**2. Transformation in red seaweeds**

mation mediated by *A. tumefaciens* has become the most commonly used method to transmit

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Since not all plant cells are susceptible to infection by *A. tumefaciens*, other methods were developed and are available in plants. Particle bombardment [13], which is also referred to as microprojectile bombardment, particle gun or biolistics, makes use of DNA-coated gold particles, which enables the transient and stable transformation of almost any type of cell, regardless of rigidity of the cell wall, and is thus extensively used for land plants. For proto‐ plasts, electroporation is well employed, for which a high-voltage electrical pulse temporarily disturbs the phospholipid bilayer of the plasma membrane, allowing cells to take up plasmid DNAs [14,15]. In addition, the polyethylene glycol (PEG)-mediated transformation system is also thought to affect the plasma membrane and induce the uptake of DNAs into cells [15,16] and is almost exclusively applied with the moss *Physcomitrella patens* and liverwort *Marchantia polymorpha* [17,18]. Therefore, several kinds of genetic transformation methods are now

Seaweeds are photosynthetic macroalgae, the majority of which live in the sea, and are usually divided into green, red and brown algae. Traditionally, all classes of seaweeds are known as human foods especially in Asian countries; for instance, red algae are known as Nori and brown algae are called Konbu and Wakame in Japan. In addition, red and brown algae are utilized as the sources of industrially and medically valuable compounds such as phycoery‐ thrin, n-3 polyunsaturated fatty acids, porphyran, ager and carrageenan from red algae, and fucoxantine, fucoidan and alginate from brown algae [19-22]. Thus, to make new strains carrying advantageous characteristics benefiting industry and medicine, researchers have worked hard since the early 1990s to establish methods of genetic transformation in seaweeds [20,23,24]. However, the process is very difficult, and most of the early studies were reported in conference abstracts without the accompanying manuscript publication [25-28]. This situation has hampered us from gaining an understanding of gene functions in various physiological regulations and also a utilization of seaweeds in biotechnological applications.

Transformation can be divided into genetic (stable) and transient transformations under the control of the genes introduced into cells. In genetic transformation, genes introduced by genetic recombination are maintained in the genome through generations of cells, whereas in transient transformation, rapid loss of introduced foreign genes is usually observed. Estab‐ lishing the genetic transformation system requires four basal techniques: an efficient gene transfer system, an efficient expression system for foreign genes, an integration and targeting system to deliver the foreign gene into the genome, and a selection system for transformed cells. It is notable that the transient transformation system is completed by the first two of the four required systems. In this respect, the development of an efficient and reproducible transient transformation system is the most critical step to establishing a genetic transforma‐

The current progress in establishing of both transient and genetic transformation systems in macroalgae is reviewed here. Although high-quality review articles for algal transformation have been published previously [20,23,24], I believe addressing the recent activity in seaweed transformation provides valuable information for seaweed molecular biologists and breeding scientists. Since considerable technical improvement was recently made in red seaweeds

DNA fragment into higher plants [12].

Applications

324

available in land green plants.

tion system in seaweeds.

### **2.1. Pioneer studies for transient transformation**

As far as I know, Donald P. Cheney is the pioneer in researching red algal transformation. He and his colleague performed transient transformation of the red alga *Kappaphycus alvarezii* using particle bombardment [25], which was the first report about the transient transformation of seaweeds (Table 1). In this case, the *Escherichia coli uidA* gene encoding β-glucuronidase (GUS) was expressed as a reporter under direction of the cauliflower mosaic virus (CaMV) 35S promoter (*CaMV 35S-GUS* gene). Since the GUS expression can be visualized as a blue color following treatment with X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) and also be quantified by fluorometric analysis [31,32], this reporter gene is widely used in land green plants having no background of the GUS activity [33,34]. In addition, the *CaMV 35S* promoter is heterologously used in land green plants as a strong constitutive and non-tissue-specific transcriptional regulator [35,36]. Therefore, it is a natural choice for the selection of the *CaMV35S-GUS* gene by pioneers for initial trials of seaweed transformation.

To date, studies have been mainly focused on *Porphyra* species because of their economical values. As shown in Table 1, expression of the *CaMV 35S-GUS* gene was previously observed in *P. miniata*, *P. tenera* and *P. yezoensis*[37-42], all of which were performed by electoroporation using protoplasts. Kuang et al. [38] also tested the particle bombardment of the *CaMV 35S-GUS* gene in *P. yezoensis* and got positive results. Moreover, the availability of mammaliantype simian virus 40 (*SV40*) promoter was reported to express the *E. coli lacZ* reporter gene, encoding β-galactosidase cleaving colorless substrate X-gal (5-bromo-4-chloro-3-indolyl-βgalactopyranoside) to produce a blue insoluble product [43], in *P. haitanensis*, *Gracilaria chagii* and *K. alvarezii* by electroporation or particle bombardment [44,45].

### **2.2. Recent improvement of the transient transformation system in** *Porphyra*

As noted above, pioneer experiments of red algal transient transformation were performed using plant viral *CaMV 35S RNA* and animal viral *SV40* promoters in combination with *GUS* and *lacZ* reporter genes (Table 1). The *CaMV 35S* and *SV40* promoters are typical eukaryotic class II promoters with a TATA box and thus are generally employed to drive transgenes in dicot plant and animal cells, respectively [46,47]. However, we have found that the TATA box is not usually found in the core promoters of *P. yezoensis* genes (unpublished observation), and we thus proposed that there were differences in the promoter structure and transcriptional regulation of protein-coding genes between red algae and dicot plants. Indeed, we recently observed quite low activity of the *CaMV 35S* promoter and the *GUS* reporter gene in *P. yezoensis* gametophytec cells [29,30,48]. These observations are completely opposite from the results in previous reports using the *CaMV 35S* promoter [25,37-41]. As a result, the transient transformation system in red seaweeds has recently been improved by resolving this problem.

*2.2.1. Optimization of codon usage in the reporter gene*

enable the efficient expression of this gene in *P. yezoensis* cells.

*2.2.2. Employment of endogenous strong promoters*

Inefficient expression of foreign genes in the green alga *Chlamydomonas reinhardtii* is often due to the incompatibility of the codon usage in the gene's coding regions [49-51]. Expressed sequence tag (EST) analysis of *P. yezoensis*reveals that the codons in *P. yezoensis* nuclear genes frequently contain G and C residues especially in their third letters, by which means the GC content reaches a high of 65.2% [52]. Since bacterial *GUS* and *lacZ* reporter genes have AT-rich codons, the incompatibility of codon usage, which generally inhibits the effective use of transfer RNA by rarely used codons in the host cells, thus decreasing the efficiency of the translation [53], might be responsible for the poor translation efficiency of foreign genes in *P. yezoensis* cells. It is therefore possible that modification of codon usage in the *GUS* gene would

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Accordingly, the codon usage of the *GUS* reporter gene was adjusted to that in the nuclear genes of *P. yezoensis* by introducing silent mutations [48], by which unfavorable or rare codons in the *GUS* reporter gene were exchanged for favorable ones without affecting amino acid sequences. The resultant artificially codon-optimized *GUS* gene was designated *PyGUS*, and its GC content was increased from 52.3% to 66.6% [48]. When the *PyGUS* gene directed by the *CaMV 35S* promoter was introduced into *P. yezoensis* gametophytic cells by particle bombard‐ ment, low but significant expression of the *PyGUS* gene was observed by histochemical detection and GUS activity test, indicating enhancement of the expression level of the *GUS* reporter gene [29,30,48]. Optimization of the codon usage of the reporter gene is therefore one

The *CaMV 35S* promoter has very low activity in cells of green microalgae such as *Dunaliella salina* [54], *Chlorella kessleri* [55] and *Chlorella vulgaris* [56] and no activity in *C. reinhardtii* cells [57-59]. Thus, a low level of *PyGUS* expression under the direction of the *CaMV 35S* promoter is likely to be caused by the low activity of this promoter in *P. yezoensis* cells. A hint to overcoming this problem was that employment of strong endogenous promoters such as the *β-Tub*, *RbcS2* and *Hsp70* promoters results in the efficient expression of foreign genes in microalgae [60-65]. Therefore, it is likely that efficient expression of the *PyGUS* reporter gene

By comparison with steady-state expression levels by reverse transcription-polymerase chain reaction (PCR), we found two genes strongly expressed in *P. yezoensis*: genes encoding glyceraldehyde-3-phosphate dehydrogenase (PyGAPDH) and actin 1 (PyAct1) [29]. When the *PyGUS* gene fused with the 5' upstream regions of these genes were introduced into gameto‐ phytic cells by particle bombardment, cells expressing the reporter gene and GUS enzymatic activity were dramatically increased [48,66]. These results indicate that employment of endogenous strong promoters is another important factor necessary for high-level expression of the reporter gene in *P. yezoensis* cells. In addition, the original *GUS* gene was not activated by *PyGAPDH* or *PyAct1* promoter [29,30,48], demonstrating that the *PyGUS* gene and endog‐ enous strong promoter have a synergistic effect on the efficiency of the expression in *P. yezoensis* cells (Figure 1A). Therefore, the combination of endogenous strong promoters with

of the important factors for successful expression in *P. yezoensis* cells [29,30,48].

in *P. yezoensis* cells is caused by the recruitment of endogenous strong promoters.


*\*Porphyra species* used are *P. yezoensis, P. tenera, P. okamurae, P. onoi, P. variegate* and *P. pseudolinearis.*

**Table 1.** Transformation in red seaweeds.

### *2.2.1. Optimization of codon usage in the reporter gene*

**Species Status of**

Applications

326

Porphyra tenera Porphyra yezoensis

Porphyra species\* Bangia fuscopurpurea

Porphyra species\* Bangia fuscopurpurea **expression**

*Porphyra yezoensis* transient electroporation CaMV 35S

*Porphyra yezoensis* transient particle bombardment CaMV 35S

*Porphyra yezoensis* stable *Agrobacterium-*mediated

*Porphyra yezoensis* stable *Agrobacterium-*mediated

**Table 1.** Transformation in red seaweeds.

*Porphyra yezoensis* transient Electroporation

**Gene transfer method Promoter Marker or Reporter Ref.**

β-tubulin

PyGAPDH

PyGAPDH

transient particle bombardment PtHSP70 PyGUS [87]

CaMV 35S GUS [38]

GUS [42]

PyGUS [48]

[71]

[86]

[131]

ZsYFP, sGFP(S65T)

sGFP(S65T)

CaMV 35S GUS [26]

(unknown) (unknown) [28]

EGFP

PyGUS [85]

*Kappaphycus alvarezii* transient particle bombardment CaMV 35S GUS [25] *Porphyra miniata* transient electroporation CaMV 35S GUS [37]

*Porphyra tenera* transient electroporation CaMV 35S GUS [39] *Porphyra yezoensis* transient electroporation rbcS GUS [40] *Porphyra yezoensis* transient electroporation CaMV 35S GUS [41]

*Gracilaria changii* transient particle bombardment SV40 lacZ [44] *Porphyra haitanensis* transient SV40 CAT [128] *Porphyra yezoensis* transient electriporation SV40 CAT, GUS [129] *Porphyra yezoensis* transient electroporation Rubisco GUS, sGFP(S65T) [130]

*Porphyra yezoensis* transient particle bombardment PyAct1 PyGUS [66] *Porphyra yezoensis* transient particle bombardment PyAct1 AmCFP [70]

*Porphyra yezoensis* transient particle bombardment PyAct1 AmCFP, ZsGFP,

gene transfer

gene transfer

*\*Porphyra species* used are *P. yezoensis, P. tenera, P. okamurae, P. onoi, P. variegate* and *P. pseudolinearis.*

*Porphyra haitanensis* stable glass bead agitation SV40 lacZ

*Porphyra leucostica* stable ekectroporation CaMV 35S lacZ [27]

*Kappaphycus alvarezii* stable particle bombardment SV40 lacZ [45]

*Gracilaria changii* stable particle bombardment SV40 lacZ [91] *Gracilaria gracilis* stable particle bombardment SV40 lacZ [92]

transient particle bombardment PtHSP70

transient particle bombardment PyAct1 PyGUS

particle bombardment

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Inefficient expression of foreign genes in the green alga *Chlamydomonas reinhardtii* is often due to the incompatibility of the codon usage in the gene's coding regions [49-51]. Expressed sequence tag (EST) analysis of *P. yezoensis*reveals that the codons in *P. yezoensis* nuclear genes frequently contain G and C residues especially in their third letters, by which means the GC content reaches a high of 65.2% [52]. Since bacterial *GUS* and *lacZ* reporter genes have AT-rich codons, the incompatibility of codon usage, which generally inhibits the effective use of transfer RNA by rarely used codons in the host cells, thus decreasing the efficiency of the translation [53], might be responsible for the poor translation efficiency of foreign genes in *P. yezoensis* cells. It is therefore possible that modification of codon usage in the *GUS* gene would enable the efficient expression of this gene in *P. yezoensis* cells.

Accordingly, the codon usage of the *GUS* reporter gene was adjusted to that in the nuclear genes of *P. yezoensis* by introducing silent mutations [48], by which unfavorable or rare codons in the *GUS* reporter gene were exchanged for favorable ones without affecting amino acid sequences. The resultant artificially codon-optimized *GUS* gene was designated *PyGUS*, and its GC content was increased from 52.3% to 66.6% [48]. When the *PyGUS* gene directed by the *CaMV 35S* promoter was introduced into *P. yezoensis* gametophytic cells by particle bombard‐ ment, low but significant expression of the *PyGUS* gene was observed by histochemical detection and GUS activity test, indicating enhancement of the expression level of the *GUS* reporter gene [29,30,48]. Optimization of the codon usage of the reporter gene is therefore one of the important factors for successful expression in *P. yezoensis* cells [29,30,48].

#### *2.2.2. Employment of endogenous strong promoters*

The *CaMV 35S* promoter has very low activity in cells of green microalgae such as *Dunaliella salina* [54], *Chlorella kessleri* [55] and *Chlorella vulgaris* [56] and no activity in *C. reinhardtii* cells [57-59]. Thus, a low level of *PyGUS* expression under the direction of the *CaMV 35S* promoter is likely to be caused by the low activity of this promoter in *P. yezoensis* cells. A hint to overcoming this problem was that employment of strong endogenous promoters such as the *β-Tub*, *RbcS2* and *Hsp70* promoters results in the efficient expression of foreign genes in microalgae [60-65]. Therefore, it is likely that efficient expression of the *PyGUS* reporter gene in *P. yezoensis* cells is caused by the recruitment of endogenous strong promoters.

By comparison with steady-state expression levels by reverse transcription-polymerase chain reaction (PCR), we found two genes strongly expressed in *P. yezoensis*: genes encoding glyceraldehyde-3-phosphate dehydrogenase (PyGAPDH) and actin 1 (PyAct1) [29]. When the *PyGUS* gene fused with the 5' upstream regions of these genes were introduced into gameto‐ phytic cells by particle bombardment, cells expressing the reporter gene and GUS enzymatic activity were dramatically increased [48,66]. These results indicate that employment of endogenous strong promoters is another important factor necessary for high-level expression of the reporter gene in *P. yezoensis* cells. In addition, the original *GUS* gene was not activated by *PyGAPDH* or *PyAct1* promoter [29,30,48], demonstrating that the *PyGUS* gene and endog‐ enous strong promoter have a synergistic effect on the efficiency of the expression in *P. yezoensis* cells (Figure 1A). Therefore, the combination of endogenous strong promoters with

codon optimized reporter genes is critical for successful transient transformation in *Porphyra* species [29,30]. The established procedure of transient transformation is schematically represented in Figure 2.

### *2.2.3. Application of the transient transformation for using fluorescent proteins*

The *GUS* reporter gene is usually used to monitor gene expression *in planta*; however, visualization of the reporter products requires cell killing. Reporters that function in liv‐ ing cells have also been established to date with fluorescent proteins used most common‐ ly. The green fluorescent protein (GFP) has the advantage over other reporters for monitoring subcellular localization of proteins in living cells, because its fluorescence can be visualized without additional substrates or cofactors [67]. At present, there are GFP variants with non-overlapping emission spectra such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein, which allows multicolor imaging in cells [68,69].

**Figure 1.** Efficient expression of PyGUS and fluorescent proteins by the transeint transformation with circular expression plasmids in *P. yezoensis* gametophytic cells. (A) Expression of the codon-optimized *PyGUS* reporter gene under the direc‐ tion of the actin 1 (*PyAct1*) promoter. Blue histochemically stained cells are PyGUS expression cells. Scale bar corresponds to 100 μm. (B) Expression of humanized AmCFP and plant-adapted sGFP(S65T). Gametophytic cells transiently transformed with expression plasminds containng *AmCFP* or *sGFP(S65T)* gene under the control of the *PyAct1* promoter. Left and right

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**Figure 2.** The established procedure of transeient transformation in *P. yezoensis.* A circular expression plasmid is bom‐ barded into *P. yezoensis* gametophytic cells using the Bio-Rad PDS-1000/He after coating of gold particles with the plasmid. Expression of the reporter gene is observed after cultivation of the bombareded gametophyte under dark for two days; for *PyGUS* reporter gene, histochemical staining with X-gluc solution and fluorometric analysis of enzymatic activity are performed; for fluorescent reporter genes, bombarded sanples are examined with fluorescent microscopy.

panels show bright field and fluorescence images, respectively. Scale bar corresponds to 5 μm.

Until recently, there was no report about the successful expression of fluorescent pro‐ teins in seaweeds. However, based on an efficient transient transformation system in *P. yezoensis*, fluorescent reporter systems have recently been established in *P. yezoensis* [29,30,70,71]. The humanized fluorescent protein genes, AmCFP, ZsGFP, and ZsYFP (Clontech) and the plant-adapted GFP(S65T) [72], the GC contents of which are as high as 63.7%, 62.8%, 61.9% and 61.4%, respectively, were strongly expressed in gametophytic cells under the direction of the *PyAct1* promoter using the particle bombardment method [71] (see Figure 1B).

The analysis of subcellular localization of cellular molecules was available using humanized and plant-adapted fluorescent reporters. The first successful attempt at achieving this process was to monitor the plasma membrane localization of phosphoinositides in *P. yezoensis* [70]. Phosphoinositides (PIs), whose inositol ring has hydroxyl groups at positions D3, D4 and D5 for phosphorylation, constitute a family of structurally related lipids, PtdIns-monophosphates [PtdIns3P, PtdIns4P and PtdIns5P], PtdIns-bisphosphates [PtdIns(3,4)P2, PtdIns(3,5)P2 and PtdIns(4,5)P2] and PtdIns-trisphosphate [PtdIns(3,4,5)P3], all of which are detectable in plants except for PtdIns(3,4,5)P3 [73,74]. Although the PIs are a minority among membrane phos‐ pholipids, they play important roles in regulating multiple processes of development and cell responses to environmental stimuli in land plants and green algae [74,75]. Recently, Li et al. [76,77] demonstrated that PIs are involved in the establishment of cell polarity in *P. yezoensis* monospores. The Pleckstrin homology (PH) domain, a PI-binding module, each part of which has individual substrate specificity, is usually used to monitor PIs *in vivo* by fusion with a fluorescent protein [78-80]. For instance, the PH domains from human phospholipase Cδ1 (PLCδ1) are employed for the detection of PtdIns(4,5)P2 [81], whereas that from the v-akt murine thymoma viral oncogene homolog 1 (Akt1) has dual specificity in the detection of both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 [82]. Because of this substrate specificity, we were able to visualize PtdIns(3,4)P2 and PtdIns(4,5)P2 at the plasma membrane with humanized AmCFP and ZsGFP fused to the PH domains from PLCδ1 and Akt1 via the direction of the *PyAct1* promoter [70].

codon optimized reporter genes is critical for successful transient transformation in *Porphyra* species [29,30]. The established procedure of transient transformation is schematically

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

The *GUS* reporter gene is usually used to monitor gene expression *in planta*; however, visualization of the reporter products requires cell killing. Reporters that function in liv‐ ing cells have also been established to date with fluorescent proteins used most common‐ ly. The green fluorescent protein (GFP) has the advantage over other reporters for monitoring subcellular localization of proteins in living cells, because its fluorescence can be visualized without additional substrates or cofactors [67]. At present, there are GFP variants with non-overlapping emission spectra such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein, which allows multicolor

Until recently, there was no report about the successful expression of fluorescent pro‐ teins in seaweeds. However, based on an efficient transient transformation system in *P. yezoensis*, fluorescent reporter systems have recently been established in *P. yezoensis* [29,30,70,71]. The humanized fluorescent protein genes, AmCFP, ZsGFP, and ZsYFP (Clontech) and the plant-adapted GFP(S65T) [72], the GC contents of which are as high as 63.7%, 62.8%, 61.9% and 61.4%, respectively, were strongly expressed in gametophytic cells under the direction of the *PyAct1* promoter using the particle bombardment method

The analysis of subcellular localization of cellular molecules was available using humanized and plant-adapted fluorescent reporters. The first successful attempt at achieving this process was to monitor the plasma membrane localization of phosphoinositides in *P. yezoensis* [70]. Phosphoinositides (PIs), whose inositol ring has hydroxyl groups at positions D3, D4 and D5 for phosphorylation, constitute a family of structurally related lipids, PtdIns-monophosphates [PtdIns3P, PtdIns4P and PtdIns5P], PtdIns-bisphosphates [PtdIns(3,4)P2, PtdIns(3,5)P2 and PtdIns(4,5)P2] and PtdIns-trisphosphate [PtdIns(3,4,5)P3], all of which are detectable in plants except for PtdIns(3,4,5)P3 [73,74]. Although the PIs are a minority among membrane phos‐ pholipids, they play important roles in regulating multiple processes of development and cell responses to environmental stimuli in land plants and green algae [74,75]. Recently, Li et al. [76,77] demonstrated that PIs are involved in the establishment of cell polarity in *P. yezoensis* monospores. The Pleckstrin homology (PH) domain, a PI-binding module, each part of which has individual substrate specificity, is usually used to monitor PIs *in vivo* by fusion with a fluorescent protein [78-80]. For instance, the PH domains from human phospholipase Cδ1 (PLCδ1) are employed for the detection of PtdIns(4,5)P2 [81], whereas that from the v-akt murine thymoma viral oncogene homolog 1 (Akt1) has dual specificity in the detection of both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 [82]. Because of this substrate specificity, we were able to visualize PtdIns(3,4)P2 and PtdIns(4,5)P2 at the plasma membrane with humanized AmCFP and ZsGFP fused to the PH domains from PLCδ1 and Akt1 via the direction of the *PyAct1*

*2.2.3. Application of the transient transformation for using fluorescent proteins*

represented in Figure 2.

Applications

328

imaging in cells [68,69].

[71] (see Figure 1B).

promoter [70].

**Figure 1.** Efficient expression of PyGUS and fluorescent proteins by the transeint transformation with circular expression plasmids in *P. yezoensis* gametophytic cells. (A) Expression of the codon-optimized *PyGUS* reporter gene under the direc‐ tion of the actin 1 (*PyAct1*) promoter. Blue histochemically stained cells are PyGUS expression cells. Scale bar corresponds to 100 μm. (B) Expression of humanized AmCFP and plant-adapted sGFP(S65T). Gametophytic cells transiently transformed with expression plasminds containng *AmCFP* or *sGFP(S65T)* gene under the control of the *PyAct1* promoter. Left and right panels show bright field and fluorescence images, respectively. Scale bar corresponds to 5 μm.

**Figure 2.** The established procedure of transeient transformation in *P. yezoensis.* A circular expression plasmid is bom‐ barded into *P. yezoensis* gametophytic cells using the Bio-Rad PDS-1000/He after coating of gold particles with the plasmid. Expression of the reporter gene is observed after cultivation of the bombareded gametophyte under dark for two days; for *PyGUS* reporter gene, histochemical staining with X-gluc solution and fluorometric analysis of enzymatic activity are performed; for fluorescent reporter genes, bombarded sanples are examined with fluorescent microscopy.

Moreover, subcellular localization of transcription factors was also visualized in *P. ye‐ zoensis*. When complete open reading frames (ORFs) of transcription elongation factor 1 (PyElf1) and multiprotein bridging factor 1 (PyMBF1) from *P. yezoensis* were fused to AmCFP or ZsGFP, nuclear localization of these fusion proteins was observed in gameto‐ phytic cells, which was confirmed by overlapping of fluorescent signals with SYBR Gold staining of the nucleus [71]

with the importance of two critical factors for transient transformation in red seaweeds, adjustment of the codon usage in reporter genes and employment of a strong endogenous

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The other important message gleaned from this experimental data is the efficient heterologous activation of *PyGAPDH* and *PtHSP70* promoters in *P. tenera* and *P. yezoensis*, respectively [85, 87]. For the genetic transformation, the target site for recombination is usually determined by the DNA sequence of genes desired for disruption or modification. Thus, it is better to exclude a possibility of homologous recombination at the DNA region corresponding to the promoter sequence used for expression of the reporter gene that is usually sandwiched between two different DNA sequences from the objective gene or its flanking regions. To avoid incorrect recombination at the promoter region, it is critical to employ heterologous promoters, whose sequence has low homology to the genome sequence of the host, to direct the expression of reporter genes. It is therefore possible that *PyGAPDH* and *PtHSP70* promoters are useful for genetic transformation in *P. tenera* and *P. yezoensis*, respectively. The number of promoters acting for heterologous reporter gene expression in red algae must be increased to develop a

The successful genetic transformation in red alga has been established only in unicellular algae [20,88]. The first report described chloroplast transformation in the unicellular red alga *Porphyridium* sp. through integration of the gene encoding AHAS(W492S) into the chloroplast genome by homologous recombination, resulting in sulfometuron methyl (SMM) resistance at a high frequency in SMM-resistant colonies [89]. The next report was of stable nuclear transformation in the unicellular red alga *Cyanidioschyzon merolae*, for which the uracil auxotrophic mutant lacking the *URA5.3* gene was used for the genetic background to isolate mutants with uracil prototrophic by employing the wild-type *URA5.3* gene fragment as a

Table 1 shows preliminary experiments with red seaweeds. The first was by Cheney et al. [26], who introduced the *CaMV 35S-GUS* and *CaMV 35S-GFP* genes in *P. yezoensis* genome via an *Agrobacterium*-mediated transformation system. In addition, they transformed *P. yezoensis* with a bacterial nitroreductase gene via an *Agrobacterium*-mediated method [28] and *P. leucosticte* monospores with an unknown gene by electroporation [27]. However, these reports appeared on conference abstracts and thus details of experimental procedures are unknown. In related work, the genetic transformation of *Gracilaria* species was recently reported [91,92], in which integration of the *SV40-lacZ* gene was checked by PCR using genomic DNAs prepared from particle-bombarded seaweeds; however, selection of transformed cells was not performed. Taken together, these preliminary experiments are not enough to conclude the establishment of genetic transformation in red seaweeds, meaning that the genetic transformation system

As mentioned above, procedures of integration and targeting of foreign genes into the genome and selection of transformed cells must be developed for establishing the genetic transformation system, although other requirements such as an efficient gene transfer

sophisticated system for red algal genetic transformation.

**2.3. Towards genetic transformation in red seaweeds**

has not yet been fully established in red macroalgae.

promoter.

selection maker [90].

With the successfull visualization of subcellular localization of cellular molecules, the transient transformation system developed in *P. yezoensis* appearst to be powerful tool to analyze functions of genes and cellular components [29,30].

### *2.2.4. Applicability of the P. yezoensis transient transformation system in other red seaweeds*

As described above, both the adjustment of codon usage of the reporter gene according to algal preference and the employment of the strong endogenous promoters are impor‐ tant for providing highly efficient and reproducible expression of the reporter gene in *P. yezoensis*. In addition to Bangiophyceae like *Porphyra* species, Florideophyceae are also known, including a number of industrially important species such as *Gracilaria* and *Geli‐ dium* as sources of agar and *Chondrus* and *Kappaphycus* as sources of carrageenan. Thus, the establishment of a genetic manipulation system for both Bangiophyceae and Florideo‐ phyceae other than *P. yezoensis* is awaited. EST analysis of *P. haitanensis* revealed that the GC content of the ORFs in this alga was as high as that in *P. yezoensis*, and analysis of the *GAPDH* gene from a Florideophycean alga *Chondrus crispus* showed a high GC con‐ tent (approximately 60%) in the coding region [83,84], which is consistent with the codon preference in *P. yezoensis*. Since efficient expression of the *GAPDH-PyGUS* gene has re‐ cently been confirmed in *P. tenera* [85], the applicability of the *P. yezoensis* transient gene expression system in other red seaweeds is expected. Indeed, using the *PyGUS* and *sGFP(S65T)* reporter genes under the direction of the *PyAct1* promoter, efficient expres‐ sion of *PyGUS* and *sGFP(S65T)* genes was observed in Bangiophyceae including *P. ten‐ era, P. okamurae, P. psedolinearis* and *Bangia fuscopurpurea*, although the expression efficiency varied among species [86]. Thus, the transient transformation system devel‐ oped in *P. yezoensis* is widely applicable in Bangiophycean red algae [29,30,86].

No expression of the reporter genes was seen in Florideophyceae [29,30,86]. Since the availa‐ bility of the *P. yezoensis* promoter is responsible for this deficiency in gene expression, it is important to employ the 5' upstream region of the suitable endogenous gene from Florideo‐ phycean algae. Alternatively, it is possible that the efficiency of plasmid transfer by bombard‐ ment parameters is reduced by the cell wall and thus the size of the gold particles, target distance, acceleration pressure and/or amount of DNA per bombardment should be adjusted.

Taken together, *PyGUS* and *sGFP(S65T)* genes act synergistically with the *PyAct1* promoter as a heterologous promoter for transient transformation in Bangiophycean algae. Recently, the same synergistic effect was found in *P. tenera*; that is, Son et al. [85] clearly indicated that the heat shock protein 70 (*PtHSP70*) promoter from *P. tenera* can activate the *PyGUS* gene in gametophytic cells of this alga. Moreover, the *PtHSP70-PyGUS* gene was expressed in *P. yezoensis, P. okamurae, P. psedolinearis* and *B. fuscopurpurea* [85,87]. These findings are consistent with the importance of two critical factors for transient transformation in red seaweeds, adjustment of the codon usage in reporter genes and employment of a strong endogenous promoter.

The other important message gleaned from this experimental data is the efficient heterologous activation of *PyGAPDH* and *PtHSP70* promoters in *P. tenera* and *P. yezoensis*, respectively [85, 87]. For the genetic transformation, the target site for recombination is usually determined by the DNA sequence of genes desired for disruption or modification. Thus, it is better to exclude a possibility of homologous recombination at the DNA region corresponding to the promoter sequence used for expression of the reporter gene that is usually sandwiched between two different DNA sequences from the objective gene or its flanking regions. To avoid incorrect recombination at the promoter region, it is critical to employ heterologous promoters, whose sequence has low homology to the genome sequence of the host, to direct the expression of reporter genes. It is therefore possible that *PyGAPDH* and *PtHSP70* promoters are useful for genetic transformation in *P. tenera* and *P. yezoensis*, respectively. The number of promoters acting for heterologous reporter gene expression in red algae must be increased to develop a sophisticated system for red algal genetic transformation.

### **2.3. Towards genetic transformation in red seaweeds**

Moreover, subcellular localization of transcription factors was also visualized in *P. ye‐ zoensis*. When complete open reading frames (ORFs) of transcription elongation factor 1 (PyElf1) and multiprotein bridging factor 1 (PyMBF1) from *P. yezoensis* were fused to AmCFP or ZsGFP, nuclear localization of these fusion proteins was observed in gameto‐ phytic cells, which was confirmed by overlapping of fluorescent signals with SYBR Gold

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

With the successfull visualization of subcellular localization of cellular molecules, the transient transformation system developed in *P. yezoensis* appearst to be powerful tool to analyze

As described above, both the adjustment of codon usage of the reporter gene according to algal preference and the employment of the strong endogenous promoters are impor‐ tant for providing highly efficient and reproducible expression of the reporter gene in *P. yezoensis*. In addition to Bangiophyceae like *Porphyra* species, Florideophyceae are also known, including a number of industrially important species such as *Gracilaria* and *Geli‐ dium* as sources of agar and *Chondrus* and *Kappaphycus* as sources of carrageenan. Thus, the establishment of a genetic manipulation system for both Bangiophyceae and Florideo‐ phyceae other than *P. yezoensis* is awaited. EST analysis of *P. haitanensis* revealed that the GC content of the ORFs in this alga was as high as that in *P. yezoensis*, and analysis of the *GAPDH* gene from a Florideophycean alga *Chondrus crispus* showed a high GC con‐ tent (approximately 60%) in the coding region [83,84], which is consistent with the codon preference in *P. yezoensis*. Since efficient expression of the *GAPDH-PyGUS* gene has re‐ cently been confirmed in *P. tenera* [85], the applicability of the *P. yezoensis* transient gene expression system in other red seaweeds is expected. Indeed, using the *PyGUS* and *sGFP(S65T)* reporter genes under the direction of the *PyAct1* promoter, efficient expres‐ sion of *PyGUS* and *sGFP(S65T)* genes was observed in Bangiophyceae including *P. ten‐ era, P. okamurae, P. psedolinearis* and *Bangia fuscopurpurea*, although the expression efficiency varied among species [86]. Thus, the transient transformation system devel‐

*2.2.4. Applicability of the P. yezoensis transient transformation system in other red seaweeds*

oped in *P. yezoensis* is widely applicable in Bangiophycean red algae [29,30,86].

No expression of the reporter genes was seen in Florideophyceae [29,30,86]. Since the availa‐ bility of the *P. yezoensis* promoter is responsible for this deficiency in gene expression, it is important to employ the 5' upstream region of the suitable endogenous gene from Florideo‐ phycean algae. Alternatively, it is possible that the efficiency of plasmid transfer by bombard‐ ment parameters is reduced by the cell wall and thus the size of the gold particles, target distance, acceleration pressure and/or amount of DNA per bombardment should be adjusted. Taken together, *PyGUS* and *sGFP(S65T)* genes act synergistically with the *PyAct1* promoter as a heterologous promoter for transient transformation in Bangiophycean algae. Recently, the same synergistic effect was found in *P. tenera*; that is, Son et al. [85] clearly indicated that the heat shock protein 70 (*PtHSP70*) promoter from *P. tenera* can activate the *PyGUS* gene in gametophytic cells of this alga. Moreover, the *PtHSP70-PyGUS* gene was expressed in *P. yezoensis, P. okamurae, P. psedolinearis* and *B. fuscopurpurea* [85,87]. These findings are consistent

staining of the nucleus [71]

Applications

330

functions of genes and cellular components [29,30].

The successful genetic transformation in red alga has been established only in unicellular algae [20,88]. The first report described chloroplast transformation in the unicellular red alga *Porphyridium* sp. through integration of the gene encoding AHAS(W492S) into the chloroplast genome by homologous recombination, resulting in sulfometuron methyl (SMM) resistance at a high frequency in SMM-resistant colonies [89]. The next report was of stable nuclear transformation in the unicellular red alga *Cyanidioschyzon merolae*, for which the uracil auxotrophic mutant lacking the *URA5.3* gene was used for the genetic background to isolate mutants with uracil prototrophic by employing the wild-type *URA5.3* gene fragment as a selection maker [90].

Table 1 shows preliminary experiments with red seaweeds. The first was by Cheney et al. [26], who introduced the *CaMV 35S-GUS* and *CaMV 35S-GFP* genes in *P. yezoensis* genome via an *Agrobacterium*-mediated transformation system. In addition, they transformed *P. yezoensis* with a bacterial nitroreductase gene via an *Agrobacterium*-mediated method [28] and *P. leucosticte* monospores with an unknown gene by electroporation [27]. However, these reports appeared on conference abstracts and thus details of experimental procedures are unknown. In related work, the genetic transformation of *Gracilaria* species was recently reported [91,92], in which integration of the *SV40-lacZ* gene was checked by PCR using genomic DNAs prepared from particle-bombarded seaweeds; however, selection of transformed cells was not performed. Taken together, these preliminary experiments are not enough to conclude the establishment of genetic transformation in red seaweeds, meaning that the genetic transformation system has not yet been fully established in red macroalgae.

As mentioned above, procedures of integration and targeting of foreign genes into the genome and selection of transformed cells must be developed for establishing the genetic transformation system, although other requirements such as an efficient gene transfer

system and an efficient expression system for foreign genes have been resolved by devel‐ oping the transient transformation system in Bangiophyceae [29,30]. Regarding the unre‐ solved points, knowledge about the selection of transformed cells is now accumulating. Selection marker genes are required to distinguish between transformed cells and nontransformed cells, since successful integration of a foreign gene into the host genome usually occur in only a small percentage of transfected cells. These genes confer new traits to any transformed target strain of a certain species, thus enabling the transformed cells to survive on medium containing the selective agent, where non-transformed cells die. Genes with resistance to the aminoglycoside antibiotics, which bind to ribosomal subunits and inhibit protein synthesis in bacteria, eukaryotic plastids and mitochondria [93], are generally used as selection markers. For example, the antibiotics hygromycin and geneticin (G418) are frequently used as selection agents with the hygromycin phos‐ photransferase (*hptII*) gene to inactivate hygromycin via an ATP-dependent phosphoryla‐ tion [94] and the neomycin phosphotransferase II (*nptII*) gene to detoxify neomycin, G418 and paromomycin [93], respectively. In the green alga *Chlamydomonas reinhardtii,* the hy‐ gromycin phosphotransferase (*aph7"*) gene from *Streptomyces hygroscopicus* and the ami‐ noglycoside phosphotransferase *aphVIII* (*aphH*) gene from *S. rimosus* had been reported as selectable marker genes for hygromycin and paromomycin, respectively, with similari‐ ty in the codon usage [95-97]. The *aphH* gene from *S. rimosus* is also applicable to the multicellular green alga *Volvox carteri* as a paromomycin-resistance gene [97,98]. In the diatom *Phaeodactylum tricornutum*, the expressed chloramphenicol acetyltransferase gene (*CAT*) detoxifies chloramphenicol [99], and the *nptII* gene confers resistance to the amino‐ glycoside antibiotic G418 [64]. Likewise, the *nptII* gene gives resistance to the antibiotic G418 in the diatoms *Navicula saprophila* and *Cyclotella cryptica* [100]. However, it is un‐ known what kinds of antibiotics-based selection marker genes are available for red sea‐ weeds, since red algae usually have strong resistance to antibiotics.

**3. Transformation in brown seaweeds**

promoter is active in the transient transformation [103].

**expression**

**Species Status of**

**Table 2.** Transformation in brown seaweeds.

According to Qin et al. [103], trials of genetic engineering in brown seaweeds have been started by transient expression of the *GUS* reporter gene under direction of the *CaMV 35S* promoter by particle bombardment in *Laminaria japonica* and *Undaria pinnatifida*, which were first performed in 1994 by them. Descriptions of related experiments were published later [104,105]. Qin et al. then focused on the establishment of genetic transformation in brown seaweeds and provided successful reports of genetic transformation in *L. japonica* [103,106]. Genetic transformation was performed by particle bombardment only and ex‐ pression of a reporter gene was driven by the *SV40* promoter that is usually used for gene expression in mammalian cells (Table 2). This promoter represented non-tissue and -cell specificity for expression of the *E. coli lacZ* reporter gene [105]. Promoters from maize ubiq‐ uitin, algal adenine-methyl transfer enzyme and diatom fucoxanthin chlorophyll a/c-bind‐ ing protein (*FCP*) genes are also useful for transient expression of the *GUS* gene, and the *FCP* promoter is also employable for the genetic transformation [107]. Interestingly, there has been no successful genetic transformation using the *CaMV 35S* promoter, although this

Despite the reports of successful genetic transformation, there was no experiment using antibiotics-based selection of transformants in brown seaweeds. Although the susceptibility of brown seaweeds to antibiotics has not been well studied, it was reported that *L. japonica* was sensitive to chloramphenicol and hygromycin, but not to ampicillin, streptomycin, kanamycin, neomycin or G418 [103,106]. Since hygromycin is more effective than chloramphenicol [103,106], it is necessary to confirm the utility of the *SV40-hptII* gene for the selection of

*Laminaria japonica* transient particle bombardment CaMV 35S GUS [103] *Laminaria japonica* stable particle bombardment SV40 GUS [105]

*Laminaria japonica* stable particle bombardment FCP GUS [107] *Laminaria japonica* stable particle bombardment SV40 HBsAg [113] *Laminaria japonica* stable particle bombardment SV40 Rt-PA [114] *Laminaria japonica* stable particle bombardment SV40 bar [114] *Undaria pinnatifida* transient particle bombardment CaMV 35S GUS [103] *Undaria pinnatifida* transient particle bombardment SV40 GUS [104]

**Gene transfer method Promoter Marker or**

AMT

**Reporter**

Current Advances in Seaweed Transformation

http://dx.doi.org/10.5772/52978

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GUS [107]

**Ref.**

transformants to fully establish the genetic transformation system in kelp.

*Laminaria japonica* transient particle bombardment CaMV 35S, UBI,

Recently, the sensitivity of *P. yezoensis* gametophytes to ampicillin, kanamycin, hygromycin, geneticin (G418), chloramphenicol and paromomycin was investigated, and lethal effects of these antibiotics on gametophytes were observed at more than 2.0 mg mL-1 of hygromycin, chloramphenicol and paromomycin and 1.0 mg mL-1 of G418, whereas *P. yezoensis* gameto‐ phytes were highly resistant to ampicillin and kanamycin [101]. Although these concentrations are in fact very high in comparison with the cases for the red alga *Griffithsia japonica* and the green alga *C. reinhardtii* that were highly sensitive to 50 μg mL-1 and 1.0 μg mL-1 of hygromycin [96,102], these four antibiotics and corresponding resistance genes are suitable for the selection of genetically transformed cells from *P. yezoensis* gametophytes. According to these findings, it is necessary to confirm whether *P. yezoensis* gametophytes will obtain antibiotic tolerance by introducing plasmid constructs containing the antibiotic-resistance genes mentioned above. In this case, optimization of codon usage and the employment of strong endogenous promoter are expected for functional expression of the antibiotic resistance genes, according to the knowledge from the transient transformation system [29,30]. Such efforts could effectively contribute to the establishment of the genetic transformation system in red seaweeds in the near future.
