Evolution and functional diversification of fructose bisphosphate aldolase genes in photosynthetic marine diatoms

Abstract

Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with β-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.

FIG. 1.

Localization of plastid FBA-EYFP fusion proteins. (A) FBAC1-EYFP, (B) FBAC2-EYFP, (C) FBAC5-EYFP. On the left light microscopy image, chlorophyll autofluorescence is shown in red and YFP fluorescence in green. Bars, 2 μm.

FIG. 2.

Colocalization of plastid FBA-YFP with CA1-CFP. (A) FBAC1-EYFP + CA1-CFP, (B) FBAC5-EYFP + CA1-CFP. Chlorophyll autofluorescence is shown in red, YFP fluorescence in green, and CFP fluorescence in blue. Left image: CFP fluorescence, center: YFP fluorescence, right image: combined fluorescence. Bars, 2 μm.

FIG. 3.

Localization of Phaeodactylum tricornutum FBAC5 by transmission electron microscopy. A clone expressing the FBAC5_EYFP fusions was fixed, and the intracellular EYFP was immunolocalized by means of an anti-GFP antibody. The antibody bound strongly to an intraplastidial structure in FBAC5_EYFP transformant cells. Chloroplasts (C) and pyrenoids (Pyr) and gold particles detected with electron microscopy are indicated. Unlabeled cells did not show any labeling (data not shown).

FIG. 4.

Protein maximum likelihood phylogeny of class I FBA. Numbers at the nodes indicate approximate likelihood-ratio test support values.

FIG. 5.

Protein maximum likelihood phylogeny of class II A FBA. Numbers at the nodes indicate approximate likelihood-ratio test support values.

FIG. 6.

Localization of FBA3-EYFP fusion proteins. On the left light microscopical image center: YFP fluorescence in yellow and chlorophyll autofluorescence is shown in red. YFP fluorescence is shown in green on the right in order to more easily distinguish the chloroplast. Bars, 2 μm.

FIG. 7.

Localization of FBA4. (A) FBA4-EYFP fusion; YFP fluorescence is shown in yellow. (B) EYFP-FBA4 fusion; YFP fluorescence is shown in green, chlorophyll autofluorescence in red. Bars, 2 μm.

FIG. 8.

Protein maximum likelihood phylogeny of class I bacterial-like cytosolic FBA. Numbers at the nodes indicate approximate likelihood-ratio test support values.

FIG. 9.

Expression of Phaeodactylum tricornutum FBA genes according to their normalized frequency of observation in a range of cDNA libraries representing different environmental conditions (Maheswari et al. 2010). Normalized expression values were obtained by dividing the number of FBA ESTs in the different libraries by the total number of sequences generated for the different libraries. The R value was defined in Stekel et al. (2000), an R value above 12 indicates nonspurious differences in the EST frequency across libraries (i.e., differential expression). The features of the 15 libraries have been published elsewhere (Allen et al. 2008; Sapriel et al. 2009; Maheswari et al. 2010). Si− = f/2 with artificial seawater, no Si added; Si+ = 350 μM metasilicate; Oval = Oval strain CCAP1052/1B; Triradiate = Triradiate strain NEPCC640; Blue light = Cells grown in the dark for 48 h and subjected to blue light for 1 h; Tropical = Tropical strain CCMP633 grown at a suboptimal temperature of 18 °C; N replete = 1.12 mM chemostat culture; NO3 starved = 50 μM in chemostat culture for 12 days; Urea adapted = 50 μM Urea; Fe deplete = 5 nM Iron; Low DD = 0.5 μg/ml 2E,4E-decadienal for 6 h; High DD = 5 μg/ml 2E,4E-decadienal for 6 h (for a full description of culture conditions, see Maheswari et al. 2010).

FIG. 10.

FBA activity in Phaeodactylum tricornutum (UTEX646) cells grown for 5 days in Fe deplete and replete medium. Enzymatic activity is given as mU/mg total cellular soluble protein. The measurements were performed in either presence or absence of EDTA to measure class I FBA activity or total FBA activity, respectively.