Photoinhibition Resistance in the Red Alga Porphyra perforata: The Role of Photoinhibition Repair

Photoinhibition resistance exhibited by the high intertidal red alga Porphyra perforata relative to its subtidal congener Porphyra nereocystis was examined using the protein synthesis inhibitor chloramphenicol to separate the damage and repair components of photoinhibition. Under photoinhibitory conditions, the rates of both damage to and replacement of photoinhibition-sensitive proteins was much higher in P. nereocystis than in P. perforata. Thus, photoinhibition resistance in P. perforata appears to be due to a reduced rate of photoinhibition damage rather than to an accelerated rate of photoinhibition repair. Reduction of photoinhibition damage in P. perforata may be by means of biophysical processes which increase the radiationless decay of excitation to heat in photosystem II. Alternatively, the photoinhibition-sensitive proteins in P. perforata may have slight structural alterations that improve their stability or they may be protected by enzyme systems that quench radicals formed by overexcitation of photosystem II. Reduction of the damage component of photoinhibition is a reasonable way to limit photoinhibition in P. perforata during the severe desiccation and exposure to full sun that occur simultaneously during daily low tides, conditions under which the protein synthesis required for photoinhibition repair could not occur.     

The effects of osmotic tissue dehydration and air drying on morphology and energy transfer in two species of porphyra

Studies were conducted to document the effects on morphology and energy transfer in photosynthesis of severe tissue dehydration induced either by air-drying or by immersing the tissues of two Porphyra species in hyperosmotic solutions. These studies showed that the dehydration-tolerant intertidal alga, Porphyra perforata J.Ag., was almost unaffected by either of these treatments, while the dehydration-sensitive Porphyra nereocystis Anders. was damaged similary by both treatments. Damage to that sensitive species was characterized by ruptured organelles as seen by interference microscopy as well as by increased fluorescence emission at 682 nanometers emanating from allophycocyanin. These results suggest that a disruption of energy transfer between allophycocyanin and chlorophyll a occurs because of the damage to membranes following tissue dehydration, and that the increase in the yield of phycobilin fluorescence is a good indicator of these phenomena. Thus, air-drying and osmotic-dehydration appear to have similar physiological consequences in a dehydration-sensitive alga but almost no effect in a tolerant species.     

An evaluation of species relationships in the Porphyra perforata complex (Bangiales,Rhodophyta) using starch gel electrophoresis

Traditional morphological features have formed the basis for distinguishing species of Porphyra. Among these features are number of cell layers, number of chloroplasts per cell, arrangement of reproductive structures on the thallus, and overall morphology. Chromosome number and chromosome morphology have helped corroborate some species identities. A survey of northeast Pacific species of Porphyra using starch gel electrophoresis of 15 soluble proteins has shown that electrophoretic banding patterns provide a reliable diagnostic tool for species identification. Data from starch gel electrophoresis are presented to confirm the identities of species formerly associated with the Porphyra perforata species-complex in British Columbia and northern Washington. Porphyra abbottae, P. fallax, P. kanakaensis, and P. torta are recognized as distinct species, and Porphyra sanjuanensis is synonymized with P. perforata.     

Radiationless transitions as a protection mechanism against photoinhibition in higher plants and a red alga

Exposure of the red alga Porphyra perforata or leaves of Phytolacca americana and Echinodorus sp. to white light equivalent to full sunlight for short periods induced large decreases of variable fluorescence measured at 695 nm at 77K. This change was not produced by photoinhibition but rather appeared to result from an inorease in the rate constant of radiationless transition in the reaction centers of photosystem II. It is proposed that this increase is related to the formation of the high energy state which serves as a photoprotective mechanism in plants.     

Protoplast isolation and regeneration in the marine red alga Porphyra nereocystis

We have developed a method for isolating viable protoplasts from the blade phase of the epiphytic marine red alga Porphyra nereocystis Anderson, using a two-step enzymatic digestion with commercially available enzymes. The first step uses papain, the second step uses abalone acetone powder. The method is rapid and gives a high yield of viable protoplasts. In liquid culture in enriched seawater medium, the protoplasts can undergo regeneration along three pathways: they directly form filaments resembling the conchocelis phase of Porphyra; they form calli with relatively thick-walled, pigmented cells; and they indirectly form blades from the edges of these calli. Porphyra nereocystis protoplasts also may serve as an alternative propagation method in aquaculture and be useful for studies of cell-wall formation, cell division, and thallus differentiation. They may also be used in somatic selection, somatic hybridization and gene-transfection experiments.Abbreviations AAP abalone acetone powder - PAP papain - FDA fluorescein diacetate This paper is dedicated to the memory of the late Dr. Munenao Kurogi (1921–1988), Professor Emeritus of Hokkaido UniversityThis research was supported by the Washington Sea Grant Program (National Oceanic and Atmospheric Administration). We thank Professor Y. Fujita (Nagasaki University, Japan), Professor S.-J. Wang (Shanghai University of Fisheries, P.R. China) and Dr. H. Kito (Seikai Regional Fisheries Research Laboratory, Nagasaki, Japan) for sharing their experience with Porphyra protoplast production with us. We thank J.S. Charleston for expert technical assistance in preparation of the electron-microscopy specimens. We also thank Dr. S.K. Herbert and John Carrier (Friday Harbor Laboratories) and Dr. John Merrill and D. Gillingham (American Sea Vegetable Co. and Applied Algal Research, Seattle) for collections of P. nereocystis.     

Conchospore production and seasonal occurrence of some Porphyra species (Bangiales,Rhodophyta) in Washington State

The leafy thalli of species of the marine red algal genus Porphyra grow rapidly but persist for a relatively short time on rocky intertidal or subtidal substrata or as epiphytes on other marine plants. In most species, the large, short-lived leafy thalli alternate with small, presumably perennial, filamentous conchocelis plants. Depending on the species of northeastern Pacific Porphyra, photoperiod and temperature are important regulators of conchospore formation and release. Data from laboratory studies of conchospore formation and release in five Washington species of Porphyra (P. abottae, P. nereocystis, P. perforata, P. pseudolanceolata and P. torta) indicate that conchospores are most likely to be released at a time that precedes the appearance of the leafy thalli in the field.     

Resistance to the herbicide paraquat and increased tolerance to photoinhibition are not correlated in several weed species

Photoinhibition was examined in paraquat-resistant and paraquat-susceptible biotypes of Hordeum glaucum Steud., Hordeum leporinum Link., Arctotheca calendula (L.) Levyns., and Conyza bonariensis (L.) Cronq. Plants were photoinhibited at low temperature, and the extent of photoinhibition determined by O₂ evolution and 77 K fluorescence. No difference in the degree of photoinhibition was detected between paraquat-resistant and paraquat-susceptible biotypes for any of the species examined. C. bonariensis plants were also photoinhibited by treatment without CO₂ at either 21% (volume/volume) O₂ or 4% (volume/volume) O₂, and again no difference was observed between the paraquat-resistant and paraquat-susceptible biotypes in reduction of the ratio of variable fluorescence to maximal fluorescence. This is in contrast to a recent report (MAK Jansen, Y Shaaltiel, D Kazzes, O Canaani, S Malkin, J Gressel, [1989] Plant Physiol 91: 1174-1178 in which it was claimed that a paraquat-resistant biotype of C. bonariensis was more tolerant of photoinhibition than a paraquat-susceptible biotype. We conclude that paraquat-resistant biotypes of these plant species are not more tolerant of photoinhibition when compared with the paraquat-susceptible biotypes.     

MOLECULAR ANALYSIS REVEALS CRYPTIC DIVERSITY IN PORPHYRA (RHODOPHYTA)1

The small subunit ribosomal RNA (SSU rRNA) gene was amplified from 15 species of the red alga Porphyra and digested with restriction enzymes to generate data for species identification. The subset of species selected for phylogenetic analysis was P. cuneiforms (Setchell et Hus) Krishnamurthy, P. nereocystis anderson, P. schizophylla Hollenberg et Abbott, P. thuretii Setchell et Dawson and Porphyra 1674. Bangia sp. was used as an out-group. Restriction sites were mapped and used as characters in parsimony and maximum likelihood analysis. The phylogenetic hypotheses generated were compared statistically to possible alternative phylogenies based on traditional morphological taxonomic characters. The results indicate that the current subgenera in Porphyra do not represent monophyletic groups and that traditional morphological and ecological taxonomic characters alone may not be adequate for definitive species identification and cannot be relied on as an indication of Porphyra have large insertions in the SSU gene that are apparently splicesd from the final SSU rRNA molecule. The possible character, distribution and potential significance of these putative introns are discussed.     

Photoinhibition of photosynthesis in the cyanobacterium Microcystis aeruginosa

We have examined characteristics of the photoinhibition of photosynthesis which occur in the unicellular cyanobacterium Microcystis aeruginosa, following exposure to photon fluence rates in excess of those required for growth. Photoinhibition occurs following exposure of cells to a photon fluence rate of 1,000 μmol m^-2 s^-1, which is manifested as a decrease in either light-limited CO₂ fixation or light-saturated CO₂-dependent O₂ evolution. The extent and rapidity of this photoinhibition is greatly enhanced under CO₂-depleted conditions. Experiments in which cultures were sparged with different gases indicate that photoinhibition is not an obvious consequence of elevated O₂ tensions, unlike the photooxidative bleaching of photosynthetic pigments. Comparative studies on the photoinactivation of CO₂-dependent O₂ evolution and of the methyl viologen-dependent Mehler reaction, in whole cells, indicate that a primary site of light damage is within the photosynthetic electron-transport reactions and that carbon fixation is initially unaffected.     

A COMPARISON OF PHOTOSYNTHETIC ELECTRON TRANSPORT RATES IN MACROALGAE MEASURED BY PULSE AMPLITUDE MODULATED CHLOROPHYLL FLUOROMETRY AND MASS SPECTROMETRY

The relationship between whole chain photosynthetic electron transport and PSII activity was investigated in Porphyra columbina (Montagne) (Rhodophyta), Ulva australis (Areschoug) (Chlorophyta), and Zonaria crenata ( J. Agardh) (Phaeophyta). Mass spectrometric measurements of gross O₂ evolution and gross O₂ uptake were combined with simultaneous measurement of pulse-modulated chl fluorescence under a range of irradiances and inorganic carbon (C_i) concentrations. At light-limiting irradiance, a good correlation between gross O₂ evolution and the electron transport rate (ETR) calculated from chl fluorescence ((F_m′− F_s)/F_m′) was found in the optically thin species (Ulva and Porphyra). The calculated ETR was equivalent to the theoretical electron requirement in these species but overestimated gross O₂ evolution in the thicker species Zonaria. In saturating light, especially when C_i availability was low, ETR overestimated gross O₂ evolution in all species. Excess electron flow could not be accounted for by an increase in gross O₂ uptake; thus neither Mehler-ascorbate-peroxidase reaction nor the photosynthetic carbon oxidation cycle were enhanced at high irradiance or low C _i. Alternative explanations for the loss of correlation include cyclic electron flow around PSII that may be engaged under these conditions or nonphotochemical energy quenching within PSII centers. The loss of correlation between ETR and linear photosynthetic electron flow as irradiance increased from limiting to saturating or at low C_i availability and in the case of optically thick thalli limits the application of this technique for measuring photosynthesis in macroalgae.     

Responses of Two CAM Species to Different Irradiances during Growth and Susceptibility to Photoinhibition by High Light

Two CAM species, Kalanchoë daigremontiana Hamet et Perrier and Hoya carnosa (L.) R. Br., were grown under a range of five photon flux area densitites (PFD) and then characterized. Significant acclimation to shade was indicated by progressive decreases in leaf thickness, rates of respiratory O₂ uptake, light compensation point, maximum rates of photosynthetic O₂ evolution, nocturnal acid accumulation, and δ¹³C values, and increases in chlorophyll concentration and absolute levels of room temperature (25°C) and 77K fluorescence. Quantum yields (as measured by O₂ exchange) and the ratio of variable 77K fluorescence over the maximum yield (F_v/F_m) were relatively constant across the treatments. The only significant deviation from the above characteristics was in H. carnosa grown under full glasshouse PFD, where it apparently experienced photoinhibition. Following a photoinhibitory treatment, K. daigremontiana exhibited increases in the light compensation point and progressively greater reductions in the quantum yield, maximum photosynthetic rate, F_v/F_m, and the variable component of room temperature fluorescence with increasing shade during growth. Thus although Crassulacean acid metabolism plants can adjust to shaded conditions, they are susceptible to photoinhibition when exposed to higher PFD than that experienced during growth.     

The Susceptibility of Photosynthesis to Photoinhibition and the Capacity of Recovery in High and Low Light Grown Cyanobacteria, Anacystis nidulans

The susceptibility of photosynthesis to photoinhibition and the rate of its recovery were studied in the cyanobacterium Anacystis nidulans grown at a low (10 micromoles per square meter per second) and a high (120 micromoles per square meter per second) photosynthetically active radiation. The rate of light limited photosynthetic O₂ evolution was measured to determine levels of photoinhibition and rates of recovery. Studies of photoinhibition and recovery with and without the translation inhibitor streptomycin demonstrated the importance of a recovery process for the susceptibility of photosynthesis to photoinhibition. We concluded that the approximately 3 times lower susceptibility to photoinhibition of high light than of low light grown cells, significantly depended on high light grown cells having an approximately 3 times higher recovery capacity than low light grown cells. It is suggested that these differences in susceptibility to photoinhibition and recovery depends on high light grown cells having a higher turnover rate of photosystem II protein(s) that is(are) the primary site(s) of photodamage, than have low light grown cells. Furthermore, we demonstrated that photoinhibition of A. nidulans may occur under physiological light conditions without visible harm to the growth of the cell culture. The results give support for the hypotheses that the net photoinhibitory damage of photosystem II results from the balance between the photoinhibitory process and the operation of a recovery process; the capacity of the latter determining significant differences in the susceptibility of photosynthesis to photoinhibition of high and low light grown A. nidulans.     

Rapid turnover of the D1 reaction-center protein of photosystem II as a protection mechanism against photoinhibition in a moss,Ceratodon purpureus (Hedw.) Brid.

Susceptibility of a moss,Ceratodon purpureus (Hedw.) Brid., to photoinhibition and subsequent recovery of the photochemical efficiency of PSII was studied in the presence and absence of the chloroplast-encoded protein-synthesis inhibitor lincomycin.Ceratodon had a good capacity for repairing the damage to PSII centers induced by strong light. Tolerance against photoinhibition was associated with rapid turnover of the D1 protein, since blocking of D1 protein synthesis more than doubled the photoinhibition rate measured as the decline in the ratio of variable fluorescence to maximal fluorescence (F_v/F_max). Under exposure to strong light in the absence of lincomycin a net loss of D1 protein occurred, indicating that the degradation of damaged D1 protein inCeratodon was rapid and independent of the resynthesis of the polypeptide. The result suggests that synthesis is the limiting factor in the turnover of D1 protein during photoinhibition of the mossCeratodon. The level of initial fluorescence (F_o) correlated with the production of inactive PSII centers depleted of D1 protein. The higher the F_o level, the more severe was the loss of D1 protein seen in the samples during photoinhibition. Restoration of F_v/F_max at recovery light consisted of a fast and slow phase. The recovery of fluorescence yield in the presence of lincomycin, which was added at different times in the recovery, indicated that the chloroplast-encoded protein-synthesis-dependent repair of damaged PSII centers took place during the fast phase of recovery. Pulse-labelling experiments with [³⁵S]methionine supported the conclusion drawn from fluorescence measurements, since the rate of D1 protein synthesis after photoinhibition exceeded that of the control plants during the first hours under recovery conditions.     

Photoinhibition of Photosynthesis in Broken Chloroplasts as a Function of Electron Transfer Rates during Light Treatment

Photoinhibition was studied in osmotically broken chloroplasts isolated from spinach leaves (Spinacia oleracea L.). Both whole chain electron transport (measured as ferricyanide-dependent O₂ evolution in the presence of NH₄Cl) and photosystem II activity (measured as O₂ evolution in the presence of either silicomolybdate plus 3-(3,4-diphenyl)-1,1 dimethylurea or parabenzoquinone) showed similar decreases in activity in response to a photoinhibitory treatment (8 minutes of high light given in the absence of an electron acceptor other than O₂). Photosystem I activity was less affected. Photoinhibition of silicomolybdate reduction was largely reversible by an 8 minute dark incubation following the light treatment. Decreasing the O₂ concentration during photoinhibition below 2% increased photoinhibition of whole chain electron transport. Addition of superoxide dismutase to the reaction medium did not affect photoinhibition. Photoinhibition of both photosystem I and photosystem II activity increased as the rate of electron transfer during the treatment increased, and was largely prevented when 3-(3,4-diphenyl)-1,1-dimethylurea was present during the photoinhibition period. Noncyclic photophosphorylation was decreased as a consequence of whole chain electron transfer photoinhibition. Since diphenyl carbazide added after light treatment did not relieve photoinhibition of dichlorophenol indophenol reduction, we conclude that the site of inhibition is located within or near the photosystem II reaction center.     

Photoinhibition of CO(2)-Dependent O(2) Evolution by Intact Chloroplasts Isolated from Spinach Leaves

Intact spinach (Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO₂-free buffer at 21% O₂, showed a decrease (30-70%) in CO₂-dependent O₂ evolution and ¹⁴CO₂ uptake. This photoinhibition was observed only when the O₂ concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO₂ concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO₂ concentration was increased to 30 micromolar. Addition of small quantities of P-glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose-1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O₂ evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO₂ concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O₂ evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O₂-dependent photoinhibition of light saturated CO₂ fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O₂ for photoinhibition of CO₂ fixation capacity in isolated chloroplasts may be explained by an effect of O₂ in allowing metabolic depletion of Calvin cycle intermediates.     

Two mechanisms of recovery from photoinhibition in vivo: Reactivation of photosystem II related and unrelated to D1-protein turnover

Recovery (at 20° C) of spinach (Spinacia oleracea L.) leaf sections from photoinhibition of photosynthesis was monitored by means of the fluorescence parameter F_V/F_M of intact leaf tissue and of PSII-driven electron-transport activity of isolated thylakoids. Different degrees of photoinactivation of PSII were obtained by preillumination in ambient air (at 4 or 20° C), CO₂-free air or at low and high O₂ levels (2 or 41 %) in N₂. The kinetics of recovery exhibited two distinct phases. The first phase usually was completed within about 20-60 min and was most pronounced after preillumination in low O₂. The slow phase proceeded for several hours leading to almost complete reactivation of PSII. Preincubation of the leaves with streptomycin (SM), which inhibits chloroplast-encoded protein synthesis, inhibited the slow recovery phase only, indicating the dependence of this phase on resynthesis of the reaction-centre protein, D1. The fast recovery phase remained largely unaffected by SM. Both phases were strongly but not totally dependent on irradiation of the leaf with low light. When SM was absent, net degradation of the D1 protein could neither be detected upon photoinhibitory irradiation nor during following incubation of the leaf sections in low light or darkness. In the presence of SM, net D1 degradation was seen and tended to increase with O₂ concentration during photoinhibition treatment. Based on these data, we suggest that photoinactivation of PSII in vivo occurs in at least two steps. From the first step, reactivation appears possible in low light without D1 turnover (fast recovery phase). Action of oxygen then may lead to a second step, in which the D1 protein is affected and reactivation requires its removal and replacement (slow phase).Abbreviations Chl chlorophyll - F₀, F_M and F_V initial, maximum total and maximum variable chlorophyll fluorescence yield, respectively - PFD photon flux density - SM streptomycin We thank Professor P. Böger (Department of Plant Physiology and Biochemistry, University of Konstanz, Germany) for a gift of D1-specific antibodies. The paper contains part of the thesis work of J.L. The study was supported by the Deutsche Forschungs-gemeinschaft (SFB 189).     

Inter- and intrapopulation genetic variation in species ofPorphyra (Rhodophyta: Bangiales) from British Columbia and adjacent waters

Starch gel electrophoresis of 17 proteins has provided data on inter- and intrapopulation genetic variation in 20 species ofPorphyra occurring in British Columbia and adjacent areas.P. cuneiformis andP. nereocystis showed no within species variation, even over ranges of more than 1000 km. Populations ofP. abbottae, P. fallax, P. fucicola, P. gardneri andP. schizophylla were characterized by fixation for certain alleles. The number of polymorphic loci in a population ranged from zero to nine, depending on the species. Six species had populations that were polymorphic at just a single locus. Only two species (P. mumfordii andP. pseudolanceolata) had populations that were polymorphic at more than three loci. These levels of genetic variation are lower than those reported for populations of JapanesePorphyra species. Eleven taxa were polymorphic for 6-phosphogluconate dehydrogenase, the most variable enzyme. No within species polymorphisms were detected for bromoperoxidase, lactate dehydrogenase, superoxide dismutase or phycoerythrin. Possible evidence for the chimeric nature of the thallus was observed only inP. mumfordii.     

Chlorophyll fluorescence and photoinhibition in a tropical rainforest understory plant

The data presented here deal with the effects of high-light exposure on the 77 K fluorescence characteristics of Elatostema repens. It is shown that the decrease of the variable fluorescence during the treatment is biphasic. The reactions responsible for the first phase of fluorescence quenching are saturated under 700 mol photon m^-2 s^-1 and insensitive to streptomycin, whereas those responsible for the second phase are not yet saturated under 700 mol photon m^-2 s^-1 and sensitive to streptomycin. It is concluded that only the second phase of fluorescence quenching is associated with photoinhibitory processes. Rate and amplitude of recovery from photoinhibition are maximum under very low light (3.5 mol photon m^-2 s^-1), and very small at a moderate light (160 mol photon m^-2 s^-1) which does not cause photoinhibition. It is concluded that recovery processes are inhibited during photoinhibition. It is suggested that they could be associated with damage occuring on the oxidizing side of PSII.Abbreviations F_o, F_v, F_m initial, variable and maximum fluorescence, respectively - PFD photon flux density - PS II photosystem II     

Porphyra nereocystis: A dual-daylength seaweed

Conchospores from the perennial conchocelis phase of the annual, epiphytic, marine red alga Porphyra nereocystis Anderson, which in nature lives on the large annual kelp Nereocystis luetkeana (Mertens) Postels et Ruprecht, are released in culture only in response to a short-day photoperiod treatment followed by a long-day treatment. Each treatment requires a minimum of three to four weeks and is enhanced by lower temperature during the second photoperiod treatment. To our knowledge P. nereocystis is the first known dualdaylength seaweed and requires a short-day-longday treatment for completion of its life cycle. This stringent environmental control of its reproduction appears to be an adaptation to coordinate conchospore production with the seasonal availability of its host kelp Nereocystis.Abbreviations LD long day - SD short day - SLD shortlong day