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Seasonal changes in the xanthophyll cycle and antioxidants in sun-exposed and shaded parts of the crown of Cryptomeria japonica in relation to rhodoxanthin accumulation during cold acclimation.

Xanthophyll rhodoxanthin, which is present in sun-exposed needles of certain gymnosperms in winter, may have a photoprotective role during long-term cold acclimation. To examine how cold acclimation processes vary within tree crowns and to examine putative correlations between xanthophyll cycle pigments (VAZ), rhodoxanthin and the water-water cycle in photoprotection, we monitored seasonal changes in the activities of two key antioxidant enzymes (ascorbate peroxidase (APX) and glutathione reductase (GR)), pigment composition and chlorophyll fluorescence parameters in sun and shade needles of crowns of the gymnosperm Cryptomeria japonica D. Don. Although APX and GR activities in both sun and shade needles were higher in winter than in summer when assayed at 20 degrees C, differences between seasons were less pronounced when enzymatic activities in summer and winter were assayed at 20 and 5 degrees C, respectively. These results suggest that increases in the potential activity of antioxidant enzymes in winter is an adaptation that helps counterbalance reductions in absolute enzyme activity caused by low temperature, and thus allows the photoprotective capacity of the water-water cycle in C. japonica to be maintained at a roughly constant value throughout the year. In shade needles, the concentration of VAZ increased in winter, but no rhodoxanthin accumulated. Photosynthetic activity was maintained in winter. In sun needles, however, the electron transport rate (ETR) and photochemical quenching (q(P)) decreased to their lowest values in December, just before the accumulation of rhodoxanthin, which coincided with the highest amount of VAZ. Changes in rhodoxanthin concentration mirrored changes in VAZ concentration from January to March. Winter values of ETR and q(P) were comparable with summer values after accumulation of rhodoxanthin, indicating that rhodoxanthin may play a more important role than the VAZ cycle in protecting the photosynthetic apparatus from photodamage in winter. Photosynthetic activity may be modulated, as a result of the interception of light by rhodoxanthin, to match the extent to which absorbed light energy can be utilized in winter when the VAZ cycle is unable to operate effectively because of low temperatures.

Acclimatization↗

Photosynthetic acclimation to dynamic changes in environmental conditions associated with deciduous overstory phenology in Daphniphyllum humile, an evergreen understory shrub.

Photoprotective responses during photosynthetic acclimation in Daphniphyllum humile Maxim, an evergreen understory shrub that grows in temperate deciduous forests, were examined in relation to changes in light availability and temperature caused by the seasonal dynamics of canopy leaf phenology. Gradual increases in irradiance in the understory from summer to autumn as overstory foliage senesced were accompanied by increased concentrations of xanthophyll cycle pigments (VAZ) in understory leaves. The chlorophyll (Chl) a/b ratio in understory leaves also increased from summer to autumn, reflecting the change in ratio of the light-harvesting antenna to the reaction center. However, low temperatures following overstory leaf fall reduced Rubisco activity. In contrast, the photosynthetic capactiy of leaves of D. humile growing at the forest border, which was higher in summer than that of leaves of understory plants, decreased in autumn. In autumn, Fv/Fm ratios decreased and concentrations of zeaxanthin (Z) and especially antheraxanthin (A) increased in leaves of both forest-border and understory plants. Although VAZ was twice as high in leaves of forest-border than of understory plants, NPQ was similar in both. We conclude that leaves of understory plants are able to acclimate to seasonal changes in light and temperature by varying their photosynthetic and photoprotective functions, thereby taking advantage of the favorable light conditions caused by overstory leaf fall.

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Tradeoff between shade adaptation and mitigation of photoinhibition in leaves of Quercus mongolica and Acer mono acclimated to deep shade.

We investigated susceptibility to photoinhibition in leaves acclimated to different light regimes in intermediately shade-tolerant Japanese oak (Quercus mongolica Fisch. ex Turcz. var. crispula (Blume) Ohashi) and shade-tolerant Japanese maple (Acer mono Maxim. var. glabrum (Lév. et Van't.) Hara), to elucidate adaptability to gap formation in leaves differing in shade acclimation. We hypothesized that there is a tradeoff between shade adaptation and capacity to mitigate photoinhibition associated with leaf morphology. We simultaneously measured chlorophyll fluorescence and gas exchange in seedlings that had been grown in full sunlight (open), 10% of full sun (moderate shade) and 5% of full sun (deep shade). Shade-tolerant A. mono adapted to deep shade through changes in leaf morphology, lowering its leaf mass per area (LMA), but Q. mongolica showed little change in LMA between moderate and deep shade. Photochemical quenching (qP) did not differ between species in full sunlight and moderate shade; however, in deep shade, qP of Q. mongolica was higher than that of A. mono, suggesting that Q. mongolica grown in deep shade is less susceptible to photoinhibition at gap formation. This is consistent with the finding that chronic photoinhibition 3 days after the transfer to full sunlight, indicated by the decrease in maximum photochemical efficiency, Fv/Fm, at predawn, was less in deep-shade-grown Q. mongolica than in deep shade-grown A. mono. In deep shade, the electron transport rate (ETR) of Q. mongolica was higher than that of A. mono, whereas thermal energy dissipation through photosystem II antennae, indicated by non-photochemical quenching, was lower in Q. mongolica than in A. mono. In deep shade, the greater ETR capacity in Q. mongolica in association with higher LMA and higher leaf N content could contribute to maintaining high qP and mitigating photoinhibition. These results indicate that, by maintaining a high electron transport capacity even in deep shade, the gap-dependent and intermediate-shade-tolerant Q. mongolica trades improved shade adaptation for higher growth potential when a gap event occurs.

Acclimatization↗

Noninvasive assessment of cerebral oxygenation during acclimation to hypobaric hypoxia.

Factors regulating cerebral tissue PO2 (PtO2) are complex. With the increased use of clinical PtO2 monitors, it has become important to elucidate these mechanisms. The authors are investigating a new methodology (electron paramagnetic resonance oximetry) for use in monitoring cerebral PtO2 in awake animals over time courses of weeks. The authors used this to study cerebral PtO2 in rats during chronic acclimation to hypoxia predicting that such acclimation would cause an increase in PtO2 because of increases that occur in capillary density and oxygen carrying capacity. The average PtO2 between 7 and 21 days was increased by 228% over controls.

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Cooler butterflies lay larger eggs: developmental plasticity versus acclimation.

We use a full factorial design to investigate the effects of maternal and paternal developmental temperature, as well as female oviposition temperature, on egg size in the butterfly Bicyclus anynana. Butterflies were raised at two different temperatures and mated in four possible sex-by-parental-temperature crosses. The mated females were randomly divided between high and low oviposition temperatures. On the first day after assigning the females to different temperatures, only female developmental temperature affected egg size. Females reared at the lower temperature laid larger eggs than those reared at a higher temperature. When eggs were measured again after an acclimation period of 10 days, egg size was principally determined by the prevailing temperature during oviposition, with females ovipositing at a lower temperature laying larger eggs. In contrast to widely used assumptions, the effects of developmental temperature were largely reversible. Male developmental temperature did not affect egg size in either of the measurements. Overall, developmental plasticity and acclimation in the adult stage resulted in very similar patterns of egg size plasticity. Consequently, we argue that the most important question when testing the significance of acclamatory changes is not at which stage a given plasticity is induced, but rather whether plastic responses to environmental change are adaptive or merely physiological constraints.

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Photosynthetic acclimation is reflected in specific patterns of gene expression in drought-stressed loblolly pine.

Because the product of a single gene can influence many aspects of plant growth and development, it is necessary to understand how gene products act in concert and upon each other to effect adaptive changes to stressful conditions. We conducted experiments to improve our understanding of the responses of loblolly pine (Pinus taeda) to drought stress. Water was withheld from rooted plantlets of to a measured water potential of -1 MPa for mild stress and -1.5 MPa for severe stress. Net photosynthesis was measured for each level of stress. RNA was isolated from needles and used in hybridizations against a microarray consisting of 2173 cDNA clones from five pine expressed sequence tag libraries. Gene expression was estimated using a two-stage mixed linear model. Subsequently, data mining via inductive logic programming identified rules (relationships) among gene expression, treatments, and functional categories. Changes in RNA transcript profiles of loblolly pine due to drought stress were correlated with physiological data reflecting photosynthetic acclimation to mild stress or photosynthetic failure during severe stress. Analysis of transcript profiles indicated that there are distinct patterns of expression related to the two levels of stress. Genes encoding heat shock proteins, late embryogenic-abundant proteins, enzymes from the aromatic acid and flavonoid biosynthetic pathways, and from carbon metabolism showed distinctive responses associated with acclimation. Five genes shown to have different transcript levels in response to either mild or severe stress were chosen for further analysis using real-time polymerase chain reaction. The real-time polymerase chain reaction results were in good agreement with those obtained on microarrays.

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Plasma Membrane Lipids Associated with Genetic Variability in Freezing Tolerance and Cold Acclimation of Solanum Species.

Simultaneous comparisons were made between a freezing-tolerant, cold-acclimating (CA) wild potato species (Solanum commersonii) and a freezing-sensitive, nonacclimating (NA) cultivated species (Solanum tuberosum). Comparative studies allowed differentiation of plasma membrane lipid changes associated with increased freezing tolerance following CA from lipid changes that can result from metabolic adjustment to reduced temperature during CA. Following CA treatment lipid changes found in both the NA and CA species included a decrease in palmitic acid, an increase in unsaturated to saturated fatty acid ratio, an increase in free sterols, an increase in sitosterol, and a slight decrease in cerebrosides. Lipid changes detected only in the acclimating species included an increase in phosphatidylethanolamine, a decrease in sterol to phospholipid ratio, an increase in linoleic acid, a decrease in linolenic acid, and an increase in acylated steryl glycoside to steryl glycoside ratio. These changes were either absent or opposite in the NA species, suggesting an association of these lipid changes with CA. Furthermore, the lipid changes associated with increased freezing tolerance during CA were distinct from lipid differences between the two species in the NA state.

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Exogenous Abscisic Acid Mimics Cold Acclimation for Cacti Differing in Freezing Tolerance.

The responses to low temperature were determined for two species of cacti sensitive to freezing, Ferocactus viridescens and Opuntia ficus-indica, and a cold hardy species, Opuntia fragilis. Fourteen days after shifting the plants from day/night air temperatures of 30/20[deg]C to 10/0[deg]C, the chlorenchyma water content decreased only for O. fragilis. This temperature shift caused the freezing tolerance (measured by vital stain uptake) of chlorenchyma cells to be enhanced only by about 2.0[deg]C for F. viridescens and O. ficus-indica but by 14.6[deg]C for O. fragilis. Also, maintenance of high water content by injection of water into plants at 10/0[deg]C reversed the acclimation. The endogenous abscisic acid (ABA) concentration was below 0.4 pmol g-1 fresh weight at 30/20[deg]C, but after 14 d at 10/0[deg]C it increased to 84 pmol g-1 fresh weight for O. ficus-indica and to 49 pmol g-1 fresh weight for O. fragilis. Four days after plants were sprayed with 7.5 x 10-5 M ABA at 30/20[deg]C, freezing tolerance was enhanced by 0.5[deg]C for F. viridescens, 4.1[deg]C for O. ficus-indica, and 23.4[deg]C for O. fragilis. Moreover, the time course for the change in freezing tolerance over 14 d was similar for plants shifted to low temperatures as for plants treated with exogenous ABA at moderate temperatures. Decreases in plant water content and increases in ABA concentration may be important for low-temperature acclimation by cacti, especially O. fragilis, which is widely distributed in Canada and the United States.

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Short-day potentiation of low temperature-induced gene expression of a C-repeat-binding factor-controlled gene during cold acclimation in silver birch.

Development of winter hardiness in trees is a two-stage process involving sequential perception of distinct environmental cues, short-day (SD) photoperiod and low temperature (LT). We have shown that both SD and LT are recognized by leaves of silver birch (Betula pendula cv Roth) leading to increased freezing tolerance, and thus leaves can be used as an experimental model to study the physiological and molecular events taking place during cold acclimation. To obtain a molecular marker for the acclimation process in birch we cloned a gene, designated Bplti36, encoding a 36-kD acidic SK2 type of dehydrin. The gene was responsive to LT, drought, salt, and exogenous abscisic acid. This responsiveness to abiotic stresses and abscisic acid was retained when Bplti36 was introduced to Arabidopsis (Arabidopsis thaliana). The LT induction of the gene appeared to be under the control of the C-repeat-binding factor pathway as suggested by the presence of several C-repeat/dehydration-responsive element/LT-responsive elements in the Bplti36 promoter and its constitutive expression in C-repeat-binding factor overproducing Arabidopsis. In birch SD photoperiod at normal-growth temperature did not result in significant induction of Bplti36. However, preexposure to SD followed by LT treatment resulted in a remarkable increase in Bplti36 transcript accumulation as compared to LT-treated plants grown at long-day photoperiod. This suggests that SD photoperiod potentiates the LT response by conditioning the leaf tissue to be more responsive to the LT stimulus.

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Acclimation of the Photosynthetic Apparatus to Growth Irradiance in a Mutant Strain of Synechococcus Lacking Iron Superoxide Dismutase.

The acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus sp. PCC 7942 lacking detectable iron superoxide dismutase activity was studied. The growth of the mutant was inhibited at concentrations of methyl viologen 4 orders of magnitude smaller than those required to inhibit the growth of the wild-type strain. An increased sensitivity of photosynthetic electron transport near photosystem I (PSI) toward photooxidative stress was also observed in the mutant strain. In the absence of methyl viologen, the mutant exhibited similar growth rates compared with those of the wild type, even at high growth irradiance (350 [mu]E m-2 s-1) where chronic inhibition of photosystem II (PSII) was observed in both strains. Under high growth irradiance, the ratios of PSII to PSI and of [alpha]-phycocyanin to chlorophyll a were less than one-third of the values for the wild type. In both strains, cellular contents of chlorophyll a, [alpha]-phycocyanin, and [beta]-carotene, as well as the length of the phycobilisome rods, declined with increasing growth irradiance. Only the cellular content of the carotenoid zeaxanthin seemed to be independent of growth irradiance. These results suggest an altered acclimation to growth irradiance in the sodB mutant in which the stoichiometry between PSI and PSII is adjusted to compensate for the loss of PSI efficiency occurring under high growth irradiance. Similar shortening of the phycobilisome rods in the sodB mutant and wild-type strain suggest that phycobilisome rod length is regulated independently of photosystem stoichiometry.

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Growth at Low Temperature Mimics High-Light Acclimation in Chlorella vulgaris.

Structural and functional alterations to the photosynthetic apparatus after growth at low temperature (5[deg]C) were investigated in the green alga Chlorella vulgaris Beijer. Cells grown at 5[deg]C had a 2-fold higher ratio of chlorophyll a/b, 5-fold lower chlorophyll content, and an increased xanthophyll content compared to cells grown at 27[deg]C even though growth irradiance was kept constant at 150 [mu]mol m-2 s-1. Concomitant with the increase in the chlorophyll a/b ratio was a lower abundance of light-harvesting polypeptides in 5[deg]C-grown cells as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by western blotting.The differences in pigment composition were found to be alleviated within 12 h of transferring 5[deg]C-grown cells to 27[deg]C. Furthermore, exposure of 5[deg]C-grown cells to a 30-fold lower growth irradiance (5 [mu]mol m-2 s-1) resulted in pigment content and composition similar to that in cells grown at 27[deg]C and 150 [mu]mol m-2 s-1. Although both cell types exhibited similar measuring-temperature effects on CO2-saturated O2 evolution, 5[deg]C-grown cells exhibited light-saturated rates of O2 evolution that were 2.8-and 3.9-fold higher than 27[deg]C-grown cells measured at 27[deg]C and 5[deg]C, respectively. Steady-state chlorophyll a fluorescence indicated that the yield of photosystem II electron transport of 5[deg]C-grown cells was less temperature sensitive than that of 27[deg]C-grown cells. This appears to be due to an increased capacity to keep the primary, stable quinone electron acceptor of photosystem II (QA) oxidized at low temperature in 5[deg]C- compared with 27[deg]C-grown cells regardless of irradiance. We conclude that Chlorella acclimated to low temperature adjusts its photosynthetic apparatus in response to the excitation pressure on photosystem II and not to the absolute external irradiance. We suggest that the redox state of QA may act as a signal for this photosynthetic acclimation to low temperature in Chlorella.

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Acclimation of Soybean Nodules to Changes in Temperature.

This study examines how O2 status, respiration rate, and nitrogenase activity of soybean (Glycine max) nodules acclimate to short-term (<30 min) temperature change from 20 to 15[deg]C or from 20 to 25[deg]C. Acclimation responses were compared between nodules on uninhibited plants and nodules that were severely O2 limited by exposure to Ar:O2. In uninhibited nodules the decrease in temperature caused a rapid inhibition of nitrogenase activity followed by partial recovery, whereas in Ar:O2-inhibited nodules the temperature decrease caused a minor stimulation followed by a gradual decline in nitrogenase activity. In contrast, the temperature increase caused a gradual increase in nitrogenase activity in uninhibited nodules, and an initial inhibition followed by a rapid rise in Ar:O2-inhibited nodules. In both uninhibited and Ar:O2-inhibited nodules, temperature had only minor effects on the degree to which nitrogenase activity was limited by O2 supply, but nodule permeability to O2 diffusion was greater at 25[deg]C, and less at 15[deg]C, than that measured at 20[deg]C. On the basis of these data, we propose that temperature change alters the nodule's respiratory demand and that the observed changes in nodule permeability occur to maintain control over the infected cell O2 concentration as the O2 demand increases at high temperature or decreases at low temperature.

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Role of Abscisic Acid in Drought-Induced Freezing Tolerance, Cold Acclimation, and Accumulation of LT178 and RAB18 Proteins in Arabidopsis thaliana.

To study the role of abscisic acid (ABA) in development of freezing tolerance of Arabidopsis thaliana, we exposed wild-type plants, the ABA-insensitive mutant abi1, and the ABA-deficient mutant aba-1 to low temperature (LT), exogenous ABA, and drought. Exposure of A. thaliana to drought stress resulted in a similar increase in freezing tolerance as achieved by ABA treatment or the initial stages of acclimation, suggesting overlapping responses to these environmental cues. ABA appears to be involved in both LT- and drought-induced freezing tolerance, since both ABA mutants were impaired in their responses to these stimuli. To correlate enhanced freezing tolerance with the presence of stress-specific proteins, we characterized the accumulation of RAB18 and LTI78 in two ecotypes, Landsberg erecta and Coimbra, and in the ABA mutants during stress response. LT- and drought-induced accumulation of RAB18 coincided with the increase in freezing tolerance and was blocked in the cold-acclimation-deficient ABA mutants. In contrast, LT178 accumulated in all genotypes in response to LT and drought and was always present when the plants were freezing tolerant. This suggests that development of freezing tolerance in A. thaliana requires ABA-controlled processes in addition to ABA-independent factors.

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Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage.

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1-3-linked fucose.

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Acclimation of Foliar Antioxidant Systems to Growth Irradiance in Three Broad-Leaved Evergreen Species.

The protective role of leaf antioxidant systems in the mechanism of plant acclimation to growth irradiance was studied in Vinca major, Schefflera arboricola, and Mahonia repens, which were grown for several months at 20, 100, and 1200 [mu]mol photons m-2 s-1. As growth irradiance increased, several constituents of the "Mehler-peroxidase" pathway also increased: superoxide dismutase, ascorbate peroxidase, glutathione reductase, ascorbate, and glutathione. This occurred concomitantly with increases in the xanthophyll cycle pool size and in the rate of nonphotochemical energy dissipation under steady-state conditions. There was no evidence for photosystem II overreduction in plants grown at high irradiance, although the reduction state of the stromal NADP pool, estimated from measurements of NADP-malate dehydrogenase activity, was greater than 60% in V. major and S. arboricola. Ascorbate, which removes reactive O2 species generated by O2 photoreduction in the chloroplast and serves as a reductant for the conversion of the xanthophyll cycle pigments to the de-epoxidized forms A plus Z, generally exhibited the most dramatic increases in response to growth irradiance. We conclude from these results that O2 photoreduction occurs at higher rates in leaves acclimated to high irradiance, despite increases in xanthophyll cycle-dependent energy dissipation, and that increases in leaf antioxidants protect against this potential oxidative stress.

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Localization of expression of three cold-induced genes, blt101, blt4. 9, and blt14, in different tissues of the crown and developing leaves of cold-acclimated cultivated barley

Tissues expressing mRNAs of three cold-induced genes, blt101, blt14, and blt4.9, and a control gene, elongation factor 1alpha, were identified in the crown and immature leaves of cultivated barley (Hordeum vulgare L. cv Igri). Hardiness and tissue damage were assessed. blt101 and blt4.9 mRNAs were not detected in control plants; blt14 was expressed in control plants but only in the inner layers of the crown cortex. blt101 was expressed in many tissues of cold-acclimated plants but most strongly in the vascular-transition zone of the crown; blt14 was expressed only in the inner layers of the cortex and in cell layers partly surrounding vascular bundles in the vascular-transition zone; expression of blt4.9, which codes for a nonspecific lipid-transfer protein, was confined to the epidermis of the leaf and to the epidermis of the older parts of the crown. None of the cold-induced genes was expressed in the tunica, although the control gene was most strongly expressed there. Thus, the molecular aspects of acclimation differed markedly between tissues. Damage in the vascular-transition zone of the crown correlated closely with plant survival. Therefore, the strong expression of blt101 and blt14 in this zone may indicate a direct role in freezing tolerance of the crown.

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Induction of acclimative proteolysis of the light-harvesting chlorophyll a/b protein of photosystem II in response to elevated light intensities

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

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Acclimation of Arabidopsis leaves developing at low temperatures. Increasing cytoplasmic volume accompanies increased activities of enzymes in the Calvin cycle and in the sucrose-biosynthesis pathway.

Photosynthetic and metabolic acclimation to low growth temperatures were studied in Arabidopsis (Heynh.). Plants were grown at 23 degrees C and then shifted to 5 degrees C. We compared the leaves shifted to 5 degrees C for 10 d and the new leaves developed at 5 degrees C with the control leaves on plants that had been left at 23 degrees C. Leaf development at 5 degrees C resulted in the recovery of photosynthesis to rates comparable with those achieved by control leaves at 23 degrees C. There was a shift in the partitioning of carbon from starch and toward sucrose (Suc) in leaves that developed at 5 degrees C. The recovery of photosynthetic capacity and the redirection of carbon to Suc in these leaves were associated with coordinated increases in the activity of several Calvin-cycle enzymes, even larger increases in the activity of key enzymes for Suc biosynthesis, and an increase in the phosphate available for metabolism. Development of leaves at 5 degrees C also led to an increase in cytoplasmic volume and a decrease in vacuolar volume, which may provide an important mechanism for increasing the enzymes and metabolites in cold-acclimated leaves. Understanding the mechanisms underlying such structural changes during leaf development in the cold could result in novel approaches to increasing plant yield.

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