cDNA Microarrays as a Tool for Identification of Biomineralization Proteins in the CoccolithophoridEmiliania huxleyi(Haptophyta)

Abstract

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up-or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase ( Coccolithophores are a diverse group of calcifying unicellular marine algae, which includes the extremely abundant species Emiliania huxleyi. This group of phytoplankton has captured the attention of scientists from diverse disciplines, particularly because of the important roles it plays in the cycling of carbon and sulfur in the marine environment. The fixation of CO 2 via photosynthesis and the production of CO 2 in the calcification process affect global carbon cycling and thus link coccolithophores with climate and the chemical balance between atmosphere, hydrosphere, and geosphere (20). Furthermore, the striking morphological diversity of the more than 200 species of extant coccolithophores (13) and the seemingly endless range of fossil coccolith types (40) provide a unique resource with which to study biodiversity and speciation in the marine environment (23, 24). The biomineralized skeletons or "coccoliths" (Fig. 1A) produced by these microscopic organisms have also captured the attention of materials scientists interested in the microscale and nanoscale fabrication of three-dimensional biomineralized structures for novel applications related to telecommunications, optoelectronics, and medicine (31, 37).Although hypotheses continue to be debated in the literature, the function of the calcite skeletons and the role of biomineralization in coccolithophores remain unresolved. Knowledge of the mechanisms of calcification in coccolithophores is equally poor. Coccolithogenesis takes place within the coccolith vesicle, a specialized apparatus derived from the Golgi body, in a highly regulated and reproducible manner. The molecules involved in the acquisition and transport of calcium ions and inorganic carbon within the cell and those involved in the precipitation o...

Light-Dependent Transcriptional Regulation of Genes of Biogeochemical Interest in the Diploid and Haploid Life Cycle Stages of Emiliania huxleyi

“…Concerning gpa, the upregulation observed in calcifying cells contrasts with the downregulation reported by Quinn et al (21). It must be pointed out, however, that in that study, calcifying cells were obtained by inducing a phosphorus limitation.…”

“…In this paper, we take advantage of the fact that the haplodiploid life cycle of E. huxleyi involves an alternation between calcified, coccolithbearing diploid cells and organic scale-bearing motile haploid cells, each of which can exist independently of the other and reproduce vegetatively (8). This approach has the considerable advantage of maintaining the two life stages of E. huxleyi under identical culture conditions.While the large subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase), rbcL, is well known for its central role in photosynthetic carbon fixation and has already been shown in a pilot field experiment to relate successfully to photosynthesis measurements (1), research aimed at identifying key genes and proteins involved in calcification is still in its infancy.In the last few years, several novel molecular tools have been used to investigate calcification and coccolithogenesis (4,6,17,21,27). Results from these studies of coccolithophores as well as those carried out with other calcifying organisms were used to select genes.…”

“…In the last few years, several novel molecular tools have been used to investigate calcification and coccolithogenesis (4,6,17,21,27). Results from these studies of coccolithophores as well as those carried out with other calcifying organisms were used to select genes.…”

“…However, this area can now be intensively investigated thanks to the ever-growing number of analyses of expressed sequence tags (6,10,21,27) and the whole-genomeannotation effort (Joint Genome Institute).…”

Ocean acidification alters skeletogenesis and gene expression in larval sea urchins

“…Cossins & Crawford 2005, Gracey 2007) as well as to gauge response to changing environmental conditions (e.g. Place et al 2008), including the identification of genes involved in biomineralization (Quinn et al 2006).…”