A high-affinity phosphate transporter gene, TcPHO, was isolated from a growth-dependent subtracted cDNA library of the marine unicellular alga Tetraselmis chui. The full-length cDNA of TcPHO obtained by 5 and 3 rapid amplification of cDNA ends was 1,993 bp long and encoded an open reading frame consisting of 610 amino acids. The deduced amino acid sequence of TcPHO exhibited 51.6 and 49.8% similarity to the amino acid sequences of PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa, respectively. In addition, hydrophobicity and secondary structure analyses revealed 12 conserved transmembrane domains that were the same as those found in PHO89 and PHO4. The expression of TcPHO mRNA was dependent on phosphate availability. With a low-phosphate treatment, the TcPHO mRNA concentration increased sharply to 2.72 fmol g of total RNA؊1 from day 1 to day 2 and remained at this high level from days 2 to 4. Furthermore, rescue treatment with either phosphate or p-nitrophenyl phosphate effectively inhibited TcPHO mRNA expression. In contrast, TcPHO mRNA expression stayed at a low level (range, 0.25 to 0.28 fmol g of total RNA ؊1 ) under low-nitrate conditions. The expression pattern suggests that TcPHO can be used as a molecular probe for monitoring phosphorus stress in T. chui.

The present study using zebrafish as a model explores the role of isotocin, a homolog of oxytocin, in controlling ion regulatory mechanisms. Double-deionized water treatment for 24 h significantly stimulated isotocin mRNA expression in zebrafish embryos. Whole-body Cl−, Ca2+, and Na+ contents, mRNA expressions of ion transporters and ionocyte-differentiation related transcription factors, and the number of skin ionocytes decreased in isotocin morphants. In contrast, overexpression of isotocin caused an increase in ionocyte numbers. Isotocin morpholino caused significant suppression of foxi3a mRNA expression, while isotocin cRNA stimulated foxi3a mRNA expressions at the tail-bud stage of zebrafish embryos. The density of P63 (an epidermal stem cell marker)-positive cells was downregulated by isotocin morpholinos and was upregulated by isotocin cRNA. Taken together, isotocin stimulates the proliferation of epidermal stem cells and differentiation of ionocyte progenitors by regulating the P63 and Foxi3a transcription factors, consequently enhancing the functional activities of ionocytes.

A novel death-specific gene, ScDSP, was obtained from a death stage subtraction cDNA library of the diatom Skeletonema costatum. The full length of ScDSP cDNA was 921 bp in length, containing a 699-bp open reading frame encoding 232 amino acids and two stretches of 66 and 156 bp in the 5 and 3 untranslated regions, respectively. Analysis of the peptide structure revealed that ScDSP contained a signal peptide domain, a transmembrane domain, and a pair of EF-hand motifs. When S. costatum grew exponentially at a rate of 1.3 day ؊1 , the ScDSP mRNA level was at 2 mol ⅐ mole 18S rRNA ؊1 . In contrast, when the culture entered the death phase with a growth rate decreasing to 0.5 day ؊1 , ScDSP mRNA increased dramatically to 668 mol ⅐ mole 18S rRNA ؊1 , and a high degree of DNA fragmentation was simultaneously observed. Under the influence of a light-dark cycle, ScDSP expression in both exponential and stationary phases clearly showed a diel rhythm, but the daily mean mRNA level was significantly higher in the stationary phase. Our results suggest that ScDSP may play a role in the molecular mechanism of self-destructive autolysis in phytoplankton under stress.Events of mass cell loss usually occur after algal blooms. In addition to physical damage, such as sedimentation or herbivore grazing, the self-destructive lysis of stressed cells is also considered to be a main cause for the decline of phytoplankton blooms (6, 12). Brussaard et al. (6) first used esterase activity to demonstrate that the self-destructive lysis of cells was the major loss factor accounting for 75% of the decline of a Phaeocystis bloom. Dissolved organic material released by lysed phytoplankton is subsequently utilized by heterotrophic bacteria and enters the microbial loop to support regenerated production in marine ecosystems (1,3,6). Such massive autolysis of phytoplanktonic cells is usually triggered by external stress factors such as nutrient starvation (4), light limitation (4, 39), and pathogenic virus infection (15). Nevertheless, the molecular regulatory mechanisms involved in cell lysis have seldom been investigated in algal cells.Several genes are known to have a close association with self-destructive lysis in unicellular organisms. When the budding yeast Saccharomyces cerevisiae enters the stationary phase, a series of genes, including doa4, ctt1, ppn1, sod1, sod2, and so on, are expressed to maintain intracellular homeostasis, and a deficiency in any of these gene functions can cause the loss of viability (14, 16). Additionally, apoptosis-like death has also been observed in aging yeast cells. The cause of this event is considered to be facilitated by a novel metacaspase encoded by yca1 (19,30). In phytoplankton, increasing numbers of studies have pointed out that light limitation, nutrient starvation, or the accumulation of reactive oxygen species can induce an apoptosis-like syndrome, such as cell shrinkage, blebbing, chromatin condensation, and the formation of nuclear DNA fragmentation. Through biochemical and immunological assays...

Nitrate transporter genes (Nrt2) encode high-affinity nitrate transporters in marine phytoplankton, and their transcript levels are potential markers of nitrogen deficiency in eukaryotic phytoplankton. For the proper interpretation of measured Nrt2 transcript abundances, a relative expression assay was proposed and tested in Isochrysis galbana Parke (Prymnesiophyceae) and Thalassiosira pseudonana (Hust.) Hasle et Heimdal (Bacillariophyceae). The minimal transcript levels of Nrt2 genes were achieved by the addition of 100 μM ammonium, which led to a rapid decline in Nrt2 transcripts in 10-30 min. Experiments using a concentration series revealed that the effective dosage of ammonium to create a minimal transcript level of ∼1 μmol · mol(-1) 18S rRNA was ≥25 μM in both species. On the other hand, the addition of l-methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, enhanced the Nrt2 transcript level in I. galbana but did not affect that in T. pseudonana. Nitrogen deprivation was used as an alternative means to create maximal Nrt2 transcript levels. By transferring cells into N-free medium for 24 h, Nrt2 transcript levels increased to ∼90 μmol · mol(-1) 18S rRNA in I. galbana, and to ∼800 μmol · mol(-1) 18S rRNA in T. pseudonana. The degree of nitrogen deficiency thus can be determined by comparing original Nrt2 transcript levels with the minimal and maximal levels.