**2. Results**

To determine the sulphation and desulphation of galactan, transcript expressions of *carbohydrate sulfotransferase* in charge of addition sulphate groups and *galactose-6 sulfurylase* for removing sulphate groups were determined according to development stage of thalli and to the presence of reproductive structures (cystocarps; Figure 3).

### *2.1. Gene Expression for Stages of Thalli Development*

Expression levels of each of two gene sequences encoding *sulfotransferase* and *sulfurylase* for infertile, fertilised and fertile thalli are shown (Figure 4). Expressions for *sulfotransferase*, *ST1* and *ST2*, exhibit similar behaviour (Figure 4A). In addition, no significative differences in levels of expression are reported between different stages of development of the thalli (Figure 4A).

Different transcript expressions are shown for *sulfurylase* depending on the development stage of the thalli. Thus, when transcript expression for *sulfurylase 1* (*SY1*) was compared to that of infertile thalli, *SY1* was overexpressed for fertilised thalli and down expressed for fertile thalli (Figure 4B). Meanwhile the transcript expression for *SY2* was significantly down-expressed for fertilised and fertile thalli, compared to infertile thalli (Figure 4B). Significant differential expression is also reported when *sulfurylase SY1* and *SY2* from fertilised thalli are compared.

**Figure 3.** Schematic showing different development stages of thalli (fertilised and fertile thalli) and cystocarps maturation (well-developed and fully-developed cystocarps) of *Grateloupia imbricata*. Infertile thalli are thalli without cystocarps in all axes of the same individual.

### *2.2. Gene Expression for the Maturation of Reproductive Structures (Cystocarps)*

Expression levels of *sulfotransferase* (*ST1* and *ST2*) in the presence of fully-developed cystocarps show significant over-expression compared to the absence of cystocarps and well-developed cystocarps (Figure 5A,B).

Regarding *sulfurylase*, expression levels of *SY1* and *SY2* show different behaviours. On the one hand, *sulfurylase SY1* is over-expressed in well-developed cystocarps in comparison with fully-developed cystocarps (Figure 6A).

On the other hand, *sulfurylase SY2* is expressed significantly in the presence of fully-developed cystocarps. Thus, this expression was related to the maturity of the cystocarps (Figure 6B).

### *2.3. Gene Expression Encoding Stress Proteins during Cystocarp Maturation*

Levels of the expression of *Cytochrome P450* and *WD 40* follow a similar pattern (Figure 7). No significant differences were found in the *Cytochrome P450* and *WD 40* expression levels in thalli with well-developed cystocarps when compared to those without cystocarps (dotted line, Figure 7). The gene transcript expression is only significant for *WD40* in fully-developed cystocarps in comparison with well-developed cystocarps (Figure 7).

**Figure 4.** Normalised expression signal during three thalli development stages (infertile, fertilised and fertile thalli) of *Grateloupia imbricata* for (**A**) two *carbohydrate sulfotransferase* (*Sulfotransferase*, *ST1* and *ST2*); (**B**) two *galactose-6 sulfurylase* (*Sulfurylase SY1* and *SY2*). Data in bars are the mean ± SD, *n* = 4. Different symbols mean significant differences (*p* < 0.01) between infertile thalli and fertilised and fertile thalli (\*), between two gene sequences 1 and 2 (֮)), and between different stages of development (-).

**Figure 5.** Normalised expression for *carbohydrate sulfotransferase* during two stages of cystocarps maturation (well-developed and fully-developed cystocarps) of *Grateloupia imbricata* for (**A**) *Sulfotransferase*, *ST1*; (**B**) *Sulfotransferase*, *ST2*. Data in bars are the mean ± SD, *n* = 4. \* significant differences (*p* < 0.01) between samples without cystocarps and at different maturation stages. Dotted line corresponds to transcript expression from pooled thalli containing cystocarps at different stages of development and thalli without cystocarps (*n* = 8).

**Figure 6.** Normalised expression for *galactose-6 sulfurylase* during two stages of cystocarps maturation (well-developed and fully-developed cystocarps) of *Grateloupia imbricata* for (**A**) *Sulfurylase SY1*; (**B**) *Sulfurylase SY2*. Data in bars are the mean ± SD, *n* = 4. Different symbols mean significant differences (*p* < 0.01) between samples without cystocarps and at different maturation stages (\*), and between different stages of cystocarps maturation for the same gene (-). Dotted line corresponds to transcript expression from pooled thalli containing cystocarps at different stages of development and thalli without cystocarps (*n* = 8).

**Figure 7.** Normalised expression for *Cytochrome P450* and *WD40* during two stages of cystocarps maturation (well-developed and fully-developed cystocarps) of *Grateloupia imbricata* Data in bars are the mean ± SD, *n* = 4. Different symbols mean significant differences (*p* < 0.01) between thalli with cystocarps and those without them (\*), and between different stages of cystocarps maturation for the same gene (-). Dotted line corresponds to gene transcript expression of thalli without cystocarps (*n* = 8).
