Isolation and functional characterization of an ammonium transporter gene, PyAMT1, related to nitrogen assimilation in the marine macroalga Pyropia yezoensis (Rhodophyta).

Ammonium and nitrate are the primary nitrogen sources in natural environments, and are essential for growth and development in photosynthetic eukaryotes. In this study, we report on the isolation and characterization of an ammonium transporter gene (PyAMT1) which performs a key function in nitrogen (N) metabolism of Pyropia yezoensis thalli. The predicted length of PyAMT1 was 483 amino acids (AAs). The AA sequence included 11 putative transmembrane domains and showed approximately 33-44% identity to algal and plant AMT1 AA sequences. Functional complementation in an AMT-defective yeast mutant indicated that PyAMT1 mediated ammonium transport across the plasma membrane. Expression analysis showed that the PyAMT1 mRNA level was strongly induced by N-deficiency, and was more highly suppressed by resupply of inorganic-N than organic-N. These results suggest that PyAMT1 plays important roles in the ammonium transport system, and is highly regulated in response to external/internal N-status.

Noninvasive evaluation of heavy metal uptake and storage in micoralgae using a fluorescence resonance energy transfer-based heavy metal biosensor.

We have developed a fluorescence resonance energy transfer (FRET)-based heavy metal biosensor for the quantification of bioavailable free heavy metals in the cytoplasm of the microalga Chlamydomonas reinhardtii. The biosensor is composed of an end-to-end fusion of cyan fluorescent protein (CFP), chicken metallothionein II (MT-II), and yellow fluorescent protein (YFP). In vitro measurements of YFP/CFP fluorescence emission ratios indicated that the addition of metals to the purified biosensor enhanced FRET between CFP and YFP, consistent with heavy metal-induced folding of MT-II. A maximum YFP/CFP FRET ratio of 2.8 was observed in the presence of saturating concentrations of heavy metals. The sensitivity of the biosensor was greatest for Hg2+ followed by Cd2+≈Pb2+>Zn2+>Cu2+. The heavy metal biosensor was unresponsive to metals that do not bind to MT-II (Na+ and Mg2+). When expressed in C. reinhardtii, we observed a differential metal-dependent response to saturating external concentrations (1.6 mm) of heavy metals (Pb2+>Cd2+) that was unlike that observed for the isolated biosensor (in vitro). Significantly, analysis of metal uptake kinetics indicated that equilibration of the cytoplasm with externally applied heavy metals occurred within seconds. Our results also indicated that algae have substantial buffering capacity for free heavy metals in their cytosol, even at high external metal concentrations.

Isolation and characterization of a SEPALLATA-like gene, ZjMADS1, from marine angiosperm Zostera japonica.

In flowering plants, floral homeotic MADS-box genes, which constitute a large multigene family, play important roles in the specification of floral organs as defined by the ABCDE model. In this study, a MADS-box gene, ZjMADS1, was isolated and characterized from the marine angiosperm Zostera japonica. The predicted length of the ZjMADS1 protein was 246 amino acids (AA), and the AA sequence was most similar to those of the SEPALLATA (SEP) subfamily, corresponding to E-function genes. Southern blot analysis suggested the presence of two SEP3-like genes in the Z. japonica genome. ZjMADS1 mRNA levels were extremely high in the spadices, regardless of the developmental stage, compared to other organs from the reproductive and vegetative shoots. These results suggest that the ZjMADS1 gene may be involved in spadix development in Z. japonica and act as an E-function gene in floral organ development in marine angiosperms.

Isolation and characterization of a single-copy actin gene from a sterile mutant of Ulva pertusa (Ulvales, Chlorophyta).

We constructed a cDNA library from sterile Ulva pertusa (Ulvales, Chlorophyta), and isolated and characterized a full-length cDNA clone encoding actin. The actin (ACT) cDNA consisted of 1487 nucleotides (nt) and had an open reading frame (ORF) encoding a polypeptide of 377 amino acid (AA) residues. The ACT gene had one intron in the 5'-untranslated region and three introns in the coding region. Transcription started 26 nt downstream of the putative TATA box. A potential polyadenylation signal, TGTAG, was located 100 nt downstream of the terminator codon, TAG. Amino acid alignment with actins from various algae and land plants showed that sterile U. pertusa actin was more similar to actins from Chlorophyta, Phaeophyta, Euglenophyta, and higher plants (over 76.9%) than to actins from Rhodophyta. Southern blot analysis indicated that the sterile U. pertusa genome has only a single actin-encoding gene. Thalli grown on a 12D/12L photoperiod increased in surface area some two-fold over 24 h regardless of the nutritional conditions. The growth rate of thalli during the light period was significantly higher than that during the dark period. Northern hybridization indicated that the expression of actin mRNA was induced and repressed by the light and dark treatments, respectively. These results suggest that the U. pertusa cell division cycle has a periodicity of 24 h and that the ACT gene is highly transcribed during cell growth and development in the light period.