transient increase in cytokinin concentrations, supporting a role for endogenous cytokinins in photomorphogenesis (Uheda and Kuraishi 1977, Qamaruddin and Tillberg 1989); however another study using the cytokininresistance, ckr1, mutation suggests that photomorphogenesis can take place without a functioning cytokinin-response pathway (Su and Howell 1995). The strongest evidence supporting a role of cytokinins in photomorphogenesis has come from investigation of intact seedlings of wild-type and photomorphogenic mutants of Arabidopsis. Cytokinin treatment of dark-grown wild-type seedlings induces developmental changes similar to the phenotypic characteristics of the constitutive photomorphogenic mutants det1, det2 and amp1. The cytokinins N 6 ( 2 -isopentenyl)adenine (ip), kinetin and benzylaminopurine (BAP) each inhibited hypocotyl elongation, promoted expansion of cotyledons and leaves, promoted chloroplast maturation, and increased transcription of genes normally up-regulated by blue and red light (Chory et al. 1991, Chory et al. 1994). While amp1 mutants have six-fold higher cytokinin concentrations than wild type (Chaudhury et al. 1993), the det1 and det2 mutants do not have elevated cytokinin levels, but rather have increased sensitivity to exogenous cytokinins as measured by tissue culture and leaf senescence bioassays (Chory et al. 1994). Cytokinin induction of photomorphogenic responses has been demonstrated in dicots (Miller 1956), monocots (Flores and Tobin 1986) and gymnosperms (Qamaruddin and Tillberg 1989). Cytokinins also regulate developmental patterning in mosses, including caulonema initial formation and bud assembly (reviewed by Schumaker and Dietrich 1998), but do not appear to be able to substitute for light in bryophyte development. While the effects of cytokinins in bryophyte development have been well studied, little is known about the role of cytokinins in pteridophytes. Cytokinins have been identified in fern gametophytic (Schraudolf and Fischer 1979) and sporophytic (Yamane et al. 1983) stages, but have only a slight effect on 1 Corresponding author: , spiro@bucknell.edu; Fax,

2 Cytokinin-induced photomorphogenesis in Ceratopteris 1253 Fig. 1 Representative images of 11 d light-grown (A) and dark-grown gametophytes cultured in the absence of cytokinins (B, C), or in the presence of 100 nm BAP (D, E). Panels C and E are enlargements of panels B and D respectively in order to show cytokinin-induced formation of the notch meristem (n) and differentiation of rhizoid initials (r) within the subapical cells (1,2). The enlarged images also show the cytokinin-induced broadening of the meristem resulting from an increase in periclinal cell division, which was measured by the number of cells across the meristematic region, and shows the cytokinin-mediated inhibition of elongation of the cells below the meristem. In all panels bar = 250 µm. developmental patterning in light-grown ferns, attributed to an increase in mitosis (reviewed by Raghavan 1989). In this study we investigated the role of cytokinins in the development of dark-grown gametophytes of the tropical fern Ceratopteris richardii. In the past decade the gametophytic stage of Ceratopteris has been used as a model system for photomorphogenesis, in part because of its simple anatomy and rapid response to low levels of both blue and red light in initiating the transition from filamentous to prothallial growth (Cooke et al. 1995, Murata et al. 1997). Additionally, Ceratopteris is a useful genetic system in that it has a short life cycle, the ability to produce large quantities of spores, and forms morphologically distinct male and hermaphroditic gametophytes that allows for crossing of desired strains (reviewed by Hickok et al. 1995, Banks 1999). Recent studies have focused on hermaphroditic gametophytes, which show a more pronounced light-induced alteration in developmental patterning than that seen in males. The Her1 strain, which forms only hermaphrodites and lacks the ability to form male gametophytes (Warne et al. 1988), has been particularly useful for these studies (Murata et al. 1997, Murata and Sugai 2000). In the light, hermaphrodites develop a notch

3 1254 Cytokinin-induced photomorphogenesis in Ceratopteris Fig. 2 Gametophytes were grown in liquid medium containing 500 nm BAP (horizontal hatching), 125 nm BAP (open bars), 30 nm BAP (diagonal hatching) and no addition (solid bars). The spores were allowed to germinate in the light for 24 h before being placed in the dark for an additional 6 10 d. The length of the gametophytes and width across the apical region were measured; the length-to-width ratio of the light-grown control was approximately 1 : 1. Values are shown as mean with standard error for a total of 100 gametophytes (25 gametophytes each from four separate experiments). meristem that forms a heart-shaped prothallus of approximately equal length and width bearing both archegonia and antheridia (Fig. 1A), while in the dark, these gametophytes form a convex apical meristem atop a highly elongate, asexual, multiseriate filament only four to six cells wide (Fig. 1B, C). Exposure of dark-grown gametophytes to light initiates a number of developmental events that are part of the transition from filamentous to prothallial growth. These include a reduction in the gametophyte length-to-width ratio that is accomplished by an inhibition in cell elongation, induced by brief exposure to blue light, and an alteration of the meristem morphology to form a broadened notch meristem, induced by both blue and red light (Murata et al. 1997). Light also induces the formation of rhizoid initials in the cells immediately below the meristem, a low-fluence phytochrome response that is initiated by red and inhibited by far-red light (Murata and Sugai 2000). Thus, as in angiosperms, photomorphogenesis in Ceratopteris gametophytes involves a significant alteration in developmental patterning that is accomplished by changes in the rate and orientation of cell division, cell elongation and cell differentiation. However, in contrast to angiosperms, dark-grown Ceratopteris gametophytes contain fully developed chloroplasts and show only a slight light-induced elevation in chlorophyll content (Cooke et al. 1993). In this study we investigated the effect of exogenous cytokinins on the development of Ceratopteris gametophytes towards the goal of characterizing the evolutionary conservation in the role of this hormone. While cytokinins have only a slight effect on light-grown gametophytes, we show that they induce, at sub-nanomolar concentrations, a large alteration in Table 1 growth Cytokinin-induced alteration in cell division and cell Parameter No cytokinin 125 nm BAP Prothallus width (µm) 174 ± ± 3.4 No. cells across width 8.2 ± ± 0.29 Cell width (µm) a 21.8 ± ± 0.30 Prothallus length (µm) 2,973.3 ± 35 1,659.2 ± 31 No. cells along length a 12.8 ± ± 0.95 No. elongated cells b 7.8 ± ± 0.24 Length basal cells (µm) c ± ± 9.4 Contributions of cell division and cell growth to the cytokinin-induced alterations in the morphology of day 11 dark-grown Ceratopteris gametophytes. All values are shown as mean ± standard error for 30 gametophytes from three experiments. a No significant differences were seen between treated and untreated gametophytes for cell width or number of cells along length. The values of all other measurements were highly significantly different between cytokinin-treated and untreated gametophytes. b A cell was considered to be elongated if its length was at least twice its width. c The length of the two basal-most cells was used as these cells had completed elongation. the developmental patterning of dark-grown gametophytes that is correlated with photomorphogenesis. Results Timing of cytokinin-induced developmental events in darkgrown gametophytes The first set of studies focused on the timing of cytokinininduced photomorphogenic events in dark-grown Her1 Ceratopteris gametophytes. The Her1 strain, which lacks the ability to form males (Warne et al. 1988), produces only asexual multiseriate filaments in the dark and hermaphroditic prothalli in the light and has been shown to be particularly useful for investigating photomorphogenesis (Murata et al. 1997, Murata and Sugai 2000). Spores were soaked in the dark for 3 days before plating in basal growth media or media supplemented with nm BAP. Preliminary experiments demonstrated that cytokinins could not substitute for light in the initiation of germination; therefore, spores were exposed to light for 24 h before being placed in the dark for an additional 6 ( day 7 ) to 10 ( day 11 ) days. On days 7 11 gametophytes were analyzed for four developmental responses correlated with photomorphogenesis (Murata et al. 1997): a reduction in cell elongation, an increase in meristem width, conversion of the meristem from apical to notch morphology and the formation of rhizoid initials in the subapical cells. Gametophytes grown in the light have a notch meristem that gives rise to the characteristic heart-shaped prothallus of approximately equal length and width (Fig. 1A). Gametophytes grown in the dark in the absence of cytokinins grew as extensively elongated multiseriate filaments possessing a nar-

4 Cytokinin-induced photomorphogenesis in Ceratopteris 1255 Fig. 3 Gametophytes were grown in the dark as described for Fig. 2 in liquid medium containing 500 nm BAP (horizontal hatching), 125 nm BAP (open bars), 30 nm BAP (diagonal hatching), and no addition (solid bars),. Gametophytes were scored for the presence of a notch meristem as defined by a concave surface in which two distinct prothallial lobes were developing. All of the light-grown gametophytes possessed notch meristems. Values are shown as mean with standard error for a total of 100 gametophytes (25 gametophytes each from four separate experiments). row apical meristem (Fig. 1B, C). The length-to-width ratio of the untreated dark-grown gametophytes increased from approximately 14 : 1 on day 7 to approximately 17 : 1 on days 8 11, while gametophytes treated with BAP at any of the concentrations tested ( nm) had a consistent length-to-width ratio of approximately 7 : 1 (Fig. 2). The reduction in the length-towidth ratio of the cytokinin-treated gametophytes was due to an increase in width of the meristematic region as well as a decrease in gametophyte length (Fig. 1D, E), both processes characteristic of the light-induced initiation of prothallial development. Measurements of cell number and cell size in day 11 gametophytes (Table 1) showed that the increased meristematic width resulted from an increase in periclinal cell division, rather than a change in cell size, while the decrease in gametophyte length resulted from a decrease in the degree of cell elongation and the number of elongated cells (those with a length at least twice their width), rather than an alteration in anticlinal cell divisions. The rate of periclinal and anticlinal cell division was determined by counting the number of cells across the meristem or along the length of the longest cell file, respectively. The degree of cell elongation was measured in the two basal-most cells since these cells were fully elongated and typically were the longest cells within the gametophyte. Addition of BAP to dark-grown gametophytes also resulted in a change in the pattern of cell division within the meristematic region resulting in a higher proportion of gametophytes bearing notch rather than apical meristems. A meristem was considered to have a notch morphology if it possessed a concave surface in which two distinct prothallial lobes were developing (Fig. 1E). Notch meristems were never observed in Fig. 4 Gametophytes were grown in the dark as described for Fig. 2 in liquid medium containing 500 nm BAP (horizontal hatching), 125 nm BAP (open bars), 30 nm BAP (diagonal hatching), and no addition (solid bars),. Gametophytes were scored for the number of rhizoids formed in cells 1 and 2 (see Fig. 1). Light-grown gametophytes form rhizoids from basal prothallus cells only. Values are shown as mean with standard error for a total of 100 gametophytes (25 gametophytes each from four separate experiments). untreated dark-grown gametophytes on day 7 or day 8, but were present in as many as 8% of untreated gametophytes on days BAP treatment, at nm, induced a steady increase in the percentage of gametophytes bearing notch meristems starting on day 8, such that by day 11 approximately 60% of the gametophytes had notch meristems (Fig. 3). The induction of notch meristem formation occurred more slowly and required higher BAP concentrations than observed for the alteration in overall gametophyte morphology described above. The cytokinin-induced notch meristems in dark-grown gametophytes were never observed to form archegonia, while lightgrown Her1 gametophytes invariably form notch meristems with one or more archegonia by day 8. The cytokinin-induced formation of rhizoid initials in the marginal subapical cells (Fig. 1E) followed a similar time course to that observed for the induction of notch meristems, beginning on day 8 and increasing steadily through day 11. Untreated dark-grown gametophytes formed few rhizoid initials at any time point, an average of no more than 0.15 initials per gametophyte (Fig. 4). All tested concentrations of BAP induced a highly significant increase in rhizoid initial formation starting at day 8 and becoming most pronounced by day 11 up to a maximum of 0.9 initials per gametophyte. There was no significant difference in the effect of BAP within the concentration range tested, suggesting that this effect was saturated by concentrations at least as low as 30 nm. These results indicate that cytokinins induce an alteration in the rate and pattern of cell division, cell elongation and cell differentiation similar to the effects induced by light. This photomorphogenic developmental patterning increases with continued exposure to cytokinin treatment, producing a highly

5 1256 Cytokinin-induced photomorphogenesis in Ceratopteris Fig. 5 Each form of cytokinin tested induces a decrease in the length-to-width ratio of dark-grown gametophytes at sub-nanomolar concentrations. Experiments were carried out as described for Fig. 2 except that all measurements were made on day 11. Gametophytes were incubated in liquid medium containing varying concentrations of BAP (circles), kinetin (triangles) or ip (diamonds), or in the absence of cytokinins (dashed line). Values are shown as mean with standard error for a total of 100 gametophytes (25 gametophytes each from four separate experiments). pronounced effect by day 11. Additionally, each of the developmental responses appeared to be saturated by nanomolar concentrations of BAP. Further characterization of cytokinininduced developmental responses focused on day 11 gametophytes using a broad concentration range of three cytokinins. The cytokinins BAP, kinetin and ip each induce photomorphogenic developmental patterning at sub-nanomolar concentrations Dark-grown day 11 gametophytes were used to generate a concentration response curve for each of the above characterized developmental responses. The activity of one naturally occurring cytokinin, ip, and two synthetic cytokinins, BAP and kinetin, was tested over a range from 1 pm to 100 nm ( M). At the lowest concentration tested (10 12 M) each form of cytokinin induced a small, but highly significant reduction in the ratio of gametophyte length to width (Fig. 5). At each concentration BAP was more active than ip and kinetin, which had roughly equivalent activities. Untreated gametophytes had a length-to-width ratio of approximately 17 : 1, while at saturating concentrations BAP (10 nm), ip (100 nm) and kinetin (100 nm) induced a ratio of approximately 7.4 : 1. Compared with the effects on the overall morphology, a considerably higher concentration of cytokinin (1 nm for BAP and kinetin, 10 nm for ip) was required to induce a significant conversion of the meristem morphology from apical to notch (Fig. 6). However, at 10 nm each form of cytokinin induced a large increase in notch meristem formation. Again BAP was the most active form, inducing notch meristem formation in up to 67% of gametophytes, while only 4% of untreated gametophytes had notch meristems. Fig. 6 Each form of cytokinin tested induces an increase in the formation of notch meristems at sub-nanomolar concentrations. Experiments were carried out as described for Fig. 3 except that all measurements were made on day 11. Gametophytes were incubated in liquid medium containing varying concentrations of BAP (circles), kinetin (triangles) or ip (diamonds), or in the absence of cytokinins (dashed line). Values are shown as mean with standard error for a total of 100 gametophytes (25 gametophytes each from four separate experiments). Fig. 7 Sub-nanomolar concentrations of each form of cytokinin tested induces an increase in the formation of rhizoids from the first two cells of the subapical region (cells 1 and 2). Experiments were carried out as described above with all measurements made on day 11. Gametophytes were incubated in liquid medium containing varying concentrations of BAP (circles), kinetin (triangles) or ip (diamonds), or in the absence of cytokinins (dashed line). Values are shown as mean with standard error for a total of 100 gametophytes (25 gametophytes each from four separate experiments). The formation of rhizoid initials within the subapical cells was the most sensitive of the developmental responses (Fig. 7). Untreated dark-grown gametophytes produced an average of 0.18 initials per gametophyte, but each form of cytokinin even at the lowest concentration tested (1 pm) induced more than a two-fold increase in rhizoid initials, which was highly significant. Again, BAP was the most active analog and at 1 nm

6 Cytokinin-induced photomorphogenesis in Ceratopteris 1257 Table 2 Effect of light and cytokinin treatment on chlorophyll synthesis Treatment Chlorophyll a [mg (g dry weight) 1 ] a Chlorophyll b [mg (g dry weight) 1 ] a Ratio a/b Dark, no cytokinin 3.89 ± ± ± 0.05 Dark, 100 nm BAP 4.39 ± ± ± 0.06 Light, no cytokinin 9.85 ± ± ± 0.16 Chlorophyll was extracted in 90% acetone (v/v) from 11 d gametophytes that were grown in continuous dark in the absence or presence of BAP, or in continuous light (100 µe m 2 s 1 ). Values are mean ± standard error for six treatments from three separate experiments. a Light, but not cytokinin treatment, induced a highly significant difference in chlorophyll a and chlorophyll b content. No significant differences were observed in the ratio of chlorophyll a / chlorophyll b between the three treatments. Table 3 Effect of cytokinins on developmental patterning in light-grown gametophytes Treatment Prothallus length (µm) a Prothallus width (µm) Length to width ratio a Notch meristem (%) No cytokinin 90.9 ± ± ± nm BAP 74.4 ± ± ± Gametophytes were grown for 11 d in continuous light (100 µe m 2 s 1 ) in the absence or presence of 100 nm BAP. Values are shown as mean ± standard error for a total of 195 gametophytes from four separate experiments. a There was a highly significant difference between treated and untreated gametophytes for length and length-to-width ratio. No significant differences were found for other measurements. induced a saturating effect of more than a five-fold increase in the average number of rhizoid initials. Kinetin and ip induced a similar saturating effect, but only at a 10- to 100-fold higher concentration. Our results indicate that each of the tested forms of cytokinin is active in each photomorphogenic effect at nanomolar to sub-nanomolar concentrations. In general BAP is the most active and ip the least active of the tested forms. Cytokinin does not increase chlorophyll content of dark-grown gametophytes We were interested to determine whether cytokinins induce physiological changes in addition to the observed developmental alterations; specifically we tested the ability of cytokinins to increase the levels of chlorophyll, as has been shown in dark-grown seedlings. In dark-grown angiosperm seedlings, cytokinins have been shown to substitute for light in the induction of chlorophyll synthesis by activating protochlorophyllide oxidoreductase (Kusnetsov et al. 1998). In contrast to angiosperms, chloroplast development and chlorophyll synthesis are not light regulated in ferns; indeed, dark-grown gametophytes possess fully developed chloroplasts and have only a moderately lower level of chlorophyll than light-grown gametophytes (Raghavan 1989, Cooke et al. 1993). The ability of a saturating cytokinin treatment (100 nm BAP) to stimulate chlorophyll synthesis in dark-grown Ceratopteris gametophytes was compared with the effect of light. Gametophytes were cultured as described above, but at a six-fold higher density. On day 11 the gametophytes were collected and their chlorophyll extracted with acetone. BAP treatment of dark-grown gametophytes did not significantly alter the concentration of chlorophyll a, chlorophyll b or the ratio of chlorophyll a/b. Light induced more than a two-fold increase in the concentration of both chlorophyll a and chlorophyll b, but had no significant effect on the ratio of chlorophyll a/b (Table 2). The effects of cytokinins on developmental patterning are specific to dark-grown gametophytes Previous studies of cytokinin activity in fern gametophytes have focused on light-grown plants and have found little to no effect even at relatively high concentrations (reviewed by Raghavan 1989). It was not clear whether the discrepancy between these results and our observations was due to differences in the species or in the growth conditions. To address this question, the effect of cytokinins on developmental patterning was investigated in day 11 light-grown cultures of Ceratopteris. Cytokinins induced only a minimal effect on developmental patterning of light-grown gametophytes even at concentrations that caused a saturating effect on dark-grown gametophytes. Light-grown gametophytes treated with 100 nm BAP for 11 d displayed a small, but highly significant decrease in prothallus length and length-to-width ratio, but showed no significant difference in prothallus width, or meristem morphology (Table 3); in the day 11 light-grown cultures both treated and untreated gametophytes consistently formed sexually mature heart-shaped hermaphrodites. Treatment with 10 nm BAP did not induce any significant alteration in the development of light-grown gametophytes (data not shown). This indicates that, at least at the concentrations tested, the effects of cytokinins on developmental patterning are specific to dark-grown gametophytes.

7 1258 Cytokinin-induced photomorphogenesis in Ceratopteris Discussion Our experiments demonstrate that cytokinins can induce photomorphogenic development in dark-grown gametophytes of C. richardii. To our knowledge this is the first study to demonstrate a role for cytokinins in developmental patterning in ferns. Previous studies focusing on light-grown gametophytes found only slight effects of cytokinin treatment; our results support these findings and demonstrate that the role of cytokinins in developmental patterning, at least at nanomolar concentrations, is specific to dark-grown gametophytes. These developmental processes mimic the normally light-induced transition from filamentous to prothallial growth, which involves an alteration in the rate and pattern of cell division, cell elongation and cell differentiation. At concentrations as low as M, BAP, kinetin and ip each induced the formation of rhizoid initials in subapical cells, reduced cell elongation and increased periclinal cell division resulting in a decrease in the length-to-width ratio of dark-grown gametophytes. Somewhat higher concentrations (10 9 to 10 8 M) induced the conversion of the meristem from apical to notch morphology. Cytokinin-treatment induces developmental patterning similar to that induced by both red and blue light (Murata et al. 1997, Murata and Sugai 2000), but can only partially substitute for the effect of prolonged light treatment, in that it does not allow for the formation of sexually mature heart-shaped hermaphrodites nor for light-independent spore germination. The formation of rhizoid initials in subapical cells is a red/ far-red reversible phytochrome response, which was induced more than two-fold in dark-grown gametophytes at M of each of the cytokinins tested, making this the most sensitive of the cytokinin-induced responses. While rhizoid formation was shown to be a low-fluence response, tip growth requires exposure to light at relatively high irradiance, suggesting that rhizoid formation and elongation involve separate signal transduction pathways (Murata and Sugai 2000); our evidence that cytokinins can initiate the formation, but not the elongation of rhizoid initials provides additional evidence for separate pathways. Each of the cytokinins tested, at concentrations as low as M, inhibited prothallus cell elongation, which has been shown to be a low-fluence blue-light-mediated response (Murata et al. 1997). Both blue and red light can induce notch meristem formation, although the fluence requirement for this response has not been characterized (Murata et al. 1997). The alteration of meristem morphology was the least sensitive of the cytokinin effects investigated, requiring a concentration of at least 10 9 M to induce a significant effect. Further experiments would be required to determine whether there is a correlation between cytokinin concentration and the fluence required to induce these photomorphogenic responses. The results of this paper indicate an evolutionarily conserved role for cytokinins in regulating photomorphogenesis. Addition of cytokinins to dark-grown angiosperm and gymnosperm seedlings induces photomorphogenic development including inhibition of stem elongation, promotion of cotyledon and leaf expansion, and induction of chloroplast maturation and chlorophyll synthesis (Chory et al. 1994). The cytokinin-induced alteration in developmental patterning in seed plants involves inhibition of elongation and promotion of lateral expansion similar to that induced by cytokinins in darkgrown Ceratopteris. Additionally in seed plants as in Ceratopteris the cytokinin-induced effects are specific to dark-grown plants (Su and Howell 1995) and can substitute for both red and blue light treatments (Chory et al. 1991). The reason why cytokinins have limited effects in light-grown seed plants as well as in our system may be that the cytokinin levels are saturated due to light-mediated biosynthesis. There are some distinct differences between the effects of cytokinins on Ceratopteris and those reported for flowering plants. For example, cytokinins stimulate chlorophyll synthesis in dark-grown angiosperms (Kusnetsov et al. 1998), but this effect was not observed in Ceratopteris gametophytes. Angiosperm seedlings grown in the dark are etiolated, but quickly turn green upon exposure to light, largely due to the action of light-dependent protochlorophyllide oxidoreductase. In contrast, most non-flowering plants, including Ceratopteris, express both light-independent and light-dependent forms of protochlorophyllide oxidoreductase and are able to synthesize chlorophyll in the dark (reviewed by Fujita 1996, Schoefs and Franck 2003). Indeed, we observed only slightly more than a two-fold increase in chlorophyll content in light- versus darkgrown gametophytes, and no cytokinin-induced increase in chlorophyll content. Another difference between our observed effects and those reported for seedlings is the concentration required to induce a saturating effect. For example, inhibition of hypocotyl elongation in Arabidopsis was saturated only in the presence of µm cytokinin (Chory et al. 1994), while 1,000-fold lower concentrations induced saturating effects in Ceratopteris. This may be due to a difference in the effective concentrations reaching the target tissues; in seedlings the cytokinins must be taken up by the roots and transported to the hypocotyl and cotyledons where they have their effect, while in gametophytes the responding cells are in direct contact with the cytokinins. Indeed, cytokinins are active at nanomolar concentrations in the inhibition of root elongation and the stimulation of root-hair elongation in Arabidopsis where the responding cells are in direct contact with the cytokinin solution (Su and Howell 1992). While exogenous cytokinins induce photomorphogenic developmental patterning in dark-grown Ceratopteris gametophytes, the role of endogenous cytokinins remains to be investigated. Light induces cytokinin synthesis in dark-grown angiosperm and gymnosperm seedlings providing correlative data on the role of cytokinins in photomorphogenesis; however, the strongest evidence supporting an endogenous role of cytokinins in seedling photomorphogenesis comes from the use of photomorphogenic mutants of Arabidopsis. Constitutive

8 Cytokinin-induced photomorphogenesis in Ceratopteris 1259 photomorphogenic mutants have been found to have an increased endogenous cytokinin concentration (Chaudhury et al. 1993) or increased sensitivity to cytokinin treatment (Chory et al. 1994). Recently a Ceratopteris mutant, dkg1, that was originally identified by its dark-germinating phenotype (Scott and Hickok 1991) has been shown to be constitutively photomorphogenic (Kamachi et al. 2004). Under continuous dark growth dkg1 has increased prothallus width and subapical rhizoid formation compared to dark-grown wild type, and forms meristems with archegonia. Experiments that combine the use of photomorphogenic mutants, such as dkg1, with quantification of cytokinin levels under varying light exposures will help us understand the endogenous role of cytokinins in Ceratopteris and the evolutionary conservation of signal transduction pathways involved in photomorphogenesis. Materials and Methods Plant material and culture Spores of C. richardii strain Her1 (Carolina Biological Supply, Burlington, NC, U.S.A.) were soaked in sterile distilled water in the dark at 23 C for 3 d, before being plated at a density of approximately 30 spores per ml in 10 ml of liquid C-fern medium (Carolina Biological Supply; Klekowski 1969), ph 6.0, in glass 60 mm culture dishes. Gametophytes were grown at 28 C under continuous lighting (Sylvania cool white fluorescent; 100 µe m 2 s 1 ) for 24 h to satisfy the light requirement for germination. At this time dark-grown cultures were wrapped in two layers of aluminum foil and transferred to a dark growth chamber at 28 C for an additional 6 10 days (days 7 11), while light-grown cultures were left in continuous light for an additional 10 d (day 11). Hormone treatments The synthetic cytokinins, BAP and kinetin, and the naturally occurring cytokinin, ip, each obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.), were added to the media just prior to plating spores. Each of these cytokinins was dissolved in a minimal amount of 0.5 M KOH before dilution and sterilization using 0.2 µm syringedriven filters (Millex-GS; Millipore, Billerica, MA, U.S.A.). The cytokinin solutions, even at the highest concentration, had no effect on the ph of the growth media. Growth analysis Whole unstained gametophytes were examined with a compound microscope. Size was determined using an ocular micrometer; gametophyte length was measured from the spore case to the most apical position along the axis that lies parallel to the cell files and the width was measured at the widest point perpendicular to the cell files. In dark-grown gametophytes the apical meristem was invariably the widest point. Additionally, dark-grown gametophytes were scored for the number of rhizoid initials formed in the marginal subapical cells (cells 1 and 2, compare Fig. 1C and E) and all gametophytes were scored for meristem morphology (notch or apical meristem). Gametophytes were considered to have a notch meristem if the meristematic region had a concave surface in which cell divisions were initiating the development of two distinct prothallial lobes (compare Fig. 1C and E). Chlorophyll measurements Gametophytes were grown in liquid culture as described above, except at a density of approximately 180 gametophytes per ml. Both dark- and light-grown gametophytes were collected at day 11 using100 µm nylon cell strainers (Becton Dickinson, Franklin Lakes, NJ), blotted dry, and transferred to 1 ml 90% (v/v) acetone. Chlorophyll was extracted overnight at 4 C, the gametophytes were centrifuged and the supernatant measured in a spectrophotometer using the equations of Jeffrey and Humphrey (1975): chlorophyll a (µg ml 1 ) = (A 664 ) 1.93 (A 647 ) and chlorophyll b (µg ml 1 ) = (A 647 ) 5.5 (A 664 ). Dry weight was determined after vacuum desiccation of the extracted gametophytes along with the supernatant. Data collection and statistical analysis For analysis of developmental patterning at least 25 gametophytes were measured per treatment in four separate experiments, except where noted. For determination of chlorophyll content three separate experiments were carried out with duplicates for each treatment. All data are represented as mean ± standard error. Means were compared by a two-tailed t-test. Throughout the paper differences are designated as significant for P < 0.05, highly significant for P < and not significant for P > References Banks, J.A. (1999) Gametophyte development in ferns. Annu. 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