Biochemical and physiological processes associated with the differential ozone response in ozone-tolerant and sensitive soybean genotypes.

Biochemical and physiological traits of two soybean [Glycine max (L.) Merr.] genotypes differing in sensitivity to ozone (O3 ) were investigated to determine the possible basis for the differential response. Fiskeby III (O3 -tolerant) and Mandarin (Ottawa) (O3 -sensitive) were grown in a greenhouse with charcoal-filtered air for 4 weeks, then treated with O3 for 7 h·day(-1) in greenhouse chambers. Mandarin (Ottawa) showed significantly more leaf injury and hydrogen peroxide (H2 O2 ) and superoxide (O2 (-) ) production compared with Fiskeby III. Peroxidase activity in Mandarin (Ottawa) was 31% higher with O3 but was not significantly different in Fiskeby III. Ozone did not affect superoxide dismutase or glutathione reductase activities, or leaf concentrations of glutathione or ascorbic acid. Thus, variation in O3 response between Fiskeby III and Mandarin (Ottawa) was not explained by differences in antioxidant enzymes and metabolites tested. Ethylene emission from leaves declined in Fiskeby III following O3 exposure but not in Mandarin (Ottawa). Ozone exposure reduced quantum yield (ΦPSII ), electron transport rate (ETR) and photochemical quenching (qp ) in Mandarin (Ottawa) more than in Fiskeby III, indicating that efficiency of energy conversion of PSII and photosynthetic electron transport was altered differently in the two genotypes. Short-term exposure to O3 had minimal effects on net carbon exchange rates of both soybean cultivars. A trend toward higher stomatal conductance in Mandarin (Ottawa) suggested stomatal exclusion might contribute to differential O3 sensitivity of the two genotypes. Increased sensitivity of Mandarin (Ottawa) to O3 was associated with higher H2 O2 and O2 (-) production compared with Fiskeby III, possibly associated with genotype differences in stomatal function or regulation of ethylene during the initial phases of O3 response.

Perchlorate content of plant foliage reflects a wide range of species-dependent accumulation but not ozone-induced biosynthesis.

Perchlorate (ClO4(-)) interferes with uptake of iodide in humans. Emission inventories do not explain observed distributions. Ozone (O3) is implicated in the natural origin of ClO4(-), and has increased since pre-industrial times. O3 produces ClO4(-)in vitro from Cl(-), and plant tissues contain Cl(-) and redox reactions. We hypothesize that O3 exposure may induce plant synthesis of ClO4(-). We exposed contrasting crop species to environmentally relevant O3 concentrations. In the absence of O3 exposure, species exhibited a large range of ClO4(-) accumulation but there was no relationship between leaf ClO4(-) and O3, whether expressed as exposure or cumulative flux (dose). Older, senescing leaves accumulated more ClO4(-) than younger leaves. O3 exposed vegetation is not a source of environmental ClO4(-). There was evidence of enhanced ClO4(-) content in the soil surface at the highest O3 exposure, which could be a significant contributor to environmental ClO4(-).

High ozone increases soil perchlorate but does not affect foliar perchlorate content.

Ozone (O) is implicated in the natural source inventory of ClO, a hydrophilic salt that migrates to groundwater and interferes with the uptake of iodide in mammals, including humans. Tropospheric O is elevated in many urban and some rural areas in the United States and globally. We previously showed that controlled O exposure at near-ambient concentrations (up to 114 nL L, 12-h mean) did not increase foliar ClO. Under laboratory conditions, O has been shown to oxidize Cl to ClO. Plant tissues contain Cl and exhibit responses to O invoking redox reactions. As higher levels of O are associated with stratospheric incursion and with developing megacities, we have hypothesized that exposure of vegetation to such elevated O may increase foliar ClO. This would contribute to ClO in environments without obvious point sources. At these high O concentrations (up to 204 nL L, 12-h mean; 320 nL L maximum), we demonstrated an increase in the ClO concentration in surface soil that was linearly related to the O concentration. There was no relationship of foliar ClO with O exposure or dose (stomatal uptake). Accumulation of ClO varied among species at low O, but this was not related to soil surface ClO or to foliar ClO concentrations following exposure to O. These data extend our previous conclusions to the highest levels of plausible O exposure, that tropospheric O contributes to environmental ClO through interaction with the soil but not through increased foliar ClO.

Demonstration of a diel trend in sensitivity of Gossypium to ozone: a step toward relating O3 injury to exposure or flux.

Plant injury by ozone (O3) occurs in three stages, O3 entrance through stomata, overcoming defences, and attack on bioreceptors. Concentration, deposition, and uptake of O3 are accessible by observation and modelling, while injury can be assessed visually or through remote sensing. However, the relationship between O3 metrics and injury is confounded by variation in sensitivity to O3. Sensitivity weighting parameters have previously been assigned to different plant functional types and growth stages, or by differentially weighting O3 concentrations, but diel and seasonal variability have not been addressed. Here a plant sensitivity parameter (S) is introduced, relating injury to O3 dose (uptake) using three independent injury endpoints in the crop species, Pima cotton (Gossypium barbadense). The diel variability of S was determined by assessment at 2h intervals. Pulses of O3 (15 min) were used to assess passive (constitutive) defence mechanisms and dose was used rather than concentration to avoid genetic or environmental effects on stomatal regulation. A clear diel trend in S was apparent, with maximal sensitivity in mid-afternoon, not closely related to gas exchange, whole leaf ascorbate, or total antioxidant capacity. This physiologically based sensitivity parameter provides a novel weighting factor to improve modelled relationships between either flux or exposure to O3, and O3 impacts. This represents a substantial improvement over concentration- or phenology-based weighting factors currently in use. Future research will be required to characterize the variability and metabolic drivers of diel changes in S, and the performance of this parameter in prediction of O3 injury.

Functional role of anthocyanins in high-light winter leaves of the evergreen herb Galax urceolata.

High-light leaves of the evergreen herb Galax urceolata exhibit a striking color change from green to red during winter months due to anthocyanin synthesis in outermost mesophyll cells. Here we investigate three possible functions of this color change. To test the hypothesis that anthocyanins function as light attenuators, maximum photosystem II efficiency (F(v)/F(m)) of red and green leaves was measured during and after exposure to wavelengths either strongly or poorly absorbed by anthocyanin. To determine whether anthocyanins elevate radical-scavenging capacity, antioxidant activity of red and green leaves was assessed using the alpha,alpha-diphenyl-beta-picrylhydrazyl assay. Nonstructural carbohydrate levels were analyzed to test the hypothesis that anthocyanins function as a carbon sink. Declines in F(v)/F(m) under white and green light were significantly greater for green than red leaves, but were comparable under red light. Anthocyanin content positively correlated with antioxidant activity. Although levels of anthocyanins did not appear to be related to nonstructural carbohydrate concentration, high levels of sugars may be necessary for their photo-induction. Results suggest that anthocyanins function as light attenuators and may also contribute to the antioxidant pool in winter leaves.

Growth and yield responses of snap bean to mixtures of carbon dioxide and ozone.

Elevated CO2 concentrations expected in the 21st century can stimulate plant growth and yield, whereas tropospheric O3 suppresses plant growth and yield in many areas of the world. Recent experiments showed that elevated CO2 often protects plants from O3 stress, but this has not been tested for many important crop species including snap bean (Phaseolus vulgaris L.). The objective of this study was to determine if elevated CO2 protects snap bean from O3 stress. An O3-tolerant cultivar (Tenderette) and an O3-sensitive selection (S156) were exposed from shortly after emergence to maturity to mixtures of CO2 and O3 in open-top field chambers. The two CO2 treatments were ambient and ambient with CO2 added for 24 h d(-1) resulting in seasonal 12 h d(-1) (0800-2000 h EST) mean concentrations of 366 and 697 microL L(-1), respectively. The two O3 treatments were charcoal-filtered air and nonfiltered air with O3 added for 12 h d(-1) to achieve seasonal 12 h d(-1) (0800-2000 h EST) mean concentrations of 23 and 72 nL L(-1), respectively. Elevated CO2 significantly stimulated growth and pod weight of Tenderette and S156, whereas elevated O3 significantly suppressed growth and pod weight of S156 but not of Tenderette. The suppressive effect of elevated O3 on pod dry weight of S156 was approximately 75% at ambient CO2 and approximately 60% at elevated CO2 (harvests combined). This amount of protection from O3 stress afforded by elevated CO2 was much less than reported for other crop species. Extreme sensitivity to O3 may be the reason elevated CO2 failed to significantly protect S156 from O3 stress.

Identification of a novel isoform of the chloroplast-coupling factor alpha-subunit.

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF1) alpha-subunit. Antibodies for the 64-kD protein also recognized the alpha-subunit of CF1. Both the 64-kD protein and the 61-kD CF1 alpha-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF1 alpha-subunit. The 64-kD protein accumulated in response to high irradiance (1000 mumol photons m-2 s-1) and declined in response to low irradiance (80 mumol photons m-2 s-1) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF1 alpha-subunit found only in monocots. Analysis of purified CF1 complexes showed that the 64-kD protein represented up to 15% of the total CF1 alpha-subunit.

Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast.

Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll](-1) s(-1). Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I](-1). In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll](-1) s(-1), respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I](-1). No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b6f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.

Effects of natural shade on soybean thylakoid membrane composition.

The effect of natural shade on chloroplast thylakoid membrane activity and composition was examined for soybean (Glycine Max. cv. Young) grown under field conditions. Plots with high (10 plants m(-1) row) or low (1 plant m(-1) row) plant density were established. Expanding leaves were tagged at 50, 58 and 65 days after planting (DAP). At 92 DAP, tagged leaves were used as reference points to characterize canopy light environments and isolate thylakoid membranes. Light environments ranged from a photosynthetic photon flux density (PPFD) of 87% of full sun to a PPFD of 10% of full sun. The decline in PPFD was accompanied by an increase in the far-red/red (735 nm/645 nm) ratio from 0.9 to approximately six. The major effects of shade on chloroplast thylakoid membranes were a reduction in chloroplast coupling factor and a shift in light-harvesting capacity from Photosystem I to Photosystem II. Photosynthetic electron transport capacity was not affected by differences in PPFD, but was 20 to 30% higher in the 1 plant m(-1) row treatment. The plant density effect on electron transport was associated with differences in plastocyanin concentration, suggesting that plastocyanin is a limiting factor in soybean. Shade did not have a significant effect on the concentration of Photosystem II, Cyt b6f, or Photosystem I complexes.

Effect of growth irradiance on plastocyanin levels in barley.

Plastocyanin levels in barley (Hordeum vulgare cv Boone) were found to be dependent on growth irradiance. An immunochemical assay was developed and used to measure the plastocyanin content of isolated thylakoid membranes. Barley grown under 600 µmole photons m(-2)s(-1) contained two- to four-fold greater quantities of plastocyanin per unit chlorophyll compared with plants grown under 60 µmole photons m(-2)s(-1). The plastocyanin/Photosystem I ratio was found to be 2 to 3 under high irradiance compared with 0.5 to 1.5 under low irradiance. The reduced plastocyanin pool size in low light plants contributed to a two-fold reduction in photosynthetic electron transport activity. Plastocyanin levels increased upon transfer of low light plants to high irradiance conditions. In contrast, plastocyanin levels were not affected in plants transferred from high to low irradiance, suggesting that plastocyanin is not involved in the acclimation of photosynthesis to shade.

Novel light-regulated chloroplast thylakoid membrane protein.

A 64 kilodalton chloroplast membrane polypeptide was dependent on growth irradiance with 10-fold greater quantities of the protein present in barley (Hordeum vulgare) grown under 500 micromoles of photons per square meter per second compared with growth at 50 micromoles per square meter per second. The concentration of the protein was sensitive to changes in irradiance, with a slow time course for the response (days) similar to other reported light acclimation processes. The polypeptide also was observed in maize (Zea mays), oats (Avena sativa), and wheat (Triticum aestivum), but not in soybean (Glycine max Merr). The 64 kilodalton polypeptide did not correspond to any thylakoid membrane protein with an assigned function, so its structural or regulatory role is not known.

Physiological effects of sublethal atrazine on barley chloroplast thylakoid membranes.

This study was conducted to more clearly define the physiological effects of PS II herbicides on chloroplast thylakoid membrane activity and composition. Barley (Hordeum vulgare L. cv Boone) was grown in hydroponic culture at 20°C in a growth chamber with a light intensity of 500 µmole photons m(-2) s(-1). Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine), a Photosystem II herbicide, was supplied continuously via the roots to 7-day-old plants. Atrazine concentrations greater than 0.07 ppm (0.32 µM) were associated with decreased leaf chlorophyll (chl), lowered chl a/b ratio, inhibition of chloroplast electron transport, and plant death within 1 to 2 weeks. Atrazine at 0.07 ppm was defined as sublethal because no toxic effects were observed. Sublethal atrazine induced a decrease in chl a/b ratio with no effect on leaf chl content. Photosynthetic electron transport was either unaffected in fully expanded leaves or slightly stimulated in expanding leaves by treatment of intact plants with 0.07 ppm atrazine. The major effect of sublethal atrazine was on the chl-protein complex composition. Sublethal atrazine increased the level of the Photosystem II light-harvesting complex (LHC-II) and lowered the level of the CP1a Photosystem I complex relative to controls. The numbers of Photosystem II and Photosystem I reaction centers and cytochrome b 6/f complexes per unit chl were not affected by sublethal atrazine. The overall result was an atrazine-induced redistribution of light-harvesting chl from Photosystem I to Photosystem II with no effect on the number of thylakoid membrane-protein complexes associated with electron transport.

Response of soybean photosynthesis and chloroplast membrane function to canopy development and mutual shading.

The effect of natural shading on photosynthetic capacity and chloroplast thylakoid membrane function was examined in soybean (Glycine max. cv Young) under field conditions using a randomized complete block design. Seedlings were thinned to 15 plants per square meter at 20 days after planting. Leaves destined to function in the shaded regions of the canopy were tagged during early expansion at 40 days after planting. To investigate the response of shaded leaves to an increase in available light, plants were removed from certain plots at 29 or 37 days after tagging to reduce the population from 15 to three plants per square meter and alter the irradiance and spectral quality of light. During the transition from a sun to a shade environment, maximum photosynthesis and chloroplast electron transport of control leaves decreased by two- to threefold over a period of 40 days followed by rapid senescence and abscission. Senescence and abscission of tagged leaves were delayed by more than 4 weeks in plots where plant populations were reduced to three plants per square meter. Maximum photosynthesis and chloroplast electron transport activity were stabilized or elevated in response to increased light when plant populations were reduced from 15 to three plants per square meter. Several chloroplast thylakoid membrane components were affected by light environment. Cytochrome f and coupling factor protein decreased by 40% and 80%, respectively, as control leaves became shaded and then increased when shaded leaves acclimated to high light. The concentrations of photosystem I (PSI) and photosystem II (PSII) reaction centers were not affected by light environment or leaf age in field grown plants, resulting in a constant PSII/PSI ratio of 1.6 +/- 0.3. Analysis of the chlorophyll-protein composition revealed a shift in chlorophyll from PSI to PSII as leaves became shaded and a reversal of this process when shaded leaves were provided with increased light. These results were in contrast to those of soybeans grown in a growth chamber where the PSII/PSI ratio as well as cytochrome f and coupling factor protein levels were dependent on growth irradiance. To summarize, light environment regulated both the photosynthetic characteristics and the timing of senescence in soybean leaves grown under field conditions.

Acclimation of barley to changes in light intensity: chlorophyll organization.

Barley seedlings (Hordeum vulgare L. cv. Boone) were grown at 20°C with a 16h/8h light/dark cycle of either high (H) intensity (550 µmole m(-2) s(-1)) or low (L) intensity (55 µmole m(-2) s(-1)) white light. Plants were transferred from high to low (H → L) or low to high (L → H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod and a 3 cm apical segment removed for analysis. H control plants had greater chlorophyll (Chl) per leaf area and higher Chl a/b ratios than L controls. Analysis of Chl-protein complexes revealed that H and L plants had the same percentage of total Chl (62-65%) associated with Photosystem II (PS II), but that the organization of Chl within PS II was different. H plants contained lower levels of light-harvesting complex (LHC-II) and higher levels of the PS II complex CPa compared with L plants. Leaf Chl content and Chl organization within PS II were sensitive to changes in light intensity. In H → L plants, leaf Chl content decreased, Chl a/b ratio decreased, and a redistribution of Chl from CPa to LHC-II occurred during acclimation to low light. Acclimation of L → H plants to high light involved an increase in leaf Chl content, an increase in Chl a/b ratio, and a decrease in LHC-II. In contrast, the level of photosystem I related Chl-protein complexes (CP1 + CP1a) was similar in all light treatments. The light acclimation process occurred slowly over a period of 6 to 8 d in H → L and L → H plants.

Acclimation of barley to changes in light intensity: photosynthetic electron transport activity and components.

Barley seedlings (Hordeum vulgare L. Boone) were grown at 20°C with 16 h/8 h light/dark cycle of either high (H) intensity (500 µmole m(-2) s(-1)) or low (L) intensity (55 µmole m(-2) s(-1)) white light. Plants were transferred from high to low (H → L) and low to high (L → H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod. Thylakoid membranes were isolated from 3 cm apical segments and assayed for photosynthetic electron transport, Photosystem II (PS II) atrazine-binding sites (QB), cytochrome f(Cytf) and the P-700 reaction center of Photosystem I (PS I). Whole chain, PS I and PS II electron transport activities were higher in H than in L controls. QB and Cytf were elevated in H plants compared with L plants. The acclimation of H → L plants to low light occurred slowly over a period of 7 days and resulted in decreased whole chain and PS II electron transport with variable effects on PS I activity. The decrease in electron transport of H → L plants was associated with a decrease in both QB and Cytf. In L → H plants, acclimation to high light occurred slowly over a period of 7 days with increased whole chain, PS I and PS II activities. The increase in L → H electron transport was associated with increased levels of QB and Cytf. In contrast to the light intensity effects on QB levels, the P-700 content was similar in both control and transferred plants. Therefore, PS II/PS I ratios were dependent on light environment.

Light intensity regulates the accumulation of the major light-harvesting chlorophyll-protein in greening seedlings.

Etiolated pea (Pisum sativum [L.] cv Progress 9) and barley (Hordeum vulgare [L.] cv Boone) seedlings greened under either low (40 microeinsteins per square meter per second) or high (550 microeinsteins per square meter per second) intensity light were analyzed for chlorophyll (Chl) content and the levels of mRNA and protein for the major light-harvesting chlorophyll (Chl)-protein of photosystem II (LHC-II). Low intensity plants accumulated Chl more rapidly than high intensity plants. Both single radial immunodiffusion analysis and mild sodium dodecyl sulfate-polyacrylamide gel electrophoresis green gels showed that low intensity plants also accumulated LHC-II protein more rapidly than high intensity plants, following a kinetic pattern similar to the total Chl data. In contrast, LHC-II mRNA levels appeared to be independent of LHC-II protein levels although pea and barley LHC-II mRNA exhibited different light intensity responses. The absence of coordination between LHC-II mRNA and protein levels suggested that the biosynthesis of LHC-II in greening seedlings is not limited by mRNA. A correlation (better than the 0.01 significance level) between LHC-II protein accumulation and Chl accumulation was found for both pea and barley. The accumulation of LHC-II protein was not linked to the development of photosynthetic electron transport. These results and the similar effect of light intensity on Chl content and LHC-II protein levels suggested that the availability of Chl may limit LHC-II protein accumulation in greening seedlings.

Regulation of light-harvesting chlorophyll protein biosynthesis in greening seedlings : a species comparison.

The biosynthesis of the chlorophyll a/b binding protein associated with photosystem II (LHC-II) was characterized during light-induced greening of etiolated barley (Hordeum vulgare [L.] cv Boone), maize (Zea mays [L.] Pioneer 3148), pea (Pisum sativum [L.] cv Progress 9), and soybean (Glycine max [L.] Merr. cv Ransom 2). Northern blot analysis revealed that pea LHC-II mRNA was present in dark-grown seedlings and accumulated rapidly within 1 hour following illumination with white light. In contrast, the accumulation of LHC-II mRNA was delayed in barley and soybean until 2 to 4 hours after illumination began. Single radial immunodiffusion analysis revealed that LHC-II polypeptides began to accumulate in all species between 4 and 8 hours although the protein was present in detectable levels at earlier times in certain species. In a pattern similar to the LHC-II protein accumulation, chlorophyll accumulated at increased rates between 4 and 8 hours of greening in all species following an initial delay. The absence of coordination between LHC-II mRNA and LHC-II protein accumulation that was clearly observed in pea suggested that transcription is not the factor that limits LHC-II complex formation during chloroplast development. The accumulation of chlorophyll and LHC-II protein appeared to coincide suggesting that chlorophyll biosynthesis may be a factor that limits LHC-II complex formation.

Chlorophyll-protein complex composition during chloroplast development: A species comparison.

Barley, maize, pea, soybean, and wheat exhibited differences in chlorophyll a/b ratio and chlorophyll-protein (CP) complex composition during the initial stages of chloroplast development. During the first hours of greening, the chlorophyll a/b ratios of barley, pea, and wheat were high (a/b≥8) and these species contained only the CP complex of photosystem I as measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. A decrease in chlorophyll a/b ratio and the observation of the CP complexes associated with photosystem II and the light-harvesting apparatus occurred at later times in barley, pea, and wheat. In contrast, maize and soybean exhibited low chlorophyll a/b ratios (a/b<8) and contained the CP complexes of both photosytem I and the light-harvesting apparatus at early times during chloroplast development. The species differences were not apparent after 8 h of greening. In all species, the CP complexes were stabilized during the later stages of chloroplast development as indicated by a decrease in the percentage of chlorophyll released from the CP complexes during detergent extraction. The results demonstrate that CP complex synthesis and accumulation during chloroplast development may not be regulated in the same way in all higher plant species.

Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings.

The time course for the observation of intact chlorophyll-protein (CP) complexes during barley chloroplast development was measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. The procedure required extraction of thylakoid membranes with sodium bromide to remove extrinsic proteins. During the early stages of greening, the proteins extracted with sodium bromide included polypeptides from the cell nucleus that associate with developing thylakoid membranes during isolation and interfere with the separation of CP complexes by electrophoresis. Photosystem I CP complexes were observed before the photosystem II and light-harvesting CP complexes during the initial stages of barley chloroplast development. Photosystem I activity was observed before the photosystem I CP complex was detected whereas photosystem II activity coincided with the appearance of the CP complex associated with photosystem II. Throughout chloroplast development, the percentage of the total chlorophyll associated with photosystem I remained constant whereas the amount of chlorophyll associated with photosystem II and the light-harvesting complex increased. The CP composition of thylakoid membranes from the early stages of greening was difficult to quantitate because a large amount of chlorophyll was released from the CP complexes during detergent extraction. As chloroplast development proceeded, a decrease was observed in the amount of chlorophyll released from the CP complexes by detergent action. The decrease suggested that the CP complexes were stabilized during the later stages of development.