Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis

Megumu Takahashi
Graduate School of Fisheries Sciences
Hokkaido University
Hakodate 041-8611, Japan

Toshiki Uji
Graduate School of Fisheries Sciences
Hokkaido University
Hakodate 041-8611, Japan

Naotsune Saga
Faculty of Fisheries Sciences
Hokkaido University
Hakodate 041-8611, Japan

Koji Mikami*
Faculty of Fisheries Sciences
Hokkaido University
Hakodate 041-8611, Japan
E-mail:komikami@fish.hokudai.ac.jp

*Corresponding author

Financial support: This study was supported in part by a grant from the Sumitomo Foundation (to KM) and by Grants-in-Aid for Scientific Research (C; no. 21580213) and a National Project "Knowledge Cluster Initiative" (Innovative Stage, "Hakodate Marine Bio Industrial Cluster-Green Innovation of UMI") from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to NS).

Keywords: allantoin, protoplast, P. yezoensis, regeneration, transient gene expression.

Despite the recent progress of transient gene expression systems in a red alga Porphyra yezoensis by particle bombardment, a stable transformation system has yet to establish in any marine red macrophytes. One of the reasons of the difficulty in genetic transformation in red algae is the lack of systems to select and isolate transformed cells from gametophytic blades. Thus, toward the establishment of the stable transformation system in P. yezoensis, we have developed a procedure by which transiently transformed gametophytic cells were prepared from particle bombarded-gametophytic blade as regeneratable protoplasts. Using mixture of marine bacterial enzymes, yield of protoplasts was high as reported elsewhere; however, these protoplasts did not develop. In contrast, protoplasts prepared from gametophytes treated with allantoin were normally developed, in which the overexpression of a β-glucuronidase reporter gene had no effect on the regeneration of protoplasts. Therefore, the use of allantoin in protoplast preparation sheds a new light on the realization of an efficient isolation and selection of study transformed cells from gametophytic blades.

Porphyra yezoensis is recently received a great attention as a most promising model macrophyte for physiological and molecular biological studies in marine red algae (Saga and Kitade, 2002; Waaland, 2004). Physiological study of P. yezoensis has been enhanced by our establishment of the laboratory culture system of the P. yezoensis strain TU-1 (Kuwano et al. 1996). For example, the ability of constant harvestigation of monospores derived from gametophytic blades enabled us to examine how cell polarity is determined during migration and following development in monospores (Li et al. 2008; Li et al. 2009).

In contrast, until recently, experimental systems for analyzing regulation and function of genes has yet to establish in any marine macrophyte, which prevented the progress of molecular biological studies in multicellular algae. However, we have recently succeeded in the efficient expression of the modified β-glucuronidase (PyGUS) gene, whose codon usage had been adapted to that of P. yezoensis, and humanized cyan and green fluorescent protein (AmCFP and ZsGFP) genes by transient transformation of gametophytic blade using particle bombardment (Fukuda et al. 2008; Mikami et al. 2009; Uji et al. 2009). Such a technical development is expected to contribute to progress studies on subcellular localization of protein and regulation of the gene expression in P. yezoensis. Indeed, a plasma membrane localization of Pleckstrin homology (PH) domains from human proteins was correctly observed using PH domain-AmCFP fusion gene (Mikami et al. 2009) and nuclear localization of P. yezoensis transcription factors was also confirmed with AmCFP and ZsGFP (Uji et al. 2009).

Despite the development of transient gene expression systems in P. yezoensis, the fine analysis of gene function requires methods for manipulation of genome via the stable transformation. To develop the stable transformation procedure in P. yezoensis, efficient isolation and selection systems of transformed gametophytic cells are indispensable. In the present study, we developed a method to isolate transiently transformed gametophytic cells as regeneratable protoplasts from particle bombarded-gametophytic blades. Our results indicate that allantoin treatment, which was originally reported by Mizuta et al. (2003), is useful for preparation of transformed protoplasts from gametophytes. This is the first report of the regeneration of transiently transformed protoplasts in red algae.

Cultivation of gametophyte of P. yezoensis in the enriched sealife (ESL) medium was performed as described previously (Li et al. 2008). Protoplasts were prepared by two different methods. The first is a treatment of 5.0 mg gametophytic blade with 1.0 ml of AMX solution containing 20 mM 2-(N-Morpholino) ethanesulfonic acid (MES) (pH 7.5), 0.5 M mannitol, 4 mg of β-agarase, 4 mg of β-1,4-mannanase and 4 mg of β-1,3-xylanase (all of which parchased for Yakult Co Ltd, Tokyo, Japan), essentially according to Araki et al. (1994) with modification as using gametophytic blades pre-treated with 1.0 ml of 5% papain solution [the ESL medium containing 20 mM MES (pH 7.5) and 0.5 M mannitol] for 60 min. The second employs allantoin as described previously (Mizuta et al. 2003) expect for culture in the ESL medium with 60 µmol m^-2 s^-1 irradiance (10 L: 14 D cycle). After cultivation in allantoin-containing ESL medium for 3 weeks, gametophytic blades were homogenized in Downce type homogenizer to release protoplasts. For both methods, resultant protoplasts were filtered through 50 µm of nylon mesh and centrifuged at 1,000 rpm for 5 min, then resolved in 10 ml of the ESL medium. The total number of collected protoplasts was counted three times with a Thoma hemacytometer.

To construct a plasmid to express the PyGUS reporter gene, the Cauliflower mosaic virus 35S RNA promoter was released from p35S-PyGUS (Fukuda et al. 2008) by digestion with HindIII and BamHI and then a 5’ upstream region of the actin1 gene from P. yezoensis (PyAct1), which amplified with two primers, such as HindIII-PyACT1-F1 (5'-CCCAAGCTTCCACGCTCAGAGGGTTGAAG-3') and BamHI-PyACT1-R1 (5'-CGCGGATCCGGGCTTGCTCATGGTGGC-3'), by polymerase chain reaction whose conditions were 98ºC for 10 sec followed by 30 cycles of 94ºC for 10 sec and 60ºC for 30 sec with Prime STAR HS DNA polymerase (TaKaRa, Tokyo, Japan), was inserted into the promoter-less vector after digestion with HindIII and BamHI. Resultant plasmid designated pPyAct1-PyGUS were used for transient transformation of P. yezoensis gametophytes by particle bombardment as described previously (Mikami et al. 2009). Histochemical staining of bombarded blades with 5-bromo-4-chloro-3-indolylglucuronide (X-gluc) was performed according to Fukuda et al. (2008) for β-glucuronidase (GUS) assays.

We first test the usability of two methods for preparation of protoplasts by treatment of P. yezoensis gametophytic blades with AMX solution and allantoin. Number of protoplasts isolated from 5 mg of gametophytic blades by treatment with AMX solution was 0.7 x 10⁶ cells. However, the regeneration of released protoplasts was not observed (Figure 1a). On the other hand, the recovery of protoplasts isolated from gametophytes treated with 10 mM allantoin through homogenization was 1 x 10⁶ cells per 0.1 g of gametophytic blades, 70% of which was able to regenerate as shown in Figure 1b and Figure 1c. Therefore, allantoin treatment is considered to be useful for preparation of protoplasts from transformed gametophytic blades, although recovery is 14-fold less than the method with AMX solution.

Next, we examined the isolation of transiently transformed protoplasts from allantoin-treated gametophytic blades. As shown in Figure 2a and Figure 2b, allantoin treatment did not affect on the efficiency of transient transformation of gametophytic cell; that is, the number of PyAct1-PyGUS expressing cells was 700 in average in pieces of gametophytes containing 3.0 x 10⁵ cells from both control and 10 mM allantoin-treated blades. Importantly, PyAct1-PyGUS expressing protoplasts prepared from particle bombarded gametophytic blades were regeneratable (Figure 2c-m). However, the number of PyGUS expressing cells was 40 cells per particle bombarded blade, meaning that the recovery rate of transformed protoplasts was only 6%.

In the present study, we have succeeded to isolate transiently transformed cells from particle bombarded-gametophytic blade as regeneratable protoplasts. We have already test polyethylene glycol and glass beads methods to introduce the PyGUS gene into a large amount of protoplasts from allantoin-treated blades, which had succeeded in other alga (Kindle, 1990; Ohnuma et al. 2008) as well as a method using Magnetofection (OZ biosciences); however, any protoplast expressing PyGUS reporter gene was not observed (date not shown). These findings indicated that the use of the particle bombardment is an only way to transform gametophytic cells in P. yezoensis at present. Therefore, our procedure using allantoin-treated gametophytic blades is only one way to isolate transiently transformed protoplasts from particle bombarded gametophytic blades. However, there is a problem in the recovery rate of protoplasts after homogenization of particle bombarded-gametophyte. We proposed that low efficiency of the recovery is probably due to the death of PyGUS- expressing cells during homogenization and/or the disappearance during centrifugation. Thus, it is necessary to improve the recovery rate of transformed cells from gametophytes, by which the method with allantoin is expected to contribute to the establishment of the stable transformation system in P. yezoensis in future.

ARAKI, Toshiyoshi; HAYAKAWA, Michio; TAMARU, Yutaka; YOSHIMATSU, Keiji and MORISHITA, Tatsuo. Isolation and regeneration of haploid protoplast from Bangia atropurpurea (Rodophyta) with marine bacterial enzymes. Journal of Phycology, December 1994, vol. 30, no. 6, p. 1040-1046. [CrossRef]

FUKUDA, Satoru; MIKAMI, Koji; UJI, Toshiki; PARK, Eun-Jeong; OHBA, Toshiharu; ASADA, Kiyozo; KITADE, Yukihiro; ENDO, Hirotoshi; KATO, Ikunoshin and SAGA, Naotsune. Factors influencing efficiency of transient gene expression in the red macrophyte Porphrya yezoensis. Plant Science, March 2008, vol. 174, no. 3, p. 329-339. [CrossRef]

KINDLE, Karen L. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences of the United States of America, February 1990, vol. 87, no. 3, p. 1228-1232.

KUWANO, Kazuyoshi; ARUGA, Yusho and SAGA, Naotsune. Cryopreservation of clonal gametophytic thalli of Porphyra (Rhodophyta). Plant Science, April 1996, vol. 116, no. 1, p. 117-124. [CrossRef]

LI, Lin; SAGA, Naotsune and MIKAMI, Koji. Phosphatidylinositol 3-kinase activity and asymmetrical accumulation of F-actin are necessary for establishment of cell polarity in the early development of monospores from the marine red alga Porphyra yezoensis. Journal of Experimental Botany, October 2008, vol. 59, no. 13, p. 3575-3586. [CrossRef]

LI, Lin; SAGA, Naotsune and MIKAMI, Koji. Ca²⁺ influx and phosphoinositide signalling are essential for the establishment and maintenance of cell polarity in monospores from the red alga Porphyra yezoensis. Journal of Experimental Botany, August 2009, vol. 60, no. 12, p. 3477-3489.[CrossRef]

MIKAMI, Koji; UJI, Toshiki; LI, Lin; TAKAHASHI, Megumu; YASUI, Hajime and SAGA, Naotsune. Visualization of phosphoinositides via the development of the transient expression system of a cyan fluorescent protein in the red alga Porphyra yezoensis. Marine Biotechnology, October 2009, vol. 11, no. 5, p. 563-569. [CrossRef]

MIZUTA, Hiroyuki; YASUI, Hajime and SAGA, Naotsune. A simple method to mass monospores in the thallus of Porphyra yezoensis Ueda. Journal of Applied Phycology, July 2003, vol. 15, no. 4, p. 351-353. [CrossRef]

OHNUMA, Mio; YOKOYAMA, Takashi; INOUYE, Takayuki; SEKINE, Yasuhiko and TANAKA, Kan. Polyethylene glycol (PEG)-mediated transient gene expressin in a red alga, Cyanidioschyzon merolae 10D. Plant and Cell Physiology, January 2008, vol. 49, no. 1, p. 117-120. [CrossRef]

SAGA, Naotsune and KITADE, Yukihiro. Porphyra: A model plant in marine sciences. In: Proceeding of International Commemorative Symposium 70^th Anniversary of The Japanese Society of Fisheries Science. (1^st - 5^th October, 2001, Yokohama, Japan). Supplement, 2002. p. 1075-1078.

UJI, Toshiki; TAKAHASHI, Megumu; SAGA, Naotsune and MIKAMI, Koji. Visualization of nuclear localization of transcription factors with cyan and green fluorescent proteins in the red alga Porphyra yezoensis. Marine Biotechnology. In press, 2010. [CrossRef]

WAALAND, J. Robert; STILLER, John W. and CHENEY, Donald P. Macroalgal candidates for genomics. Journal of Phycology, February 2004, vol. 40, no. 1, p. 26-33.