Mi Sook Hwang

Heat shock protein gene family of the Porphyra seriata and enhancement of heat stress tolerance by PsHSP70 in Chlamydomonas.

Heat shock proteins and molecular chaperones are key components contributing to survival in the abiotic stress response. Porphyra seriata grows on intertidal rocks exposed to dynamic environmental changes associated with the turning tides, including desiccation and heat stress. Analysis of the ESTs of P. seriata allows us to identify the nine HSP cDNAs, which are predicted to be PsHSP90, three PsHSP70, PsHSP40 and PsHSP20, and three 5′-truncated HSP cDNAs. RT–PCR results show that most of the PsHSP transcripts were detected under normal cell growth conditions as well as heat stress, with the exception of two cDNAs. In particular, PsHSP70b and PsHSP20 transcripts were upregulated by heat stress. When the putative mitochondrial PsHSP70b was introduced and overexpressed in Chlamydomonas, transformed Chlamydomonas evidenced higher rates of survival and growth than those of the wild type under heat stress conditions. Constitutive overexpression of the PsHSP70b gene increases the transcription of the HSF1 as well as the CrHSP20 and CrHSP70 gene. These results indicate that PsHSP70b is involved in tolerance to heat stress and the effects on transcription of the CrHSP20 and CrHSP70 genes.

IDENTIFICATION OF THE HIGH-TEMPERATURE RESPONSE GENES FROM PORPHYRA SERIATA (RHODOPHYTA) EXPRESSION SEQUENCE TAGS AND ENHANCEMENT OF HEAT TOLERANCE OF CHLAMYDOMONAS (CHLOROPHYTA) BY EXPRESSION OF THE PORPHYRA HTR2 GENE1

Temperature is one of the major environmental factors that affect the distribution, growth rate, and life cycle of intertidal organisms, including red algae. In an effort to identify the genes involved in the high‐temperature tolerance of Porphyra, we generated 3,979 expression sequence tags (ESTs) from gametophyte thalli of P. seriata Kjellm. under normal growth conditions and high‐temperature conditions. A comparison of the ESTs from two cDNA libraries allowed us to identify the high temperature response (HTR) genes, which are induced or up‐regulated as the result of high‐temperature treatment. Among the HTRs, HTR2 encodes for a small polypeptide consisting of 144 amino acids, which is a noble nuclear protein. Chlamydomonas expressing the Porphyra HTR2 gene shows higher survival and growth rates than the wild‐type strain after high‐temperature treatment. These results suggest that HTR2 may be relevant to the tolerance of high‐temperature stress conditions, and this Porphyra EST data set will provide important genetic information for studies of the molecular basis of high‐temperature tolerance in marine algae, as well as in Porphyra.

DNA Sequences and Identification of Porphyra Cultivated by Natural Seeding on the Southwest Coast of Korea

Nuclear SSU and ITS1 rDNA and plastid rbcL sequences were determined to identify the seven samples of Porphyra cultivated by means of natural seeding on the southwest coast of Korea and analyzed to access the phylogenetic relationships of them with the natural populations of P. tenera and P. yezoensis from Korea and Japan. SSU, rbcL and ITS1 data from 18, 21 and 31 samples, respectively, including previously published sequences were investigated in the study. Results from our individual and combined data indicated that the seven samples were all P. yezoensis and the entities except one from Muan 2 aquafarm strongly grouped together with the natural populations of P. yezoensis from the south and the west coast of Korea. The sample from Muan 2 seems to be derived from a strain of P. yezoensis introduced from Japan by Porphyra farmers, based on DNA sequence data.

Development of cyan fluorescent protein (CFP) reporter system in green alga Chlamydomonas reinhardtii and macroalgae Pyropia sp

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonasreinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropiatenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropiatenera/Chlamydomonasreinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.

A nuclear fucosyltransferase-like protein, PtFUT, from marine red alga Pyropia tenera (Rhodophyta) confers osmotic stress tolerance

Pyropia tenera is a marine red alga that grows in the intertidal zone and may lose more than 90% of its water during twice-daily low tides. Based on the differentially expressed genes (DEGs) identified from the desiccation transcriptome of P. tenera gametophyte, a complementary DNA was isolated that upregulated strongly under desiccation stress and encoded a polypeptide of 591-amino acid residues showing a sequence homology with α-1,2-fucosyltransferase, named PtFUT. Fucosyltransferases transfer an l-fucose residue to various acceptor molecules, but information of their activity in the red algae is limited. PtFUT showed upregulation in transcription in gametophytes of P. tenera under desiccation and osmotic stress conditions induced by mannitol. Transcription of the PtFUT gene was also increased by abscisic acid as well as H2O2 treatment. The presence of a signal peptide for nucleus localization and fluorescence of the PtFUT-GFP fusion proteins in tobacco protoplasts indicated that PtFUT is located in the nucleus. When PtFUT was overexpressed in Chlamydomonas, the PtFUT improved the growth of transgenic cells under osmotic stress. These results suggest that PtFUT may play a role in tolerance to desiccation stress and provide insights into the molecular functions of the fucosyltransferases and understanding the desiccation tolerance mechanisms in marine red algae growing in intertidal zones.

PsCYP1 of marine red alga Pyropia seriata (Bangiales, Rhodophyta) confers salt and heat tolerance in Chlamydomonas

Cyclophilins (CYPs) are ubiquitous in all subcellular compartments, possess peptidyl-prolyl cis-trans isomerase (PPIase) activity and are present in prokaryotes and eukaryotes. Their physiological functions are various such as protein folding, symbiotic relationships, disease or plant responses to abiotic stresses. Pyropia seriata (Bangiales, Rhodophyta) is a marine red alga that is cultivated as commercially valuable seaweed. By analyzing transcriptome data, we identified six full coding sequences of CYPs from P. seriata. Among them, only PsCYP1 showed upregulation of transcription in responded to high temperature stress. PsCYP1 belongs to the single domain form of cytosolic cyclophilin and contains the typical 12 conserved amino acid residues for PPIase activity. Despite no detected nuclear localization signal (NLS), a chimeric PsCYP1 protein with green fluorescent protein (GFP) was detected predominantly in nucleus. When PsCYP1 was introduced into the green alga, Chlamydomonas, the introduced PsCYP1 conferred salt and heat stress tolerance in transgenic Chlamydomonas. Especially the transgenic Chlamydomonas cells exhibited a salt-tolerant phenotype. The cyclophilin genes from P. seriata will facilitate future studies of the molecular function of cyclophilin, and the mechanisms of abiotic stress tolerance in red algae.

Characterization of high temperature-tolerant strains of Pyropia yezoensis

High-temperature stress related to global warming reduces the growth and productivity of seaweeds. Thus, the development of new strains is urgently required for maintaining or even enhancing the productivity of useful seaweeds such as red alga Pyropia yezoenesis in an increasingly warmer sea environment. To develop competitive high-temperature-tolerant strains of P. yezoensis (Sugwawon no. 104), we screened libraries of gamma-irradiated strains and identified high-temperature-resistant (HTR) mutants. Our results showed that HTR-1 and HTR-2 grew well at higher temperatures that inhibited the growth of the wild-type strain. The efficiency of conchosporangium maturation and conchospore release of HTR-1 was similar to or higher than the wild-type strain. Moreover, thallus growth, pigment content, photosynthetic efficiency, and monospore release from the growing thallus in HTR-1 could be maintained even at high temperature. Taken together, our data demonstrate that HTR-1 may be suitable for industrial cultivation at sea, even at elevated temperatures.

Characterization of PyGUS gene silencing in the red macroalga, Pyropia yezoensis

Transgene silencing is a common phenomenon in higher plants and microalgae. In the present study, we analyzed transgene silencing in the macroalga Pyropia yezoensis. This is the first report of transgene silencing in macroalgae. Transgenic lines were generated by transformation with the PyGUS gene and a hygromycin resistance gene (PtAph7-1). Histochemical GUS staining detected PyGUS expression in only three out of 12 lines. Southern and northern blotting, polymerase chain reaction (PCR), and reverse transcription (RT)-PCR analyses indicated that the PyGUS gene was silenced in the other 9 lines. Interestingly, PyGUS gene silencing in transgenic line 7 was repeatedly initiated at a later stage, in the developmental process from monospore to thallus through asexual reproduction. We identified genes encoding Argonaute and Dicer-like protein, the major components of the RNA-silencing pathway, in the genome and transcripts of P. yezoensis. Interestingly, we found the DDA motif in the PIWI domain of the AGO, suggesting that translational repression may be the major gene-silencing pathway in P. yezoensis. In this study, the observation of PyGUS gene silencing and identification of RNA-silencing components indicate that the gene (transgene) silencing machinery functions actively in the macroalga P. yezoensis. These results will be useful to study the regulation of gene expression and RNA-silencing mechanisms in Pyropia species and related macroalgae.