Phototropism in the Marine Red Macroalga Pyropia yezoensis

Phototropism is a response to the direction of light that guides growth orientation and determines the shape of plants to optimize photosynthetic activity. The phototropic response is present not only in terrestrial plants but also in water-living algae. However, knowledge about phototropism in Bangiophycean seaweeds is limited. Here, we examined the phototropic response of the red alga Pyropia yezoensis to elucidate the regulatory mechanism of phototropism in Bangiophyceae. When leafy gametophytes and filamentous sporophytes of P. yezoensis were cultured under directional light, phototropism was observed in the gametophytes. Conchosporangia on the sporophytes also exhibited phototropism. Phototropism was positive in the majority of gametophytes and conchosporangia but in some cases was negative. In addition, a strong phototropic response occurred under white light, whereas blue and red light elicited minor and no responses, respectively. This observation is in contrast with the phototropic response in terrestrial plants and several algae, in which blue light is responsible for positive phototropism. Surprisingly, the genome of P. yezoensis has no homologues of the photoreceptors for blue and red light, revealing differences in the regulation of phototropism between terrestrial plants and P. yezoensis. Studies on the phototropism in P. yezoensis could shed light on the evolutional divergence of phototropic responses in plants.


Introduction
Phototropism is defined as the response of plants to directional light that directs growth orientation to optimize photosynthetic activity and energy production [1] [2]. Since the discovery of the pivotal role of auxin in phototropism [3] [4], the Cholodny-Went hypothesis has been generally accepted. This hypothesis posits the lateral movement of auxin from the illuminated to the shaded side where it promotes cell elongation and curvature of the coleoptile towards the light.
The study of phototropism was extensively advanced using a genetic approach and the dicotyledon Arabidopsis thaliana [5] [6] [7]. The identification of phototropin, a plasma membrane-associated blue light receptor consisting of an N-terminal lightsensing domain with two light, oxygen or voltage (LOV) domains and a C-terminal serine/threonine kinase domain, was another major finding that furthered our understanding of the process of phototropism [8] [9] [10] [11]. To date, photoperception and activation of phototropins have been extensively analyzed, and several phototropinsignaling components such as nonphototropic hypocotyl 3 (NPH3) and phytochrome kinase substrate 1 (PKS1) have been identified [12] [13]. Moreover, red light receptor phytochromes, and a class of blue light receptors, the cryptochromes, are involved in fine-tuning phototropin activity by repression of the negative regulators ATP binding cassette B19 (ABCB19), which is an auxin efflux carrier, and root phototropism 2 (RPT2) which participates in blue light-induced phototropism [14] [15]. Thus, all classes of photoreceptors known in A. thaliana play a role in the early phase of phototropism.
The formation of the auxin gradient has also been extensively studied. Initially, three families of auxin transporters were studied: auxin resistant 1 (AUX1), the ABC transporters (specifically ABCB19), and the PIN-FORMED (PIN) family [16]. The asymmetric distribution of auxin, results from polar transport through the activity of these transporters [15] [17]. Subsequently, studies of the auxin signal transduction pathway identified the auxin receptors transport inhibitor response 1 (TIR1, also called auxin signaling F-box, AFB) and auxin binding protein 1 (ABP1), the negative regulators auxin/indole-3-acetic acid (Aux/IAA) proteins, and the auxin response factors (ARFs) [18] [19]. Cell-to-cell movement of auxin mediated by auxin transporters establishes an auxin gradient, and the auxin that accumulates in the shaded side activates a signal transduction cascade that leads to expression of genes that stimulate cell elongation at the shaded side specifically [5] [6] [7]. Thus, the molecular mechanisms that regulate phototropism are now mostly elucidated in A. thaliana.
The polarity in zygotes of brown algae is determined by the direction of light; the illuminated and shaded sides develop into vegetative and rhizoid cells, respectively [20].
Although evidence for photopolarization is limited to brown algal zygotes, these findings suggest the ability of seaweeds to recognize the direction of light. Indeed, phototropic responses have been found in water-living seaweeds: according to the excellent review by Rico and Guiry [21] for example, negative phototropism of rhizoids and positive phototropism of the thallus were observed [22]- [27]. Subsequently, phototropism responses to directional blue light were found in many species belonging to the Chlorophyceae, Phaeophyceae and Rhodophyceae [21]. Since 1996, no studies on seaweed phototropism have been reported except for the identification of a blue light receptor, aureochrome, in the photosynthetic stramenopile algae [28] [29]. Thus, detailed information on the process and regulatory mechanism of phototropism in seaweeds is limited.
Pyropia yezoensis belongs to the family Bangiophyceae and is a model species for red seaweeds [30]; the nuclear, plastid and mitochondrial genomes of this seaweed have been sequenced [31] [32] [33]. We have investigated the light-dependent release of asexual spores of P. yezoensis and the establishment of polarity in these spores [30] [34 [35] [36]. However, it remains unclear whether the establishment of polarity in asexual spores depends on the direction and color of light. Phototropism can serve as a useful model to analyze the formation of the polarized axis that determines growth direction.
Currently, in contrast to the Florideophyceae family, in which phototropism has been extensively studied [21] [37], there are only two reports on phototropism in the Bangiophyceae family, in P. yezoensis and P. tenera [38] and Porphyra umbilicalis [37]. Migita and Kim [38] documented the phototropic response in sporophytes and conchosporangia of P. yezoensis.
In this study, we examine the phototropic responses of gametophytes, sporophytes and conchosporangia of P. yezoensis. We extend the previous observations by studying the light color requirement and locating the photo-perception and bending sites through the dissection of the processes that meditate phototropic curvature. Our results help to clarify the regulatory mechanisms of phototropism and photopolarization in Bangiophycean seaweeds. In addition, to identify a potential mechanism that underlies the phototropic response, a large-scale survey of red algal EST information was performed to confirm the presence of homologs of factors that are involved in the regulation of phototropism in other eukaryotes.

Directional Light Irradiation
For unilateral light exposure, a culturing box (H 6.5 cm × W 10.5 cm × D 9.0 cm) with only one opened side was used. The box was wrapped with black paper to minimize re- Olympus, Tokyo, Japan) with a camera (DP26, Olympus). Statistical analysis was performed using t-test and was carried out between vertical (Top + Bottom) and horizontal (Left + Right) ratio of each experimental group. Furthermore, it was also carried out vertical (Top + Bottom) or horizontal (Left + Right) to each other before and after the change of light irradiation direction. Differences were reported as significant when P < 0.05.

Phototropism in Filamentous Sporophytes
When filamentous sporophytes were exposed to directional light during culture, no phototropic response was observed (Figure 1(a) and Figure 1(b)), even though Migita and Kim [38] reported positive phototropism under similar conditions. Because changing the direction of light and exposure to blue or red light did not cause phototropic growth (data not shown), we concluded that P. yezoensis sporophytes lack the capacity for phototropic response.

Phototropism in Conchosporangia
As shown in Figure 2(a), conchosporangia showed a clear phototropic response after seven days of culture under directional white light from the top, which is consistent with the data from Migita and Kim [38]. However, it is notable that both positive and negative phototropic curvature was observed, and not all conchosporangia showed a phototropic response. Indeed, 65.6% and 22.5% of the examined conchosporangia showed positive and negative phototropism, respectively, and the remaining 11.9% did not respond (Table 1 and Figure 3(a)). In addition, when exposed to blue light from the top, positive and negative responses were observed in 39.6% and 27.3% of conchosporangia respectively, whereas exposure to unilateral red light did not cause a phototropic response (Table 1 and Figure 3(a)). Thus, P. yezoensis conchoporangia respond to the direction of light. Since white light remained more effective than blue light alone (Table 1 and Figure 3  ( Figure 3(b)). These effects were not observed under exposure to red light ( Figure   2(q) and Figure 3(b)). Thus, blue light is involved in the phototropic response of conchosporangia, albeitits phototropic effect is weaker than that caused by white light.

Phototropism of Monospore Germlings
When monospores were exposed to directional white light immediately after release, germination was not photopolarized (Figure 4(a)). By contrast, germlings that were cultured for 7 d responded to the direction of white light both positively and negatively (Figure 4(b)), although, similar to conchosporangia, not all germlings showed a phototropic response (Figure 4(c)). In this case, positive and negative curvatures were observed in 43.2% and 13.4% of all germlings, respectively, and thus 43.4% germlings did not respond to directional light from the top (Table 1 and Figure   5(a)). Surprisingly, neither red nor blue light resulted in a phototropic response in germlings (Table 1 and Figure 5(a)).    (Figure 4(f)-(h)), and that the cells located above these cells are responsible for the additional bending due to illumination for more than 3 d (Figure 4(i) and Figure 4(j)), which completes the phototropic response as shown in Figure 4(k).
Interestingly, blue light reduced the number of germlings that grew toward the original direction from 31.0% to 18.5% and increased growth along the new direction from 21.7% to 35.6% (Table 1, Figure 4(l), Figure 4(m) and Figure 5(b)), whereas red light had no effect (Table 1, Figure 4(n) and Figure 5(b)). Thus, gametophytes of P. yezoensis respond to the direction of white light, and blue light might be involved in this phototropic response.

Sequence Searches for Photoreceptor Homologues
Using the amino acid sequences of phototropins, cryptochromes, phytochromes, superchrome and aureochrome as queries, a BLAST search was performed against the EST databases for P. yezoensis, P. umbilicalis and P. purpurea. Surprisingly, the results indicated that these red seaweeds might lack homologs of any of the photoreceptors that have been identified in green plants and brown seaweeds. These find-

Discussion
In the present study, we observed phototropism in gametophytes and conchosporangia, but not in sporophytes, of the marine red seaweed P. yezoensis. The phototropic response was observed in the tip cells of conchosporangia and in the cells close to the holdfast of the gametophytes (Figure 2 and Figure 4). There are, however, two unique features of phototropism in P. yeziensis. The first is that, although positive phototropism occurred in most cases, negative phototropism was also observed in both gametophytes and conchosporangia (Table 1, Table 2, Figure 3 and Figure 5). The second is that phototropic curvature was not observed in all examined gametophytes and conchosporangia (Table 1, Table 2, Figure 3 and Figure 5), indicating differences in the sensitivity to the light direction between individual organisms. It remains unclear whether these unique phototropic features have a significant biological function in the growth of P. yezoensis. Interestingly, our results in Figure 1 are inconsistent with those of Migita and Kim [38], who reported positive phototropism in sporophytes. The lack of phototropic response in sporophytes in our study might be due to the fact that the response to the light direction could prevent boring of sporophytes into shells. Moreover, although white light effectively induced phototropic curvature, conchosporangia and gametophytes showed a weak receptivity to blue light ( Table 1, Table 2 and Figures 2-5), which is inconsistent with the general consensus that blue light plays a central role in phototropism in seaweeds [21]. Taken together, our results therefore indicate the presence of novel mechanisms that regulate phototropism in P. yezoensis. The uniqueness of this phototropic machinery in P. yezoensis is supported by the results of the homology searches for photoreceptor genes. The blue light-receptor phototropin, which recognizes and absorbs blue light through LOV domains, plays a central role in the phototropic response in terrestrial plants [8] [10] [11]. However, the genomes of P. yezoensis have no homologue of this LOV domain-containing photoreceptor. In addition, a large scale-EST survey did not identify homologues of other photoreceptors present in terrestrial plants and brown seaweeds. Because white and blue light, but not red light, promote phototropic bending in gametophytes and conchosporangia, unknown photoreceptors might be present in P. yezoensis and may function as regulators whose activity is not sufficient to mediate phototropism in all examined individuals. These findings also indicate differences in regulatory mechanisms of phototropism between terrestrial plants and P. yezoensis. The difference in phototropism between vascular plants and conchosporangia of P. yezoensis could be related to lateral cell-to-cell interaction. Phototropism in the multicellular architecture in vascular plants requires differential growth between cells in the illuminated and shaded sides [1] [2] [5] [6] [7], whereas phototropism in the single conchosporangia tip cell requires differences in growth rate between the illuminated and shaded sides within a single cell. To date, single cell-based phototropism has been observed in tip-growing protonemal cells of the mosses Ceratodon purpureus and Physcomitrella patens [43] [44] [45]. Moreover, in C. purpureus the red light-receptor phytochrome is involved in phototropism in the tip cells through re-organization of microfilaments (MFs) [46] [47] [48]. Although red light is not required for phototropism in P. yezoensis conchosporangia (Table 1, Table 2, Figure 2(q) and Figure 3), it is necessary to address whether light-dependent re-organization of MFs is involved in bending of the tip cell of P. yezoensis conchosporangia.
We also observed phototropism at an early developmental phase of gametophytes that were composed of a tandem array of cells along the apical-basal axis (Figure 4). Although bending requires differential growth between the illuminated and shaded sides, it is still unclear whether sensing the light direction and asymmetrical elongation are mediated by a single cell or spatially separated cells. Moreover, our findings clearly demonstrate that the ability to recognize the light direction is acquired at a multi-cellular stage during early development of gametophytes from monospores (compare Figure 4(a)-(c)). Thus, identification of the cell(s) capable of perceiving the light signal and bending due to asymmetrical growth is important to elucidate the mechanism of phototropism in gametophytes.
Although the asymmetrical distribution of the phytohormone auxin between the illuminated and shaded sides is critical for phototropism in terrestrial plants [1] [2] [5] [6] [7], it is unknown whether auxin acts as a regulator of phototropism in P. yezoensis, and whether auxin is asymmetrically distributed in the bending cell in conchosporangia and gametophytes. The gravitropic response was attenuated by inhibitors of auxin transporters in the tip cell of C. purpureus protonemata [49]. In addition, endogenous auxin is present in P. yezoensis [50]. Thus, it is interesting to examine whether auxin is involved in phototropism in conchosporangia and gametophytes of P. yezoensis. There are in fact no homologues of factors involved in auxin biosynthesis, transport and signal transduction in P. yezoensis [50], and it is possible that auxin, if involved, meditates phototropism in P. yezoensis through an unknown mechanism.
As shown in Figure 2(n)-(q) and Figure 4(l)-(n), it was unexpectedly observed that the color of the conchosporangia and monospore germlings changed to green and bright purple under red and blue light, respectively (summarized in Supplemental Figure 1). Such light responses are very similar to the complementary chromatic adaptation (CCA) found in chromatically-adapting prokaryotes such as the cyanobacterium Fremyella diplosiphon [51] [52] [53]. CCA is a process of adaptation to differences in light color by modification of the pigment composition in the light-harvesting phycobilisomes, in which exposure to green and red light alter the cellular color to red and green, respectively. Because the photosynthetic machinery of P. yezoensis includes phycobilisomes, it is likely that CCA is conserved in the phycobilisome-containing marine photosynthetic eukaryotes such as the red seaweeds. Confirmation of this possibility remains to be addressed.

Conclusion
Our work demonstrates that leafy gametophytes and conchosporangia of P. yezoensis perceive and respond to the direction of light both positively and negatively, although not all individuals respond to light and blue light has a weaker potential to produce phototropic response than white light. In addition, P. yezoensis has no homologs of any light receptors found in terrestrial plants. Because these characteristics are specific to this seaweed, this study of phototropism in P. yezoensis sheds light on the evolutionary divergence of photomorphogenesis in plants.