Modulation of photosynthesis and respiration in guard and mesophyll cell protoplasts by oxygen concentration

Stomatal movement is an energetic oxygen-requiring process. In the present study, the effect of oxygen concentration on mitochondrial respiratory activity and red-light-dependent photosynthetic oxygen evolution by Vicia faba and Brassica napus guard cell protoplasts was examined. Comparative measurements were made with mesophyll cell protoplasts isolated from the same species. At air saturated levels of dissolved oxygen in the protoplast suspension media, respiration rates by mesophyll protoplasts ranged from 6 to 10μmoles O₂ mg^?1 chl h^?1, while guard cell protoplasts respired at rates of 200–300 μmoles O₂ mg chl^?1 h^?1, depending on the species. Lowering the oxygen concentration below 50–60 mmol m^?3 resulted in a decrease in guard cell respiration rates, while rates by mesophyll cell protoplasts were reduced only at much lower concentrations of dissolved oxygen. Rates of photosynthesis in mesophyll cell protoplasts isolated from both species showed only a minor reduction in activity at low oxygen concentrations. In contrast, photosynthesis by guard cell protoplasts isolated from V. faba and B. napus decreased concomitantly with respiration. Oligomycin, an inhibitor of oxidative phos-phorylation, reduced photosynthesis in mesophyll cell protoplasts by 27–46% and in guard cell protoplasts by 51–58%. The reduction in both guard cell photosynthesis and respiration following exposure to low oxygen concentrations suggest close metabolic coupling between the two activities, possibly mediated by the availability of substrate for respiration associated with photosynthetic electron transport activity and subsequent export of redox equivalents.     

EFFECTS OF LIGHT AND TEMPERATURE ON NET PHOTOSYNTHESIS AND DARK RESPIRATION OF GAMETOPHYTES AND EMBRYONIC SPOROPHYTES OF MACROCYSTIS PYRIFERA1

The rates of net photosynthesis as a function of irradiance and temperature were determined for gametophytes and embryonic sporophytes of the kelp, Macrocystis pyrifera (L.) C. Ag. Gametophytes exhibited higher net photosynthetic rates based on oxygen and pH measurements than their derived embryonic sporophytes, but reached light saturation at comparable irradiance levels. The net photosynthesis of gametophytes reached a maximum of 66.4 mg O₂ g dry wt^?1 h^?1 (86.5 mg CO₂ g dry wt^?1 h^?1), a value approximately seven times the rate reported previously for the adult sporophyte blades. Gametophytes were light saturated at 70 μE m^?2 s^?1 and exhibited a significant decline in photosynthetic performance at irradiances 140 μE m^?1 s^?1. Embryonic sporophytes revealed a maximum photosynthetic capacity of 20.6 mg O₂ g dry wt^?1 h^?1 (25.3 mg CO₂ g dry wt^?1 h^?1), a rate about twice that reported for adult sporophyte blades. Embryonic sporophytes also became light saturated at 70 μE m^?2 s^?1, but unlike their parental gametophytes, failed to exhibit lesser photosynthetic rates at the highest irradiance levels studied; light compensation occurred at 2.8 μE m^?2 s^?1. Light-saturated net photosynthetic rates of gametophytes and embryonic sporophytes varied significantly with temperature. Gametophytes exhibited maximal photosynthesis at 15° to 20° C, whereas embryonic sporophytes maintained comparable rates between 10° and 20° C. Both gametophytes and embryonic sporophytes declined in photosynthetic capacity at 30° C. Dark respiration of gametophytes was uniform from 10° to 25° C, but increased six-fold at 30° C; the rates for embryonic sporophytes were comparable over the entire range of temperatures examined. The broader light and temperature tolerances of the embryonic sporophytes suggest that this stage in the life history of M. pyrifera is well suited for the subtidal benthic environment and for the conditions in the upper levels of the water column.     

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO₃^?) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO₃^? by the surface‐bound enzyme carbonic anhydrase (CA_ext). Here, we examined other putative HCO₃^? uptake mechanisms in M. pyrifera under pH_T 9.00 (HCO₃^?: CO₂ = 940:1) and pH_T 7.65 (HCO₃^?: CO₂ = 51:1). Rates of photosynthesis, and internal CA (CA_int) and CA_ext activity were measured following the application of AZ which inhibits CA_ext, and DIDS which inhibits a different HCO₃^? uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO₃^? uptake by M. pyrifera is via an AE protein, regardless of the HCO₃^?: CO₂ ratio, with CA_ext making little contribution. Inhibiting the AE protein led to a 55%–65% decrease in photosynthetic rates. Inhibiting both the AE protein and CA_ext at pH_T 9.00 led to 80%–100% inhibition of photosynthesis, whereas at pH_T 7.65, passive CO₂ diffusion supported 33% of photosynthesis. CA_int was active at pH_T 7.65 and 9.00, and activity was always higher than CA_ext, because of its role in dehydrating HCO₃^? to supply CO₂ to RuBisCO. Interestingly, the main mechanism of HCO₃^? uptake in M. pyrifera was different than that in other Laminariales studied (CA_ext‐catalyzed reaction) and we suggest that species‐specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO₃^?:CO₂ due to ocean acidification.     

PHOTOSYNTHETIC PROPERTIES OF PROTOPLASTS,AS COMPARED WITH THALLI,OF ULVA FASCIATA (CHLOROPHYTA)1

Protoplasts were prepared from Ulva fasciata Delile, and their photosynthetic performance was measured and compared with that of thalli discs. These protoplasts maintained maximal rates of photosynthesis as high as those of thalli (up to 300 μmol O₂·mg chlorophyll^?1·h^?1) for several hours after preparation and were therefore considered suitable for kinetic studies of inorganic carbon utilization. The photosynthetic K_1/2(inorganic carbon) at pH 6.1 was 3.8 μM and increased to 67, 158, and 1410 μM at the pH values 7.0, 7.9, and 8.9, respectively. Compared with these protoplasts, thalli had a much lower affinity for CO₂ but approximately the same affinity for HCO₃^?. Comparisons between rates of photosynthesis and the spontaneous dehydration of HCO₃^? (at 50 μM inorganic carbon) revealed that photosynthesis of both protoplasts (which lacked apparent activity of extracellular/surface-bound carbonic anhydrase) and thalli (which were only 25% inhibited by the external carbonic anhydrase inhibitor acetazolamide) could not be supported by CO₂ formation in the medium at the higher pH values, indicating HCO₃^? uptake. Since both protoplasts and thalli were sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulfonate, we suggest that HCO₃^? transport was facilitated by the membrane-located anion exchange protein recently reported to function in certain Ulva thalli. These findings suggest that the presence of a cell wall may constitute a diffusion barrier for CO₂, but not for HCO₃^?, utilization under natural seawater conditions.     

STUDIES OF VANDADIUM-BROMOPEROXIDASE USING SURFACE AND CORTICAL PROTOPLASTS OF MACROCYSTIS PYRIEFERA (PHAEOPHYTA)1

Aqueous Tri-SO₄ buffer (pH 8.3) extracts of cortical and surface protoplasts of Macrocystis pyrifera (L.) C. Ag. Catalyzed the bromination of monochlorodimedone (2-chloro-5, 5-dimethyl-1, 3-dimedone, MCD) in the presence of hydrogen peroxide and bromide. The apparent bromo-peroxidase activity as measured by the bromination of MCD was inhibited by the presence of endogenous compounds which are probably polyphenolics compounds (i.e. polymers of phloroglucinol) or other inhibitors. The bromoperoxidase activity of the protoplast extracts increased substantially when the extracts were washed extensively with Tris-SO₄ buffer (pH 8.3) by ultrafiltration. The bromoperoxidase activity of both surface and cortical protoplast extracts was dependent on the presence of vanadium, indicating that the bromoperoxidase present in cortical and surface cells of M. pyrifera is vanadium-bromoperoxidase. Halogenated compounds constitute one of the most significant classes of marine natural products. Since bromoperoxidases are assumed to be involved in the biosynthesis of these compounds, elucidation of the location of BrPO with in the algal tissue is important.     

Analysis of viability and cell types of macroalgal protoplasts using flow cytometry

The ability to rapidly distinguish viable sub-populations of cells within populations of macroalgal protoplast isolations was demonstrated using flow cytometry. Viable protoplasts from Ulva sp. and Porphyra perforata J. Ag. were distinguished from non-viable protoplasts based on differential fluorescein accumulation. The identities of cortical and epidermal protoplasts from Macrocystis pyrifera (L.) C. Ag. were inferred based on light-scattering and chlorophyll a autofluorescence. Three cell types could be distinguished among protoplasts released from thalli of P. perforata based on chlorophyll a and phycoerythrin autofluorescence. Mixed protoplast populations of Ulva sp. and P. perforata were also discernable based on relative chlorophyll a and phycoerythrin autofluorescence. The ability to screen heterogenous protoplast populations rapidly, combined with the cell sorting capabilities of many flow cytometers, should prove valuable for seaweed biotechnology.     

Photosynthesis in chloroplasts rapidly isolated from primary leaves of oat (Avena sativa L.): effects of orthophosphate, metal ion and chelators

Abstract A simple mechanical method for the rapid isolation of chloroplasts with high rates of photosynthesis from young leaves of oat (Avena sativa L.) was described. The photosynthetic activity of these chloroplasts was stable for at least 2 h with rates of CO₂-dependent O₂ evolution of 30–40 μmol g ¹ Chl s ¹. The photosynthetic properties of these chloroplasts were similar to those reported for spinach and pea chloroplasts isolated by mechanical disruption. The pH optimum for photosynthetic O₂ evolution was pH 7.6. The induction time was 0.5–2 min. Maximal rates of photosynthetic O₂ evolution in these chloroplast preparations were obtained in the absence of both divalent cations and EDTA. Addition of divilent cations strongly inhibited photosynthesis which could be partially restored by the subsequent addition of EDTA. But when these cations were not present in the assay medium the addition of EDTA greater than 1 mol m ³ decreased photosynthetic activity. The optimal orthophosphate concentration required for photosynthesis in these chloroplast preparations was 0.2–0.3 mol m ³. In contrast, the addition of pyrophosphate either in the light or dark inhibited photosynthesis. In a comparative study, chloroplasts were also isolated from oat and wheat (Triticum aestivum L., cultivar Hybrid C306) protoplasts. These chloroplast preparations were found to have properties similar to those determined for oat chloroplasts isolated by the mechanical method reported above.     

Isolation of mesophyll protoplasts from mature leaves of soybeans

A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO₃⁻-dependent photosynthesis; greater than 75 micromoles O₂ evolved per milligram chlorophyll per hour at 25°C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.     

CARBON ALLOCATION IN MACROCYSTIS PYRIFERA (PHAEOPHYTA): INTRINSIC VARIABILITY IN PHOTOSYNTHESIS AND RESPIRATION1

Measurements of net photosynthesis (PS, O₂ evolution), dark respiration (R, O₂ consumption), and light and dark carbon fixation (¹⁴C) were conducted on whole blades, isolated blade discs, sporophylls, apical scimitars and representative portions of stipe and holdfast of the giant kelp Macrocystis pyrifera L.C. Ag. On a dry weight basis, highest net PS rates were observed in apical scimitar segments and whole blades (3.81 and 3.07 mgC · g dry wt^?1· h^?1, respectively), followed by sporophylls (1.42 mgC·g dry wt^?1· h^?1) and stipe segments (0.15 mgC·g dry wt^?1· h^?1). No PS capacity was observed in holdfast material. Respiration rates showed similar ranking ranging from 1.22 mgC·g dry wt^?1·h^?1 for apical scimitar to 0.18–0.22 mgC·g dry wt^?1· h^?1 for holdfast material. Considerable within blade variability in both PS and R was also found. Steepest PS and R gradients on both an areal and weight basis were found within immature blades followed by senescent and mature blade material. Highest net PS rates were associated with the blade tips ranging from 3.08 (mature blades) to 10.3 mgC·dry wt^?1·h^?1 (immature blades). Highest rates of R generally occurred towards the basal portions of blades and ranged from 1.03–1.80 mgC·g dry wt^?1·h^?1 for immature blades. The variability within and between blades was high, with coefficients of variation approaching 50%. The observed patterns can be related to the decreasing proportionment of photosynthetic tissue and increasing proportionment of structural tissue as occurs from the blade tip to the blade base. Rates of light carbon fixation (LCF) revealed longitudinal profiles similar to oxygen measurements for the different blade types, with the absolute rates being slightly lower. Patterns of dark carbon fixation (DCF) were less easily interpreted. Highest rates of DCF (0.04–0.06 mgC·g dry wt^?1·h^?1) occurred at the basal portions of immature and senescent blades. Longitudinal profiles of total chlorophyll (a + c) on both an areal and weight basis were very similar to the profiles of PS. Normalized to chlorophyll a, PS displayed an unusual longitudinal profile in immature tissue; however, such profiles for mature and senescent tissues were similar to those for PS on an areal basis. It was demonstrated that it is difficult, if not impossible, to select single tissue discs that are representative of whole blades. The metabolic longitudinal profiles reveal a characteristic developmental pattern; the previous working definitions of immature, mature, and senescent blades, based on morphology and frond position thus have a physiological basis.     

Markedly low requirement of added CO2 for photosynthesis by mesophyll protoplasts of pea (Pisum sativum): possible roles of photorespiratory CO2 and carbonic anhydrase

Mesophyll protoplasts of pea required only 74.1 μM CO₂ for maximal photosynthesis, unlike chloroplasts, which required up to 588 μM CO₂. Such a markedly low requirement for CO₂ could be because of an internal carbon source and/or a CO₂ concentrating mechanism in mesophyll protoplasts. Ethoxyzolamide (EZA), an inhibitor of internal carbonic anhydrase (CA) suppressed photosynthesis by mesophyll protoplasts at low CO₂ (7.41 μM) but had no significant effect at high CO₂ (741 μM). However, acetazolamide, another inhibitor of CA, did not exert as much dramatic effect as EZA. Three photorespiratory inhibitors, aminoacetonitrile or glycine hydroxamate (GHA) or aminooxyacetate inhibited markedly photosynthesis at low CO₂ but not at high CO₂. Inhibitors of glycolysis or tricarboxylic acid cycle (NaF, sodium malonate) or phosphoenolpyruvate carboxylase (3,3‐dichloro‐2‐dihydroxy phosphinoyl‐methyl‐2‐propenoate) had no significant effect on photosynthesis. The CO₂ requirement of protoplast photosynthesis and the sensitivity of photosynthesis to EZA were much higher at low oxygen (65 nmol ml^?1) than that at normal oxygen (212 nmol ml^?1). In contrast, the inhibitory effect of photorespiratory inhibitors on protoplast photosynthesis was similar in both normal and low oxygen medium. The marked elevation of glycine/serine ratio at low O₂ or in presence of GHA confirmed the suppression of photorespiratory decarboxylation by GHA. While demonstrating interesting difference between the response of protoplasts and chloroplasts to CO₂, we suggest that photorespiration could be a significant source of CO₂ for photosynthesis in mesophyll protoplasts at limiting CO₂ and at atmospheric levels of oxygen. Obviously, carbonic anhydrase is essential to concentrate or retain CO₂ in mesophyll cells.     

PHOTOSYNTHETIC PHYSIOLOGY AND CHEMICAL COMPOSITION OF SPORES OF THE KELPS MACROCYSTIS PYRIFERA,NEREOCYSTIS LUETKEANA,LAMINARIA FARLOWII,AND PTERYGOPHORA CALIFORNICA (PHAEOPHYCEAE)1

Recently released spores of the kelps Macrocystis pyrifera (L.) C. Ag., Nereocystis luetkeana (Mert.) Post. and Rupr., Laminaria farlowii Setch., and Pterygophora californica Rupr. had different levels of net photosynthesis. Spore-specific photosynthesis–irradiance relationships were similar in many respects for M. pyrifera, N. luetkeana, and L. farlowii spores. All three species had low rates of net light-saturated photosynthesis. In contrast, spores of P. californica had higher photosynthetic potential and overall net photosynthesis than the other three species. On a cell carbon basis, however, photosynthetic rates in N. luetkeana spores were similar to those of P. californica spores and higher than those of M. pyrifera spores. Chlorophyll a content of spores varied 10-fold among species. The rank order of significant differences in chlorophyll a content was P. californica > L. farlowii > N. luetkeana > M. pyrifera. As a result, chlorophyll-specific measurements suggest M. pyrifera and N. luetkeana spores had much higher quantum efficiency and photosynthetic potential than either P. californica or L. farlowii spores. Maternal carbon and nitrogen investment significantly differed in spores of M. pyrifera, N. luetkeana, and P. californica with P. californica > M. pyrifera > N. luetkeana. Carbon content in spores of each of these three species increased by about 30% during 12 h of saturating irradiance. We suggest that the photosynthetic capabilities of and maternal investment in spores may be related to the spore as a unit of dispersal, to the reproductive ecology of the parental sporophytic stages, and to the growth and physiology of the germling gametophyte stages.     

Abiotic influences on bicarbonate use in the giant kelp,Macrocystis pyrifera,in the Monterey Bay

In the Monterey Bay region of central California, the giant kelp Macrocystis pyrifera experiences broad fluctuations in wave forces, temperature, light availability, nutrient availability, and seawater carbonate chemistry, all of which may impact their productivity. In particular, current velocities and light intensity may strongly regulate the supply and demand of inorganic carbon (Ci) as substrates for photosynthesis. Macrocystis pyrifera can acquire and utilize both CO₂ and bicarbonate (HCO₃^?) as Ci substrates for photosynthesis and growth. Given the variability in carbon delivery (due to current velocities and varying [DIC]) and demand (in the form of saturating irradiance), we hypothesized that the proportion of CO₂ and bicarbonate utilized is not constant for M. pyrifera, but a variable function of their fluctuating environment. We further hypothesized that populations acclimated to different wave exposure and irradiance habitats would display different patterns of bicarbonate uptake. To test these hypotheses, we carried out oxygen evolution trials in the laboratory to measure the proportion of bicarbonate utilized by M. pyrifera via external CA under an orthogonal cross of velocity, irradiance, and acclimation treatments. Our Monterey Bay populations of M. pyrifera exhibited proportionally higher external bicarbonate utilization in high irradiance and high flow velocity conditions than in sub‐saturating irradiance or low flow velocity conditions. However, there was no significant difference in proportional bicarbonate use between deep blades and canopy blades, nor between individuals from wave‐exposed versus wave‐protected sites. This study contributes a new field‐oriented perspective on the abiotic controls of carbon utilization physiology in macroalgae.     

Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O₂ evolution of about 100 to 150 micromoles of O₂ per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO₃ with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O₂ per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.

Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.

Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO₃ and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma     

Seawater pH,and not inorganic nitrogen source,affects pH at the blade surface of Macrocystis pyrifera: implications for responses of the giant kelp to future oceanic conditions

Ocean acidification (OA), the ongoing decline in seawater pH, is predicted to have wide‐ranging effects on marine organisms and ecosystems. For seaweeds, the pH at the thallus surface, within the diffusion boundary layer (DBL), is one of the factors controlling their response to OA. Surface pH is controlled by both the pH of the bulk seawater and by the seaweeds' metabolism: photosynthesis and respiration increase and decrease pH within the DBL (pH_DBL), respectively. However, other metabolic processes, especially the uptake of inorganic nitrogen (N_i; NO₃^? and NH₄⁺) may also affect the pH_DBL. Using Macrocystis pyrifera, we hypothesized that (1) NO₃^? uptake will increase the pH_DBL, whereas NH₄⁺ uptake will decrease it, (2) if NO₃^? is cotransported with H⁺, increases in pH_DBL would be greater under an OA treatment (pH = 7.65) than under an ambient treatment (pH = 8.00), and (3) decreases in pH_DBL will be smaller at pH 7.65 than at pH 8.00, as higher external [H⁺] might affect the strength of the diffusion gradient. Overall, N_i source did not affect the pH_DBL. However, increases in pH_DBL were greater at pH 7.65 than at pH 8.00. CO₂ uptake was higher at pH 7.65 than at pH 8.00, whereas HCO₃^? uptake was unaffected by pH. Photosynthesis and respiration control pH_DBL rather than N_i uptake. We suggest that under future OA, Macrocystis pyrifera will metabolically modify its surface microenvironment such that the physiological processes of photosynthesis and N_i uptake will not be affected by a reduced pH.     

Importance of AOX pathway in optimizing photosynthesis under high light stress: role of pyruvate and malate in activating AOX

The present study shows the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under high light (HL). The responses of photosynthesis and respiration were monitored as O₂ evolution and O₂ uptake in mesophyll protoplasts of pea pre‐incubated under different light intensities. Under HL (3000 µmol m^?2 s^?1), mesophyll protoplasts showed remarkable decrease in the rates of NaHCO₃‐dependent O₂ evolution (indicator of photosynthetic carbon assimilation), while decrease in the rates of respiratory O₂ uptake were marginal. While the capacity of AOX pathway increased significantly by two fold under HL, the capacity of cytochrome oxidase (COX) pathway decreased by >50% compared with capacities under darkness and normal light (NL). Further, the total cellular levels of pyruvate and malate, which are assimilatory products of active photosynthesis and stimulators of AOX activity, were increased remarkably parallel to the increase in AOX protein under HL. Upon restriction of AOX pathway using salicylhydroxamic acid (SHAM), the observed decrease in NaHCO₃‐dependent O₂ evolution or p‐benzoquinone (BQ)‐dependent O₂ evolution [indicator of photosystem II (PSII) activity] and the increase in total cellular levels of pyruvate and malate were further aggravated/promoted under HL. The significance of raised malate and pyruvate levels in activation of AOX protein/AOX pathway, which in turn play an important role in dissipating excess chloroplastic reducing equivalents and sustenance of photosynthetic carbon assimilation to balance the effects of HL stress on photosynthesis, was depicted as a model.     

THE EFFECT OF SEAWEED DIETS ON GROWTH OF GREEN ABALONE,HALIOTIS FULGENS,FROM BAJA CALIFORNIA,MEXICO

Two abalone species: green Haliotis fulgens and yellow Halioti corrugata represent nearly 97% of the total production in the Mexican abalone fishery. It has been assumed that abalone feed on the kelp algae Macrocystis pyrifera. Regional hatcheries use this species as a main source of natural food. M. pyrifera does not occur at the southern limit of the distribution of abalone species along the Baja California Peninsula. In this study, growth rates of juveniles H. fulgens, 17.3 ± 2.2 mm shell length and 0.4 ± 0.2 g body weight, were evaluated. Juveniles were fed with common species in the benthic environments inhabited by abalone along the western coast of Baja California during 191 days. Three diets were based on algae: palm kelp, Eisenia arborea, giant kelp, M. pyrifera and Gelidium robustum, and one on seagrass, Phyllospadix torreyi. Shell length and body growth rates varied between 21.5 μm day^?1 and 2.2 mg day^?1 for E. arborea and between 45.9 μm day^?1 and 6.7 mg day^?1 for M. pyrifera. Higher specific growth rates (SGR) in length and weight were determined for M. pyrifera: 0.2% and 0.7% day^?1. Significant differences between values of juveniles fed M. pyrifera with the rest of the diets were found. The highest mortality (21%) was in juveniles fed the red algae G. robustum.     

PHOTOSYNTHETIC RESPONSE OF THE GIANT KELP MACROCYSTIS PYRIFERA (PHAEOPHYCEAE) TO ULTRAVIOLET RADIATION1

Solar ultraviolet radiation (UVA + UVB) impairs photosynthesis in marine algae. Canopy blades of the giant kelp Macrocystis pyrifera (L.) C. Agardh are exposed to high levels of solar UV in the field. To determine the effects of UV radiation on photosynthesis in the giant kelp and to identify sites of UV damage, O₂ evolution, reaction center organization, light harvesting, and energy transfer efficiency were measured in canopy blades that had been exposed to elevated levels of UV in the laboratory. UV treatment reduced both the light-saturated rate and the light-limited rate of photosynthesis by 50% but produced no significant change in the rate of dark respiration. A significant impairment of photosystem II (PSII) reaction center function was observed, suggesting that PSII is a major site of damage in chromophytes. Reduced quantum efficiency of photosynthesis and loss of energy transfer from light-harvesting pigments (fucoxanthin, chlorophyll a, and chlorophyll c) to PSII indicate that the major light-harvesting complex of M. pyrifera, the fucoxanthin-chlorophyll protein complex (FCPC), was another site of UV damage. These measures provide the first evidence of a direct effect of UV radiation on specific sites in the photosynthetic apparatus of chromophytes and indicate that in situ fluorescence excitation analysis may be a simple means to detect UV stress in algae.     

CO2 is the main inorganic C species entering photosynthetically active leaf protoplasts of the freshwater macrophyte Ranunculus penicillatus ssp. pseudofluitans

Submerged aquatic macrophytes growing in water where free CO₂ is unavailable (above pH 8·2) must use mechanisms to supply external dissolved inorganic carbon in a form available to chloroplasts (CO₂). Active transport of HCO₃^– across the plasmalemma has not been proven to be widespread in aquatic macrophytes and catalytic conversion of HCO₃^– to CO₂ is the usual supply mechanism in submerged macrophytes. The interaction of leaf form and function in this respect was investigated in the linear, submerged leaves of Ranunculus penicillatus (Dumort.) Bab ssp. pseudofluitans (Syme) S.Webster. Viable protoplasts were isolated using a mixture of cell wall degrading enzymes optimized for this species. Protoplast viabilities greater than 80% after 5 h of isolation were achieved. Photosynthetic rates of isolated protoplasts were comparable with that of intact plant tissue. Results of carbon isotopic disequilibrium experiments showed that CO₂ was the preferred species of dissolved inorganic carbon for photosynthesis by protoplasts and that HCO₃^– which predominates in the plant’s natural environment mainly contributes by supplying CO₂ outside the cells.     

GAMETOPHYTIC GROWTH BY MACROCYSTIS PYRIFERA (PHAEOPHYTA) IN RESPONSE TO VARIOUS IRON AND ZINC CONCENTRATIONS1

Batch culturing experiments were conducted to examine effects on Macrocystis pyrifera (L.) C. A. Agardh gametophytic growth of various iron and zinc concentrations in the chemically defined, artificial seawater medium, Aquil. A least squares fit of experimental data represented the relative importance of these micronutrient effects. Optimal iron and zinc concentrations in Aquil were estimated at 340 nM (7 × 10^?11 nM as [Fe³⁺]) and 135 nM (6 × 10^?2 nM as [Zn²⁺]), respectively.