C. Barry Osmond

Two components of onset and recovery during photoinhibition of Ulva rotundata

Short-term (up to 5 h) transfers of shade-adapted (100 μmol · m−2 · s−1) clonal tissue of the marine macroalga Ulva rotundata Blid. (Chlorophyta) to higher irradiances (1700, 850, and 350 μmol · m−2 · s−1) led to photoinhibition of room-temperature chlorophyll fluorescence and O2 evolution. The ratio of variable to maximum (Fv/Fm) and variable (Fv) fluorescence, and quantum yield (ϕ) declined with increasing irradiance and duration of exposure. This decline could be resolved into two components, consistent with the separation of photoinhibition into energy-dissipative processes (photoprotection) and damage to photosystem II (PSII) by excess excitation. The first component, a rapid decrease in Fv/Fm and in Fv, corresponds to an increase in initial (Fo) fluorescence and is highly sensitive to 1 mM chloramphenicol. This component is rapidly reversible under dim (40 μmol · m−2 · s−1) light, but is less reversible with increasing duration of exposure, and may reflect damage to PSII. The second (after 1 h exposure) component, a slower decline in Fv/Fm and Fv with declining Fo, appears to be associated with the photoprotective interconversion of violaxanthin to zeaxanthin and is sensitive to dithiothreitol. The accumulation of zeaxanthin in U. rotundata is very slow, and may account for the predominance of increases in Fo at high irradiances.

Photoacclimation and photoinhibition in Ulva rotundata as influenced by nitrogen availability

Clonal tissue of the marine chlorophyte macroalga, Ulva rotundata Blid., was transferred from 100 to 1700 μmol photons · m−2 · s−1 under limiting (1.5 μM NH4+maximum, N/P=2) and sufficient (15 μM NH4+maximum, N/P=20) nitrogen supply at 18° C and 11 h light-13 h darkness daily. Photoinhibition was assayed by light-response curves (photosynthetic O2 exchange), and chlorophyll fluorescence at 77 K and room temperature. Daily surface-area growth rate (μSA) in N-sufficient plants increased sixfold over 3 d and was sustained at that level. During this period, respiration (Rd) doubled and light-saturated net photosynthesis capacity (Pm) increased by nearly 50%, indicating acclimation to high light. Quantum yield (ϕ) decreased by 25% on the first day, but recovered completely within one week. The ratio of variable to maximum fluorescence (Fv/Fm) also decreased markedly on the first day, because of an increase in initial fluorescence (Fo) and a decrease in Fm, and partially recovered over several days. Under the added stress of N deficiency, μSA accelerated fivefold over 4 d, despite chronic photoinhibition, then declined along with tissue-N. Respiration doubled, but Pm decreased by 50% over one week, indicating inability to acclimate to high light. Both ϕ and Fv/Fm decreased markedly on the first day and did not significantly recover. Changes in Fo, Fm and xanthophyll-cycle components indicate concurrent photodamage to photosystem II (PSII) and photoprotection by thermal deexcitation in the antenna pigments. Increasing μSA coincided with photoinhibition of PSII. Insufficient diel-carbon balance because of elevated Rd and declining Pm and tissue-N, rather than photochemical damage per se, was the apparent proximate cause of decelerating growth rate and subsequent tissue degeneration under N deficiency in U. rotundata.

Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress, and evidence for zeaxanthin-associated photoprotection

The function of photosystem (PS)II during desiccation and exposure to high photon flux density (PFD) was investigated via analysis of chlorophyll fluorescence in the desert resurrection plant Selaginella lepidophylla (Hook. and Grev.) Spring. Exposure of hydrated, physiologically competent stems to 2000 μmol · m−2 · s−1 PFD caused significant reductions in both intrinsic fluorescence yield (FO) and photochemical efficiency of PSII (FV/FM) but recovery to pre-exposure values was rapid under low PFD. Desiccation under low PFD also affected fluorescence characteristics. Both FV/FM and photochemical fluorescence quenching remained high until about 40% relative water content and both then decreased rapidly as plants approached 0% relative water content. In contrast, the maximum fluorescence yield (FM) decreased and non-photochemical fluorescence quenching increased early during desiccation. In plants dried at high PFD, the decrease in FV/FM was accentuated and FO was reduced, however, fluorescence characteristics returned to near pre-exposure values after 24-h of rehydration and recovery at low PFD. Pretreatment of stems with dithiothreitol, an inhibitor of zeaxanthin synthesis, accelerated the decline in FV/FM and significantly increased FO relative to controls at 925 μmol · m−2 · s−1 PFD, and the differences persisted over a 3-h low-PFD recovery period. Pretreatment with dithiothreitol also significantly decreased non-photochemical fluorescence quenching, increased the reduction state of QA, the primary electron acceptor of PSII, and prevented the synthesis of zeaxanthin relative to controls when stems were exposed to PFDs in excess of 250 μmol · m−2 · s−1. These results indicate that a zeaxanthin-associated mechanism of photoprotection exists in this desert pteridophyte that may help to prevent photoinhibitory damage in the fully hydrated state and which may play an additional role in protecting PSII as thylakoid membranes undergo water loss.

Contrasting patterns of photosynthetic acclimation and photoinhibition in two evergreen herbs from a winter deciduous forest

The relationship between the microclimate within an Oak-Hickory forest and photosynthetic characters of two resident evergreen herbs with contrasting leaf phenologies was investigated on a monthly basis for 1 full year. Heuchera americana has leaf flushes in the spring and fall, with average leaf life spans of 6–7 months. Hexastylis arifolia produces a single cohort of leaves each spring with a leaf life span of 12–13 months. We predicted that among evergreen plants inhabiting a seasonal habitat, a species for which the frequency of leaf turnover is greater than the frequency of seasonal extremes would have a greater annual range in photosynthetic capacity than a species that only produced a single flush of leaves during the year. Photosynthetic parameters, including apparent quantum yield, maximum photosynthetic capacity (Pmax), temperature of maximum photosynthesis, photochemical efficiency of PSII and leaf nitrogen (N) and chlorophyll concentrations, were periodically measured under laboratory conditions in leaves sampled from natural populations of both species. Mature leaves of both species acclimated to changing understory conditions with the mean seasonal differences being significantly greater for Heuchera than for Hexastylis. Area based maximum photosynthetic rates at 25°C were approximately 250% and 100% greater in winter leaves than summer leaves for Heuchera and Hexastylis respectively. Nitrogen concentrations were highest in winter leaves. Chlorophyll concentrations were highest in summer leaves. Low Pmax/N values for these species suggest preferential allocation of leaf nitrogen into non-photosynthetic pools and/or light-harvesting function at the expense of photosynthetic enzymes and electron transport components. Despite the increase in photosynthetic capacity, there was evidence of chronic winter photoinhibition in Hexastylis, but not in Heuchera. Among these ecologically similar species, there appears to be a trade-off between the frequency of leaf production and the balance of photosynthetic acclimation and photoinhibition.

INORGANIC CARBON LIMITATION OF PHOTOSYNTHESIS IN ULVA ROTUNDATA (CHLOROPHYTA)1

A computerized oxygen electrode Astern was used to make rapid and accurate measurements of photosynthetic light and dissolved inorganic carbon (DIC) response cures with a macroalga. Ulva rotundata Blid. was grown in an outdoor, continuous flow system in seawater under sunlight or 9% of sunlight at Beaufort, North Carolina. The light compensation points in the shade‐ and sun‐grown plants, measured in seawater, were at photon flux densities (PFDs) of 16 and 27 μmol. Photons·m−2·s−1, respectively but the quantum yield of O2 evolution was not significantly different. Rates of photosynthesis in seawater per unit area of thallus under saturating light and rates of dark respiration were about 1.5‐fold higher in sun‐ than in shade‐grown plants. The concentration of DIC in seawater (approximately 2 mM) limited photosynthesis at absorbed PFDs above 60–70 μmol photons·m−2·s−1 Addition of 20 mM inorganic carbon had no effect on quantum yield but caused about a 1.5‐fold increase in the light‐saturated photosynthetic rate in both shade‐ and sun‐grown Ulva. The effect of DIC supplementation was greatest in plants grown in October and least in plants grown in June. The light‐ and DIC‐saturated rate of photosynthesis in seawater was similar to the maximum rate obtained by exposing Ulva to 10% CO2, in the gas phase. The carbon isotope values (δ13C, reflecting the 13C/12C ratio compared to a standard) of Ulva grown in the same seawater supply were dependent on light and agitation. Samples from Beaufort Inlet were more negative (δ13C value, −20.03‰) than those grown in bright light with agitation (δ13C value, −17.78‰ outdoors; −17.23‰ indoors), which may indicate DIC supply limited carbon uptake in seawater.

Very high light resistant mutants of Chlamydomonas reinhardtii : Responses of Photosystem II, nonphotochemical quenching and xanthophyll pigments to light and CO 2

We have isolated very high light resistant nuclear mutants (VHLR) in Chlamydomonas reinhardtii, that grow in 1500–2000 μmol photons m−2 s−1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHLR mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L*) from which another class of VHLR mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHLR mutants was independent of CO2 availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 μmol photons m−2 s−1) was strongly depressed when all genotypes were grown in 5% CO2. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO2, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid ΔpH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO2 at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L* and VHLR mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO2, possibly because they were unable to maintain sufficiently high ΔpH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHLR phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHLR mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.

Biophysical, Biochemical, and Physiological Characterization ofChlamydomonas reinhardtii Mutants with Amino Acid Substitutions at the Ala251 Residue in the D1 Protein That Result in Varying Levels of Photosynthetic Competence

The QBbinding site of the D1 reaction center protein, located within a stromal loop between transmembrane helices IV and V formed by residues Ile219 to Leu272, is essential for photosynthetic electron transport through photosystem II (PSII). We have examined the function of the highly conserved Ala251D1 residue in this domain in chloroplast transformants ofChlamydomonas reinhardtii and found that Arg, Asp, Gln, Glu, and His substitutions are nonphotosynthetic, whereas Cys, Ser, Pro, Gly, Ile, Val, and Leu substitutions show various alterations in D1 turnover, photosynthesis, and photoautotrophic growth. The latter mutations reduce the rate of QA to QB electron transfer, but this is not necessarily rate-limiting for photoautotrophic growth. The Cys mutant divides and evolves O2 at wild type rates, although it has slightly higher rates of D1 synthesis and turnover and reduced electron transfer between QA and QB. O2 evolution, D1 synthesis, and accumulation in the Ser, Pro, and Gly mutants in high light is reduced, but photoautotrophic growth rate is not affected. In contrast, the Ile, Val, and Leu mutants are impaired in photoautotrophic growth and photosynthesis in both low and high light and have elevated rates of D1 synthesis and degradation, but D1 accumulation is normal. While rates of synthesis/degradation of the D1 protein are not necessarily correlated with alterations in specific parameters of PSII function in these mutants, bulkiness of the substituted amino acids is highly correlated with the dissociation constant for QB in the seven mutants examined. These observations imply that the Ala251 residue plays a key role in D1 protein.

Interpretations of gradients in δ13C value in thick photosynthetic tissues of plants with Crassulacean acid metabolism

In Ceropegia dichotoma, Crassula argentea, Esheveria colorata, Kalanchoë beharensis, Opuntia ficus-indica, Sansveria stuckyi and Opuntia inermis the carbon-isotope ratio (δ13C) of tissues close to the epidermis is 2–4.3‰ more negative than those in the centre of the leaf or cladode. The greatest change in δ13C value occurs between the epidermal layer and the layer of mesophyll tissue immediately underneath. Analysis of major metabolic and structural components in successive layers of Crassula argentea grown under controlled environmental conditions conducive to Crassulacean acid metabolism confirmed that much of the variation in δ13C values of bulk carbon is caused by differences in chemical composition. Thus the steep gradient in δ13C value at the epidermis reflects, in part, the contribution of more-negative δ13C values of lipids in these tissues. Moreover, during nocturnal CO2 fixation the amount of malic acid synthesised decreases with depth and the δ13C value of the methanol-soluble fraction is less negative with distance away from the upper epidermis. These results are consistent with diffusion limitation to CO2 uptake in these thick leaf tissues, which also contributes to the observed gradients in δ13C value.

The 18O of water in the metabolic compartment of transpiring leaves.

Publisher Summary Photosynthesis in terrestrial plants is invariably associated with transpiration of water with associated isotopic fractionation. Water at the site of photosynthetic metabolism is enriched in 18 O relative to the water that is transpired. Diffusion of water is the principal mechanism of mixing operating in a leaf, and this relatively slow process is opposed by a large net flux of liquid water to the evaporating surfaces. This chapter discusses a set of experiments done in the phytotron. The chapter delineates a series of corrections to earlier data sets. The corrections do not alter the conclusion that leaf metabolism in transpiring leaves of sunflower—Helianthus annuus cv. giant mammoth—occurs in a water fraction the oxygen isotopic composition of which is distinctly less positive than that predicted for water at the sites of evaporation. The isotopic exchange of CO 2 between the leaves and the atmosphere is dynamic and beset with species-dependent, capacitance-related effects on isotopic gradients within leaves.