Partial separation of pigments into an acetone extract for Chl and carotenoids and a phosphate buffered saline (PBS) extract for phycobiliproteins, combined with narrower absorption peaks in solution, allowed for a quantitative analysis of Chl and carotenoids. Moreover, the PBS extract enabled better assessment of relative changes between phycobiliproteins due to the removal of overlapping absorption of Chl and carotenoids.
The acetone extracts revealed a slow decrease of the Chl/carotenoid ratio until one day before the end of the experiments, and a steep decrease of this ratio on the last day of the experiment (Fig. 4b). The most dramatic change induced by Fe-limitation, however, was the appearance of high concentrations of keto-carotenoids (mainly echinenone), which were only a very minor component in Fe-replete cultures (Fig. 4b).
Since no direct normalisation per cell is possible for extracts, we used the observation from the in vivo spectra (see above) showing constant phycoerythrin content throughout the experiment, and normalised the PBS extract spectra to this peak. Spectra of PBS extracts differed from the single-cell in vivo spectra mainly in two ways. First, while in the in vivo spectra phycourobilin increased only slightly (Fig. 4a), in the PBS extracts a peak at about 504 nm, close to the normal absorption of phycourobilin (497 nm), dominated the spectra 18 and 20 days after the start of iron limitation (Fig. 4c). Additionally, the PBS extract spectra showed that the phycocyanin (peak at 620 nm) decreased under iron limitation (Fig. 4c).
Phycocyanin could not be analysed in the single-cell absorption spectra due to overlapping of the phycocyanin peak with the side peak of the red absorbance band (Qy band) of Chl a.
Fig. 4. Effect of iron limitation on pigment composition. a) Single-cell in vivo absorption spectroscopy of Trichodesmium filaments. Each graph represents the average of all single-cell spectra measured on all five time points of the daily activity cycle, and the average of both replicate experiments. For the absorption spectrum of the Fe-replete cells, additionally all days of the measurement were averaged. Background was corrected only by setting OD750 to zero. No normalization was done. b) Analysis of Chl and carotenoid composition in acetone extracts. Averages and standard errors of all samples measured between 12 and 20 days after start of iron limitation, altogether six samples from two independent experiments for each data point shown. Values for “chlorophylls” are the sum of Chl a and all Chl a degradation products (chlorophyllide a, pheophytin a, and oxidation products). c) Changes in phycobilisome composition of Trichodesmium under iron limitation analysed by absorption spectra of extracts in phosphate buffered saline. Each spectrum shown represents the average of 5 or 10 samples taken at the 5 different time points of the daily cycle. Since the single cell in vivo spectra, where artefacts of extraction can be excluded, showed constant phycoerythrin per cell (see Fig. 4a), the spectra shown here are normalised to the phycoerythrin peak of the Fe-replete cultures.
Iron limitation in Trichodesmium
Western blot analysis of protein expression
In the Fe-replete cultures dinitrogenase reductase NifH was consistently abundant for 19 days of sampling with the highest expression level assayed at early afternoons (Fig. 5a). Strongly reduced levels of NifH were found in samples collected in the mornings and evenings, which is consistent with the diurnal rhythms reported for this protein earlier (Church et al., 2005). In contrast, iron limitation led to a very rapid decline of NifH levels, corresponding with declining nitrogenase activity (compare Figs. 2 and 5). Approximately 60% lower NifH level was measured already at the 9th day in Fe-limited culture collected at the early afternoon as compared with the Fe-replete controls (Fig. 5a and b). Only traces of this protein were detected in cultures exposed to prolonged iron limitation. Yet, in the Fe-limited cultures, both NifH expression and in vivo nitrogenase activity partially recovered at the end of the experiments. Western blots with antibodies against the subunit C of PS I (PsaC, Fig. 5a) and the D1 protein of PS II (not shown), both containing iron as a cofactor, showed no measurable change in the amount of these proteins under iron limitation stress (Fig. 5a and b). The band of PsaC is at about 47 kDa, indicating that in Trichodesmium this protein forms covalently linked trimers as they are known also from many other organisms. In accordance with the in vivo absorption spectra (Fig. 4a), phycoerythrin expression remained constant until about 12 days after the start of iron limitation. On the following eight days of the experiments, however, the amount of regular (16 kDa) phycoerythrin decreased and an additional, larger (20 kDa) band reacting with the phycoerythrin antibody appeared (Fig. 5a and b). On the last day of the experiments, only the 20 kDa band was present in immunoblots with phycoerythrin antibody (Fig. 5a and b), while the single cell in vivo spectra (Fig. 4a) still showed the normal phycoerythrin absorption peak, and a phycourobilin-like peak was found in the PBS extract spectra (Fig. 4c).
Fig. 5. Western blot analysis of protein expression under iron limitation stress in Trichodesmium. a) Western blot analysis using antibodies directed against dinitrogenase reductase (NifH), subunit C of PSI (PsaC) and phycoerythrin (PE). Equal protein was loaded per lane (6 µg total protein). Samples were taken daily just before the onset of the light period (M, morning), in the middle of the light period (A, afternoon), and directly after the end of the light period (E, evening). The days are separated by vertical lines on the blot. The evening sample of 8th day was lost. The asterisk marks the 20 kDa protein band that accumulated during iron limitation and reacted with phycoerythrin antibody. b) Quantification of Western blot signals shown in (a). The maximal value of control and Fe-limited samples was set as 100%. Although the chemistry behind any detection on Western blots is not a linear function of protein abundance, the results of background-corrected densitometric scanning shown here clearly do provide an indication of increase/decrease trends.
Biophysics of photosynthesis