Axes and cotyledons of recalcitrant seeds of Castanea sativa Mill. exhibit contrasting responses of respiration to drying in relation to desiccation sensitivity

Oxidative damage originating from uncontrolled metabolism is thought to be responsible for the sensitivity to drying in recalcitrant seeds. This study compares the responses of respiration to drying and the loss of membrane integrity in isolated axes and cotyledons of the recalcitrant seeds of Castanea sativa Mill. Electron spin resonance spectroscopy of two nitroxide spin probes introduced into the seed tissues was used to assess the cytoplasmic viscosity and the membrane permeability during fast and slow drying. Drying rates had not effect on the rise in viscosity in axes and cotyledons. In both tissues, the cytoplasmic viscosity during drying remained constant at 0.2 Poise until 1.6 g water/g DW (g/g), thereafter it increased exponentially. Axes were found to be more tolerant to drying than cotyledons: membranes showed minor changes in their permeability during drying and 50% viability was retained in dried axes containing 0.12 g/g. In contrast, plasma membranes in cotyledons lost their integrity below 0.6 g/g, regardless of the drying rate. Drying axes and cotyledons exhibited contrasting responses of their metabolism to drying. At the onset of drying, the rates of O2 uptake declined rapidly in drying axes. However, respiration in drying cotyledons sequentially increased to c. 1.4-fold at 1.2 g/g then decreased concomitantly with the loss of membrane integrity. The respiratory quotients (CO2 output/O2 input) remained constant around 0.9 until the loss of membrane integrity, then rose to 2.8. As a symptom of mitochondrial injury, the levels of reduction of cytochromes were assessed in situ in fresh and dried cotyledons using light spectroscopy. The levels of reduced cytochrome c and aa3 were lower in dried C. sativa cotyledons than in dried orthodox cotyledons of cowpea, indicating that a disruption in the electron transport chains may have occurred during drying. Desiccation sensitivity in recalcitrant seeds may be due to the inability to actively depress their metabolism during drying, thereby increasing the chances of initiating peroxidative damage during drying.Key words: Castanea satia, membrane permeability, recalcitrant seed, respiration, viscosity.      

Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the ¹⁸O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.     

Oxidative stress in Nostoc flagelliforme subjected to desiccation and effects of exogenous oxidants on its photosynthetic recovery

The metabolism of reactive oxygen species in Nostoc flagelliforme and effects of exogenous oxidants on its photosynthetic recovery were investigated to obtain insight into oxidative stress in desiccation and its possible damaging impact on photosynthetic apparatus. No ascorbate was detected with ascorbate oxidase in N. flagelliforme. Superoxide dismutase (SOD) remained active even after three years drying storage and its activity was 78% of that in fully recovered samples. The SOD activity decreased during desiccation or in drying storage. Intracellular active oxygen production was studied by incubating samples in BG₁₁ medium for 2 h and measuring the oxidation of 2,7 -dichlorohydrofluorescein diacetate. The production rate was 38.11 nmol DCF_g (d.wt)^-1 h^-1 in dried field samples and was significantly higher than in fully recovered or air-dried samples. The balance between intracellular active oxygen production and the defense systems mightbreak down in air-dried and dried field samples. Treatment with exogenous oxidants slowed the photosynthetic recovery especially with singlet oxygen. Oxidative stress might play an important role in desiccationinduced damages to the photosynthetic apparatus.     

Mapping Intercellular CO2 Mole Fraction (Ci) in Rosa rubiginosa Leaves Fed with Abscisic Acid by Using Chlorophyll Fluorescence Imaging : Significance of Ci Estimated from Leaf Gas Exchange

Imaging of photochemical yield of photosystem II (PSII) computed from leaf chlorophyll fluorescence images and gas-exchange measurements were performed on Rosa rubiginosa leaflets during abscisic acid (ABA) addition. In air ABA induced a decrease of both the net CO₂ assimilation (A_n) and the stomatal water vapor conductance (g_s). After ABA treatment, imaging in transient nonphotorespiratory conditions (0.1% O₂) revealed a heterogeneous decrease of PSII photochemical yield. This decline was fully reversed by a transient high CO₂ concentration (7400 μmol mol⁻¹) in the leaf atmosphere. It was concluded that ABA primarily affected A_n by decreasing the CO₂ supply at ribulose-1,5-bisphosphate carboxylase/oxygenase. Therefore, the A_n versus intercellular mole fraction (C_i) relationship was assumed not to be affected by ABA, and images of C_i and g_s were constructed from images of PSII photochemical yield under nonphotorespiratory conditions. The distribution of g_s remained unimodal following ABA treatment. A comparison of calculations of C_i from images and gas exchange in ABA-treated leaves showed that the overestimation of C_i estimated from gas exchange was only partly due to heterogeneity. This overestimation was also attributed to the cuticular transpiration, which largely affects the calculation of the leaf conductance to CO₂, when leaf conductance to water is low.     

Desiccation sensitivity and successful cryopreservation of oil seeds of European hazelnut (Corylus avellana)

The sensitivity of a Polish provenance of dormant Corylus avellana seeds to extreme desiccation and cryopreservation in liquid nitrogen (LN, ?196°C) was investigated. No sensitivity to desiccation was observed as seeds readily germinated and exhibited seedling emergence, even when the critical water content (WC) of seeds (nuts devoid of pericarp) was reduced to 0.027 g H₂O/g dry mass, g g^?1 by drying. Results of germination and seedling emergence tests indicated that seeds tolerated cooling in LN when desiccated to a WC in the range of 0.05–0.10 and 0.08–0.10 g g^?1, respectively. The results of this study demonstrate the feasibility of long‐term cryopreservation of European hazelnut seeds. As the seeds of this species have been classified in several different categories (orthodox, suborthodox and recalcitrant), based on their response to desiccation and low temperature, the assignment of the seeds of hazelnut to a specific category is provided and discussed.     

Enrichment of High-Affinity CO Oxidizers in Maine Forest Soil

Carboxydotrophic activity in forest soils was enriched by incubation in a flowthrough system with elevated concentrations of headspace CO (40 to 400 ppm). CO uptake increased substantially over time, while the apparent K_m (_appK_m) for uptake remained similar to that of unenriched soils (<10 to 20 ppm). Carboxydotrophic activity was transferred to and further enriched in sterile sand and forest soil. The _appK_ms for secondary and tertiary enrichments remained similar to values for unenriched soils. CO uptake by enriched soil and freshly collected forest soil was inhibited at headspace CO concentrations greater than about 1%. A novel isolate, COX1, obtained from the enrichments was inhibited similarly. However, in contrast to extant carboxydotrophs, COX1 consumed CO with an _appK_m of about 15 ppm, a value comparable to that of fresh soils. Phylogenetic analysis based on approximately 1,200 bp of its 16S rRNA gene sequence suggested that the isolate is an α-proteobacterium most closely related to the genera Pseudaminobacter, Aminobacter, and Chelatobacter (98.1 to 98.3% sequence identity).     

Fourier Transform Infrared Microspectroscopy Detects Changes in Protein Secondary Structure Associated with Desiccation Tolerance in Developing Maize Embryos

Isolated immature maize (Zea mays L.) embryos have been shown to acquire tolerance to rapid drying between 22 and 25 d after pollination (DAP) and to slow drying from 18 DAP onward. To investigate adaptations in protein profile in association with the acquisition of desiccation tolerance in isolated, immature maize embryos, we applied in situ Fourier transform infrared microspectroscopy. In fresh, viable, 20- and 25-DAP embryo axes, the shapes of the different amide-I bands were identical, and this was maintained after flash drying. On rapid drying, the 20-DAP axes had a reduced relative proportion of α-helical protein structure and lost viability. Rapidly dried 25-DAP embryos germinated (74%) and had a protein profile similar to the fresh control axes. On slow drying, the α-helical contribution in both the 20- and 25-DAP embryo axes increased compared with that in the fresh control axes, and survival of desiccation was high. The protein profile in dry, mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in the 20-DAP embryo axes and much less so in the 25-DAP axes. After slow drying, low plasma membrane permeability ensued in both the 20- and 25-DAP axes. We conclude that slow drying of excised, immature embryos leads to an increased proportion of α-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress.     

Effects of Hypoxia on 13NH4+ Fluxes in Rice Roots : Kinetics and Compartmental Analysis

Techniques of compartmental (efflux) and kinetic influx analyses with the radiotracer ¹³NH₄⁺ were used to examine the adaptation to hypoxia (15, 35, and 50% O₂ saturation) of root N uptake and metabolism in 3-week-old hydroponically grown rice (Oryza sativa L., cv IR72) seedlings. A time-dependence study of NH₄⁺ influx into rice roots after onset of hypoxia (15% O₂) revealed an initial increase in the first 1 to 2.5 h after treatment imposition, followed by a decline to less than 50% of influx in control plants by 4 d. Efflux analyses conducted 0, 1, 3, and 5 d after the treatment confirmed this adaptation pattern of NH₄⁺ uptake. Half-lives for NH₄⁺ exchange with subcellular compartments, cytoplasmic NH₄⁺ concentrations, and efflux (as percentage of influx) were unaffected by hypoxia. However, significant differences were observed in the relative amounts of N allocated to NH₄⁺ assimilation and the vacuole versus translocation to the shoot. Kinetic experiments conducted at 100, 50, 35, and 15% O₂ saturation showed no significant change in the K_m value for NH₄⁺ uptake with varying O₂ supply. However, V_max was 42% higher than controls at 50% O₂ saturation, unchanged at 35%, and 10% lower than controls at 15% O₂. The significance of these flux adaptations is discussed.     

Water Content, Raffinose, and Dehydrins in the Induction of Desiccation Tolerance in Immature Wheat Embryos

Desiccation tolerance is initiated in wheat (Triticum aestivum L.) embryos in planta at 22 to 24 d after anthesis, at the time that the embryo water content has decreased from about 73% fresh weight (2.7 g water/g dry weight) to about 65% fresh weight (1.8 g water/g dry weight). To determine if desiccation tolerance is fully induced by the loss of a relatively small amount of water, detached wheat grains were treated to reduce the embryo water content by just a small amount to approximately 69% (2.2 g water/g dry weight). After 24 h of such incipient water loss, subsequently excised embryos were able to withstand severe desiccation, whereas those embryos that had not previously lost water could not. Therefore, a relatively small decrease in water content for only 24 h acts as the signal for the development of desiccation tolerance. Embryos that were induced into tolerance by a 24-h water loss had no detectable raffinose. The oligosaccharide accumulated at later times even in embryos of detached grains that had not become desiccation tolerant, although tolerant embryos (i.e. those that previously had lost some water) contained larger amounts of the carbohydrate. It is concluded that desiccation tolerance and the occurrence of raffinose are not correlated. Immunodetected dehydrins accumulated in embryos in planta as desiccation tolerance developed. Detachment of grains induced the appearance of dehydrins at an earlier age, even in embryos that had not been made desiccation tolerant by incipient drying. It is concluded that a small reduction in water content induces desiccation tolerance by initiating changes in which dehydrins might participate but not by their interaction with raffinose.     

Developmental and Environmental Effects on the Expression of the C3-C4 Intermediate Phenotype in Moricandia arvensis

Cellular anatomy and expression of glycine decarboxylase (GDC) protein were studied during leaf development of the C₃-C₄ intermediate species Moricandia arvensis. Leaf anatomy was initially C₃-like and the number and profile area of mitochondria in the bundle-sheath cells were the same as those in adjacent mesophyll cells. Between a leaf length of 6 and 12 mm there was a bundle-sheath-specific, 4-fold increase in the number of mitochondrial profiles, followed by a doubling of their individual profile areas as the leaves expanded further. Subunits of GDC were present in whole-leaf extracts before the anatomical development of bundle-sheath cells. Whereas the GDC H-protein content of leaves increased steadily throughout development, the increase in GDC P-protein was synchronous with the development of mitochondria in the bundle sheath. The P-protein was confined to bundle-sheath mitochondria throughout leaf development, and its content in individual mitochondria increased before the anatomical development of the bundle sheath. Anatomical and biochemical attributes of the C₃-C₄ character were present in the cotyledons and sepals but not in other photosynthetic organs/tissues. In leaves and cotyledons that developed in the dark, the expression of the P-protein and the organellar development were reduced but the bundle-sheath cell specificity was retained.     

Estimating the Excess Investment in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Leaves of Spring Wheat Grown under Elevated CO2

Wheat (Triticum aestivum L.) was grown under CO₂ partial pressures of 36 and 70 Pa with two N-application regimes. Responses of photosynthesis to varying CO₂ partial pressure were fitted to estimate the maximal carboxylation rate and the nonphotorespiratory respiration rate in flag and preceding leaves. The maximal carboxylation rate was proportional to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content, and the light-saturated photosynthetic rate at 70 Pa CO₂ was proportional to the thylakoid ATP-synthase content. Potential photosynthetic rates at 70 Pa CO₂ were calculated and compared with the observed values to estimate excess investment in Rubisco. The excess was greater in leaves grown with high N application than in those grown with low N application and declined as the leaves senesced. The fraction of Rubisco that was estimated to be in excess was strongly dependent on leaf N content, increasing from approximately 5% in leaves with 1 g N m⁻² to approximately 40% in leaves with 2 g N m⁻². Growth at elevated CO₂ usually decreased the excess somewhat but only as a consequence of a general reduction in leaf N, since relationships between the amount of components and N content were unaffected by CO₂. We conclude that there is scope for improving the N-use efficiency of C₃ crop species under elevated CO₂ conditions.     

Lack of Cytochrome c in Mouse Fibroblasts Disrupts Assembly/Stability of Respiratory Complexes I and IV

Cytochrome c (cyt c) is a heme-containing protein that participates in electron transport in the respiratory chain and as a signaling molecule in the apoptotic cascade. Here we addressed the effect of removing mammalian cyt c on the integrity of the respiratory complexes in mammalian cells. Mitochondria from cyt c knockout mouse cells lacked fully assembled complexes I and IV and had reduced levels of complex III. A redox-deficient mutant of cyt c was unable to rescue the levels of complexes I and IV. We found that cyt c is associated with both complex IV and respiratory supercomplexes, providing a potential mechanism for the requirement for cyt c in the assembly/stability of complex IV.The mitochondrial electron transport chain consists of four multisubunit complexes, namely, NADH-ubiquinone oxidoreductase (complex I),² succinate-ubiquinone oxidoreductase (complex II), ubiquinone-cytochrome c oxidoreductase (complex III), and cytochrome c oxidase (complex IV, COX). Cytochrome c (cyt c) shuttles electrons from oxidative phosphorylation complex III to complex IV. Electrons are transferred from reduced cyt c sequentially to the Cu_A site, heme a, heme a₃, and Cu_B binuclear center in the complex IV before being finally transferred to molecular oxygen to generate water (1). Respiratory complexes are assembled into supercomplexes (also called respirasomes). These contain complex I bound to dimeric complex III and a variable copy number of complex IV (2).In Saccharomyces cerevisiae, cyt c is encoded by two genes: CYC1 and CYC7. Mutagenesis studies in yeast have shown that cyt c is required for the assembly of COX (3, 4). In yeast lacking both the cyt c genes (CYC1 and CYC7), COX assembly was absent. It was also shown that cyt c is only structurally required for COX assembly, because a catalytic mutant of cyt c (W65S) was sufficient to bring about near normal levels of COX. However, because yeast lacks complex I, they could not analyze the role of cyt c in the assembly/stability of complex I. Mammals possess two different isoforms of cyt c encoded on different chromosomes: the somatic (cyt cS)- and testis (cyt cT)-specific isoforms. In mouse, the cDNAs bear 74% homology, whereas the proteins possess 86% identity with most dissimilarity in the C terminus.Cardiolipin (CL) is an anionic phospholipid present almost exclusively in the mitochondrial membranes and constitutes 25% of its total phospholipids (5). Work from several laboratories showed that CL is essential for the membrane anchorage of the respiratory supercomplexes. CL has two main roles in the mitochondrial structure and function, namely, stabilization of mitochondrial membranes and specific interactions with proteins. CL deficiency results in inefficient energy transformation by oxidative phosphorylation, swelling of mitochondria, decreased ATP/oxygen ratio, and reduced membrane potential (6, 7). In accordance, in S. cerevisiae lacking CL synthase, the supercomplex comprising complexes III and IV is unstable (8). Assembly mutants of COX had significantly reduced CL synthase activity, whereas assembly mutants of respiratory complex III and complex V showed less inhibition (9). Subsequently, the proton gradient across the inner mitochondrial membrane was found to be important for CL formation and that CL synthase was stimulated by alkaline pH at the matrix side (10). In this study, we investigated the role of cyt c depletion on CL levels by examining its content and composition in cyt c null cells.Here we aimed to answer the following questions: What is the role of cyt c in the assembly and maintenance of the different respiratory complexes in mammals? Are there changes in the content/composition of lipids in the cyt c-ablated cells? Analysis of mouse fibroblasts revealed that cyt c is essential for the assembly/stability of COX, and a catalytically mutant form of cyt c cannot rescue the COX defect in the cyt c null cells. CL and triacylglycerols showed significant differences in the cyt c null cells, both in content and composition.     

Accumulation of trehalose in response to desiccation and salt stress in the terrestrial cyanobacterium Nostoc commune

Changes in photosynthetic activity and trehalose levels in field‐isolated, natural colonies of the terrestrial cyanobacterium Nostoc commune responding to desiccation and salt stress were investigated. As the water content decreased in N. commune colonies during desiccation, photosynthetic O₂‐evolving activity decreased and no activity was detected in desiccated colonies. A high level of O₂ evolution was restored in the colonies as they absorbed atmospheric moisture, indicating that only a small amount of water is required for reactivation of photosynthesis. No detectable trehalose was found in fully hydrated N. commune colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air‐dried colonies. Moreover, a 0.2 M NaCl treatment also induced trehalose accumulation to a level equivalent to that by desiccation. Photosynthetic O₂ evolution was inhibited by 0.2 M NaCl, indicating that N. commune can tolerate only low levels of salt. These results suggest that cessation of photosynthesis and trehalose accumulation occur in response to both matric water stress (desiccation) and osmotic water stress (high salt concentration), and that while trehalose may be a less effective osmoprotective compound than others, it is important for the extreme tolerance to desiccation observed in terrestrial cyanobacterium.     

The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii

The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO₂ and about 90% with ambient CO₂. In addition, it is likely that pyrenoidal Rubisco is active in CO₂ fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO₂-fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO₂ fixation in C. reinhardtii and other unicellular algae containing CO₂-concentrating mechanisms.     

Fate of pGFP-Bearing Escherichia coli O157:H7 in Ground Beef at 2 and 10°C and Effects of Lactate, Diacetate, and Citrate

Although beef has been implicated in the largest outbreaks of Escherichia coli O157:H7 infection in the United States, studies on the fate of this pathogen have been limited. Problems in such studies are associated with detection of the pathogen at levels considerably lower than the levels of the competing microorganisms. In the present study, a green fluorescent protein-expressing E. coli O157:H7 strain was used, and the stable marker allowed us to monitor the behavior of the pathogen in ground beef stored aerobically from freshness to spoilage at 2 and 10°C. In addition, the effects of sodium salts of lactate (SL) (0.9 and 1.8%), diacetate (SDA) (0.1 and 0.2%), and buffered citrate (SC) (1 and 2%) and combinations of SL and SDA were evaluated. SC had negligible antimicrobial activity, and SL delayed microbial growth, while SDA and SL plus SDA were most inhibitory to the total-aerobe population in the meat. At 2°C, the initial numbers of E. coli O157:H7 (3 and 5 log₁₀ CFU/g) decreased by ~1 log₁₀ CFU/g when spoilage was manifest (>7 log₁₀ CFU of total aerobes/g), irrespective of the treatment. There was no decline in the numbers of the pathogen during storage at 10°C. Our results showed that the pathogen was resistant to the salts tested and confirmed that refrigerated meat contaminated with the pathogen remains hazardous.     

Decreased GA1 Content Caused by the Overexpression of OSH1 Is Accompanied by Suppression of GA 20-Oxidase Gene Expression

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA₁ was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA₅₃ and GA₂₀, precursors of GA₁, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA₂₀, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA₁ and GA₂₀ contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA₁₉, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA₁ by suppressing GA 20-oxidase expression.     

What affects the desiccation tolerance threshold of Brazilian Eugenia (Myrtaceae) seeds?

Desiccation sensitive (DS) seeds are shed at high water contents (WC) and metabolically active, but WC thresholds vary broadly among species even in the same genus. Eugenia is an important ecological genus that has high occurrence in several Brazilian morphoclimatic domains. In this study, we assessed seed desiccation tolerance of five Eugenia species collected in specific meteorological conditions. We reported the species geographical ranges and verified the rainfall and temperature of species sites in the year prior to seed collection. We also assessed initial WC, seed germination and vigor and seedling growth upon desiccation. Eugenia uniflora was the widest spread among the five species, while E. astringens was the most restricted. In this specific study, widespread species showed a higher WC threshold than restricted species. In the same way, the WC of fresh seeds was not correlated to the desiccation tolerance threshold. Seed desiccation tolerance was species dependent and correlated with the environmental status of seed collection sites. Wetter and warmer conditions were correlated to the E. uniflora higher DS threshold. Low rainfall and temperature corresponded to a lower desiccation sensitivity of E. astringens seeds. Seeds of the five species lost half viability between 0.44 and 0.25 g H₂O g DW^??1 and after 65–270 h of desiccation. Our results indicate that abiotic factors impact plant populations during the seed production season and can drive seed desiccation tolerance threshold and physiological behavior. These results should be taken into account in ex-situ plant conservation programs and tropical species management.

     

Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development

Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from ≈80% “stage 1” to ≈55% “stage 2”. When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K_m ≈ 9 moles per cubic meter; V_max ≈ 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k_i ≈ 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V_max from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2-cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure.     

Overexpression of Iron Superoxide Dismutase in Transformed Poplar Modifies the Regulation of Photosynthesis at Low CO2 Partial Pressures or Following Exposure to the Prooxidant Herbicide Methyl Viologen

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H₂O₂, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO₂ assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O₂) and at low (1%) O₂. CO₂-response curves for photosynthesis showed similar net CO₂-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO₂ (Ci) values below 200 μL L⁻¹ but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O₂ concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 μmol m⁻² s⁻¹) a progressive decrease in the ratio of variable (F_v) to maximal (F_m) fluorescence was observed with decreasing temperature. At 6^oC the high-light-induced decrease in the F_v/F_m ratio was partially prevented by low O₂ but values were comparable in all lines. Methyl viologen caused decreased F_v/F_m ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.     

Expression of Tobacco Carbonic Anhydrase in the C4 Dicot Flaveria bidentis Leads to Increased Leakiness of the Bundle Sheath and a Defective CO2-Concentrating Mechanism

Flaveria bidentis (L.) Kuntze, a C₄ dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO₂ assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O₂ concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O₂ levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O₂ concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C₄ CO₂-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO₂ for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.