# Click and Sea: using bioorthogonal click chemistry to visualise seaweed cell walls

Wendy Delpont, Godfrey Neutelings, Zoë A Popper

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## To cite this version:

Wendy Delpont, Godfrey Neutelings, Zoë A Popper. Click and Sea: using bioorthogonal click chemistry to visualise seaweed cell walls. Annals of Botany, In press, 10.1093/aob/mcaf103. hal-05110367

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TECHNICAL ARTICLE

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**Click & Sea: using bioorthogonal click chemistry to visualise seaweed cell walls**

Wendy f. Delpont¹*, Godfrey Neutelings², Zoë a. Popper¹

¹ Botany and Plant Science, School of Natural Science, College of Science and Engineering,

University of Galway, Galway, Ireland, H91 TK33; ² Univ. Lille, CNRS, UMR 8576 - UGSF -

Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France

*For correspondence: wendy.delpont31@gmail.com

**Running title:** Click chemistry of seaweed cell walls

Received: 07/01/2025

Returned for revision: 06/05/2025

Editorial decision: 15/05/2025

**Keywords:** cell wall – seaweed – polysaccharides – bioorthogonal click chemistry – strain-promoted alkyne-azide cycloaddition (SPAAC)

**Abstract**

*Objectives*

The study of seaweed cell walls, including their metabolism and composition, is crucial to monitor and understand their adaptation to climate change. Microscopy-based techniques that facilitate studies of seaweed cell walls *in situ*, including staining and immunolabelling, exist but have

© The Author(s) 2025. Published by Oxford University Press on behalf of Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
1 significant limitations, including that only a few monoclonal antibodies have been developed
2 towards seaweed cell wall components. Furthermore, not all seaweed cell wall components have
3 been fully described. This makes *in situ* studies focussed on the metabolism of seaweed cell walls
4 particularly challenging. Here, we present a method for labelling seaweed cell walls by
5 incorporating chemical reporters *in muro*, followed by their association with fluorophores by click
6 chemistry.

7 • *Methods*

8 Two different species of seaweed, representing different groups, i.e. the red seaweed *Phycodrys*
9 *rubens* and the green seaweed *Ulva* spp. were selected for their abundance on the Irish coast and
10 because they have relatively thin tissues, a feature likely to facilitate uptake and labelling using
11 monosaccharides reporters. We selected three different activated sugars (fucose, galactose and
12 glucose analogues) on the basis that they are major components of seaweed polysaccharides. Small
13 sections of the seaweeds were incubated with the activated sugars, and their uptake and
14 incorporation were visualised by attachment to the fluorescent probe AF488 and imaged using
15 confocal microscopy.

16 • *Results*

17 After incubation with the activated sugars, the seaweed cell walls, i.e. the contours of the cells,
18 and, to a lesser extent, some cell organelles, fluoresced at 517 nm (emission wavelength of AF488),
19 suggesting that the seaweed had incorporated the activated sugars. Fiji software was used to
20 remove nonspecific fluorescence (autofluorescence of the seaweed and non-specific binding of the
21 fluorochrome), with the final images suggesting good and specific incorporation of the
22 fluorochrome by the seaweeds. More interestingly, the fluorescence was primarily associated with
1 the cell walls, implying that the activated sugars were predominantly incorporated into cell wall
2 components (most likely either polysaccharides or glycoproteins).

3 • Conclusion

4 To date, bioorthogonal click chemistry has not been applied to seaweed but represents a useful tool
5 for phycologists to better understand seaweed cell wall composition and dynamics, for example,
6 throughout the seaweed life cycle or in the face of stresses (biotic or abiotic), including those
7 resulting from climate change.

8

9 INTRODUCTION

10 The ability to look inside living tissue, specifically inside cells, has always intrigued researchers
11 and has long been a subject of study. However, it can be difficult to recognise and differentiate the
12 cell components. Increasingly sophisticated methods have been developed over the years, enabling
13 the identification of specific molecules: from histological tissue staining in the 1770s (Alturkistani
14 et al., 2016) to immunofluorescence targeting specific parts of polysaccharides and proteins
15 (Coons et al., 1941) to protein labelling via genetics (Chalfie et al., 1994), and, more recently, the
16 integration of fluorescent tags into the genome via genome editing (Schwinn et al., 2018).

17

18 Seaweeds are predominantly marine photosynthetic organisms. They are multicellular organisms
19 typically occurring in coastal locations and organised into rock-fixed thalli, although some, such
20 as *Sargassum*, may be pelagic. Some seaweeds can be very small, couple of cm, while can be up
21 to 70 meters long. However, despite similarities, including habitat, multicellularity, and the ability
22 to photosynthesise and produce sulphated polysaccharides, seaweeds are not a single group but
23 rather include several lineages. Seaweeds are enormously diverse and possess many distinct
1 features. However, they are frequently grouped according to their pigmentation into one of three

2 taxa: green (Chlorophyta), red (Rhodophyta) and brown (Phaeophyceae). In this manuscript, we

3 will focus on their cell wall compositions, a feature that is sufficiently diverse that it has been used

4 as an important character to define individual taxa (Stace, 1989). The green seaweed cell wall is

5 mainly composed of cellulose microfibrils (made up of D-glucose residues), making up 15-30%

6 of the total dry weight of the seaweed (Wahlström et al., 2020). Hemicellulose structures, which

7 play a cohesive role in the wall, are also found. The main hemicelluloses present in green seaweed

8 are xyloglucans, mannans and β-(1-3)-glucans. Other polysaccharides, called ulvans, are found

9 only in the Chlorophyta. Ulvans are sulphated polysaccharides composed mainly of L-rhamnose
10 and glucuronate chains to which D-xylose, iduronic acid, D-mannose, D-galactose and fucose
11 residues may be added. Ulvans are made up of a disaccharide repeat, whose composition can vary.

10

11

12 However, there are two main combinations of repeats: type A and type B. Type A is called
13 ulvanobiuronic acid 3-sulphate (A3S) and is made up of rhamnose-3-sulphate and glucuronic acid

13

14 or iduronic acid. Type B is called ulvanobiuronic acid 3-sulphate (B3S) and is made up of
15 rhamnose-3-sulphate and xylose (Lahaye & Robic, 2007). Carbohydrates are stored as starch in

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16 green seaweed, like in land plants. This starch is formed of two polymers, amylose and
17 amylopectin, composed of D-anhydroglucopyranose (Prabhu et al., 2019).

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18 Other molecules, such as glycoproteins, including arabinogalactan proteins (AGPs) and extensins,
19 are also present in the cell walls of green seaweed. AGPs and extensins also occur in green land

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20 plants (Clarke et al., 1979). However, green seaweed AGPs and extensins have structural features
21 that are seaweed-specific, such as a unique sugar residue profile and the presence of sulfated sugars

20

22 that are not present in most land plants (Estevez et al., 2009; Liu et al., 2016; Přerovská et al.,
23 2021).

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In red seaweed cell walls, cellulose microfibrils constitute only 1–10% of the total dry weight of the seaweed (Ross, 1953) and may be replaced by either glucomannans or xylans, depending on the species. Similarly to ulvans in the Chlorophyta (green seaweed), red seaweed possesses specific polysaccharides, carrageenans and agars. Carrageenans are linear sulphated galactans composed of repeat units of α-D-galactopyranose and β-D-galactopyranose residues. There are fifteen different types of carrageenans known based on their degree of sulfation and the location of sulfate side groups. Nevertheless, only three carrageenans are commonly found: the ι- (iota), κ-(kappa) and λ- (lambda) carrageenans (Lahaye, 2001; Stiger-Pouvreau et al., 2016). Agar is composed of two different types of molecules called agarose and agarpectin. Both molecules are made up of disaccharide repeat units composed of-D-galactopyranose and anhydro-3,6-α-L-galactopyranose (Usov, 2011). Agarose is a linear polysaccharide, and agarpectin is a branched polysaccharide. Some sulphate groups may be attached to the agarpectin structure, which confers a degree of sulfation to the molecule (Lahaye & Rochas, 1991). Red seaweed also stores carbohydrates in the form of starch. However, this starch is different to the starch found in green seaweed and land plants and is called floridean starch. Although floridean starch is composed of a highly branched succession of α-(1→4) linked glucose units, (Ball et al., 2011) it differs from the starch found in land plants by its composition and degree of branching.

Seaweed cell walls, similarly to those of land plants, play important roles in cell structure, cell growth and the maintenance of cell homeostasis. The cell wall is also a physical barrier involved in interacting with the environment and its various stresses and in cell-cell communication (Kloareg & Quatrano, 1988; Popper et al., 2011). As a result, their cell wall compositions are diverse (Table 1), and many seaweed are commercially important, predominantly because of their constituent polysaccharides that are largely used for their gelling and viscosifying properties in
biomedical, pharmaceutical, cosmetic (Cardozo et al., 2007; Pereira, 2018), food and feed, agricultural (Craigie, 2011), textiles (Lomartire et al., 2022) industries as well as for biofuels (Sharmila et al., 2021) and bioremediation (Ibrahim, 2011; Leandro et al., 2020; Makkar et al., 2016; Ścieszka & Klewicka, 2019). In 2023, the market for seaweed products was valued at 5.3 billion USD, and forecasts predict an average annual growth of 6.4% to reach a total market of 7.3 billion USD by 2028 (MarketandMarkets, 2023). Although used in a wide range of fields and carrying considerable weight in the world markets, the components and dynamics of seaweed cell walls are not well studied in *situ*.

Different techniques have been developed to study the cell walls and their components in seaweed. Specific histological stains, including alcian blue, calcofluor white or concanavalin A, can be used to observe and follow the metabolism of cell wall components. For example, Martone et al., in 2010, observed the growth of the red seaweed *Calliarthron cheilosporioides* using calcofluor white (Martone, 2010). Other techniques, such as immunolabeling and the development of specific monoclonal antibodies, for example, to different alginate structures, have allowed researchers to look at the development of zygotes in the brown seaweed *Fucus* (Torode et al., 2016).

Despite the progress made in understanding seaweeds using these methods, there are many limitations, including that seaweed cell walls are highly diverse and that their components, even some of those that are amongst the most widely used, such as fucoidans, have not yet been fully characterised. A few monoclonal antibodies that were developed against land plant cell wall components can recognise features present in brown (Raimundo et al., 2016) and green seaweeds (Estevez et al. 2009; Fernández et al. 2010). However, the development of highly specific monoclonal antibodies that label structures present in seaweed cell wall polysaccharides, giving insight into their location and metabolism, has been very limited. This is especially the case in
1 comparison to the large array of different monoclonal antibodies available against plant cell wall

2 components (Duffieux et al., 2020; Pattathil et al., 2010). Currently, only a few monoclonal

3 antibodies are available that were specifically developed against seaweed cell wall components.

4 These include the range of monoclonal antibodies, commercially available from SeaProbes

5 (https://www.sb-roscoff.fr/en/seaprobes), that recognise and bind to alginates and fucoidans found

6 in brown seaweed (Torode et al., 2015, 2016). These antibodies recognise alginates with different

7 sulfation levels and possess different ratios of glucuronic and mannuronic acids (Torode et al.,

8 2015, 2016) and have enabled improved knowledge of zygote development in brown seaweed

9 (Torode et al., 2015, 2016). Monoclonal antibodies have also been made to ulvans (Rydahl et al.,

10 2017). Many more seaweed cell wall polysaccharide-specific monoclonal antibodies were

11 developed, predominantly to brown seaweed polysaccharides (Jones et al., 1988; Vreeland, 1970;

12 Vreeland & Laetsch, 1989) but also to carrageenans from red seaweeds (Vreeland et al., 1992).

13 Unfortunately, the majority of these antibodies are no longer commercially available to the

14 scientific community (Raimundo et al., 2016), and information regarding the localisation and

15 dynamics of seaweed cell wall polysaccharides, despite increasing in recent years (Estevez et al.

16 2009; Fernández et al. 2010, Torode et al., 2015, 2016, Raimundo et al., 2015, 2026), remains

17 scarce (Raimundo et al., 2016), particularly for red seaweeds.

18 Red seaweed cell walls, despite the significant commercial importance of their polysaccharides,

19 are comparatively less well characterised than green and brown seaweed cell walls, and there

20 remains a distinct lack of available tools for studying their *in situ* dynamics, e.g., in response to

21 climate change.

22 In the 1960s, Huisgen (Huisgen, 1961) developed the concept of 1,3-dipolar cycloadditions, which

23 highly inspired K. B. Sharpless, who first used the term “click chemistry” (Kolb et al., 2001). His
1 laboratory and that of Morten Meldal have developed copper-catalyzed regioselective chemical
2 associations of the Huisgen cycloaddition of azides and terminal alkynes to form 1,2,3-triazoles.

3 They were awarded the Nobel Prize in Chemistry in 2022, along with Carolyn Bertozzi, who, in
4 the late 1990s, described the possibility of carrying out such reactions under biological conditions
5 inside cells. These reactions are called bioorthogonal, meaning they occur rapidly in aqueous
6 solutions, in the presence of biological molecules, at low concentrations, and are extremely
7 selective, as they only react with a single functional group. The reactions resulting from these
8 works are azide-alkyne cycloadditions depending (CuAAC: copper-catalysed azide-alkyne
cycloaddition) or not (SPAAC: Strain Promoted Alkyne-Azide Cycloaddition) on copper. In the
9 first case, the copper(I) ion lowers the activation energy, making the reaction highly efficient and
10 selective, and in the second case, the strain in the cyclooctyne drives the reaction with the azide,
11 bypassing the need for copper (Agard et al., 2004). The use of click chemistry approaches is
12 generally straightforward from a technical point of view, with the only possible difficulties being
13 the mobility of reporters across cell interfaces and verification of their compatibility with cellular
14 metabolism. The real challenge, however, remains their synthesis when they are not commercially
15 available, as is the case for lignin precursors (Morel et al., 2022).

17 Although CuAAC and SPAAC have been successfully applied to study plant cell walls (Anderson
18 et al., 2012; Lion et al., 2017; Morel et al., 2022; Simon et al., 2018), it appears not to have been
19 applied to seaweed cell walls. In this article, we adapted and applied the SPAAC technique to
20 seaweed to label their cell wall components. The seaweed species were chosen because of their
21 availability on the Irish coast so that the material used could be as fresh (and as metabolically
22 active) as possible and for the thinness of their tissues, which typically is associated with better
23 uptake and integration of the reagents used in the click chemistry reactions. The green seaweed,
Ulva, was additionally chosen as it has been sequenced (De Clerck et al., 2018), and its cell wall
components are relatively well-characterised (Bothwell et al., 2022; Lahaye & Robic, 2007). The
red seaweed, *Chondrus crispus*, has also been sequenced (Collén et al., 2013) and has commercial
importance in part derived from its high concentration of carrageenans. The gametophyte and
sporophyte can also be distinguished since the gametophyte generation exhibits autofluorescence
(Fournet et al., 1993).

To the best of our knowledge, click chemistry has not previously been applied to seaweed. We
tested the integration of three modified sugar residues that commonly occur in cell wall
components (fucose, galactose, glucose). We were then able to observe the cell components that
had integrated the modified sugar residues by reacting them to attach a fluorescent dye and
visualising using confocal microscopy.

## 13 METHODS

### 15 Collection of seaweed

Seaweeds were collected at low tide in May 2023 at An Spidéal, Co. Galway, Ireland (53°14' 33"
N - 9°18'23" W). This collection site is predominantly sandy, surrounded by sandstone rocks in
which there are intertidal pools at low tide.

Two species of seaweed were collected: one red seaweed (*Rhodophyta*), *Phycodrys rubens*, and
one green seaweed (*Chlorophyta*) *Ulva spp* (Fig.1). All the seaweeds collected were visually
ungrazed or damaged, adult in appearance, and clean with no visible parasites.

After collection, the seaweed samples were transported to the laboratory in a sealed plastic bag
containing seawater. In the laboratory, the samples were washed using clean seawater to remove
any particles of sand and placed into 50 mL Falcon tubes containing clean seawater. The samples were stored at 10°C for approximately 20 hours, followed by storage at 4°C for 40 hours. The seaweed samples were then cut into small square sections (approximately 20 mm by 20 mm) using dissecting scissors.

5

## *Sugar reporters*

The ability of the seaweed sections to uptake and incorporate three common monosaccharide reporters was tested. The monosaccharide reporters were 1,2,3,4-Tetra-*O*-acetyl-6-azido-L-fucopyranose (TF608, Synthose, Concord, Canada), 1,3,4,6-Tetra-*O*-acetyl-2-azido-2-deoxy-D-galactopyranose (TL709, Synthose, Concord, Canada), and 1,3,4,6-Tetra-*O*-acetyl-2-azido-2-deoxy-D-glucopyranose (TG713, Synthose, Concord, Canada) (Fig.2). These three modified monosaccharides contain an azide moiety, which allows another molecule, previously linked by an alkyl moiety, to attach to them together and form a triazole. In addition, the peracetylated forms were chosen to allow transport inside the cells (Anderson & Wallace, 2012).

## *Fluorochrome*

For the modified sugars to bind to an azide group, a molecule containing an alkyl group must be present. To visualise the modified sugars, they must be linked to a probe that can be seen under the microscope. Fluorescent probes have typically been used for click chemistry (Mbua et al., 2011; Simon et al., 2018; Subramanian et al., 2014). The DBCO-AF488 (dibenzocyclooctyne bound to the fluorescent probe AF488) was used in this study (Fig.3). We chose to use AF488 to avoid interference from strong autofluorescence. Autofluorescence from seaweed would, most likely, be derived from chlorophyll a. Therefore, the selection of the fluorescent probe AF488 limits autofluorescence from chlorophyll a since AF488 emits at a wavelength of 517 nm, i.e. in
1 the green part of the spectrum, whereas chlorophyll a emits between 685-690 nm, in the red part

2 of the spectrum.

3

4 **Reporter integration**

5 The experimental protocol is summarised in Figure 4. Three fragments of each seaweed species

6 were placed in the wells of 24-well microplates with 500 µL of 1X PBS (Phosphate-buffered

7 saline) solution. The seaweed sections were incubated in 300 µL of 10 mM of the reporter solution

8 at room temperature, under agitation at 300 rpm for either 24- or 48 hours. Two controls were

9 carried out: (1) to determine autofluorescence of seaweed cell components using confocal

10 microscopy (no reporter, no fluorochrome) and (2) non-specific binding of the fluorochrome (no

11 reporter, + fluorochrome).

12

13 *Fluorochrome integration*

14 After either 24- or 48 hours, the reporter solution was removed, and the seaweed sections were

15 washed three times using 500 µL of 1X PBS solution. The sections were then incubated at room

16 temperature, in the dark, in a 5 µM DCBO-AF488 (Jena Bioscience, Jena, Germany) solution for

17 90 minutes, with 300 rpm agitation (Thermo-Shaker, PHMP-4). After the addition of the probe, all

18 the following steps were carried out in the dark to limit the degradation of the probe.

19

20 *Tissue washing*

21 Tissue washing was carried out by incubating the sections in methanol 70% v/v for 1 hour, in the

22 dark, with 300 rpm agitation, in the dark. This step aimed to remove the fluorophores non-
1 specifically bound to the cells and fix the cells to allow better observation. The samples were

2 washed three times with 500 µL of 1X PBS solution.

3

4 *Confocal microscopy*

5 Before observation, the samples were placed in a drop of Fluoromount-G® (Thermo Fisher

6 Scientific) between a slide and a coverslip and observed using a confocal microscope ZEISS LSM

7 740. The excitation and emission wavelengths for Alexa Fluor® 488-Azide were 494 and 517 nm,

8 respectively. Microscope settings (gain, laser intensity, pinhole size) were kept constant throughout

9 a given set of experiments to adjust all images in the same way and achieve comparable

10 visualisation of image fluorescence intensities. The images were captured and then analysed and

11 processed using the Fiji software (Schindelin et al., 2012). Each measurement was performed on

12 three independent fragments.

13

14 *Supplementary Analysis: Validation of Seaweed Cell Components*

15 To better understand and provide additional analytical insights into the images obtained through

16 click chemistry, particularly regarding the intracellular structure of seaweed, iodine and Toluidine

17 Blue O (TBO) staining were performed following the observation of the click chemistry images.

18 *Ulva spp* and *Phycodrys rubens* were collected on November 15th, 2024, at An Spidéal (Co.

19 Galway, Ireland).

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1 *Iodine staining*

2 Iodine staining (or Lugol test) was performed on plant cells to localise starch. However, cell wall
3 polysaccharides such as xyloglucans (Yuguchi et al., 2005), carrageenans (which stain pale
4 purple), and agars (Flint, 1990) can also be stained. No publications report the staining of ulvan
5 by the Lugol test. However, xyloglucan is present in the cell walls of green seaweeds, which
6 suggests that their cell walls may be stained by the Lugol test (Wahlström et al., 2020).

7 Iodine staining is made possible by binding iodine and potassium iodide within sugar chains, 8 specifically within the amylose helices of starch (Minick et al., 1991). To carry out iodine staining, 9 *Ulva spp.* and *Phycodrys rubens* sections were incubated with one drop of Lugol solution (Sigma 10 Aldrich). Following staining, the seaweed tissues were mounted on a microscope slide and covered 11 with a coverslip prior to observation under a light microscope (Optika B-293 and camera Optika 12 C-P6).

13

14 *TBO staining*

15 Toluidine Blue O (TBO) staining was performed to visualise the cell walls, chloroplasts, and 16 starch. The TBO staining method highlights acidic components in cells and tissues. TBO binds 17 specifically to acidic groups, such as sulfates, carboxylates, and phosphates, that are commonly 18 found in polysaccharides, nucleic acids, and glycoproteins (O’Brien et al., 1964). To perform TBO 19 staining, 0.05% toluidine blue O powder was dissolved in 50 mM sodium acetate buffer (pH 5). A 20 drop of the staining solution was applied to the seaweed tissue and incubated for 2 minutes at room 21 temperature. The tissue was then rinsed with distilled water and mounted between a slide and 22 coverslip for microscopic observation (Optika B-293 and camera Optika C-P6). 23

23
# 1 RESULTS

# 2 *Effects of incubation time on labelling intensities*

3 No specific protocols have been previously established for chemical reporter detection in seaweed.

4 We, therefore, incubated fragments of *Ulva spp* and *Phycodrys rubens* in 10 µM N₃-reporters for
5 24 and 48 h without any permeabilising agent to estimate the optimal time for incorporation. The
6 overall results are summarised in Table 2. For *Ulva*, the 48-hour incubation suggests that the longer
7 the incubation, the greater the signal (Fig.5). This is particularly correlated with more distinct
8 labelling of the cell walls. In *Phycodrys*, the signal intensity also increased over time, but in the
9 case of fucose, the signal intensity within the cell wall remained low at 24 and 48 h (Fig.7, image
10 of 24-hour reporter incubation available in supplementary figures 1–2).

11

12 *Incorporation of monosaccharide reporters in *Ulva spp***

13 A first control lacking both reporters and probes (R-F-) shows a total absence of signal in the
14 thallus, indicating that there is no or undetectable autofluorescence of pigments or other cellular
15 components at excitation/emission wavelengths of DCBO AF 488 (Fig.5a, f, k). A second control
16 incubated only with the fluorophore (R-F+) exhibited a very weak fluorescent signal (Fig.5b, g, l).
17 This suggests that there is no strong non-specific binding of the probes

18 The signal observed with the fucose reporter (fucose, F+) is intense (Fig.5c, h, m) but does not
19 appear to be uniform in all the cells (x20) compared to glucose labelling (Glucose, R+F+), which
20 appears to be more regular (Fig.5e, j, o). The signal derived from the galactose reporter (Galactose,
21 R+F+) is visible in most of the seaweed cells and is more easily visualised at higher magnifications
22 (Fig.5i) magnification x63 and magnification x63x3 (Fig.5n). Signals observed at high
1 magnification reveals a strong signal for all the reporters at the level of the cell walls but also the
2 inter-cell level.

3 It was possible to quantify and compare the signals obtained with the three reporters since, in each
4 case, the same bioorthogonal click chemistry reaction occurred between an azide group on the
5 reporter and DBCO anchored on the AF488 fluorophore. In addition, all the settings on the
6 confocal microscope were conserved between each sample. Values were obtained by subtracting
7 the background from the signal.

8 Quantification was carried out on the cell walls and the internal space of the cells (Fig. 6). For
9 *Ulva* spp., significant differences in incorporation levels were observed, with a higher rate for
10 glucose, followed by fucose, and finally galactose in the cell wall.

11

12 *Incorporation of monosaccharide reporters in Phycodrys rubens*

13 Using the (R-F-) control, it was possible to exclude the presence of autofluorescence in the
14 *Phycodrys* samples (Fig 7a, f, k). In contrast to the *Ulva* samples, however, we were able to observe
15 the presence of aspecific fluorescence deriving from the presence of residual probes in the tissue
16 as suggested by the (R-F+) control (Fig 7b, g, l). In the (R+F+) samples, the signal intensities were
17 much higher (Fig.7 c, d, e, h, I, j, m, n, o). This suggests that the seaweed can specifically take up
18 and incorporate the reporters. Each of the three different monosaccharides were located in the
19 internal part of the cells, and, as for *Ulva* spp, it was interesting to observe the presence of clear
20 labelling on the cell walls (Fig.7m, n, o). In both compartments, the incorporation measured for
21 glucose was the highest among the three reporters tested (Fig. 6). The comparison between
22 galactose and fucose showed a higher signal for the first in the cell wall and the second inside the
23 cells.
1

2 *Light microscopy of the intracellular structure of Ulva spp.*

3 Following the staining of *Ulva* spp. with Lugol's solution, numerous black-staining spots of ~1–3 µm in diameter were observed (arrow number 1, Fig. 8e). On average, around seven of these black-staining spots were present in each cell. Additionally, larger spots, ~10-15 µm in diameter, surrounded by a brown-staining membrane, are visible (arrow number 2, Fig. 8e). The cell walls are also visible and were stained light brown (Fig. 8e).

8 TBO staining was also carried out on *Ulva*. The cell walls are stained pink/purple (Fig. 8f), indicating that they consist predominantly of polysaccharides. Additionally, structures with distinct contours were observed inside the cells (Fig. 8f) and are likely to be cell organelles, including the vacuole, nucleus, mitochondria, and peroxisome (Blomme et al., 2020).

12

13 *Light microscopy of the intracellular structure of Phycodrys rubens*

14 Iodine staining (Lugol test) is known to stain floridean starch grains (Yu et al., 2021). Iodine staining of *Phycodrys rubens* resulted in dark-staining spots of ~5-10 µm in diameter at the periphery of each cell in the stained tissue (arrow number 1, Fig. 9e). There were approximately three brown-staining spots present in each cell. We also observed the presence of oval structures (5 µm long and 15 µm wide) that were lightly stained brown with the Lugol's stain (arrow number 3, Fig. 9e). There, therefore, appear to be at least two distinct populations of cell organelles that stain with iodine in *Phycodrys*: 1) smaller, dark brown staining, and 2) larger organelles that are more lightly stained (arrow number 3, Fig. 9e). Another structure is more clearly observed

1

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1 following iodine staining, arrow number 4, and appears to be a connexion between two cells (Fig.

2 9f).

3

4 We also attempted to stain *Phycodrys* sections with toluidine blue O stain to visualise the cell

5 walls, chloroplasts, and starch. However, the red pigment, primarily phycobiliprotein, in

6 *Phycodrys* masked any TBO-staining, and as a result, it was impossible to observe any distinct

7 intracellular staining. In contrast, *Phycodrys* cell walls were stained blue-green and could be

8 clearly visualised (Fig. 9f).

9

10 DISCUSSION

11 *Using bioorthogonal click chemistry to monitor the production of cell wall components*

12 We have explored the use of the bioorthogonal click chemistry in two species of seaweeds (from

13 the Chlorophyta and Rhodophyta phyla) by testing the metabolic incorporation of three different

14 modified sugars with the primary objective of incorporating them into cell wall polymers. Plant

15 cell walls contain mainly polysaccharides, and when a secondary cell wall is present, also lignin.

16 Each polymer is synthesised by enzymes located in different compartments, which can make the

17 incorporation of chemical reporters more or less difficult. Lignin precursors, namely *p*-coumaryl

18 alcohol, coniferyl alcohol and sinapyl alcohol, are synthesised in the cytoplasm and transported to

19 the cell wall, where peroxidases and laccases oxidise them before being incorporated into the

20 growing polymer (Zhao et al., 2013). To hijack these natural monomers, an exogenous supply of

21 tagged surrogates on plant sections, meaning directly on the cell wall is sufficient to induce the

22 incorporation (Morel et al., 2022). Concerning polysaccharides, metabolic labelling using sugar

23 analogues compatible with click chemistry makes it necessary to set up conditions such that the
reporters will enter the living cells through the cell wall and plasma membrane and then integrate

the synthetic machinery in the Golgi apparatus level for pectins and hemicelluloses or be activated

in the cytoplasm for cellulose synthesis. This was first reported by the use of an alkynylated fucose

analogue, which was successfully incorporated into the pectic rhamnogalacturonan-I (RG-I)

within the cell walls of *Arabidopsis thaliana* roots (Anderson et al., 2012). Rhamnogalacturonan-

II (RG-II) labelling in this same organ but also in tobacco (*Nicotiana tabacum* L. cv Bright Yellow-

2) cell suspension cultures was also demonstrated later on by using 8-azido-3-deoxy-D-manno-

oct-2-ulosonic acid, an azido derivative of Kdo (Kdo-N3) (Dumont et al., 2016). Kdo-N3 was also

later incorporated into fast-growing, tip-polarized expanding pollen tubes (Ropitaux et al., 2022).

Studies using click chemistry approaches have been carried out on microalgae in very specific

contexts. CuAAC click chemistry associated with LC-MS was used to detect polyether toxins

(prymnesins) produced by *Prymnesium parvum*, which is associated with harmful seaweed blooms

(Hems et al., 2018). Other works have led to the production of microalgae-based microrobot

functionalised by click chemistry with angiotensin-converting enzyme 2 (ACE2) receptor against

the SARS-CoV-2 spike protein (Zhang et al., 2021). But to the best of our knowledge, the

bioorthogonal click chemistry technique has never been reported in seaweed in the context of

understanding biological mechanisms.

*Tracking analogues of monosaccharides in green and red seaweeds using a bioorthogonal click*

*chemistry approach*

*Ulva* spp, commonly known as sea lettuce, is an excellent model organism for studying green

seaweed because of its simple and versatile life cycle and is a major component of marine food

networks. It can form seaweed blooms (e.g., green tides) in response to nutrient pollution,
1 providing a model to study eutrophication. It also exhibits high morphological plasticity,

2 responding to environmental cues by altering its shape and structure (Wichard et al., 2015).

3 Galactose, fucose and glucose, tagged with azide groups, were detected after their association with
4 a fluorescent probe by click chemistry. As described in the higher plant models, the peracetylated
5 forms were able to cross the plasma membrane and were detected in different sub-cellular
6 locations.

7 The strong labelling observed in the cell wall with the glucose reporter can be explained by the
8 presence of (1) cellulose and (2) ulvans (structure detail in the introduction) within the cell wall of
9 Ulva. Both of these polymers are primarily composed of glucose residues, and their biosynthesis
10 could incorporate the glucose reporter. In previous studies, fucose has been shown to represent the
11 second-highest molar ratio in green seaweed monosaccharide composition, exceeding 20%, in
12 polysaccharides isolated from *Ulva linza* (formerly known as *Enteromorpha linza*) and *Ulva
13 fasciata* (Barakat et al., 2022; Zhang et al., 2013). Ulvan polymers also contain low amounts of
14 galactose, which was also shown to be present within *Ulva* cell wall glycoproteins (Estevez et al.,
15 2009).

16 The observation in *Ulva* of differences in labelling between the cells present in the same tissue
17 (Fig.5) could be explained by two reasons: (1) cells at different life stages, e.g. those that are
18 entering senescence, would integrate the reporter less effectively, and/or (2) due to the seaweed
19 topology; although the surface is superficially relatively flat it may be quite rugged in parts, and
20 this would likely impact the uptake of the label.

21 The ultrastructure of *Ulva* has already been described. The seaweed is composed of two layers of
22 cells, containing one chloroplast with multiple pyrenoids, a large vacuole and a thick cell wall (Hu
23 et al., 2022; Messyasz et al., 2013). The results of the Lugol staining confirm the presence of starch
1 inside the seaweed cells, as evidenced by the black-staining spots (Fig. 8e, arrow number 1). In
2 several cells, the nucleus is visualised as larger spots surrounded by a brown membrane (Fig. 8e,
3 arrow number 2)The cell walls are stained light brown due to the presence of ulvans, a major
4 polysaccharide in the cell walls of green seaweed, including *Ulva*.

5 These results correlate well with the observations made using click chemistry, where strong
6 intracellular labelling was observed, particularly with the glucose reporter (Fig. 5e, j, o). This
7 staining, with iodine, and labelling, with the glucose reporter, is likely to be associated with starch
8 granules. Starch granules are composed of amylose and amylopectins, for which the major
9 monosaccharide component is glucose (Fig.5e, j, o, Supplementary Figure 1l) (Prabhu et al., 2019).

10 However, it can be observed that glucose labelling in starch grains is not as bright as expected.
11 This could be due to a stressful environment for the cells, affecting starch production (Zachleder
12 & Brányiková, 2014).

13 For the fucose (Fig.5c, h, m) and galactose (Fig.5d, i, n) reporters, we hypothesise that the
14 intracellular labelling observed represents cellular components involved in the synthesis of cell
15 wall sugars, such as the endoplasmic reticulum and the Golgi apparatus. The peripheral localisation
16 of these components could also be explained by variations in osmotic pressure, which might
17 influence their position within the cells. In *Ulva*, punctate fluorescence in the cell walls may be
18 due to localized differences within the cell wall, resulting in the observed fluorescence pattern.

19
20 *Phycodrys rubens* is a perennial red seaweed belonging to the *Delesseriaceae* family, commonly
21 found in the cold, temperate waters of the North Atlantic Ocean (Guiry & Guiry, 2024). It has a
22 flattened, leaf-like thallus (body) that is typically reddish in colour. Its frond has a leafy appearance
23 and is composed of a single flat layer of cells and can grow up to 3 cm wide. Analogues of
1 galactose, fucose and glucose were incorporated into the cells, and the signals were detected inside
2 the cells or in the cell wall. The labelling of cell walls by the galactose reporter is likely due to
3 carrageenans and agars, which are polysaccharides specific to the cell walls of red seaweed. The
4 galactose reporter may also label galactose-containing glycoproteins in the cell walls. Galactose
5 was previously shown to be present in very high amounts (approximately 90%) among the main
6 neutral sugars identified in soluble dietary fibre from two red seaweed, *Mastocarpus stellatus* and
7 *Gigartina pistillata* (Gómez-Ordóñez et al., 2014).

8 While fucose is not a major monosaccharide component of red seaweed carbohydrates, several
9 studies have reported its presence in these organisms. For instance, an antioxidant fucose-rich
10 sulphated polysaccharide, additionally containing α-D-Man and β-D-Gal residues, was isolated
11 from the red seaweed *Gloiopeltis tenax* (Lim & Ryu, 2009).

12 Iodine staining (Lugol reagent) effectively highlighted the presence of floridean starch in
13 *Phycodrys* cells, as evidenced by the dark spots seen at the cell periphery (Fig. 9e, arrow number
14 1), a feature previously described in the literature (Cole & Sheath, 1990). Similar structures are
15 also visible in the control image (Fig 9.d). Arrow number 2 shows the chloroplast inside the
16 cytoplasm (Fig 9e). The larger oval structures inside the cells (Fig 9e, arrow number 3), which
17 were stained light brown, are likely to be nucleus based on their staining properties (Meyer et al.,
18 2017). The structures observed between cells following staining with Lugol's reagent as well as
19 with TBO (Fig. 9e arrow number 4, arrow f) appear to be the pit plugs, which are characteristic
20 membranous structures specifically found in multicellular red seaweed. Pit plugs are also visible
21 using fucose and galactose reporters in click chemistry Fig. 7i, m, Supplementary Fig 2e (arrow
22 2). Pit plugs facilitate cell-cell communication but are not considered homologous to
plasmodesmata, which are found in land plants (Pueschel & Cole, 1982). However, despite their different origins, they perform similar roles.

We carried out TBO staining of *Phycodrys*. However, the presence of the red pigments, phycobilins and phycoerythrins, found associated with the chloroplast in many red seaweeds (French & Young, 1952, Gantt et al., 2003), interfered with the visualisation of the stain. The strong red colouration of the pigment made it difficult to distinguish intracellular features that may have stained with toluidine blue from the surrounding background. This limitation highlights the challenges when working with seaweed that contains strong pigments like phycoerythrin, which can mask other cellular details. However, blue-green staining of the cell wall with TBO could clearly be observed as a result of the binding of TBO to carrageenans that are highly sulphated (Gordon-Mills et al., 1978). TBO stains *Ulva* cell walls a different colour because, despite the presence of sulfated groups in ulvans, they contain fewer sulfated groups than carrageenans.

There is a strong correlation between the intracellular components labelled by click chemistry, TBO, and Lugol's reagent. Click chemistry using activated sucrose, glucose, and galactose (Fig. 7m, n, o) highlights the presence of unlabeled structures localised at the periphery of the cells. These structures are also observed in the control using only the fluorochrome (Fig 7l). Lugol staining allowed us to identify and confirm that these enigmatic structures are floridean granules. Additionally, click chemistry enabled the labelling of intracellular structures in a nonspecific manner, as these were visible both in the fluorochrome control (Fig. 7l) and with the activated sugars (Fig. 7m, n, o). These structures, similarly observed with Lugol and TBO staining, likely correspond to the seaweed phycobilisomes complex, composed of the phycobiliproteins pigments involved in photosynthesis found in red seaweed (Gantt, 1980). Moreover, some of the intracellular
1 structures were labelled by all of the activated sugar residues tested (Fig. 7 m, n, o), suggesting
2 that they could be either the location of carrageenans biosynthesis, i.e. within the Golgi apparatus
3 or carrageenans within transport vesicles (Vreeland et al., 1992).
4

5 *A new experimental approach to characterise cell wall dynamics during seaweed growth*
6
7 The possibility of incorporating monosaccharide-like chemical reporters into seaweed represents
8 an opportunity to extend physiological and ecological studies of these aquatic species. Uniquely,
9 this approach enables us to measure the potential of organisms to synthesise or modify their cell
10 walls.
11

10 *Ulva* is highly adaptable to various environmental conditions, such as light and temperature
11 fluctuations (Wu et al., 2018). Interestingly, the morphotype of the thalli in *Ulva* may vary in part
12 depending on the water salinity (Rybak, 2018). Indeed, tubular forms present in a very wide range
13 of saline waters, while leaf-shaped thalli are found mainly in brackish waters. Because organ
14 phenotypes are dependent on cell shapes and organisation within tissues, it is clear that approaches
15 using chemical reporters associated with bioorthogonal chemistry will be highly relevant to the
16 study of cell wall dynamics in these seaweeds.
17

17 One of the characteristics of Ulvaceae is their ability to produce large amounts of biomass over a
18 short period of time (Rybak & Gąbka, 2018) either in freshwater, even more markedly in seawater,
19 where they are known to be the cause of green tides as in the case for example, in the Yellow Sea
20 off the coasts of Jiangsu and Shandong Province, China (Zhang et al., 2019) or in the English
21 Channel and Atlantic Sea off the coasts of Brittany (Louis et al., 2023). While this proliferation is
22 largely due to an excess of nitrogenous nutrients, cell divisions require massive incorporation of
1 sugars and, therefore, sustained cell wall material production activities that could be characterised
2 by bioorthogonal click chemistry.

3 From a biotechnological point of view, the cell walls of seaweed could thus represent a target for
4 better control of their proliferation. To achieve this, it is important to characterise the regulatory
5 elements that enable their construction and then look for inhibitors. Several red seaweeds form
6 blooms and cause premature mortality in marine animals as a result of toxin production (Young et
7 al., 2022). Here, too, knowledge of the wall-forming environment is a prerequisite for finding an
8 effective solution. Additionally, red seaweeds are the primary source of agars and carrageenans,
9 which are polysaccharides widely used as gelling, stabilising, and thickening agents in food,
10 pharmaceuticals, and cosmetics. Their production is largely impacted by the photosynthetic
11 activities of the organisms, but once the monosaccharides have been synthesised, the enzymatic
12 machinery controlling their incorporation in the polymers remains the limiting factor.
13 Bioorthogonal click chemistry can make it possible to select lines that perform better than others
14 in the same way as selection in conventional agriculture.

15
16 In summary, we were able to adapt the non-toxic SPAAC click chemistry technique on green and
17 red seaweed. More specifically, we were able to reliably label seaweed cell wall components. Click
18 chemistry is a technique with numerous advantages: it is relatively simple and inexpensive to
19 perform. However, its interpretation is more complex than that of monoclonal antibodies, as the
20 activated sugar can be integrated into various components of the seaweed. While monoclonal
21 antibodies recognize specific epitopes in polymers, their use is more limited. One advantage of
22 click chemistry over antibody staining is its application in cases where polymers are not well-
23 defined (Popper, 2024).
1 This method opens many avenues of research, including determining changes in seaweed cell walls

2 related to the life cycle and various stresses (grazing, pathogen infection, etc.) that could impact

3 seaweed ecology and diversity, as well as the quality of commercially grown seaweed and the

4 products derived from them.

5

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28

29 Acknowledgements

30 We thank the TisBio and BICeL facilities (University of Lille) for the access to instruments and

31 Thomas Bance for his work on the image capture.

32 W.F.D. also thanks Clémence SIMON (UGSF-University of Lille) for her support in using Fiji

33 software. This work was supported by a Thomas Crawford Hayes Foundation award to W.F.D.
1. Additionally, we also wish to thank the Irish Department of Agriculture, Food and Marine

2. (BioCrop) for its financial support under the research stimulus award reference 2019PROG705.

3.

4. Table Legend

5. Table 1: Main polysaccharides present in cell walls of different seaweed (adapted from Popper et

6. al. 2011)

<table><thead><tr><th></th><th>Chlorophyta<br>(green seaweed)</th><th>Rhodophyta<br>(red seaweed)</th></tr></thead><tbody><tr><td>Crystalline phase: fiber<br>polysaccharides</td><td>Cellulose</td><td>Cellulose</td></tr><tr><td>Hemicellulose</td><td>Xyloglucan<br>Fucan<br>Mannan<br>Glucuronan<br>β-(1→3)-glucan</td><td>Glucomannan<br>Mannan<br>β-(1→3)-glucan<br>β-(1→4)-glucan</td></tr><tr><td>Amorphous<br>polysaccharides</td><td>Ulvan</td><td>Agar<br>Carrageenan<br>Porphyran<br>β-(1→3)-galactan<br>α-(1→3)-glucan</td></tr></tbody></table>

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8.

9.

10.

11.

12.

13.

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17.

18.
<table><caption>Table 2: Comparison of the signal strength of AF488 for *Ulva spp.* and *Phycodrys rubens* incubated for either a) 24 h or b) 48 h with the reporters for fucose (1,2,3,4-Tetra-*O*-acetyl-6-azido-L-fucopyranose), galactose (1,3,4,6-Tetra-*O*-acetyl-2-azido-2-deoxy-D-galactopyranose), or glucose (1,3,4,6-Tetra-*O*-acetyl-2-azido-2-deoxy-D-glucopyranose) prior to visualisation with DBCO-AF488 and imaging by confocal microscopy.</caption><thead><tr><th>Conditions</th><th>Incubation time</th><th><em>Ulva spp.</em></th><th><em>Phycodrys rubens</em></th></tr></thead><tbody><tr><td rowspan="2">R-/F+</td><td>24 hours</td><td>Low signal</td><td>Moderate intracellular signal</td></tr><tr><td>48 hours</td><td>Low signal</td><td>Intracellular significant signal</td></tr><tr><td rowspan="2">Fucose/F+</td><td>24 hours</td><td>Moderate signal, not diffused, labelled cell walls</td><td>Intense intracellular signal, slightly labelled cell wall</td></tr><tr><td>48 hours</td><td>High signal, diffused, labelled cell walls</td><td>Intense intracellular signal</td></tr><tr><td rowspan="2">Galactose/F+</td><td>24 hours</td><td>Moderate signal, diffused, slightly labelled cell walls</td><td>Intense intracellular signal, low labelled cell walls</td></tr><tr><td>48 hours</td><td>High signal, not too diffused, labelled cell walls</td><td>Intense intracellular signal, slightly labelled cell walls</td></tr><tr><td rowspan="2">Glucose/F+</td><td>24 hours</td><td>Moderate signal not diffused, labelled cell walls</td><td>Intense intracellular signal, low cell wall signal</td></tr><tr><td>48 hours</td><td>High signal, very diffused, intensely labelled cell walls</td><td>Intense intracellular signal, slightly labelled cell walls</td></tr></tbody></table>

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1 Figure legends

2 Figure 1: Photograph of *Ulva spp.* (left) and *Phycodrys rubens* (right), illustrating the two seaweeds studied. Photo taken by W.F.D, Galway, Ireland, October 2023.

3 Figure 2: Structure of azido activated sugar residues used as reporters of sugar residues present in seaweed cell wall polysaccharides, (a) 1,2,3,4-Tetra-*O*-acetyl-6-azido-L-fucopyranose, (b) 1,3,4,6-Tetra-*O*-acetyl-2-azido-2-deoxy-D-galactopyranose, (c) 1,3,4,6-Tetra-*O*-acetyl-2-azido-2-deoxy-D-glucopyranose. The azide part, shown in blue, can bind with an alkyl part, e.g. shown in Fig. 3, that can be used to visualise the sugars.

7 Figure 3: Structure of Dibenzocyclooctyne-Alexa Fluor 488 (DBCO-AF488). The AF488 part is shown in green, and the DBCO part is shown in red. The alkyl group, which can bind to the azide group on the modified sugar reporter, is shown in blue.

12 Figure 4: An illustrated summary of the bioorthogonal labelling strategy used in this work on seaweeds (created using BioRender.com within the agreement number SE27RFKPIS).

14 Figure 5: Labelling of green seaweed *Ulva spp.* tissues after 48-hour incubation with various reporters. Reporters and fluorochrome: a, f and k, no reporter (R-), no fluorochrome (F-) at x20, x63 and x63x3 (x63 expanded three time) respectively; b, g and l, (R-), with fluorochrome (F+) at x20, x63 and x63x3 (x63 expanded three time) respectively; c, h and m, fucose (R+) (F+) at x20, x63 and x63x3 respectively; d, i and n, galactose (R+) (F+) at x20, x63 and x63x3 respectively; e, j and o, glucose (R+) (F+) at x20, x63 and x63x3 respectively. The arrows indicate the cell walls.

20 Figure 6: Incorporation of N<sub>3</sub>-labelled monosaccharides in red and green seaweed. Reporters were incorporated for 48 hours and detected by the SPAAC reaction. Quantification was carried out by
measuring fluorescence within the cell wall or inside the cell using FIJI, and a comparison was
made between fucose, galactose and glucose surrogates. *** Anova test (p-value <0.001).

Figure 7: Labelling of tissues of the red seaweed *Phycodrys rubens* after 48-hour incubation with various reporters. Reporters and fluorochrome: a, f and k, no reporter (R-), no fluorochrome (F-) at x20, x63 and x63x3 (x63 expanded three time) respectively; b, g and l, (R-), with fluorochrome (F+) at x20, x63 and x63x3 (x63 expanded three time) respectively; c, h and m, fucose (R+) (F+) at x20, x63 and x63x3 respectively; d, i and n, galactose (R+) (F+) at x20, x63 and x63x3 respectively; e, j and o, glucose (R+) (F+) at x20, x63 and x63x3 respectively. The arrows 1 indicate the cell walls, arrows 2 shows the pit-plugs.

Figure 8: Tissues of the green seaweed *Ulva spp.* either unstained or stained with Iodine or Toluidine Blue O. The tissues were unstained and observed at ×40 and ×100 for (a, and d respectively), or stained with iodine- (b, and e) or TBO- (c, and f). Arrow 1 points to starch, and arrow 2 points to a nucleus.

Figure 9: Tissues of the red seaweed *Phycodrys rubens* either unstained or stained with Iodine or Toluidine Blue O. The tissues were unstained and observed at ×40 and ×100 for (a, and d respectively), or stained with iodine- (b, and e) or TBO- (c, and f). Arrow 1 shows the chloroplast and other plastids, arrow 2 shows several chloroplasts, arrow 3 shows the nucleus, and arrow 4 shows the pit plugs.

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