Molecular markers from different genomic compartments reveal cryptic diversity within glaucophyte species.

Glaucophytes are the least studied of the three major Archaeplastida (Plantae sensu lato) lineages. It has been largely recognized that comprehensive investigations of glaucophyte genetic and species diversity will shed light on the early evolution of photosynthetic eukaryotes. Here we used molecular phylogenetics and genetic distance estimations of diverse molecular markers to explore strain and species diversity within the glaucophyte genera Cyanophora and Glaucocystis. Single gene and concatenated maximum likelihood analyses of markers from three different genetic compartments consistently recovered similar intrageneric genetic groups. Distance analyses of plastid (psbA and rbcL) and mitochondrial (cob and cox1) genes, and the nuclear internal transcribed spacer (ITS) region, revealed substantial genetic divergence between some Cyanophora paradoxa and Glaucocystis nostochinearum strains. The genetic distances estimated between some glaucophyte strains currently considered the same species are similar or greater than divergence values calculated between different species in other unicellular algae, such as certain green algae and diatoms. The analyzed molecular markers are prospective candidates for future studies of species diversity in glaucophytes. Overall, our results unveil previously unrecognized cryptic diversity within Cyanophora and Glaucocystis species.

The dynamic surface of dividing cyanelles and ultrastructure of the region directly below the surface in Cyanophora paradoxa.

The cyanelles of glaucocystophytes are probably the most primitive of known extant plastids and the closest to cyanobacteria. Their kidney shape and FtsZ arc during the early stage of division define cyanelle division. In order to deepen and expand earlier results (Planta 227:177-187, 2007), cells of Cyanophora paradoxa were fixed with two different chemical and two different freeze-fixation methods. In addition, cyanelles from C. paradoxa were isolated to observe the surface structure of dividing cyanelles using field emission scanning electron microscopy (FE-SEM). A shallow furrow started on one side of the division plane. The furrow subsequently extended, covering the entire division circle, and then invaginated deeply, becoming clearly visible. The typical FtsZ arc was 2.3-3.4 microm long. This length matches that of the cleavage furrow observed using FE-SEM. The cyanelle cleavage furrows are from one-fourth to one-half of the circumference of the division plane. The shallow furrow that appears on the cyanelle outer surface effectively changes the division plane. Using freeze-fixation methods, the electron-dense stroma and peptidoglycan could be distinguished. In addition, an electron-dense belt structure (the cyanelle ring) was observed inside the leading edge at the cyanelle division plane. The FtsZ arc is located at the division plane ahead of the cyanelle ring. Immunogold-TEM localization shows that FtsZ is located interiorly of the cyanelle ring. The lack of an outer PD ring, together with the arch-shaped furrow, suggests that the mechanical force of the initial (arch shaped) septum furrow constriction comes from inside the cyanelle.

A carboxysomal carbon-concentrating mechanism in the cyanelles of the 'coelacanth' of the algal world, Cyanophora paradoxa?

Cyanelles are the peculiar plastids of glaucocystophyte algae that retained a peptidoglycan wall from the ancestral cyanobacterial endosymbiont. All cyanobacteria and most algae possess an inorganic carbon-concentrating mechanism (CCM) that involves a microcompartment--carboxysomes in prokaryotes and pyrenoids in eukaryotes--harboring the bulk of cellular (plastidic) Rubisco. In the case of the living fossil, Cyanophora paradoxa, the existence of a CCM was a matter of debate. Microarray data revealing 142 CO(2)-responsive genes (induced or repressed through a shift from high to low CO(2) conditions), gas exchange measurements and measurements of photosynthetic affinity provided strong support for a CCM. We favor a recent hypothesis that glaucocystophyte cyanelles as the closest cousins to cyanobacteria among plastids contain 'eukaryotic carboxysomes': bicarbonate enrichment within cyanelles should be considerably higher than in chloroplasts with their pyrenoid-based CCM. Thus, the stress-bearing function of the peptidoglycan layer, the other unique heritage, would be indispensable. An isolation method for cyanelle 'carboxysomes' was developed and the protein components other than Rubisco analyzed by MS. Rubisco activase was identified and corroborated by western blotting. The well-established cyanelle in vitro import system allows to use them as 'honorary cyanobacteria': assembly processes of supramolecular structures as phycobilisomes and carboxysomes thus can be studied after import of nucleus-encoded precursor proteins and subsequent fractionation. Even minor components can easily be tracked and a surprisingly dynamic view is obtained. Labeled pre-activase was imported into isolated cyanelles and 30% of the mature protein was found to be incorporated into the carboxysome fraction. A final decision between carboxysome or pyrenoid must await the identification of cyanelle carbonic anhydrase and, especially, the demonstration of shell proteins.

Acclimation to low [CO(2)] by an inorganic carbon-concentrating mechanism in Cyanophora paradoxa.

The glaucocystophyte Cyanophora paradoxa contains cyanelles, plastids with prokaroytic features such as a peptidoglycan wall and a central proteinaceous inclusion body. While this central body includes the majority of the enzyme ribulose 1,5-bisphosphate carboxylase/oxgenase Rubisco), the presence of a carbon-concentrating mechanism (CCM) in C. paradoxa has only been hypothesized. Here, we present physiological data in support of a CCM: CO(2) exchange activity as well as apparent affinity against inorganic carbon were found to increase under CO(2)-limiting stress. Further, expressed sequence tags (ESTs) of C. paradoxa were obtained from two cDNA libraries, one from cells grown in high [CO(2)] conditions and one from cells grown under low [CO(2)] conditions. A cDNA microarray platform assembled from 2378 cDNA sequences revealed that 142 genes significantly responded to a shift from high to low [CO(2)]. Trends in gene expression were comparable to those reported for Chlamydomonas reinhardtii and the cyanobacterium Synechocystis 6803, both possessing a CCM. Among genes regulated by [CO(2)], transcripts were identified encoding carbonic anhydrases (CAs), Rubisco activase and a putative bicarbonate transporter in C. paradoxa, likely functionally involved in the CCM. These results and the polyhedric appearance of the central body further support the hypothesis of a unique 'eukaryotic carboxysome' in Cyanophora.

Conserved relationship between FtsZ and peptidoglycan in the cyanelles of Cyanophora paradoxa similar to that in bacterial cell division.

Cyanelles of the biflagellate protist Cyanophora paradoxa have retained the peptidoglycan layer, which is critical for division, as indicated by the inhibitory effects of beta-lactam antibiotics. An FtsZ ring is formed at the division site during cyanelle division. We used immunofluorescence microscopy to observe the process of FtsZ ring formation, which is expected to lead cyanelle division, and demonstrated that an FtsZ arc and a split FtsZ ring emerge during the early and late stages of cyanelle division, respectively. We used an anti-FtsZ antibody to observe cyanelle FtsZ rings. We observed bright, ring-shaped fluorescence of FtsZ in cyanelles. Cyanelles were kidney-shaped shortly after division. Fluorescence indicated that FtsZ did not surround the division plane at an early stage of division, but rather formed an FtsZ arc localized at the constriction site. The constriction spread around the cyanelle, which gradually became dumbbell shaped. After the envelope's invagination, the ring split parallel to the cyanelle division plane without disappearing. Treatment of C. paradoxa cells with ampicillin, a beta-lactam antibiotic, resulted in spherical cyanelles with an FtsZ arc or ring on the division plane. Transmission electron microscopy of the ampicillin-treated cyanelle envelope membrane revealed that the surface was not smooth. Thus, the inhibition of peptidoglycan synthesis by ampicillin causes the inhibition of septum formation and a marked delay in constriction development. The formation of the FtsZ arc and FtsZ ring is the earliest sign of cyanelle division, followed by constriction and septum formation.

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa.

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in beta-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions.