Pigment orientation changes detected by low temperature linear dichroism spectroscopy: cold-hardening transition in phycobilisome-containing organisms

Low temperature (77K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. pee 6301, and cold-hardening in winter rye (Secale cereale L. cv. Puma). Samples were oriented for spectroscopy using the gel squeezing method (Abdourakhmanov et aI., 1979) and brought to 77K in liquid nitrogen. The linear dichroism (LD) spectra of Synechococcus 6301 phycobilisome/thylakoid membrane fragments cross-linked in light state 1 and light state 2 with glutaraldehyde showed differences in both chlorophyll a and phycobilin orientation. A decrease in the relative amplitude of the 681nm chlorophyll a positive LD peak was observed in membrane fragments in state 2. Reorientation of the phycobilisome (PBS) during the transition to state 2 resulted in an increase in core allophycocyanin absorption parallel to the membrane, and a decrease in rod phycocyanin parallel absorption. This result supports the "spillover" and "PBS detachment" models of the light state transition in PBS-containing organisms, but not the "mobile PBS" model. A model was proposed for PBS reorientation upon transition to state 2, consisting of a tilt in the antenna complex with respect to the membrane plane. Linear dichroism spectra of PBS/thylakoid fragments from the red alga, Porphyridium cruentum, grown in green light (containing relatively more PSI) and red light (containing relatively more PSll) were compared to identify chlorophyll a absorption bands associated with each photosystem. Spectra from red light - grown samples had a larger positive LD signal on the short wavelength side of the 686nm chlorophyll a peak than those from green light - grown fragments. These results support the identification of the difference in linear dichroism seen at 681nm in Synechococcus spectra as a reorientation of PSll chromophores. Linear dichroism spectra were taken of thylakoid membranes isolated from winter rye grown at 20°C (non-hardened) and 5°C (cold-hardened). Differences were seen in the orientation of chlorophyll b relative to chlorophyll a. An increase in parallel absorption was identified at the long-wavelength chlorophyll a absorption peak, along with a decrease in parallel absorption from chlorophyll b chromophores. The same changes in relative pigment orientation were seen in the LD of isolated hardened and non-hardened light-harvesting antenna complexes (LHCII). It was concluded that orientational differences in LHCII pigments were responsible for thylakoid LD differences. Changes in pigment orientation, along with differences observed in long-wavelength absorption and in the overall magnitude of LD in hardened and non-hardened complexes, could be explained by the higher LHCII monomer:oligomer ratio in hardened rye (Huner et ai., 1987) if differences in this ratio affect differential light scattering properties, or fluctuation of chromophore orientation in the isolated LHCII sample.

Kr-Ar laser Raman spectrometer for low temperature measurements /

A Czerny Mount double monochromator is used to measure Raman scattered radiation near 90" from a crystalline, Silicon sample. Incident light is provided by a mixed gas Kr-Ar laser, operating at 5145 A. The double monochromator is calibrated to true wavelength by comparison of Kr and Ar emission Une positions (A) to grating position (A) display [1]. The relationship was found to be hnear and can be described by, y = 1.219873a; - 1209.32, (1) where y is true wavelength (A) and xis grating position display (A). The Raman emission spectra are collected via C"*""*" encoded software, which displays a mV signal from a Photodetector and allows stepping control of the gratings via an A/D interface. [2] The software collection parameters, detector temperature and optics are optimised to yield the best quality spectra. The inclusion of a cryostat allows for temperatmre dependent capabihty ranging from 4 K to w 350 K. Silicon Stokes temperatm-e dependent Raman spectra, generally show agreement with Uterature results [3] in their frequency haxdening, FWHM reduction and intensity increase as temperature is reduced. Tests reveal that a re-alignment of the double monochromator is necessary before spectral resolution can approach literature standard. This has not yet been carried out due to time constraints.