Phycobiliprotein fluorescence of Nostoc punctiforme changes during the life cycle and chromatic adaptation: characterization by spectral confocal laser scanning microscopy and spectral unmixing

Abstract

Many cyanobacteria are highly adaptable to light quality, and many species undergo a complex life cycle. In this study we show that adaptive changes in the photosynthetic apparatus of cyanobacteria are not only caused by environmental, but also by developmental factors. Spectral confocal laser scanning microscopy (CLSM) was used to analyse in vivo the fluorescence spectra of the photosynthetic pigments chlorophyll a (Chl a ), allophycocyanin (APC), phycocyanin (PC) and phycoerythrin (PE) of two Nostoc punctiforme strains. Changes in pigment fluorescence emission occurred in different developmental stages. Strain 1:1-26 showed an emission maximum at 674 nm in motile hormogonia stages, whereas vegetative stages showed maxima at 658 and 575 nm. These changes were not caused by chromatic adaptation. In contrast, the second strain (1:1-26lg) showed distinct fluorescence spectra, pigment localization and clear chromatic adaptation in red light. When these properties are known, both strains can be easily distinguished by the spectral CLSM method, which also allows the localization of the pigments within single cells. To calculate the contribution of individual phycobiliproteins to the observed changes, fluorescence spectra were analysed by spectral unmixing. This allowed the mathematical estimation of fluorescence shares for the individual phycobiliproteins in different developmental stages and both before and after chromatic adaptation. It is concluded that care should be taken when characterizing cyanobacteria by differences in pigment fluorescence, because these differences are influenced not only by chromatic adaptation, but also developmental stages. Spectral CLSM offers a powerful method to study the phycobiliprotein composition in vivo .

Polyphasic approach and adaptative strategies of Nostoc cf. commune (Nostocales, Nostocaceae) growing on Mayan monuments.

“…The type and spatial localization of pigments can then be correlated to the cell type (Roldán et al 2004a). Lambda scans of vegetative cells correlated well with published spectra of extracted pigments (ong & glazeR 1988) and in vivo pigments, using similar tools and conditions (Roldán et al 2004b;WolF & scHüssleR 2005). The emission peaks and the shape of the spectra varied slightly with sample state due to environmental conditions.…”

“…Stages in the life cycle of the genus Nostoc depend on environmental conditions (Kantz & Bold 1969;mollenHaueR 1988;KomáReK & anagnostidis 1989;mollenHaueR et al 1994;dodds et al 1995;Potts 2000;WolF & scHüssleR 2005). Changes in in morphology, cell structure (Roldán et al 2006), and external sheaths or deposits are coupled to physiological changes.…”

“…In these samples, the fluorescence peaks at 645 nm and 654 nm, always present, were assigned to PC and APC, respectively (KeRänen et al 1999). Furthermore, chlorophyll a was clearly seen at the emission wavelengths of 678 nm and 686 nm (KeRänen et al 1999;WolF & scHüssleR 2005;KumaR & muRtHy 2007). In the field material, the emission spectra of both PC and APC also have a shoulder above 720 nm (KeRänen et al 1999).…”

“…Responses to drying and rewetting include: changes in the glycan layer, which protects and provides a repository for water (Hill et al 1997;Potts 2000;tamaRu et al 2005); induction of photoprotective pigments, carotenoids and external ultraviolet light (UV) (gaRcia-PicHel & castenHolz 1991;eHlingscHulz et al 1997); accumulation of non-reducing dissacharides, such as trehalosa, to stabilize drying cells (Billi & Potts 2002;saKamoto et al 2009); preservation of cell fine structure and cellular integrity during desiccation and upon rehydration (Peat & Potts 1987); and deactivation of PSII with diminution of photosynthesis and loss of PSI activity and dissipation of light energy absorbed by pigment-protein complexes (aRáoz & HädeR 1999;satoH et al 2002;HiRai et al 2004;FuKuda et al 2008). As light-harvesting pigments, cyanobacteria contain chlorophyll a, carotenoids and phycobiliproteins: phycoerythrin (PE, λ em ~ 575 nm), phycocyanin (PC, λ em ~ 645 nm) and allophycocyanin (APC, λ em ~ 658 nm) (KeRänen et al 1999, WolF & scHüssleR, 2005. The energy transfer between these pigments can be influenced by environmental factors such as temperature and light intensity (Wen et al 2005;KumaR & muRtHy 2007).…”

Transformation of Thylakoid Membranes during Differentiation from Vegetative Cell into Heterocyst Visualized by Microscopic Spectral Imaging

“…Spectral microscopy is an ideal tool to analyze physiological state and/or amounts of pigment-protein complexes under various conditions. Acquiring microscopic fluorescence spectra of individual cells is a natural extension of laser scanning confocal fluorescence microscopy, which has been applied to several types of cyanobacterial cells, including heterocysts (Peterson et al, 1981;Ying et al, 2002;Wolf and Schüssler, 2005;Kumazaki et al, 2007;Vermaas et al, 2008;Sukenik et al, 2009;Bordowitz and Montgomery, 2010;Collins et al, 2012, Sugiura andItoh, 2012). Microscopic fluorescence spectra reflect the concentration of pigment-protein complexes and the energy transfer dynamics between photosynthetic pigments.…”