Auxin Regulates Apical Stem Cell Regeneration and Tip Growth in the Marine Red Alga Neopyropia yezoensis

The red alga Neopyropia yezoensis undergoes polarized elongation and asymmetrical cell division of the apical stem cell during tip growth in filamentous generations of its life cycle: the conchocelis and conchosporangium. Side branches are also produced via tip growth, a process involving the regeneration and asymmetrical division of the apical stem cell. Here, we demonstrate that auxin plays a crucial role in these processes by using the auxin antagonist 2-(1H-Indol-3-yl)-4-oxo-4-phenyl-butyric acid (PEO-IAA), which specifically blocks the activity of the auxin receptor TRANSPORT INHIBITOR RESPONSE1 (TIR1) in land plants. PEO-IAA repressed both the regeneration and polarized tip growth of the apical stem cell in single-celled conchocelis; this phenomenon was reversed by treatment with the auxin indole-3-acetic acid (IAA). In addition, tip growth of the conchosporangium was accelerated by IAA treatment but repressed by PEO-IAA treatment. These findings indicate that auxin regulates polarized tip cell growth and that an auxin receptor-like protein is present in N. yezoensis. The sensitivity to different 5-alkoxy-IAA analogs differs considerably between N. yezoensis and Arabidopsis thaliana. N. yezoensis lacks a gene encoding TIR1, indicating that its auxin receptor-like protein differs from the auxin receptor of terrestrial plants. These findings shed light on auxin-induced mechanisms and the regulation of tip growth in plants.


Introduction
Tip growth is a highly polarized mode of growth involving the establishment of a single growing point at the apex of the cell, resulting in directional elongation [1,2]. Tip growth has been observed in a variety of eukaryotic taxa including fungi, oomycetes, green and brown algae, and terrestrial plants [1,[3][4][5][6]. Filamentous organisms exhibit two types of tip growth: the single-cell type involving polarized elongation of a single filamentous cell (such as in root hairs and pollen tubes of angiosperms) [7][8][9][10] and the hyphae type involving continual production of new tip cells through polarized elongation and the subsequent asymmetrical division of the apical cell (such as in protonemata and rhizoids of streptophyte algae, bryophytes (liverworts, hornworts, and mosses), and ferns) [4,[11][12][13][14]. The regulatory mechanisms of tip growth have been extensively investigated using root hairs and pollen tubes as single-cell models. These mechanisms involve ion flux, the asymmetric distribution of F-actin, phosphoinositides, membrane trafficking of membrane and cell wall materials, the production of reactive oxygen species, and plant hormones [15][16][17][18][19][20][21][22][23][24][25].
The hyphae of fungi and protonemata of mosses are multicellular, uniseriate, cylindrical structures whose growth occurs only in the tip cells. Tip cell elongation is restricted to the apex, and asymmetric division of an elongated tip cell produces a new apical tip cell and a basal nondividing cell [15,26,27]. Since these events occur at the apex of the growing cells, a single growing point is established at the apex and the growth direction is polarized,

Preparation of Single-Celled Conchocelis and Conchosporangium
To excise single-celled conchocelis and conchosporangium, these multicellular filamentous structures were chopped with a razor blade, filtered through a 10-µm nylon mesh to remove large pieces, and incubated in 9-cm dishes (Asnol dish 90 mm (diameter) × 20 mm (height), As One, Osaka, Japan) containing 30 mL seawater at 15 • C for 10 min. Cells whose adjacent cells disappeared were picked up with micropipettes under an Olympus IX73 light microscope equipped with an Olympus DP22 camera, transferred into 96-well plates (one cell/well containing 200 µL artificial seawater with or without chemicals, as indicated), and analyzed after 1 week of culture under the conditions described above but without aeration. The branching rate was calculated as the number of cells producing a branch as a percentage of total number of cells observed.

Observation of Naturally Produced Conchosporangia
Single-celled conchocelis were statically cultured in wells containing sterilized artificial seawater under the conditions described above except that they were aerated. Swelling tip cells of conchocelis side branches were then detected by microscopy, and their growth and side-branch formation were monitored for 7 days using an Olympus IX73 light microscope equipped with an Olympus DP22 camera. The branching rate was calculated as the number of cells producing a branch as a percentage of the total number of cells observed.

Chemical Treatment of Isolated Cells and Naturally Produced Conchosporangia
Pharmacological treatments with auxins, auxin antagonists, or 5-alkoxy-IAAs were performed by incubating isolated cells and naturally produced conchosporangium filaments at 15 • C in wells containing 200 µL artificial seawater. Auxin treatment was performed by incubating the cells for 1 week in 5, 10, 30, 50, or 100 µM IAA (Nakalai Tesque, Kyoto, Japan), NAA (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), or 2,4-D (Nakalai Tesque, Kyoto, Japan) in 200 µL artificial seawater in wells. Three types of auxin antagonists, PEO-IAA, 4-Cl-PEO-IAA, and BH-IAA [33,43], were added to the artificial seawater in the wells to generate 10, 20, 30, 40, or 50 µM solutions, followed by incubation of the cultures for 1 week. For co-treatment with auxins and PEO-IAA, different concentrations of IAA, NAA, or 2,4-D (5, 10, 20, or 30 µM) and a single concentration of PEO-IAA (30 µM) were used. Five derivatives of 5-alkoxy-IAAs, named 1a to 5a [45], were used at a concentration of 30 µM for the assays, whereas the other experiments employed 10 µM 5-alkoxy-IAAs or IAA in the presence of 30 µM PEO-IAA for 2 weeks. Cells treated with chemicals for 2 weeks in static culture were observed and photographed using an Olympus IX73 light microscope equipped with an Olympus DP22 camera to evaluate the effects of chemicals on tip growth and the formation of side branch initials. Branching rate was calculated as the number of cells producing a branch as a percentage of the total number of cells observed.

Statistical Analysis
Mean values ± SD were calculated from triplicate experiments. A statistically significant interaction was detected between the duration of incubation with various combinations of chemicals and the regeneration of apical stem cells, as determined by one-way ANOVA with the Tukey-Kramer test (p < 0.05). Significant differences for each set of treatments were determined using a cutoff value of p < 0.05.

Generation of Apical Stem Cells from Single-Celled Conchocelis to Observe Tip Growth
Since side branches develop from differentiated nondividing cells in conchocelis filaments (see Figure 1B of [47]), we reasoned that tip growth, with the production and maintenance of an apical stem cell, could be observed by examining the formation of a side branch from a single-celled conchocelis. Thus, we prepared single-celled conchocelis ( Figure 1B) by chopping sporophyte filaments ( Figure 1A) with a razor blade and examined branching from these isolated cells. As expected, approximately 90% of nonbranched single cells produced side branches after culturing for 7 days ( Figure 2G); the positions of side branch initials in the cylindrical conchocelis cells appeared to be random ( Figure 1C-E). Therefore, the single-celled conchocelis provides a novel, simple experimental system for addressing the regulatory mechanisms of tip growth of the filamentous sporophyte generation of N. yezoensis.

The Role of Auxin in the Generation and Tip Growth of Side Branches in Conchocelis
Since the exogenous application of auxin has positive effects on tip growth in terrestrial plants [28][29][30], we first examined whether tip growth from a single-celled conchocelis could be stimulated by the exogenous application of indole-3-acetic acid

The Role of Auxin in the Generation and Tip Growth of Side Branches in Conchocelis
Since the exogenous application of auxin has positive effects on tip growth in terrestrial plants [28][29][30], we first examined whether tip growth from a single-celled conchocelis could be stimulated by the exogenous application of indole-3-acetic acid (IAA). However, no effect was observed (data not shown), suggesting that if auxin is required for tip growth, an adequate amount was already present in the isolated cells.
We next addressed the role of auxin in tip growth by examining the effect of modulating the activity of the auxin receptor, since the functions of auxin receptors can be elucidated via chemical biology approaches using auxin receptor antagonists [33,43,44]. When isolated conchocelis cells were treated with the auxin receptor antagonist PEO-IAA [33], the generation and tip growth of side branches were repressed in a concentrationdependent manner ( Figure 2). The repression of branch formation by treatment with 30 µM PEO-IAA was recovered by the exogenous application of 5, 10, or 20 µM IAA ( Figure 3). Although 30 µM IAA had a negative effect on branch formation (Figure 3), this was likely an off-target effect of a high concentration of IAA on growth, as observed in terrestrial plants [48][49][50][51]. These findings indicate that auxin plays an important role in regulating tip growth and that N. yezoensis contains an auxin receptor-like protein that regulates tip growth in conchocelis filaments. (IAA). However, no effect was observed (data not shown), suggesting that if auxin is required for tip growth, an adequate amount was already present in the isolated cells. We next addressed the role of auxin in tip growth by examining the effect of modulating the activity of the auxin receptor, since the functions of auxin receptors can be elucidated via chemical biology approaches using auxin receptor antagonists [33,43,44]. When isolated conchocelis cells were treated with the auxin receptor antagonist PEO-IAA [33], the generation and tip growth of side branches were repressed in a concentration-dependent manner ( Figure 2). The repression of branch formation by treatment with 30 μM PEO-IAA was recovered by the exogenous application of 5, 10, or 20 μM IAA ( Figure 3). Although 30 μM IAA had a negative effect on branch formation ( Figure 3), this was likely an off-target effect of a high concentration of IAA on growth, as observed in terrestrial plants [48][49][50][51]. These findings indicate that auxin plays an important role in regulating tip growth and that N. yezoensis contains an auxin receptorlike protein that regulates tip growth in conchocelis filaments.   Error bars indicate the standard deviation of triplicate independent experiments (n = 3), and different lowercase letters denote significant differences in the branching rate, as determined by Tukey's test (p < 0.05) for each set of incubation times.

Role of Auxin in Tip Growth and Side Branch Production in Conchosporangia
The conchosporangium, a structure representing the conchosporophyte generation of the N. yezoensis life cycle [40], is produced on the conchocelis via the swelling of the apical cell of a side branch [52], representing a type of tip growth [47]. Since nondividing differentiated cells produce side branches ( Figure S1), we expected that single-celled conchosporangium (as well as conchocelis) would be suitable to study tip growth. However, most single cells did not produce side branches, unlike the single-celled conchocelis, as described above ( Figure 4A). Nonetheless, isolated cells derived from apical cells were able to divide and generate side branch initials for their tip growth ( Figure 4B). As shown in Figure 4B, branch formation from the conchosporangium exhibited two unique characteristics. First, the branches formed at the nondividing differentiated cell adjacent to the apical tip cell. Second, the width of the branch and the length of the nondividing cell were similar, resulting in the production of thick, cylinder-shaped outgrowths. Error bars indicate the standard deviation of triplicate independent experiments (n = 3), and different lowercase letters denote significant differences in the branching rate, as determined by Tukey's test (p < 0.05) for each set of incubation times.

Role of Auxin in Tip Growth and Side Branch Production in Conchosporangia
The conchosporangium, a structure representing the conchosporophyte generation of the N. yezoensis life cycle [40], is produced on the conchocelis via the swelling of the apical cell of a side branch [52], representing a type of tip growth [47]. Since nondividing differentiated cells produce side branches ( Figure S1), we expected that single-celled conchosporangium (as well as conchocelis) would be suitable to study tip growth. However, most single cells did not produce side branches, unlike the single-celled conchocelis, as described above ( Figure 4A). Nonetheless, isolated cells derived from apical cells were able to divide and generate side branch initials for their tip growth ( Figure 4B). As shown in Figure 4B, branch formation from the conchosporangium exhibited two unique characteristics. First, the branches formed at the nondividing differentiated cell adjacent to the apical tip cell. Second, the width of the branch and the length of the nondividing cell were similar, resulting in the production of thick, cylindershaped outgrowths.  Chopping the conchosporangia did not produce enough free apical cells to allow us to perform experiments. Thus, we improved the experimental system using single-celled conchocelis. In these structures, the formation of conchosporangia was observed in some branches after about 2 weeks of culture. This spontaneous production of conchosporangia enabled us to monitor tip growth more easily compared to examining isolated apical cells obtained by chopping. As shown in Figure 5A,B, we were able to confirm the same pattern of tip growth and branch formation in these conchosporangia as we had observed in single-celled conchosporangia ( Figure 4B). Chopping the conchosporangia did not produce enough free apical cells to allow us to perform experiments. Thus, we improved the experimental system using single-celled conchocelis. In these structures, the formation of conchosporangia was observed in some branches after about 2 weeks of culture. This spontaneous production of conchosporangia enabled us to monitor tip growth more easily compared to examining isolated apical cells obtained by chopping. As shown in Figure 5A,B, we were able to confirm the same pattern of tip growth and branch formation in these conchosporangia as we had observed in single-celled conchosporangia ( Figure 4B). We employed our novel experimental system to investigate the role of auxin in the tip growth of conchosporangia. When the tips of side branches that formed on conchocelis filaments began to swell, which we took as a signal of conchosporangium development, we treated the filaments with 5, 15, or 30 μM IAA and observed growth after 3, 5, and 7 We employed our novel experimental system to investigate the role of auxin in the tip growth of conchosporangia. When the tips of side branches that formed on conchocelis filaments began to swell, which we took as a signal of conchosporangium development, we treated the filaments with 5, 15, or 30 µM IAA and observed growth after 3, 5, and Cells 2022, 11, 2652 9 of 16 7 days of culture. In contrast to conchocelis, the tip growth of conchosporangium was accelerated by exogenously supplied auxin. The greatest increases in both the length and cell number of branches occurred following 15 µM IAA treatment, although 5 and 30 µM IAA treatments had lesser but significant effects equally ( Figure 5C,D). By contrast, treating the swelling tips of side branches with 5, 10, or 20 µM PEO-IAA resulted in the repression of tip growth in a concentration-dependent manner ( Figure 6). These findings indicate that auxin regulates tip growth in conchosporangia.
Cells 2022, 11, x 10 of 17 days of culture. In contrast to conchocelis, the tip growth of conchosporangium was accelerated by exogenously supplied auxin. The greatest increases in both the length and cell number of branches occurred following 15 μM IAA treatment, although 5 and 30 μM IAA treatments had lesser but significant effects equally ( Figure 5C,D). By contrast, treating the swelling tips of side branches with 5, 10, or 20 μM PEO-IAA resulted in the repression of tip growth in a concentration-dependent manner ( Figure 6). These findings indicate that auxin regulates tip growth in conchosporangia.

Characterization of a Unique Auxin Receptor in N. yezoensis Using IAA Derivatives
The auxin receptor antagonist PEO-IAA repressed the regeneration and tip growth of apical tip cells in side branches from both conchocelis and conchosporangium, suggesting the presence of functional auxin receptors in N. yezoensis. However, in previous studies, we failed to identify genes encoding homologs of the auxin receptor TIR1 or auxin-responsive factors (ARFs) in the N. yezoensis genome [53,54]. We therefore predicted that N. yezoensis might contain a novel, unknown auxin receptor-like protein.
To test this hypothesis, we utilized chemical biology approaches.
First, we assessed the binding of auxin to an unknown auxin receptor by examining the reversal by auxins of the repression of side branch formation by 30 μM PEO-IAA. The

Characterization of a Unique Auxin Receptor in N. yezoensis Using IAA Derivatives
The auxin receptor antagonist PEO-IAA repressed the regeneration and tip growth of apical tip cells in side branches from both conchocelis and conchosporangium, suggesting the presence of functional auxin receptors in N. yezoensis. However, in previous studies, we failed to identify genes encoding homologs of the auxin receptor TIR1 or auxin-responsive factors (ARFs) in the N. yezoensis genome [53,54]. We therefore predicted that N. yezoensis might contain a novel, unknown auxin receptor-like protein. To test this hypothesis, we utilized chemical biology approaches.
First, we assessed the binding of auxin to an unknown auxin receptor by examining the reversal by auxins of the repression of side branch formation by 30 µM PEO-IAA. The native auxin IAA exhibited potent auxin activity in the promotion of branch formation (Figure 7), whereas 5 to 30 µM 2,4-D or NAA showed weak activity in branch formation in the presence of PEO-IAA ( Figure S2). These findings indicate that both native and synthetic auxins function in N. yezoensis, demonstrating the presence of an auxin receptor-like protein in this alga. native auxin IAA exhibited potent auxin activity in the promotion of branch formation (Figure 7), whereas 5 to 30 μM 2,4-D or NAA showed weak activity in branch formation in the presence of PEO-IAA ( Figure S2). These findings indicate that both native and synthetic auxins function in N. yezoensis, demonstrating the presence of an auxin receptorlike protein in this alga. Side branch formation was observed in single-celled conchocelis cultured with each chemical for 2 weeks, and the number of conchocelis with branching was counted by microscopy observation at 2, 3, 4, 5, 6, 7, and 14 days to calculate the branching rate. (B) Activities of 5-alkoxy-IAAs. Isolated conchocelis cells were incubated in the presence of auxin derivatives 1a to 5a for 2 weeks, and the effects of these 5-alkoxy-IAAs on side branch formation were observed using a microscope at 1, 2, 3, 4, 5, 6, and 7 days. Error bars indicate the standard deviation of triplicate independent experiments (n = 3), and different lowercase letters denote significant differences in branching rate, as determined by Tukey's test (p < 0.05) for each set of incubation times.
We then examined the binding of another auxin antagonist, the PEO-IAA derivative 4-Cl-PEO-IAA [33,43], to the auxin receptor-like protein. As shown in Figure S3A, 4-Cl-l-PEO-IAA acted as an auxin antagonist for side branch formation, indicating that the IAA moiety of PEO-IAA is recognized by an unknown auxin receptor-like protein in N. Side branch formation was observed in single-celled conchocelis cultured with each chemical for 2 weeks, and the number of conchocelis with branching was counted by microscopy observation at 2, 3, 4, 5, 6, 7, and 14 days to calculate the branching rate. (B) Activities of 5-alkoxy-IAAs. Isolated conchocelis cells were incubated in the presence of auxin derivatives 1a to 5a for 2 weeks, and the effects of these 5-alkoxy-IAAs on side branch formation were observed using a microscope at 1, 2, 3, 4, 5, 6, and 7 days. Error bars indicate the standard deviation of triplicate independent experiments (n = 3), and different lowercase letters denote significant differences in branching rate, as determined by Tukey's test (p < 0.05) for each set of incubation times.
We then examined the binding of another auxin antagonist, the PEO-IAA derivative 4-Cl-PEO-IAA [33,43], to the auxin receptor-like protein. As shown in Figure S3A, 4-Cl-l-PEO-IAA acted as an auxin antagonist for side branch formation, indicating that the IAA moiety of PEO-IAA is recognized by an unknown auxin receptor-like protein in N. yezoensis. When the auxin receptor antagonist BH-IAA, which is a structural derivative distinct from PEO-IAA and 4-Cl-PEO-IAA [33,43], was employed, it showed weak auxin-antagonistic activity on side branch formation in N. yezoensis ( Figure S3B). These results indicate that an auxin receptor-like protein recognizes the common IAA moiety of these auxin antagonists.
In treating single-celled conchocelis with 30 µM PEO-IAA plus 10 µM 5-alkoxy-IAAs for 2 weeks, treatment with 1a and 2a allowed the recovery from the inhibited branching and tip growth induced by PEO-IAA, and by 10 µM IAA ( Figure 7B). However, 3a and 4a did not counteract the effects of PEO-IAA, and 5a showed additive antagonistic activity with PEO-IAA ( Figure 7B). Thus, 1a and 2a acted as auxins, while 3a, 4a, and 5a displayed auxin-antagonistic activity, indicating that 3a, 4a, and 5a act differently in N. yezoensis than in A. thaliana. Notably, although 5a apparently bound to an auxin receptor-like protein in N. yezoensis and functioned as an auxin antagonist, a previous study showed that this compound did not affect TIR1 function in A. thaliana [45]. Therefore, based on the results of our auxin structure-based experiments, we conclude that the auxin-recognition site of the auxin receptor-like protein of N. yezoensis is different from that of the A. thaliana TIR1/AFB receptor. Thus, auxin regulates side branch formation and tip growth in N. yezoensis via an unknown auxin receptor.

Discussion
We performed chemical biology studies to explore the mechanisms underpinning tip growth of the conchocelis and conchosporangium in the red seaweed N. yezoensis. Our novel experimental procedures allowed us to successfully demonstrate the critical role of auxin in tip growth of two filamentous generations in the N. yezoensis life cycle: conchocelis (sporophyte) and conchosporangium (conchosporophyte). Despite the presence of auxin in N. yezoensis and 'Bangia' sp. ESS1 [53,54], the physiological roles of auxin in Bangiales have not yet been elucidated. Thus, our results provide the first evidence for the physiological function of auxin in Bangiales. Since auxin was already shown to be involved in tip growth in the brown alga Ectocarpus siliculosus [5], it is plausible that role of auxin in regulating tip growth is conserved in seaweeds.
As shown in Figure 1, the positions of branches in single-celled conchocelis were different from those in the protonema of P. patens, whose branch initials are produced at the apical ends of filamentous differentiated cells [55]. Our findings indicate that polar regulation does not determine the branching position in isolated conchocelis cells. Since it is unknown whether the isolation of single cells affects the positioning of apical stem cell production, it is important to examine branch formation in natural filamentous conchocelis to address whether polar regulation affects branch formation in N. yezoensis.
While the conchocelis and conchosporangium both expanded via tip growth and branching in N. yezoensis, our observations indicate that their growth processes are different, especially their branching patterns. In conchocelis filaments, thin hyphae-type outgrowths are produced in nondividing mature cells distant from the tip cell in the primary filament. Since the nondividing cell is longer than the width of cells in the primary filament, branching usually involves tubular filamentous extensions, similar to branch formation in protonema of the moss P. patens [4,12,56]. By contrast, branching in conchosporangia involves the production of thick outgrowths whose diameters are nearly identical to the length of the nondividing cell (Figures 4 and 5), like in E. siliculosus [4,5]. These findings point to the different mechanisms by which branching is achieved in the conchocelis and conchosporangium: The former requires the side branch initial to be positioned some-where along the longitudinal side of the cell, but the latter does not. Although common protein factors function in tip growth in both protonema and caulonema in P. patens [56], perhaps the regulatory machinery of tip growth is basically similar but not identical in the conchocelis and conchosporangium. Thus, further characterizing the factors involved in the generation of the novel apical stem cell in branching initials will help uncover the differences in the branching systems of these two generations of the N. yezoensis life cycle.
Our results also indicate the presence of a novel, yet to be identified, auxin receptor in N. yezoensis. Genes encoding the auxin receptor TIR1/AFB and the transcription factors Aux/IAAs and ARFs have not been identified in the genomes of Bangiales species [53,54]. Thus, factors that participate in the auxin signal transduction pathway in N. yezoensis were not previously identified and appear to be novel [53,54]. Our chemical biology studies clearly demonstrated that the auxin receptor-like protein of N. yezoensis binds to the 1a to 5a versions of 5-alkoxy-IAA used in the present study; this is markedly different from A. thaliana TIR1/AFB, which does not recognize 5a [45].
Since 1a and 2a showed auxin activity and 5a, like 3a and 4a (Figure 7), acts as an auxin antagonist, it appears that the N. yezoensis auxin receptor-like protein contains one auxin-binding pocket. However, this hypothesis does not explain why treating single-celled conchocelis with PEO-IAA and 5a together had additive effects repressing side branch formation ( Figure 7B). If these molecules competitively bind to the receptor-like protein with the same affinity, the effects of their combined application should be identical to those of PEO-IAA alone. Thus, the affinity of 5a for the N. yezoensis auxin receptor-like protein should be higher than that of PEO-IAA. However, as shown in Figure 7A, PEO-IAA and 5a had identical effects in terms of antagonizing side branching. It is therefore plausible that N. yezoensis contains at least two auxin receptor-like proteins, one of which dominantly binds to PEO-IAA and another to 5a. In this case, the additive effect of these chemicals in the combined treatment of single-celled conchocelis could be explained by the combined actions of two auxin receptor-like proteins binding separately to PEO-IAA and 5a. Indeed, the TIR1/AFB families of terrestrial plants contain multiple, functionally diverse auxin receptors [57][58][59]. Like N. yezoensis [53,54], E. siliculosus also lacks any factors homologous to known auxin signal transduction components in terrestrial plants [60], indicating that the modes of action of auxin in regulating tip growth in N. yezoensis and E. siliculosus are different from those in terrestrial plants. Therefore, identifying and characterizing auxin receptors and factors involved in the auxin signal transduction pathway in seaweeds would enable us to define the novel regulatory mechanisms of auxin-directed tip growth in photosynthetic filamentous organisms.
Notably, the N. yezoensis genome also lacks homologs of the genes encoding auxin biosynthetic enzymes in A. thaliana [53,54]. Thus, the origin of the auxin in this apparently non-auxin-producing seaweed must be clarified to understand how it obtains this plant hormone to regulate tip growth in both the conchocelis and conchosporangium. A variety of epiphytic bacteria have been isolated from N. yezoensis [61][62][63][64][65][66] and other red seaweeds [67][68][69][70], some of which synthesize IAA [67,71]. Although the physiological function of bacterial IAA has not yet been confirmed, it is possible that epiphytic bacteria of N. yezoensis synthesize IAA and promote the generation of apical stem cells and their expansion for tip growth in conchocelis and conchosporangium cells. Moreover, since E. siliculosus can produce auxin [60] but hosts many types of epiphytic bacteria [72,73], the supply of auxin from epiphytic bacteria might depend on the algal species. Therefore, the origin and modes of action of auxin in N. yezoensis should be addressed to elucidate the regulatory mechanisms of tip growth in seaweeds.

Conclusions
The tip growth in both conchocelis and conchosporangia filaments was repressed by auxin antagonists and sensitivity to 5-alkoxy-IAAs was different between N. yezoensis and A. thaliana. Thus, our chemical biology studies indicated that the tip growth of filamentous generations of the N. yezoensis life cycle is regulated by auxin via a novel unknown auxin receptor-like protein. However, even though the outgrowth of secondary filaments in both generations occurs via tip growth, their branching patterns differ. In addition, it is unknown how N. yezoensis obtains auxin. To clarify the molecular basis of the auxinmediated regulation of tip growth in N. yezoensis, it is important to identify the auxin receptor and factors that mediate auxin signaling as well as auxin-producing epiphytic bacteria that might be involved in regulating tip growth.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cells11172652/s1, Figure S1. Conchosporangia of Neopyropia yezoensis. Figure S2. Reversal of the inhibitory effects of PEO-IAA by exogenous treatment with 2,4-D or NAA. Figure S3. Effects of treatment with the auxin antagonists 4-Cl-PEO-IAA and BH-IAA on the production of side branches in single-celled conchocelis.