Leaf morphological and physiological adjustments to the spectrally selective shade imposed by anthocyanins in Prunus cerasifera.

Prunus domestica L. has green leaves, whereas Prunus cerasifera Ehrh. var. atropurpurea has red leaves due to the presence of mesophyll anthocyanins. We compared morphological and photosynthetic characteristics of leaves of these species, which were sampled from shoots grafted in pairs on P. domestica rootstocks, each pair comprising one shoot of each species. Two hypotheses were tested: (1) anthocyanins protect red leaves against photoinhibition; and (2) red leaves display shade characteristics because of light attenuation by anthocyanins. Parameters were measured seasonally, during a period of increasing water stress, which caused a similar drop in shoot water potential in each species. As judged by predawn measurements of maximum PSII yield, chronic photoinhibition did not develop in either species and, despite the anthocyanic screen, the red leaves of P. cerasifera displayed lower light-adapted PSII yields and higher non-photochemical quenching than the green leaves of P. domestica. Thus, it appears that, in this system, anthocyanins afford little photoprotection. As predicted by the shade acclimation hypothesis, red leaves were thinner and had a lower stomatal frequency, area- based CO2 assimilation rate, apparent carboxylation efficiency and chlorophyll a:b ratio than green leaves. However, red leaves were similar to green leaves in conductivity to water vapor diffusion, dry-mass-based chlorophyll concentrations and carotenoid:chlorophyll ratios. The data for red leaves indicate adaptations to a green-depleted, red-enriched shade, rather than a neutral or canopy-like shade. Thus, green light attenuation by anthocyanins may impose a limitation on leaf thickness. Moreover, the selective depletion of light at wavelengths that are preferentially absorbed by PSII and chlorophyll b may lead to adjustments in chlorophyll pigment ratios to compensate for the uneven spectral distribution of internal light. The apparent photosynthetic cost associated with lost photons and reduced leaf thickness, and the absence of a photoprotective advantage, suggest that there are other, yet to be identified, benefits for permanently anthocyanic leaves of P. cerasifera.

Evidence for active cyclic electron flow in twig chlorenchyma in the presence of an extremely deficient linear electron transport activity.

Fast and slow chlorophyll fluorescence induction curves at high and low actinic visible light, post-illumination changes in fluorescence yield and reflectance changes at 820 nm induced by far-red light were used to characterize the state of PSII and PSI and their electron transport capabilities in chlorophyllous twig cortices of Eleagnus angustifolius L., while corresponding leaves served as controls. Twigs displayed low dark-adapted PSII photochemical efficiencies and particularly low linear electron transport rates when illuminated. In addition, their PSII population was characterized by a high proportion of inactive, non-Q(B)-reducing centers and an incomplete quenching of fluorescence during the slow induction phase. It is suggested that PSII in twigs is an inefficient electron donor to PSI and/or the reductive pentose phosphate cycle. Yet, in spite of this apparent PSII deficiency, pools of intermediate electron carriers and potential PSI activity were more than sufficient to support the observed linear electron transport rates. Moreover, the rate of PSI reduction upon far-red/dark transitions and the magnitude of fluorescence yield increase upon white light/dark transitions were compatible with an efficient electron flow to PSI from stromal donors in the absence of PSII activity. We conclude that corticular chlorenchyma may be actively engaged in cyclic at the expense of a linear electron flow and discuss the possible physiological significance of this finding in conjunction with the particular microenvironmental conditions encountered within twigs.

The importance of being hairy: the adverse effects of hair removal on stem photosynthesis of Verbascum speciosum are due to solar UV-B radiation.

• Here the hypothesis was tested that hairs over photosynthetic tissues afford protection against excess radiation. • The hair mat covering the epidermis of photosynthetic stems of Verbascum speciosum was easily removed by mild mechanical treatment. Thus, optical properties and chlorophyll fluorescence parameters were studied in the field in intact and de-haired stems, receiving ambient, ambient minus UV-B, or ambient minus total UV radiation through selective cut-off filters. • Optical analysis indicated that de-hairing exposes underlying tissues to appreciably higher photon fluxes in the UV but slightly higher in the visible part of the spectrum. Under full solar radiation, hair removal resulted in considerable suppression of photosynthetic electron transport rates, yet this negative effect was completely abolished by excluding the UV-B radiation band. • It is concluded that the adverse effects of hair removal on stem photosynthesis of this plant are exclusively due to UV-B radiation. This is the first field confirmation of the protective role of hairs against UV-B radiation damage.

Seasonal fluctuations in the concentration of UV-absorbing compounds in the leaves of some Mediterranean plants under field conditions.

Leaves of 14 representative Mediterranean plant species were collected on a monthly basis and assayed for UV-absorbing compounds concentration, either on an area or a dry mass basis, from 1995 to 1997. Strong seasonal fluctuations were observed in eight species (all evergreens, two phrygana, one deciduous, one summer perennial and one winter perennial). Two different patterns of changing concentrations of UV-absorbing compounds were observed. In the first, concentration of these compounds was higher in young developing leaves and concentration declined during maturation, whereas in other plants, the opposite trend was observed. These differences could be attributed to the particular leaf surface morphology of each plant. The observed seasonal fluctuations of UV-absorbing compounds seem to be more correlated to developmental processes, than to seasonal fluctuations of the naturally occurring UV-B radiation. Most of the winter perennials did not show strong fluctuations during the period of development. The concentration of these compounds varied not only on a seasonal basis among the examined plants, but between different life forms as well: during winter, examination of the leaves of 13 species showed that evergreen sclerophylls and phrygana had at least two-fold higher concentration of UV-B-absorbing compounds on a leaf area basis than winter perennials. In addition, during the same season and irrespective of life form and species, the absorbance at 300 nm per unit of mature leaf area followed an asymptotic exponential decrease when specific leaf area increased. The UV-B radiation screening capacity of the leaves of these plants is discussed in relation to each adaptive strategy.

Improved flower advertisement, pollinator rewards and seed yield by enhanced UV-B radiation in the Mediterranean annual Malcolmia maritima.

• The effects of enhanced UV-B radiation on reproductive and pollination success were investigated in the Mediterranean annual Malcolmia maritima. • Plants were exposed in the field to ambient or ambient plus supplemental UV-B radiation (biologically equivalent to a 15% ozone depletion over Patras, Greece, 38°14' N, 21°44' E) up to leaf senescence and fruit maturation. • UV-B radiation had no effect on stem and fruit biomass, anthesis time and duration and flower number. However, flower diameter, nectary volume and nectar amount per flower (but not nectar concentration) were significantly increased by supplemental UV-B radiation. In addition UV-B treated plants showed higher reproductive success (i.e. lower abortion rates and higher fruit to flower ratio) and a trend to higher pollination success (i.e. increased number of seeds per fruit). As a result, the seed yield was increased. Seed mass, seed germination and early seedling growth were not affected by UV-B treatment of mother plants. • It is suggested that the UV-B induced changes in flower attributes might have affected pollinators' behaviour in a way that improved the fitness of M. maritima.

Effects of UV-B radiation on Olea europaea: comparisons between a greenhouse and a field experiment.

We compared growth, morphology, anatomy and physiology of field-grown Olea europaea L. plants after one year with or without supplemental UV-B radiation equivalent to the increase at ground level resulting from a 15% depletion in stratospheric ozone concentration over Patras, Greece (38.3 degrees N, 29.1 degrees E). In a parallel greenhouse experiment, plants were exposed for four months to either zero UV-B radiation or UV-B radiation equivalent to that of the supplemented outdoor treatment. In the field, the only significant changes in response to supplemental UV-B radiation were an increase in adaxial epidermal thickness and a decrease in total protein concentration. In the greenhouse-grown plants, UV-B radiation caused significant increases in abaxial cuticle thickness and trichome UV-B absorbing compounds. We conclude that Olea europaea exhibits high UV-B tolerance and will not be affected by the predicted increases in UV-B radiation.

Assaying for pyruvate, orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2-0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.

Effects of glycerol on the in vitro stability and regulatory activation/inactivation of pyruvate, orthophosphate dikinase of Zea mays L.

Glycerol stabilizes the activity of pyruvate, orthophosphate dikinase extracted from darkened or illuminated maize leaves. It serves as a better protectant of activity than dithiothreitol for the active day-form and the glycerol concentration needed for full protection is inversely related to the level of protein. The night-form of the enzyme is also protected by glycerol not only against inactivation, but also against partial reactivation in storage. Glycerol does not prevent the Pi-dependent activation nor the ADP-dependent inactivation of pyruvate, orthophosphate dikinase, but the rates of both processes are substantially decreased. The ability of the inactive night-form for Pi-dependent activation is also sustained by glycerol for at least 2 h at 20°C, apparently through stabilization of the labile regulatory protein.

Regulation of C4-phosphoenolpyruvate carboxylase activity by ambient CO2.

Light activation of phosphoenolpyruvate carboxylase from the leaves of the C4 plant Setaria verticillata (L.) is more pronounced at low CO2 levels. The 2-fold activation observed at physiological ambient CO2 becomes 3.64-fold at 5 µL/L and completely abolished above 700 µL/L. When the stomata close under the influence of abscisic acid at 330 µL/L CO2, the extent of light activation is high (3.59-fold), probably because the increased diffusive resistance keeps the internal CO2 at much lower levels. Under darkness. CO2 and absicisic acid do not affect the extractable phosphoenolpyruvate carboxylase activity. Internal CO2 levels may determine phosphoenolpyruvate concentratio in the cytoplasm through the control of its utilization by phosphoenolpyruvate carboxylase. We have recently proposed (Samaras et al. 1988) that photosynthetically produced phosphoenolpyruvate could be an activator of the enzyme. It is therefore suggested that CO2 indirectly affects the activation state of phosphoenolpyruvate carboxylase by controlling the levels of phosphoenolpyruvate which may act as an activator.

Artifacts in the assay of maize leaf phosphoenolpyruvate carboxylase activity due to its instability.

When the assay of maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity is started with phosphoenolpyruvate, much lower reaction rates are obtained as compared to the enzyme-initiated reaction. The difference is due to the lability of the dilute enzyme in the absence of its substrate and is increased with incubation time in the absence of substrate or stabilizers. The activation of the enzyme by glucose-6-phosphate is overestimated with the substrate-initiated assay since a part of the apparent activation is due to stabilization of the enzymic activity by this effector during the minus-substrate preincubation. In contrast, the inhibitory effect of malate is underestimated when the reaction is started with the substrate. The enzyme-initiated assay is recommended provided that the necessary corrections for apparent activity in the absence of substrate and for inactivation during the assay at low substrate levels are made.

Effects of temperature and photosynthetic inhibitors on light activation of C4-phosphoenolpyruvate carboxylase.

Phosphoenolpyruvate carboxylase from leaves of the C4 plant Setaria verticillata (L.) Beauv. is activated by light; day levels of activity are reached after 30 minutes of illumination. Photoactivation is prevented by inhibitors of photosynthetic electron flow or of photophosphorylation and by D,L-glyceraldehyde, which inhibits the reductive pentose phosphate pathway.Although the extractable activity in the dark is not affected by temperature the photoactivation is prevented when both illumination and extraction are done under low temperature (5 C). High temperature (30 C) during either illumination or extraction is needed for activation. Once the enzyme is photoactivated at 30 C, a transfer of the leaves to 5 C does not abolish the extra activity.The results suggest that both unimpaired electron flow and photophosphorylation are prerequisites for the activation of phosphoenolpyruvate carboxylase. Low temperature apparently suppresses either the transport to the cytoplasm of a photosynthetic intermediate or the activating reaction itself. The inclusion of phosphoenolpyruvate in the extraction medium increases the night activity.On the basis of the available information, it is suggested that phosphoenolpyruvate could be the activator in vivo. In that case, the activation of phosphoenolpyruvate carboxylase would depend on internal CO2 level and prior photoactivation of both pyruvate, orthophosphate, dikinase and NADP malate dehydrogenase.

Cooperative Effects of Light and Temperature on the Activity of Phosphoenolpyruvate Carboxylase from Amaranthus paniculatus L.

The phosphoenolpyruvate carboxylase of Amaranthus paniculatus shows in vitro optimum affinity (S(0.5)) to phosphoenolpyruvate at a relatively high temperature (about 35 degrees C); even in the presence of activators, it functions efficiently only above 25 to 27 degrees C. At lower temperatures, a steep increase of activity with temperature is observed, due to the high activation energy for the catalyzed reaction. The same behavior in vivo could amplify the photoactivation of the enzyme to a large extent, since the night/day transition is soon followed by a considerable rise in leaf temperature.

Detecting Photoactivation of Phosphoenolpyruvate Carboxylase in C(4) Plants : An Effect of pH.

Photoactivation of phosphoenolpyruvate carboxylase in C(4) plants is detected more efficiently when activity is assayed at suboptimum pH (e.g. 7.2); the magnitude of the light effect is often larger at low phosphoenolpyruvate concentration.Darkness and low assay pH induce an allosteric behavior (positive cooperativity with phosphoenolpyruvate) which is relieved in light or by higher pH; thus, normal Michaelis-Menten kinetics are exhibited only when the enzyme is extracted during the day and assayed at pH 8.2.Light activation, pH, and substrate level appear to be components of a regulatory device suppressing the activity in darkness and enhancing it under light.

Photoregulation of Phosphoenolpyruvate Carboxylase in Salsola soda L. and Other C(4) Plants.

Photoactivation of phosphoenolpyruvate carboxylase was found to occur in several, though not all, C(4) species examined; Salsola soda L. was used for a detailed study of this effect of light.Activity differences between light and darkness are maximized when glycerol (25% v/v) is included in the extraction medium and in the absence of mercaptoethanol. In plants grown in the growth chamber, the night-form of the enzyme, in addition to low activity, shows a positive cooperativity (with phosphoenolpyruvate), which is gradually abolished by light of increasing intensities. This allosteric behavior is absent in plants adapted to a high light environment. Activation and deactivation, under light and darkness respectively, are quite fast, suggesting post-translational regulation. The photoactivation appears to depend on photosynthetic electron flow, since it is saturated at high photon fluxes (around 1000 microeinsteins per square meter per second) and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

Reduced Glutathione as an Effector of Phosphoenolpyruvate Carboxylase of the Crassulacean Acid Metabolism Plant Sedum praealtum D.C.

Reduced glutathione, but not mercaptoethanol or dithiothreitol, inhibits phosphoenolpyruvate carboxylase (PEPC) in desalted leaf extracts from Sedum praealtum D.C. The inhibition is more evident at low pH values (< 7.2) and becomes increasingly smaller at higher pH. In the presence of the inhibitor, the hyperbolic rate curve of night PEPC is transformed to sigmoid and the S(0.5) is increased. When the enzyme is extracted during the day, the rate curve is sigmoid and it is not changed by the inhibitor, though the S(0.5) is further increased. Oxidized glutathione is completely inactive. Levels of reduced glutathione in leaf tissue are distinctly higher in the light. A role of photosynthetically reduced glutathione in the regulation of PEPC in Crassulacean acid metabolism species appears probable.

Changes in properties of phosphoenolpyruvate carboxylase from the CAM plant Sedum praealtum D.C. upon dark/light transition and their stabilization by glycerol.

A prenounced decrease in phosphoenolpyruvate earboxylase (PEPC) activity is observed upon dark/light transition in Sedum praealtum D.C., only when glycerol is included in the extraction medium. If glycerol is omitted, the activity extracted in light is initially low, but soon reaches night levels. The stabilization of the light-induced form of the enzyme by glycerol, in crude or desalted extracts, made it possible to study its kinetic properties in comparison to those of the dark form. The behaviour towards substrate (PEP) changes from hyperbolic (dark) to sigmoid (light), S0.5 is increased and the enzymic activity becomes more sensitive to malate inhibition. Quite different activity/pH profiles are also obtained for the two forms of PEPC.It is inferred that the in vivo regulation of PEPC in CAM is effected by a concerted action of light, malate and pH shifting.

Calcium Inhibition of Pyrophosphatase in Crude Plant Extracts: IMPLICATION OF SOLUBLE CALCIUM IN C(4) PHOTOSYNTHESIS.

Assays of alkaline pyrophosphatase activity in crude plant extracts are inhibited by soluble calcium coextracted with the enzyme from leaf tissues. Calcium concentrations in most extracts are high enough to interfere seriously with the assay.All C(4) plants examined kept their soluble calcium and interference with pyrophosphatase activity at very low levels, whereas the C(3) plants covered the whole range of soluble calcium concentration. The hypothesis is advanced that low soluble calcium might be a prerequisite for high activity of pyrophosphatase and the C(4) pathway.

Enzymic nature of the protein moiety of protochlorophyllide holochrome.

The enzymic nature of the protein moiety of protochlorophyll(ide) holochrome was studied by following the fate of the [(14)C]protochlorophyll(ide) formed when dark-grown barley (Hordeum vulgare) or bean (Phaseolus vulgaris) leaves are incubated in the dark with 3 mm 4-delta-[(14)C]aminolevulinic acid. It was found that: [List: see text]Since turnover of protochlorophyll(ide) was not observed, these results show that there is a free exchange between the old "endogenous" and the new delta-aminolevulinic-acid-induced protochlorophyll(ide) molecules on the active site of the holochrome protein. These results are consistent with the hypothesis that the holochrome protein acts as an enzyme.