Yukihiro Kitade

Comparison of RNA expression profiles between the two generations of Porphyra yezoensis (Rhodophyta), based on expressed sequence tag frequency analysis

To identify gene candidates related to the morphological and physiological differences between the gametophytic and sporophytic generations of a marine red alga, Porphyra yezoensis Ueda, large‐scale expressed sequence tag (EST) analysis was conducted. A total of 10,625 5′‐end EST sequences was generated from the sporophyte cDNA library, in addition to the previously published 10,154 ESTs from the gametophyte. A total of 20,779 ESTs was clustered into 4496 nonredundant groups at the criterion of 95% identity for 50 bases. Among the EST groups, only 1013 (22.5%) groups were classified as ESTs that commonly occurred in both generations, whereas a large proportion of EST groups were identified as being unique to either the gametophyte (1940 EST groups = 43.1%) or the sporophyte (1543 EST groups = 34.3%). A statistical significance test revealed 89 and 112 highly expressed gene candidates in the gametophyte and the sporophyte, respectively. Twelve candidate genes were subjected to RT‐PCR analysis, and the differential expression was confirmed. Generation‐specific regulation of alternative splicing of a transcript was demonstrated by RT‐PCR analyses.

Porphyra: Complex Life Histories in a Harsh Environment: P. umbilicalis, an Intertidal Red Alga for Genomic Analysis

Porphyra encompasses a large group of multicellular red algae that have a prominent gametophytic phase. The complex, heteromorphic life history of species in this genus, their remarkable resilience to high light and desiccation, ancient fossil records, and value as human food (e.g., laver, nori), make Porphyra a compelling model for genome sequencing. Sequencing of the nuclear genome of Porphyra umbilicalis from the northwestern Atlantic is currently in process. The ∼270 Mb genome of this alga is much larger than that of the unicellular acidophilic Cyanidioschyzon merolae (16.5 Mb), the only rhodophyte for which there is a fully sequenced genome, and is approximately twice as large as the Arabidopsis genome. Future analyses of the P. umbilicalis genome should provide opportunities for researchers to (1) develop an increased understanding of the ways in which these algae have adapted to severe physiological stresses, (2) elucidate the molecular features of development through the complex life history, and (3) define key components required for the transition of growth from a single cell to a multicellular organism.

IDENTIFICATION OF GENES PREFERENTIALLY EXPRESSED DURING ASEXUAL SPORULATION IN PORPHYRA YEZOENSIS GAMETOPHYTES (BANGIALES, RHODOPHYTA)(1).

Asexual reproduction via archeospores in Porphyra yezoensis Ueda gametophytes is a very valuable character to nori farming; however, there is little information available on the molecular basis of the developmental process. To identify genes involved in the Porphyra asexual sporulation, we compared the gene expression profiles derived from four developmental stages of the life cycle (three from gametophytes; one from sporophytes) using cDNA macroarray, which includes 4,896 nonredundant expressed sequence tag (EST) groups. Candidate genes were screened by two different macroarray data analyses combined with reverse transcription‐PCR (RT‐PCR) analysis or Northern analysis. RT‐PCR analysis revealed that nine genes (one: similarity to 5′–3′ exoribonuclease; the other eight: no sequence similarity to known proteins) were expressed with a gametophyte (G)–specific manner, and two genes (named ASPO2608, ASPO1527) were expressed only in gametophytes that formed archeospores. The deduced amino acid sequences for the latter two genes are predicted to contain signal peptides for secretion at their N‐termini. Northern analysis revealed that expression levels of Calvin cycle genes in the gametophytic stage that formed archeospores (G‐A stage) were higher than those of the gametophyte blade with no archeospores (G‐NA stage). In the macroarray analysis based on the rank data of G‐preferentially expressed genes, which were detected in the previous P. yezoensis EST analysis, one gene encoding the cyclase associated protein (CAP) exhibited a change upwardly in the G‐A stage >1,000 ranks to the G‐NA stage. We propose that ASPO2608 and CAP may function in a signaling pathway of asexual sporulation.

Genetic polymorphism within Porphyra yezoensis (Bangiales, Rhodophyta) and related species from Japan and Korea detected by cleaved amplified polymorphic sequence analysis

We investigated genetic diversity in 10 strains of Porphyra yezoensis and related species, collected from Japan and Korea, in order to identify an appropriate crossing partner for use in various genetic analyses. Firstly, the phylogenetic relationships among the 10 strains were examined using SSU rDNA sequences. All five Japanese strains (TU-1, TU-2, TUH-25, JHS and JHU) and two of the Korean strains (KGJ and KPH) were identified as P. yezoensis while the other three Korean strains (KTY1, KTY2 and KTY3) were identified as P. tenera. Using cleaved amplified polymorphic sequence analysis, genetic polymorphisms lying in five regions of four genes (ß-tubulin, TOP2, EF-1α and V-ATPase) were examined. Twenty out of 34 restriction endonucleases revealed genetic polymorphisms between the seven strains of P. yezoensis and the three strains of P. tenera in all five regions tested. Furthermore, when they were employed to digest the fragments of TOP2 and V-ATPase, 14 enzymes discriminated the two Korean strains of P. yezoensis (KGJ and KPH) from the five Japanese strains of this species. The level of polymorphism was much higher in the amplified fragment of V-ATPase (including four introns) than in that of TOP2 without an intron. The gene regions with detected polymorphisms would be useful molecular markers for confirmation of cross-fertilization in sporophytic thalli and for construction of a linkage map. A suitable combination of the strains in the cross experiment is discussed.

Effect of DMSO on PCR of Porphyra yezoensis (Rhodophyta) gene

In order to improve the predictability ofresults of PCR with Porphyra yezoensisUeda genes, a study was made of possiblemodifications to the basic PCR protocol. DMSO used as an adjuvant considerablyincreased amplification efficiency andspecificity of PCR, the optimalconcentration being 5%. This protocolallowed for DNA templates with a high GCcontent to be amplified by PCR withoutproblem.

Inheritance pattern of chloroplast and mitochondrial genomes in artificial hybrids of Porphyra yezoensis (Rhodophyta)

To investigate inheritance of chlorplasts and mitochondria (organelles) in the marine red alga Porphyra yezoensis, cleaved amplified polymorphic sequence (CAPS) profiles of the two strains, TU-2 and KGJ, as parental gametophytes and 44 hybrid sporophytes (i.e. female of TU-2 and male of KGJ) were examined. A carbamyl phosphate synthase small subunit gene (carA) and ribosomal protein S11 gene (rps11)-small subunit ribosomal RNA gene (rns) spacer region were used as molecular markers for the chloroplast and mitochondrial DNAs, respectively. Thirty-eight of 44 (86.4%) conchocelis colonies showed maternal banding patterns, five (11.4%) colonies showed biparental bands and one (2.2%) colony showed paternal bands in CAPS analyses both with chloroplast and mitochondrial markers. These results suggest that organelles of P. yezoensis are inherited uniparentally from the female parent.

Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta).

The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.

DIURNAL CELL DIVISION REGULATED BY GATING THE G1/S TRANSITION IN ENTEROMORPHA COMPRESSA (CHLOROPHYTA)1

The cell‐cycle progression of Enteromorpha compressa (L.) Nees (=Ulva compressa L.) was diurnally regulated by gating the G1/S transition. When the gate was open, the cells were able to divide if they had attained a sufficient size. However, the cells were not able to divide while the gate was closed, even if the cells had attained sufficient size. The diurnal rhythm of cell division immediately disappeared when the thalli were transferred to continuous light or darkness. When the thalli were transferred to a shifted photoperiod, the rhythm of cell division immediately and accurately synchronized with the shifted photoperiod. These data support a gating‐system model regulated by light:dark (L:D) cycles rather than an endogenous circadian clock. A dark phase of 6 h or longer was essential for gate closing, and a light phase of 14 h was required to renew cell division after a dark phase of >6 h.

Simple differentiation of two closely related species Porphyra tenera and Porphyra yezoensis (Bangiophyceae, Rhodophyta) based on length polymorphism of actin‐related protein 4 gene (ARP4)

Genetic polymorphisms were investigated to develop a simple and rapid method to differentiate between the two closely related species, Porphyra tenera and Porphyra yezoensis. Polymerase chain reaction (PCR) using the specific primer pair of the ARP4 gene gave length polymorphic single fragments of genomic DNAs from five strains of P. tenera (Japan T-8, JTW; Korea KTY1, KTY2, KTY3) and seven strains of P. yezoensis (Japan TU-1, TU-2, TUH-25, JHU, JA-1; Korea KGJ, KPH). All strains of P. yezoensis had introns 60 bp longer than that of P. tenera. Multiple cleaved amplified polymorphic sequence (CAPS) markers were also developed to differentiate P. tenera and P. yezoensis. This is the first report of length polymorphisms that can be used to differentiate between the how species using only PCR amplification with agarose gel electrophoresis. It is expected that the length polymorphism and plentiful CAPS profiles obtained in this study will be useful in the assessment of genetic diversity within P. tenera and P. yezoensis as well as in breeding science that requires the collection of various strains of the two species.

Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1α (EF-1α), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1α. An intron is located in the 5′ untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1αtef-c (97%) than to the P. purpurea EF-1αtef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.