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Sequence comparison and expression analysis of an inferred Na+/Pi cotransporter gene in the marine diatom Skeletonema tropicum

  • Shr-Hau Hung , Yung-Hsiu Lu , Chih-Ching Chung

    Chih-Ching Chung is an associate professor at the Institute of Marine Environment and Ecology, National Taiwan Ocean University. He also serves as the director of the Center for Research Vessel Management. Chih-Ching is a marine molecular ecologist who studies the effects of environmental changes on the succession of marine picophytoplankton.

    , Chi-Yu Shih , Gwo-Ching Gong

    Gwo-Ching Gong is a life-time distinguished professor at the National Taiwan Ocean University. His research foci are chemical hydrography, nutrient dynamic, and primary productivity. He has led several integrated research projects, including Long-term Observation and Research of the East China Sea (LORECS) and Effect of Global Change on Ocean Biogeochemistry and Ecosystems in the seas surrounding Taiwan in the Northwest Pacific (ECOBEST). These projects aim to investigate the responses of marine ecosystems to the rapid climate change, ocean acidification, and increasing human disturbances.

    and Jeng Chang

    Jeng Chang is a professor at the Taiwan Ocean University. He studies diatom genes related to nitrogen and phosphorus uptake. He uses laboratory cultures to examine how the expression of a transporter gene is induced by a nutrient stress. Subsequently, expression of the same gene in a natural assemblage of diatoms becomes an indicator of physiological status that improves our understanding of ecological processes.

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From the journal Botanica Marina

Abstract

Unicellular algae have evolved to express many forms of high-affinity phosphate transporters, and homologs of these proteins are broadly distributed in yeast, fungi, higher plants, and vertebrates. In this report, an effort has been made to characterize such a transporter gene, StPHO, in the marine diatom Skeletonema tropicum. The primers used for polymerase chain reaction were designed by referring to a homologous gene in a prasinophyte, and the full-length (1692 bp) cDNA of StPHO was then cloned and sequenced. Sequence alignments and secondary structure prediction indicated that StPHO is a gene that encodes a type III Na+/Pi cotransporter (SLC20 family). To study the function of StPHO, specific concentrations of inorganic phosphate (Pi) were used to alter the physiological status of S. tropicum. In each treatment, samples were collected for the measurements of StPHO mRNA, [PO43−], cell abundance, the maximal photochemical efficiency of photosystem II (Fv/Fm), and alkaline phosphatase activity (APA). The results indicated that the ambient [PO43−] strongly affected the population growth and related physiological parameters of S. tropicum. The transcription of StPHO was fully repressed when the [PO43−] was greater than 1 μM but increased approximately 100-fold when the ambient [PO43−] decreased to 0.02 μM. Within this [PO43−] range, the regression equations are Y = −0.6644X + 0.9034 and Y = −0.5908X + 0.8054 for Pi-starved and Pi-limited treatments, respectively. This trend of gene expression suggested that StPHO plays an important role in the uptake of [PO43−], and StPHO may serve as a useful molecular biomarker for Pi-stressed diatom populations in marine ecosystems.


Corresponding author: Jeng Chang, Institute of Marine Biology, National Taiwan Ocean University, Keelung20224, Taiwan, ROC; Institute of Marine Environment and Ecology, National Taiwan Ocean University, Keelung20224, Taiwan, ROC; and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung20224, Taiwan, ROC, E-mail:

Award Identifier / Grant number: MOST 108-2811-M-019-505, MOST 109-2611-M-019-003, NSC 99-2313-B-019-002

About the authors

Chih-Ching Chung

Chih-Ching Chung is an associate professor at the Institute of Marine Environment and Ecology, National Taiwan Ocean University. He also serves as the director of the Center for Research Vessel Management. Chih-Ching is a marine molecular ecologist who studies the effects of environmental changes on the succession of marine picophytoplankton.

Gwo-Ching Gong

Gwo-Ching Gong is a life-time distinguished professor at the National Taiwan Ocean University. His research foci are chemical hydrography, nutrient dynamic, and primary productivity. He has led several integrated research projects, including Long-term Observation and Research of the East China Sea (LORECS) and Effect of Global Change on Ocean Biogeochemistry and Ecosystems in the seas surrounding Taiwan in the Northwest Pacific (ECOBEST). These projects aim to investigate the responses of marine ecosystems to the rapid climate change, ocean acidification, and increasing human disturbances.

Jeng Chang

Jeng Chang is a professor at the Taiwan Ocean University. He studies diatom genes related to nitrogen and phosphorus uptake. He uses laboratory cultures to examine how the expression of a transporter gene is induced by a nutrient stress. Subsequently, expression of the same gene in a natural assemblage of diatoms becomes an indicator of physiological status that improves our understanding of ecological processes.

Acknowledgments

We deeply appreciate Drs. L.-K. Kang, S.-P. L. Hwang, and P.-H. Hsu for technical instructions and helpful discussions. We would like to thank Dr. F.-K. Shiah for providing the FIRe fluorometer, C.-Y. Chen for assistance in measuring the phosphate concentrations, and T.-H. Kuo and X.-B. Chen for monitoring the bacterial abundance. We are grateful to the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, for assistance with DNA sequencing.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: This study was supported by the grants MOST 109-2611-M-019-003 and NSC 99-2313-B-019-002 from the Ministry of Science and Technology (Taiwan, ROC). CYS was supported through MOST 108-2811-M-019-505.

  3. Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

References

Armbrust, E.V. (2009). The life of diatoms in the world’s oceans. Nature 459: 185–192.10.1038/nature08057Search in Google Scholar PubMed

Chung, C.-C., Hwang, S.-P.L., and Chang, J. (2003). Identification of a high-affinity phosphate transporter gene in a prasinophyte alga, Tetraselmis chui, and its expression under nutrient limitation. Appl. Environ. Microbiol. 69: 754–759.10.1128/AEM.69.2.754-759.2003Search in Google Scholar PubMed PubMed Central

Chung, C.-C., Hwang, S.-P.L., and Chang, J. (2005). Cooccurrence of ScDSP gene expression, cell death, and DNA fragmentation in a marine diatom, Skeletonema costatum. Appl. Environ. Microbiol. 71: 8744–8751.10.1128/AEM.71.12.8744-8751.2005Search in Google Scholar PubMed PubMed Central

Cruz de Carvalho, M.H., Sun, H.-X., Bowler, C., and Chua, N.-H. (2016). Noncoding and coding transcriptome responses of a marine diatom to phosphate fluctuations. New Phytol. 210: 497–510.10.1111/nph.13787Search in Google Scholar PubMed

Daram, P., Brunner, S., Rausch, C., Steiner, C., Amrhein, N., and Bucher, M. (1999). Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis. Plant Cell 11: 2153–2166.10.1105/tpc.11.11.2153Search in Google Scholar PubMed PubMed Central

Dyhrman, S.T., Jenkin, B.D., Rynearson, T.A., Saito, M.A., Mercier, M.L., Alexander, H., Whitney, L.P., Drzewianowski, A., Bulygin, V.V., Bertrand, E.M., et al.. (2012). The transcriptome and proteome of the diatom Thalassiosira pseudonana reveal a diverse phosphorus stress response. PloS One 7: e33768.10.1371/journal.pone.0033768Search in Google Scholar PubMed PubMed Central

Falkowski, P.G. (2002). The ocean’s invisible forest. Sci. Am. 287: 54–61.10.1038/scientificamerican0802-54Search in Google Scholar PubMed

Falkowski, P.G., Barber, R. T., and Smetacek, V. (1998). Biogeochemical controls and feedbacks on ocean primary production. Science 281: 200–206.10.1126/science.281.5374.200Search in Google Scholar PubMed

Gallina, A.A., Chung, C.-C., and Casotti, R. (2015). Expression of death-related genes and reactive oxygen species production in Skeletonema tropicum upon exposure to the polyunsaturated aldehyde octadienal. Adv. Oceanogr. Limnol. 6: 13–20.10.4081/aiol.2015.5466Search in Google Scholar

Guillard, R.R.L. and Ryther, J.H. (1962). Studies of marine planktonic diatoms: I. Cyclotella nana hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8: 229–239.10.1139/m62-029Search in Google Scholar PubMed

Hediger, M.A., Romero, M.F., Peng, J.B., Rolfs, A., Takanaga, H., and Bruford, E.A. (2004). The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. Pflugers Arch. Eur. J. Physiol. 447: 465–468.10.1007/s00424-003-1192-ySearch in Google Scholar

Hung, S.-H., Chung, C.-C., Liao, C.-W., Gong, G.-C., and Chang, J. (2013). Sequence diversity and expression levels of Synechococcus phosphate transporter gene in the East China Sea. J. Exp. Mar. Biol. Ecol. 440: 90–99.10.1016/j.jembe.2012.11.018Search in Google Scholar

Hwang, S.-P.L., Wang, S.K., Wei, S.F., Cheng, L.-C., and Chang, J. (1999). Identification and expression pattern of DNA polymerase α gene in a marine diatom, Skeletonema costatum. Mar. Biotechnol. 1: 200–206.10.1007/PL00011768Search in Google Scholar

Kang, L.-K., Gong, G.-C., Wu, Y.-H., and Chang, J. (2015). The expression of nitrate transporter genes reveals different nitrogen statuses of dominant diatom groups in the southern East China Sea. Mol. Ecol. 24: 1374–1386.10.1111/mec.13109Search in Google Scholar

Kester, D.R., Duedall, I.W., Connors, D.N., and Pytkowicz, R.M. (1967). Preparation of artificial seawater. Limnol. Oceanogr. 12: 176–179.10.4319/lo.1967.12.1.0176Search in Google Scholar

Kumar, S., Stecher, G., Li, M., Knyaz, C., and Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35: 1547–1549.10.1093/molbev/msy096Search in Google Scholar

La Roche, J., McKay, R.M.L., and Boyd, P. (1999). Immunological and molecular probes to detect phytoplankton responses to environmental stress in nature. Hydrobiologia 401: 177–198.10.1007/978-94-011-4201-4_13Search in Google Scholar

Lu, Y.-H. (2004). Performance comparisons between polymerase chain reaction and dot blotting in differentiating phytoplankton genes from distinct species, Master’s thesis. Keelung, Taiwan, National Taiwan Ocean University.Search in Google Scholar

Martinez, P. and Persson, B.L. (1998). Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae. Mol. Gen. Genet. 258: 628–638.10.1007/s004380050776Search in Google Scholar

Młodzińska, E. and Zboińska, M. (2016). Phosphate uptake and allocation – a closer look at Arabidopsis thaliana L. and Oryza sativa L. Front. Plant Sci. 7: 1198.10.3389/fpls.2016.01198Search in Google Scholar

Pai, S.C., Yang, C.C., and Riley, J.P. (1990). Effects of acidity and molybdate concentration on the kinetics of the formation of the phosphoantimonylmolybdenum blue complex. Anal. Chim. Acta 229: 115–120.10.1016/S0003-2670(00)85116-8Search in Google Scholar

Parkhill, J.P., Maillet, G., and Cullen, J.J. (2001). Fluorescence-based maximal quantum yield for PSII as a diagnostic of nutrient stress. J. Phycol. 37: 517–529.10.1046/j.1529-8817.2001.037004517.xSearch in Google Scholar

Pedersen, B.P., Kumar, H., Waight, A.B., Risenmay, A.J., Roe-Zurz, Z., Chau, B.H., Schlessinger, A., Bonomi, M., Harries, W., Sali, A., et al.. (2013). Crystal structure of a eukaryotic phosphate transporter. Nature 496: 533–536.10.1038/nature12042Search in Google Scholar

Perry, M.J. (1976). Phosphate utilization by an oceanic diatom in phosphorus‐limited chemostat culture and in the oligotrophic waters of the central North Pacific. Limnol. Oceanogr. 21: 88–107.10.4319/lo.1976.21.1.0088Search in Google Scholar

Persson, B.L., Berhe, A., Fristedt, U., Martinez, P., Pattison, J., Petersson, J., and Weinander, R. (1998). Phosphate permeases of Saccharomyces cerevisiae. Biochim. Biophys. Acta 1365: 23–30.10.1016/S0005-2728(98)00037-1Search in Google Scholar

Rychter, A.M. and Rao, I.M. (2005). Role of phosphorus in photosynthetic carbon metabolism. In: Pessarakli, M. (Ed.). Handbook of photosynthesis. Taylor and Francis, Boca Raton, pp. 123–148.10.1201/9781420027877.ch7Search in Google Scholar

Scanlan, D.J., Mann, N.H., and Carr, N.G. (1993). The response of the picoplanktonic marine cyanobacterium Synechococcus species WH7803 to phosphate starvation involves a protein homologous to the periplasmic phosphate‐binding protein of Escherichia coli. Mol. Microbiol. 10: 181–191.10.1111/j.1365-2958.1993.tb00914.xSearch in Google Scholar PubMed

Shih, C.-Y., Kang, L.-K., and Chang, J. (2015). Transcriptional responses to phosphorus stress in the marine diatom, Chaetoceros affinis, reveal characteristic genes and expression patterns in phosphorus uptake and intracellular recycling. J. Exp. Mar. Biol. Ecol. 470: 43–54.10.1016/j.jembe.2015.05.001Search in Google Scholar

Theodorou, M.E. and Plaxton, W.C. (1993). Metabolic adaptations of plant respiration to nutritional phosphate deprivation. Plant Physiol. 101: 339–344.10.1104/pp.101.2.339Search in Google Scholar PubMed PubMed Central

Van Mooy, B.A.S., Fredricks, H.F., Pedler, B.E., Dyhrman, S.T., Karl, D.M., Koblížek, M., Lomas, M.W., Mincer, T.J., Moore, L.R., Moutin, T., et al.. (2009). Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity. Nature 458: 69–72.10.1038/nature07659Search in Google Scholar PubMed

Versaw, W.K. and Harrison, M.J. (2002). A chloroplast phosphate transporter, PHT2;1, influences allocation of phosphate within the plant and phosphate-starvation responses. Plant Cell 14: 1751–1766.10.1105/tpc.002220Search in Google Scholar PubMed PubMed Central

Virkki, L. V., Biber, J., Murer, H., and Forster, I.C. (2007). Phosphate transporters: a tale of two solute carrier families. Am. J. Physiol. Ren. Physiol. 293: 643–654.10.1152/ajprenal.00228.2007Search in Google Scholar

Wawrik, B., Paul, J.H., and Tabita, F.R. (2002). Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes. Appl. Environ. Microbiol. 68: 3771–3779.10.1128/AEM.68.8.3771-3779.2002Search in Google Scholar

Xu, H., Inouye, M., Missey, T., Collins, J.F., and Ghishan, F.K. (2002). Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes. Biochim. Biophys. Acta 1567: 97–105.10.1016/S0005-2736(02)00604-1Search in Google Scholar

Zhang, S.-F., Yuan, C.-J., Chen, Y., Chen, X.-H., Li, D.-X., Liu, J.-L., Lin, L., and Wang, D.-Z. (2016). Comparative transcriptomic analysis reveals novel insights into the adaptive response of Skeletonema costatum to changing ambient phosphorus. Front. Microbiol. 7: 1476.10.3389/fmicb.2016.01476Search in Google Scholar PubMed PubMed Central


Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/bot-2020-0037).


Received: 2020-05-29
Accepted: 2020-12-11
Published Online: 2021-01-06
Published in Print: 2021-02-23

© 2020 Walter de Gruyter GmbH, Berlin/Boston

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