Senescence-Related Changes in the Lipid Transition Temperature of Microsomal Membranes from Algae

The phase behaviour of smooth microsomal membranes from senescing cultures of Scenedesmus quadricauda has been examined by wide-angle x-ray diffraction. The algae were grown in Bristol's medium at 22°C under continuous illumination. The transition temperature, taken to be the highest temperature at which crystalline (gel) phase lipid can be detected, increased with culture age from a low of 0°C for young cultures to a high of about 70°C for 140-day-old cultures. This indicates that for young cultures the membrane lipid is entirely liquid-crystalline (fluid) at physiological temperatures, but as the cultures age portions of the lipid become crystalline. The increase in transition temperature showed a close temporal correlation with loss of chlorophyll and loss of protein per g dry weight, and can thus be construed as an index of senescence. The unsaturated to saturated fatty acid ratio of the membrane lipid, while fluctuating with culture age, did not show any consistent trend that could be related to the change in transition temperature. Thus the formation of gel phase lipid does not appear to be due to a change in fatty acid saturation.     

Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves

Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity.     

Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals

Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca²⁺ in the presence of ATP. The specific activity of ATP-dependent uptake is ~20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca²⁺ stimulation of ATP hydrolysis was not discernible above the high background of Ca²⁺-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca²⁺ concentration, suggesting that Ca²⁺ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca²⁺ at half maximal velocity of uptake (S_0.5) was 12.5 micromolar and the Hill coefficient (n_H) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca²⁺, although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca²⁺ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca²⁺ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca²⁺ into microsomal vesicles declined by ~70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S_0.5 or n_H, but V_max for ATP-dependent Ca²⁺ uptake decreased by ~40%. A similar decline in ATP-dependent uptake of Ca²⁺ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene.     

Deuterium Magnetic Resonance Studies of Senescence-Related Changes in the Physical Properties of Rose Petal Membrane Lipids

The physical properties of membrane lipids in senescing rose (Rosa hybrida L., cv Mercedes) petals were studied by deuterium nuclear magnetic resonance (2H-NMR) and fluorescence depolarization. All of the 2H-NMR spectra arising from deuterated dimyristoylphosphatidylcholine mixed with whole-lipid extracts from membranes of petals of different ages had a shape that is characteristic of liquid-crystalline lipid at 30[deg]C. Arrhenius plots of the moments of the 2H spectra and fluorescence depolarization values measured from 1,6-diphenyl hexatriene-labeled rose petal membrane lipid samples indicated that membrane lipid order increased with decreasing temperature as well as with increasing age of the petals. The latter trend is explained by previously observed increases in fatty acid saturation and increases in the sterol-to-phospholipid ratio that occur in rose petals during senescence. The 2H-NMR spectra obtained at 0[deg]C also contained quadrupolar splitting lines from lipid in the gel phase, confirming the occurrence of this phase in membranes from this tissue.     

The Effect of Putrescine on the Apoplast Ultrastructure in the Leaf Mesophyll of Mesembryanthemum crystallinum under Salinity Stress

The effects of NaCl, putrescine (Put), and the combination of two agents on the contents of free polyamines (PA), peroxidase activity, and the ultrastructure of the mesophyll apoplast were studied in the third leaf pair of Mesembryanthemum crystallinum L. plants. The NaCl solution was added to soil daily for three days (100 ml of the 100 mM solution), and plants were sprayed with the 1 mM Put solution twice per day for a six-day period. The accumulation of Put and especially spermidine (Spd) by day 3 of salinization was followed by a dramatic drop in Put and Spd contents by day 6. In contrast, the activities of soluble and ion-bound peroxidases increased following a long lag-period. Treatments with Put and NaCl plus Put considerably enhanced this rise in two peroxidase activities. An electron microscopic examination of cell walls in the control and stressed plants demonstrated that a gap developed at the middle lamella with pockets filled with amorphous polysaccharides (AP), presumably pectins. At the maximum gap width, the pockets fused with the intercellular spaces, and, in this case, the intercellular spaces also contained AP. Following salinization, AP in the apoplast swelled and expanded. Apparently the genetic determination of high AP content in M. crystallinum plants is the basis for the ability of juvenile plants to bind Na⁺ and Cl^– ions and excess PA and also to accumulate water. In the plants treated with NaCl plus Put, the number of pockets and their volume increased, and the surface of some cell walls became plated with suberin, thus providing an additional barrier for ion transport from the pockets and intercellular spaces into cells. The formation of suberin plates was correlated with the high activity of ion-bound peroxidase essential for suberin deposition. The authors presume that H₂O₂ results from PA oxidation and, in its turn, induces the activity of peroxidase involved in the suberin plate formation.     

Ascorbate in the Apoplast: Metabolism and Functions

Russian Journal of Plant Physiology - Ascorbic acid (AA) is one of the most important antioxidants and redox-active substances of plants found in the apoplast. In the form of ascorbate anion, it...     

Potassium in the Apoplast of the Root Sink Region

Using carboxyfluorescein, a fluorochrome transported along the phloem, we demonstrated that symplasmic phloem unloading in the watermelon root occurred in the basal zone of the meristem adjusting to the elongation zone. In the similar zones of maize and pumpkin roots, a high level of potassium was detected by X-ray microanalysis in the cell walls and intercellular spaces. Potassium concentration in these compartments comprised two-thirds of that in the cytoplasm. Such proportion between potassium concentrations in the cytoplasm and apoplast was characteristic of both the cortex and stele. Since potassium is a dominant osmotically active component in root tissues, such a proportion between its intracellular and apoplastic concentrations provides for a low turgor pressure in the cells of the sink region, in the phloem in particular. This might increase a turgor pressure gradient along the translocation route between source and sink tissues, which is a driving force for phloem assimilate transport.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 591–599.Original Russian Text Copyright © 2005 by Krasavina, Burmistrova, Feshchenko, Nosov.     

Age-Dependent Changes in Levels of Ascorbic Acid and Chlorogenic Acid, and Activities of Peroxidase and Superoxide Dismutase in the Apoplast of Tobacco leaves: Mechanism of the Oxidation of Chlorogenic Acid in the Apoplast

In order to understand browning in tobacco plants during aging,age-dependent changes in the levels of ascorbic acid (AA) andchlorogenic acid (CGA) and its isomers were investigated inthe apoplast and the symplast of the leaves. Also activitiesof peroxidase (POX) and superoxide dismutase (SOD) were determined.AA decreased during aging until it was no longer detectablein the apoplast, while symplastic AA remained although the leveldecreased on aging. In contrast, levels of CGA and its isomersand activity of POX in the apoplast increased on aging, whilethose in the symplast remained nearly constant in mature andold leaves. The activity of SOD in the apoplast increased duringaging, while that in the symplast decreased. Oxidation of CGAby the apoplastic solution was observed in the absence of externallyadded H₂O₂ and the oxidation was inhibited by SOD and catalase.Brown components, which contained caffeic acid moieties, accumulatedin the apoplast on aging and the components produced O–₂and H₂O₂ by autooxidation. From these results, we conclude (i)that brown components are formed in the apoplast by the CGA/POXsystem, (ii) that the H₂O₂ required for the reaction can beprovided by the CGA/POX system itself and by autooxidation ofthe brown components, and (iii) that apoplastic SOD functionsto generate H₂O₂ from apoplastically formed O–₂. (Received February 8, 1999; Accepted May 7, 1999)     

Changes Induced by Abscisic Acid and Light in the Redox State of Ascorbate in the Apoplast of Epicotyls of Vigna angularis

Levels of ascorbic acid (AA) and dehydroascorbic acid (DHA)were examined in epicotyl segments and intact epicotyls undervarious conditions. It appears that not only the redox stateof an AA-DHA system but also the level of AA plus DHA in theapoplast might be affected by growth conditions. (Received March 26, 1994; Accepted June 4, 1994)     

Diurnal Changes in Citrus Leaf Thickness, Leaf Water Potential and Leaf to Air Temperature Difference

Diurnal changes in leaf water potential and leaf thickness ofwell-watered citrus trees were found to be highly correlated.Midday decreases in leaf thickness of about 30–35 µm reflected midday decreases in leaf water potential of about1.1–1.3 MPa from predawn values. Leaf water potentialwas also correlated with changes in leaf-to-air temperaturedifference and ambient vapour pressure deficit. Leaf thicknessas well as leaf to air temperature difference could possiblybe used to monitor leaf water status continuously as an indicatorof citrus tree water stress.     

Changes in Stem and Leaf Hydraulics Preceding Leaf Shedding in Castanea Sativa L.

This paper describes changes in leaf water status and in stem, petiole and leaf blade hydraulics preceding leaf senescence and shedding in Castanea sativa L. (chestnut). Measurements of maximum diurnal leaf conductance to water vapour (g_L), minimum water potential (_L), hydraulic conductance per unit leaf surface area of stems (K_SL), petioles (K_PL) and leaf blades (K_LL) and number of functional conduits and inside diameter distribution were performed in June, September and October 1999. In September, still green leaves had undergone some dehydration as indicated by decreased g_L (by 75 %) and _L with respect to June. In the same time, K_SL decreased by 88 %, while K_PL and K_LL decreased by 50 % and 20 % of the conduits of stems and 10 % of the petioles (all belonging to the widest diameter range) were no longer functioning, causing a decrease in the theoretical flow by 82 % in stems and 27 % in petioles. Stem xylem blockage was apparently due to tyloses growing into conduits. We advance the hypothesis that the entire process of leaf shedding and winter rest may be initiated by extensive stem embolism occurring during the summer.     

淹水对玉米叶片细胞超微结构的影响

对淹水过程中玉米（Ｚｅａ ｍａｙｓ Ｌ．）叶片细胞超微结构的变化进行连续观察。淹水２ｈ后,液泡膜发生明显内陷。淹水６ｈ后,液泡膜内陷加剧,呈极度松弛状态;叶发体被膜局部向外突出一个由单层膜包裹的泡状结构。淹水１２ｈ后,液泡膜局部破裂;叶绿体被膜破坏加剧,成为一松弛的单膜结构,同时,基质类囊体出现空泡化。淹水１８ｈ后,叶绿体的破坏进一步加剧：被膜完全消失,基质类囊体开始消化;同时,线粒体膜和核膜也开     

Metabolic Regulation in the Senescing Tobacco Leaf: II. Changes in Glycolytic Metabolite Levels in the Detached Leaf

Yellowing of detached mature tobacco leaves standing in water in the dark was accompanied by a strong “climacteric rise” in respiration rate. During this period the ATP level and energy charge of the adenylate system also rose. The levels of glycolytic intermediates between glucose 1-phosphate and triose phosphates rose, those between 3-phosphoglycerate and phosphoenolpyruvate fell, and pyruvate rose. On the assumption of a drop in NAD/NADH ratio, as found by other workers in wheat leaves, the reverse crossover between triose phosphates and 3-phospholglycerate was attributed to inhibition of glyceraldehyde 3-phosphate dehydrogenase. The forward crossover between phosphoenolpyruvate and pyruvate was taken to indicate activation of pyruvate kinase, possibly by fructose diphosphate. Secondary large rises in pyruvate and fructose diphosphate occurred well after the climacteric peak had been passed. No evidence was found for participation of phosphofructokinase in metabolic control in the yellowing leaf. Possible limitations to the use of the crossover theorem in the present situation, such as changes in compartmentation and in flux through branch points, are emphasized.     

Apoplast as the site of response to environmental signals

When the life cycle of plants is influenced by various environmental signals, the mechanical properties of the cell wall are greatly changed. These signals also modify the levels and structure of the cell wall constituents and such modifications are supposed to be the cause of the changes in the wall mechanical properties. These changes in the cell wall, the major component of the apoplast, can be recognized as the response of plants to environmental signals. The analysis of the mechanism leading to the response suggests that the apoplast is involved not only in the response but also in the perception and transduction of environmental signals in concert with the receptors of signals located on the plasma membrane. Thus, the apoplast plays a principal role in the communication of plants with the outer world and enables the plants to adapt themselves and survive in the environment full of stresses.     

Apoplast as the site of response to environmental signals

When the life cycle of plants is influenced by various environmental signals, the mechanical properties of the cell wall are greatly changed. These signals also modify the levels and structure of the cell wall constituents and such modifications are supposed to be the cause of the changes in the wall mechanical properties. These changes in the cell wall, the major component of the apoplast, can be recognized as the response of plants to environmental signals. The analysis of the mechanism leading to the response suggests that the apoplast is involved not only in the response but also in the perception and transduction of environmental signals in concert with the receptors of signals located on the plasma membrane. Thus, the apoplast plays a principal role in the communication of plants with the outer world and enables the plants to adapt themselves and survive in the environment full of stresses.     

Quantifying Shape Changes and Tissue Deformation in Leaf Development

The analysis of biological shapes has applications in many areas of biology, and tools exist to quantify organ shape and detect shape differences between species or among variants. However, such measurements do not provide any information about the mechanisms of shape generation. Quantitative data on growth patterns may provide insights into morphogenetic processes, but since growth is a complex process occurring in four dimensions, growth patterns alone cannot intuitively be linked to shape outcomes. Here, we present computational tools to quantify tissue deformation and surface shape changes over the course of leaf development, applied to the first leaf of Arabidopsis (Arabidopsis thaliana). The results show that the overall leaf shape does not change notably during the developmental stages analyzed, yet there is a clear upward radial deformation of the leaf tissue in early time points. This deformation pattern may provide an explanation for how the Arabidopsis leaf maintains a relatively constant shape despite spatial heterogeneities in growth. These findings highlight the importance of quantifying tissue deformation when investigating the control of leaf shape. More generally, experimental mapping of deformation patterns may help us to better understand the link between growth and shape in organ development.The analysis of biological shapes is a field of broad interest, with diverse applications ranging from medical imaging to comparative anatomy and botany (Lestrel, 2011). Within plant biology, the wide variation of leaf shapes among species has long intrigued evolutionary biologists, physiologists, and developmental biologists alike. Leaves typically develop into flat structures that maximize photosynthetic surface, with variations that may help to facilitate gas exchange, offset water loss, improve convective cooling, increase mechanical support, or reduce resistance to physical environmental forces, for example (for review, see Tsukaya, 2006; Cronk, 2009). The processes controlling leaf shape development, therefore, are important to plant survival and biomass accumulation and hence have important agricultural implications.Understanding how leaf shape is controlled typically involves studying shape variation among related species and in shape mutants and requires tools to quantify phenotypic differences. In recent years, several sophisticated semiautomatic methods have been developed to analyze leaf shapes in terms of their two-dimensional (2D) profiles. Analyses range from simple length and width measurements and allometric ratios to statistical analysis of outline coordinates (Langlade et al., 2005; Bylesjö et al., 2008; Weight et al., 2008; Backhaus et al., 2010).Leaf shape can also be quantified in terms of a three-dimensional (3D) surface, which can range from flat to curved or ruffled, as observed in nature and in many leaf shape mutants. Approaches to 3D shape analysis based on flattened and unflattened leaf dimensions in the proximodistal and mediolateral axes have been presented by Liu et al. (2010) and Wu et al. (2007). Kaminuma et al. (2004) developed a technique for obtaining coordinates of the leaf surface in vivo, from which they measured the angle of the surface across the leaf and fit curves along the leaf proximodistal and mediolateral axes to characterize blade epinasty.These existing methods for leaf shape analysis are very useful for detecting and quantitatively describing shape differences, but they do not provide information about the underlying mechanisms that give rise to those shape differences. For example, the correspondence between evenly spaced outline points (Langlade et al., 2005; Bylesjö et al., 2008; Weight et al., 2008; Backhaus et al., 2010) is arbitrary. Furthermore, existing work has not provided an analysis of leaf shape in terms of both 2D and 3D phenotypic features together, and with the exception of Kaminuma et al. (2004), the methods are destructive and cannot be used to measure changes in shape in vivo.Differences in shapes between two organs arise through differences in how the organs grow. It is impossible, however, to ascertain from differences in final organ shape what differences in growth gave rise to them, as the possible combinations of spatial and temporal alterations in growth patterns that could be responsible for the final shape are endless. By the same token, it can be difficult to conceptualize from growth patterns how the interconnected tissues will deform and, thus, how overall organ shape will change.To address this issue, Kennaway et al. (2011) recently proposed a simulation modeling framework to investigate how a shape may locally and globally deform under the control of spatially distributed growth- and polarity-regulating substances. This framework was used to hypothesize how growth and shape deformation may be controlled in flowers (Green et al., 2010; Sauret-Güeto et al., 2013) and leaves (Kuchen et al., 2012). However, to date, shape deformation patterns have not been quantified experimentally. Here, we use data collected for a study on leaf growth (Remmler and Rolland-Lagan, 2012) to develop a method for describing 3D surface shape and shape deformations during leaf development. In particular, we generate experimental maps of tissue deformation, which offer a new approach to investigating shape differences and uncovering the link between growth and shape during development.     

Occurrence of Cello-Oligosaccharides in the Apoplast of Auxin-Treated Pea Stems

Treatment of pea (Pisum sativum L.) hypocotyl segments with indole-3-butyric acid, which promotes segment elongation, increased the solubilization of both xyloglucan and cello-oligosaccharides in the apoplast of auxin-treated pea stems. The cello-oligosaccharides were isolated from the apoplastic solution with a charcoal/Celite column and were identified as cellobiose, cellotriose, and cellotetraose after subsequent thin-layer chromatography and paper electrophoresis. Cello-oligosaccharides in the apoplastic fraction were monitored using cellobiose dehydrogenase. Both xyloglucan and cello-oligosaccharides appeared to be formed concurrently within 30 min after treatment with the auxin, and the cello-oligosaccharides increased with stem elongation even after 2 h. The total activity of cellulase did not increase for up to 4 h.