Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of photosynthesis and nitrogen fixation.

* As iron (Fe) deficiency is a main limiting factor of ocean productivity, its effects were investigated on interactions between photosynthesis and nitrogen fixation in the marine nonheterocystous diazotrophic cyanobacterium Trichodesmium IMS101. * Biophysical methods such as fluorescence kinetic microscopy, fast repetition rate (FRR) fluorimetry, and in vivo and in vitro spectroscopy of pigment composition were used, and nitrogenase activity and the abundance of key proteins were measured. * Fe limitation caused a fast down-regulation of nitrogenase activity and protein levels. By contrast, the abundance of Fe-requiring photosystem I (PSI) components remained constant. Total levels of phycobiliproteins remained unchanged according to single-cell in vivo spectra. However, the regular 16-kDa phycoerythrin band decreased and finally disappeared 16-20 d after initiation of Fe limitation, concomitant with the accumulation of a 20-kDa protein cross-reacting with the phycoerythrin antibody. Concurrently, nitrogenase expression and activity increased. Fe limitation dampened the daily cycle of photosystem II (PSII) activity characteristic of diazotrophic Trichodesmium cells. Further, it increased the number and prolonged the time period of occurrence of cells with elevated basic fluorescence (F(0)). Additionally, it increased the effective cross-section of PSII, probably as a result of enhanced coupling of phycobilisomes to PSII, and led to up-regulation of the Fe stress protein IsiA. * Trichodesmium survives short-term Fe limitation by selectively down-regulating nitrogen fixation while maintaining but re-arranging the photosynthetic apparatus.

Fast, sensitive, and inexpensive alternative to analytical pigment HPLC: quantification of chlorophylls and carotenoids in crude extracts by fitting with Gauss peak spectra.

Quantification of pigments in complex mixtures is an important task in the physiology of photosynthetic organisms, because pigment composition differs depending on the species, tissue, and physiological state. Currently available methods, however, are either limited to very few pigments (classical UV/vis spectroscopic methods), or they are time-consuming, labor intensive, or costly (e.g., HPLC). Here we describe a UV/vis spectrophotometric method that is capable of a rapid (approximately 1 min/sample) and inexpensive (<1 euro/sample) quantification of more than a dozen pigments in a crude extract, which means it is suitable for high-throughput screening applications. A detection limit of <1 pmol for each pigment allows for determining the pigment composition in only 0.5 microg of lyophilized leaves or algae. The method is based on the description of each pigment spectrum by a series of Gaussian peaks. A sample spectrum is then fitted by a linear combination of these "Gauss peak spectra" including an automatic correction of wavelength inaccuracy, baseline instability, sample turbidity, and effects of temperature/water content. Here we present the Gauss peak spectra from 350 to 750 nm for acetone solutions of all chlorophyll and carotenoid derivatives that are abundant (including conditions of Cd, Cu, or Zn stress) in leaves of higher plants, Euglena, brown algae, and various cyanobacteria like Anabaena and Trichodesmium: [Mg]-Chl a, b, c1, c2; pheophytin a, b; [Cd]-Chl a, b; [Cu]-Chl a, b; [Zn]-Chl a, b; antheraxanthin, aurochrome, beta-carotene, beta-cryptoxanthin, cis- and trans-canthaxanthin, diadinochrome (=diadinoxanthin 5,6-epoxide), cis- and trans-diadinoxanthin, diatoxanthin, cis- and trans-echinenone, fucoxanthin, lutein, myxoxanthophyll, neoxanthin, violaxanthin, and all three stereoisomers of zeaxanthin in acetone. We present extensive tests of our new quantification method for determining optimal and limiting conditions of its performance and for comparison with previous methods. Finally, we show application examples for Thlaspi fendleri (Chlorophyta), Euglena gracilisc (Euglenophyta), Ectocarpus siliculosus (Phaeophyta), and Trichodesmium erythraeum IMS101 (cyanobacteria).