Jo-Ruth Graham

ON SPECTRAL CONTROL OF PIGMENTATION IN ANACYSTIS NIDULANS (CYANOPHYCEAE)1,2

Growth of Anacystis nidulans (Richt.) Drouet & Daily in wavelengths of light predominantly absorbed by chlorophyll a causes a dramatic lowering in chlorophyll content and in the chlorophylllphycocyanin ratio. A limit to the effect is reached in far red (680 nm) light where the ratio chlorophylllphycobilinogen falls to <0.3. A special water bath for culture tubes was illuminated by tungsten‐halogen lamps through far red (>650 nm)filters; it gave the same extreme pigment ratio together with a high specific growth rate of 2.5 day‐1. Compared with normally pigmented cells there are three features which accompany the low‐chlorophyll condition of far red light. At 677 nm relative quantum efficiency increases but action decreases. Synthesis of total pigment and total cell material in far red light also decreases. These observations suggest that the low chlorophyll response to far red light reflects an incompetence rather than an adaptation.

Phycobilisome composition and possible relationship to reaction centers

The photosynthetic apparatus was studied in Anacystis nidulans wild type and in a spontaneous pigment mutant 85Y which had improved growth in far-red light (greater than 650 nm). Two phycobiliproteins, C-phycocyanin (lambda max 625) and allophycocyanin (lambda max 650), were present in a molar ratio of approximately 3:1 in the wild type and approximately 0.4:1 in the mutant. Phycobilisomes of wild type cells were larger (57 X 30 nm) than those of the mutant 85Y (28 X 15 nm). In the mutant they seemed to consist primarily of the allophycocyanin core. Fluorescence emission maxima of wild type and mutant 85Y phycobilisomes were at 680 nm (23 degrees C) and 685 nm (-196 degrees C). Excitation maxima of phycobilisomes were at 630 and 650 nm for the wild type and the mutant 85Y, respectively. The phycobilisomes of wild type cells whether grown in white or far-red light had the same size and pigment composition. A typical wild type cell in white light had a thylakoid area of 22.8 microns 2, but in far-red light the area was reduced to 13.5 microns 2, which was close to that of 85Y at 13.6 microns 2. Chlorophyll molecules per cell decreased in far-red light from 1.1 X 10(7) in wild type (white light) to 4.5 X 10(6) in mutant 85Y (far-red). The number of phycobilisomes per cell (approx 2 X 10(4)), calculated from the phycobiliprotein content and phycobilisome size, was about the same in wild type (white light) and mutant 85Y (far-red light), but the number of phycobilisomes per unit area of thylakoid was significantly greater in mutant 85Y than in wild type. The present results suggest that the phycobilisomes are linked with reaction centers and that the PSII complement (photo-system II and phycobilisome) was fully maintained in far-red light.

On the O2 flash yields of two cyanophytes

Abstract We explored O 2 flash yield in two cyanophytes, Anacystis nidulans and Agmenellum quadruplicatum . On a rate-measuring electrode, a single flash gave a contour of O 2 evolution with a peak at about 10 ms which was maximum (100) for 680 nm background light. On 625 nm illumination the peak was smaller (62) but was followed by an increased tail of O 2 attributed to enhancement of the background. After a period of darkness, repetitive flashes (5 Hz) gave a highly damped initial oscillation in individual flash yields which finally reached steady state at 94% of the yield for 680 nm illumination. When O 2 of repetitive flashes was measured as an integrated flash yield the results was distinctive and similar to that for a continuous light 1 (680 nm). An apparent inhibition of respiration which persisted into the following dark period was taken as evidence for the Kok effect. With a concentration-measuring electrode, integrated flash yield vs. flash rate showed the same nonlinear behavior as O 2 rate vs. intensity of light 1. We draw three conclusions about the two cyanophytes. (a) The plastoquinone pool is substantially reduced in darkness. (b) Because of a high ratio of reaction centers, reaction center 1 / reaction center 2, for the two photoreactions, saturating flashes behave as light 1. (c) Because repetitive flashes are light 1, they also give a Kok effect which must be guarded against in measurements designed to count reaction centers.

Spontaneous pigment mutants of Anacystis nidulans selected by growth under far-red light

Under far-red (>650 nm) illumination Anacystis nidulans grows poorly and develops a low chlorophyll content. During continued culture over many generations there are increases in growth rate and in the chlorophyll/phycocyanin ratio, usually occurring in concomitant and stepwise fashion. From such selection cultures six clones have been established which differ from the parent in pigment content and show improved growth rate in far-red light. From the evidence at hand the six clones are presumed to be spontaneous mutants selected under the photosynthetically restrictive condition of far-red illumination.

On the origins of 718 nm fluorescence from Porphyridium cruentum at 77 K.

Emission spectra and transient behavior of fluorescence in Porphyridium cruentum have been studied in search of the pathway of excitation energy from the phycobilisome to Photosystem I (PS I) of photosynthesis. For activating light at 436 nm, absorbed almost entirely by chlorophyll, fluorescence is dominated by the 718 nm band generally attributed to chlorophyll of PS I. Activating light at 550 nm, absorbed mostly by the phycobilisome, gives rise to the distinctive fluorescence band of PS II chlorophyll at 696 nm but also gives a large component at 718 nm. Analysis depends critically upon the source of emission at 718 nm under 550 nm activation: does it arise from PS I or PS IIC0 Ley and Butler (Ley, A.C. and Butler, W.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3956-3960) have proposed that the 718 nm arises mostly from PS I, to which it is transferred by spillover from PS II. We suggest a different proposition: that under 550 nm activation most of the 718 emission arises from PS II. Analysis shows that this proposition provides an alternative explanation. Using the small change in fluorescence yield observed under 436 nm activation as a monitor of excitation in PS I, we provide evidence that under 550 activation most of the 718 nm fluorescence arises from PS II.

Phycobilisome Composition and Relationship to Reaction Centers in Anacystis Nidulans

Phycobilisomes were studied in Anacystis nidulans wild type and several spontaneous mutants selected for improved growth in far-red light (>650 nm). In the mutants the phycocyanin varied in relation to allophycocyanin but there was no alteration in the antenna size in terms of chlorophylls in photosystems I and II. Phycobilisomes of wild-type contained phycocyanin and allophycocyanin in a molar ratio of 3:1. Phycobilisomes of the mutants 85Y, 19Y and 59G had respective ratios of 0.4:1, 0.7:1, and 1:1. Analysis of purified phycobilisomes on SDS-PAGE gradient gels revealed significant differences in the polypeptide pattern. A comparison of phycobilisomes of the wild-type and 85Y by electron microscopy, showed that they were larger in wild-type (57nm × 30nm) than in 85Y (28nm × 15nm). The number of phycobilisomes per cell (~2 × 104) was about the same in wild-type (white light) and 85Y (far-red light). The ratio of phycobilisome/reaction center 2 was close to 1 and was relatively constant in spite of large variations in chlorophyll and in phycocyanin content.

Variation in the polypeptide composition of phycobilisomes from Anacystis nidulans and three pigment mutants

Phycobilisomes, light harvesting antenna pigment systems, were studied from Anacystis nidulans wild type and from several spontaneous pigment mutants selected for improved growth in far-red light (>650 nm). This is the first characterization and description of polypeptide composition of phycobilisomes from spontaneous mutants (not chemically induced) of A. nidulans. The mutants had significant changes in the phycobiliprotein content relative to chlorophyll (Chl). Two phycobiliproteins, C-phycocyanin (λmax 625 nm) and allophycocyanin (λmax 650 nm) were present in a molar ratio of ∼3:1 in the wild type. In the mutants the amount of allophycocyanin (APC) per cell remained constant but the phycocyanin (PC) content varied. Phycobilisomes of the mutants 85Y, 19Y and 59G contained PC and APC in a molar ratio of 0.4:1, 0.7:1, and 1:1, respectively. Even though the phycobilisomes of mutant 85Y showed the greatest reduction in PC and consisted mostly of the APC core, the phycobilisomes were still functional and the growth rate of the 85Y cells was similar to the wild type. Fluorescence emission maxima of wild type and all the mutants were at 680 nm (23°C). Excitation maxima corresponded closely with expectations from absorption spectra. On SDS-PAGE gradient gels wild-type phycobilisomes had the usual 17–19 kD polypeptides of PC and APC, plus polypeptides at ca. 78, 56, 39, 36 and 31 kD. Mutants 19Y and 85Y, with the greatest PC deficiency were also deficient in the 36 and 39 kD polypeptides, presumably due to a decrease in peripheral rod structures. Energetically functional hybrid phycobilisomes were obtained when complexes of phycoerythrin-phycocyanin from Nostoc were mixed with dissociated phycobilisomes of wild-type and mutant 85Y. The 31 kD polypeptide is common to both species and immunologically cross reactive.