Calcium acetate induces calcium uptake and formation of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L.

During treatment of isolated, peeled leaflets of Gleditsia triacanthos with 0.5–2 mM [⁴⁵Ca]acetate, saturation of the cell-wall free space with Ca²⁺ occurred within 10 min and was followed by a period of 6–10 h during which there was no significant Ca-uptake into the protoplast, but apoplastic Ca²⁺ was periodically released into the medium. Later, Ca²⁺ was absorbed for 3–4 d at rates of up to 2.2 mol Ca²⁺·h^-1·(g FW)^-1 to final concentrations of 350 mol Ca²⁺· (g FW)^-1. The distribution of absorbed Ca²⁺ between cell wall, vacuole and Ca-oxalate crystals was determined during Ca-uptake. Wheras intact, cut leaflets deposited absorbed Ca²⁺ as Ca-oxalate in the crystal cells, peeled leaflets lacking crystal cells accumulated at least 40–50 mol·(g FW)^-1 soluble Ca²⁺ before the absorbed Ca²⁺ was precipitated as Ca-oxalate. These observations indicate that the mechanisms for the continuous uptake of Ca²⁺, the synthesis of oxalate and the precipitation of Ca²⁺ as Ca-oxalate are operational in the crystal cells of intact leaflets, but not in the mesophyll cells of peeled leaflets where they must be induced by exposure to Ca²⁺. The precipitation of absorbed Ca²⁺ as Ca-oxalate by the crystal cells of isolated Gleditsia leaflets illustrates the role of these cells in the excretion of surplus Ca²⁺ which enters normal, attached leaves with the transpiration stream.In addition to acetate, only Ca-lactate and Ca-carbonate lead to Ca-uptake, but at rates well below those observed with Ca-acetate. Other small organic anions (citrate, glycolate, glyoxalate, malate) and inorganic anions (chloride, nitrate, sulfate) did not permit Ca-uptake. Acetate-¹⁴C was rapidly absorbed during Ca-uptake, but less than 20% was incorporated into Ca-oxalate; the rest remained mostly in the soluble fraction or was metabolized to CO₂. Acetate, as a permeable weak acid, may enable rapid Ca-uptake by stimulating proton extrusion at the plasmalemma and by serving as a counterion during Ca-accumulation in the vacuole, but is unlikely to function as the principal substrate for oxalate synthesis.     

Calcium-induced patterns of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L. and Albizia julibrissin Durazz

For experimental induction of crystal cells (=crystal idioblasts) containing calcium-oxalate crystals, the lower epidermis was peeled from seedling leaflets of Gleditsia triacanthos L., exposing the crystal-free mesophyll and minor veins to the experimental solutions on which leaflets were floated for up to 10 d under continous light. On 0.3–2.0 mM Ca-acetate, increasing numbers of crystals, appearing 96 h after peeling, were induced. The pattern of crystal distribution changed with Ca²⁺-concentration ([Ca²⁺]): at low [Ca²⁺], crystals formed only in the non-green bundlesheath cells surrounding the veins, believed to have a relatively low Ca²⁺-extrusion capacity; at higher [Ca²⁺], crystals developed in up to 90% of the mesophyll cells, and at supraoptimal [Ca²⁺], large extracellular crystals formed on the tissue surface. By sequential treatments with solutions of different [Ca²⁺], the following three phases were identified in the induction of crystal cells: (1) during the initial 24-h period (adaptive aging), Ca²⁺ is not required and crystal induction is not possible; (2) during the following 48 h (induction period), exposure to 1–2 mM Ca-acetate induces the differentiation of mesophyll cells into crystal cells; (3) crystal growth begins 72 h after the start of induction. In intact leaflets of Albizia julibrissin Durazz., calcium-oxalate crystals are found exclusively in the bundle-sheath cells of the veins, but crystals were induced in the mesophyll of peeled leaflets floating on 1 mM Ca-acetate. Exposure to inductive [Ca²⁺] will thus trigger the differentiation of mature leaf cells into crystal cells; the spatial distribution of crystals is determined by the external [Ca²⁺] and by the structural and functional properties of the cells in the tissue.     

Cell and calcium oxalate crystal growth is coordinated to achieve high-capacity calcium regulation in plants

Summary Crystal idioblasts are cells which are specialized for accumulation of Ca²⁺ as a physiologically inactive, crystalline salt of oxalic acid. Using microautoradiographic, immunological, and ultrastructural techniques, the process of raphide crystal growth, and how crystal growth is coordinated with cell growth, was studied in idioblasts ofPistia stratiotes. Incorporation of⁴⁵Ca²⁺ directly demonstrated that, relative to surrounding mesophyll cells, crystal idioblasts act as high-capacity Ca²⁺ sinks, accumulating large amounts of Ca²⁺ within the vacuole as crystals. The pattern of addition of Ca²⁺ during crystal growth indicates a highly regulated process with bidirectional crystal growth. In very young idioblasts,⁴⁵Ca²⁺ is incorporated along the entire length of the needle-shaped raphide crystals, but as they mature incorporation only occurs at crystal tips in a bidirectional mode. At full maturity, the idioblast stops Ca²⁺ uptake, although the cells are still alive, demonstrating an ability to strictly regulate Ca transport processes at the plasma membrane. In situ hybridization for ribosomal RNA shows young idioblasts are extremely active cells, are more active than older idioblasts, and have higher general activity than surrounding mesophyll cells. Polarizing and scanning electron microscopy demonstrate that the crystal morphology changes as crystals develop and includes morphological polarity and an apparent nucleation point from which crystals grow bidirectionally. These results indicate a carefully regulated process of biomineralization in the vacuole. Finally, we show that the cytoskeleton is important in controlling the idioblast cell shape, but the regulation of crystal growth and morphology is under a different control mechanism.Abbreviation SEM scanning electron microscopy     

Stress-dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

When ¹⁴C-labelled abscisic acid ([¹⁴C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h^?1 m^?3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh^?l m^?3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress-dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress-induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.     

Comparative Microscopical Studies on the Patterns of Calcium Oxalate Distribution in the Needles of Various Conifer Species

The distribution of calcium oxalate crystals in various conifer needles is visualized by light and electron microscopy. Such crystals occur (1) in the vascular bundle, either intracellularly in the xylem or phloem parenchyma, or extracellularly within the radial phloem walls; (2) extracellularly on the outside of the walls of mesophyll cells which face the intercellular spaces; (3) and finally as numerous small crystals within the cell walls of the epidermal cells, especially in the cuticular layer. The development and distribution of these apoplastic crystals is described in detail. Some hypotheses are finally presented for interpretations of these unusual patterns of the crystallization of Ca-oxalate outside the vacuole. Possible evolutionary aspects of this feature among the different conifer families are also discussed.     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Cell-specific compartmentation of mineral nutrients is an essential mechanism for optimal plant productivity— another role for TPC1?

Vacuoles of different leaf cell-types vary in their capacity to store specific mineral elements. In Arabidopsis thaliana potassium (K) accumulates preferentially in epidermal and bundle sheath cells whereas calcium (Ca) and magnesium (Mg) are stored at high concentrations only in mesophyll cells. Accumulation of these elements in a particular vacuole can be reciprocal, i.e. as [K]vac increases [Ca]_vac decreases. Mesophyll-specific Ca-storage involves CAX1 (a Ca2⁺/H⁺ antiporter) and Mg-storage involves MRS2-1/MGT2 and MRS2-5/MGT3 (both Mg²⁺-transporters), all of which are preferentially expressed in the mesophyll and encode tonoplast-localised proteins. However, what controls leaf-cell [K]_vac is less well understood. TPC1 encodes the two-pore Ca²⁺ channel protein responsible for the tonoplast-localised SV cation conductance, and is highly expressed in cell-types that not preferentially accumulate Ca. Here, we evaluate evidence that TPC1 has a role in maintaining differential K and Ca storage across the leaf, and propose a function for TPC1 in releasing Ca²⁺ from epidermal and bundle sheath cell vacuoles to maintain low [Ca]vac. Mesophyll-specific Ca storage is essential to maintain apoplastic free Ca concentration at a level that does not perturb a range of physiological parameters including leaf gas exchange, cell wall extensibility and growth. When plants are grown under serpentine conditions (high Mg/Ca ratio), MGT2/MRS2-1 and MGT3/MRS2-5 are required to sequester additional Mg²⁺ in vacuoles to replace Ca²⁺ as an osmoticum to maintain growth. An updated model of Ca²⁺ and Mg²⁺ transport in leaves is presented as a reference for future interrogation of nutritional flows and elemental storage in plant leaves.     

Salinity induced differences in growth,ion distribution and partitioning in barley between the cultivar Maythorpe and its derived mutant Golden Promise

Dry matter changes and ion partitioning in two near isogenic barley cultivars Maythorpe (relatively salt sensitive) and Golden Promise (relatively salt tolerant) were studied in response to increasing salinity. Although the growth of both cultivars was significantly reduced by exposure to NaCl, the effect was greater in Maythorpe, whilst Golden Promise maintained an increased ratio of young to old leaf blade. Golden Promise maintained significantly lower Na⁺ concentrations in young expanding tissues compared with Maythorpe. Partitioning of Cl^– was evident in that both varieties maintained lower Cl^– concentrations in mesophyll than in epidermal cells. Golden Promise maintained higher K⁺/Na⁺ and Ca²⁺/Na⁺ ratios in young leaf blade and young sheath tissues than Maythorpe when exposed to salt. Differences in ion partitioning and the maintenance of higher K⁺ and Ca²⁺ to Na⁺ ratios, especially in young growing and recently expanded tissues, would appear to be important mechanisms contributing to the improved salt tolerance of Golden Promise.     

Free-space marker studies on the leaf ofZea mays L.

Summary Two free-space marker procedures (Prussian blue and lanthanum nitrate) were employed to determine the pathway(s) followed by water and solutes in the transpiration stream after their introduction into the xylem of small and intermediate bundles, and the effectiveness of the suberin lamellae of the bundle-sheath cells as a barrier to the movement of tracer ions (Fe³⁺ and La³⁺). Judged from the distribution of Prussian-blue crystals (insoluble, crystalline deposits resulting from the precipitation of ferric ions by ferrocyanide anions) and lanthanum deposits, water and the tracer ions moved readily from the lumina of the vessels into the apoplast (cell wall continuum) of the phloem and bundle-sheath cells via portions of vessel primary walls not bearing lignified secondary wall thickenings. Prussian blue and lanthanum deposits were abundant on the bundlesheath cell side of the bundle sheath/mesophyll interface but few occurred on that of the mesophyll, indicating that the suberin lamella is an effective barrier to apoplastic movement of both ferric and lanthanum ions. The presence of Prussian-blue crystals and lanthanum deposits in the compound middle lamella of the radial wall of the bundle-sheath cells indicates that the compound middle lamella provides an apoplastic pathway for transpirational water from the xylem to the evaporating surfaces of the mesophyll and epidermal cells.     

Nicotine Production by Tissue Cultures of Tobacco as Influenced by Various Culture Parameters

Assimilate efflux from vacuum-infiltrated leaf slices (spinach, barley) into a buffered solution was examined in relation to Ca^{+ +} -activity and osmotic conditions. Efflux from isolated mesophyll protoplasts and from a unicellular green alga (Eremosphaera viridis de Bary) was also measured.In the presence of Ca^{+ +}, assimilate efflux from leaf slices was small (1 to 5 % of the total carbon fixation rate, depending on osmotic conditions). Efflux was drastically stimulated by addition of Ca^{+ +} -chelators. If expressed as µmol carbon mg^-1 chlorophyll h^-1, it reached 50 % of the assimilation rate. Efflux from protoplasts or algae was slow and insensitive to Ca^{+ +} chelators at concentrations which caused fast efflux from leaf slices.Assimilate efflux from leaf slices was rather unspecific. Both in the tissue and the surrounding medium, sucrose was the most abundantly labelled compound (70 to 80 % of total soluble labelled material).A 50 % decrease of efflux was observed when turgor pressure was lowered by addition of sorbitol (200 to 300 mosmol kg^-1). At extremely high sorbitol concentrations (> 1500 mosmol kg^-1) efflux increased again and was relatively less stimulated by EDTA.It is suggested that assimilate efflux from leaf slices is mainly diffusion through open veins and/or plasmodesmata. When these symplastic connections are closed by addition of Ca^{+ +}, the remaining transmembrane flux into the apoplast is small. Thus, assimilate movement from the mesophyll to the phloem appears to be symplastic, not apoplastic as suggested in the literature.     

Palisade mesophyll cell expansion during leaf development in Zinnia elegans (Asteraceae)

Temporal and spatial patterns of palisade mesophyll cell expansion in Zinnia elegans were characterized as a basis for developing a suspension culture model for mesophyll cell expansion. Our objectives were to 1) identify the leaf regions from which cells in various stages of expansion could be selectively isolated for culture, and 2) develop a basis for comparison of rate and extent of mesophyll cell expansion in culture with that in the leaf. Palisade mesophyll cells were isolated from expanding leaves by gentle physical maceration without the use of enzymes. Isolated cells from leaves in different stages of expansion were then measured by computer image analysis. Analysis of size frequency distributions showed that unexpanded cells can be isolated from the entire blade of small leaves or the basal regions of partially expanded leaves. Fully expanded cells can be obtained from the apical and middle regions of partially expanded leaves. Within the leaf, Zinnia mesophyll cells expanded from about 400 μm² to about 2.300 μm² at an estimated rate of 160 μm² d^-1. The percent increase in cell length exceeded the percent increase in cell width. Expansion of mesophyll cells continued for 6–8 d after epidermal expansion ceased. This difference in the timing of cell expansion in epidermal and mesophyll cells indicates that different regulatory factors may be operating in these adjacent tissues and underscores the importance of investigating the regulation of mesophyll cell expansion at the cellular level.     

Developmental features of calcium oxalate crystal sand deposition inBeta vulgaris L. Leaves

Summary Sugar beet (Beta vulgaris L.) leaf has a layer of cells extended laterally between the palisade parenchyma and spongy mesophyll that develop numerous small crystals (crystal sand) within their vacuoles. Solubility studies and histochemical staining indicate the crystals are calcium oxalate. The crystals are deposited within the vacuoles early during leaf development, and at maturity the cells are roughly spherical in shape and 2 to 3 times larger than other mesophyll cells. Crystal deposition is preceeded by formation of membrane vesicles within the vacuole. The membranes are synthesizedde novo in the vacuole and have a typical trilaminate structure as viewed with the TEM. The membranes are formed within paracrystalline aggregates of tubular particles (6–8nm outer diameter) as membrane sheets, but are later organized into chambers or vesicles. Calcium oxalate is then precipitated within the membrane chambers. The tubular particles involved in membrane synthesis are usually present in the vacuoles of mature crystal cells, but in very small amounts.     

Effects of magnesium availability on the activity of plasma membrane ion transporters and light-induced responses from broad bean leaf mesophyll

Considering the physiological significance of Mg homeostasis in plants, surprisingly little is known about the molecular and ionic mechanisms mediating Mg transport across the plasma membrane and the impact of Mg availability on transport processes at the plasmalemma. In this study, a non-invasive ion-selective microelectrode technique (MIFE) was used to characterize the effects of Mg availability on the activity of plasma membrane H⁺, K⁺, Ca²⁺, and Mg²⁺ transporters in the mesophyll cells of broad bean (Vicia faba L.) plants. Based on the stoichiometry of ion-flux changes and results of pharmacological experiments, we suggest that at least two mechanisms are involved in Mg²⁺ uptake across the plasma membrane of bean mesophyll cells. One of them is a non-selective cation channel, also permeable to K⁺ and Ca²⁺. The other mechanism, operating at concentrations below 30 M, was speculated to be an H⁺/Mg⁺ exchanger. Experiments performed on leaves grown at different levels of Mg availability (from deficient to excessive) showed that Mg availability has a significant impact on the activity of plasma-membrane transporters for Ca²⁺, K⁺, and H⁺. We discuss the physiological significance of Mg-induced changes in leaf electrophysiological responses to light and the ionic mechanisms underlying this process.     

Effect of Age on Adenosine Triphosphatase Activity from Tobacco Chloroplasts

Adenosine triphosphatase activity of tobacco leaf chloroplasts in the dark was measured, using leaves of different age as determined by the position of the leaves along the stem. The activity of the chloroplast preparations strongly decreased with age, regardless of the addition of Mg²⁺ or Ca²⁺. Opposite effects of Mg²⁺ and Ca²⁺ on the activity of the chloroplasts were noted in experiments where different ratios of Mg²⁺/Ca²⁺ were applied. They were related to the age of the leaves, Ca²⁺ strongly stimulated the activity of the preparations from old leaves but was practically without effect in young, just expanded leaves. Mg²⁺ slightly stimulated the activity from old leaves while it invariably inhibited the hydrolytic activity of preparations from young leaves.     

Time-course of programmed cell death during leaf senescence in <Emphasis Type="Italic">Eucommia ulmoides</Emphasis>

Leaves of Eucommia ulmoides Oliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca²⁺-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves. Electronic Publication     

Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH

Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K⁺ concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5-7.5), allowing extremely small changes in H⁺ fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid.     

Effects of salt on growth,ion accumulation,photosynthesis and leaf anatomy of the mangrove,<Emphasis Type="Italic"> Bruguiera parviflora</Emphasis>

The effects of a range of salinity (0, 100, 200 and 400 mM NaCl) on growth, ion accumulation, photosynthesis and anatomical changes of leaves were studied in the mangrove, Bruguiera parviflora of the family Rhizophoraceae under hydroponically cultured conditions. The growth rates measured in terms of plant height, fresh and dry weight and leaf area were maximal in culture treated with 100 mM NaCl and decreased at higher concentrations. A significant increase of Na⁺ content of leaves from 46.01 mmol m^-2 in the absence of NaCl to 140.55 mmol m^-2 in plants treated with 400 mM NaCl was recorded. The corresponding Cl^- contents were 26.92 mmol m^-2 and 97.89 mmol m^-2. There was no significant alteration of the endogenous level of K⁺ and Fe²⁺ in leaves. A drop of Ca²⁺ and Mg²⁺ content of leaves upon salt accumulation suggests increasing membrane stability and decreased chlorophyll content respectively. Total chlorophyll content decreased from 83.44 g cm^-2 in untreated plants to 46.56 g cm^-2 in plants treated with 400 mM NaCl, suggesting that NaCl has a limiting effect on photochemistry that ultimately affects photosynthesis by inhibiting chlorophyll synthesis (ca. 50% loss in chlorophyll). Light-saturated rates of photosynthesis decreased by 22% in plants treated with 400 mM NaCl compared with untreated plants. Both mesophyll and stomatal conductance by CO₂ diffusion decreased linearly in leaves with increasing salt concentration. Stomatal and mesophyll conductance decreased by 49% and 52% respectively after 45 days in 400 mM NaCl compared with conductance in the absence of NaCl. Scanning electron microscope study revealed a decreased stomatal pore area (63%) in plants treated with 400 mM NaCl compared with untreated plants, which might be responsible for decreased stomatal conductance. Epidermal and mesophyll thickness and intercellular spaces decreased significantly in leaves after treatment with 400 mM NaCl compared with untreated leaves. These changes in mesophyll anatomy might have accounted for the decreased mesophyll conductance. We conclude that high salinity reduces photosynthesis in leaves of B. parviflora, primarily by reducing diffusion of CO₂ to the chloroplast, both by stomatal closure and by changes in mesophyll structure, which decreased the conductance to CO₂ within the leaf, as well as by affecting the photochemistry of the leaves.     

香樟叶肉含晶细胞季节变化研究

为探讨香樟(Cinnamomum camphora)叶肉含晶细胞超微结构的季节变化,阐明香樟叶肉中草酸钙晶体在春夏秋冬的变化规律。该研究以多年生香樟(C. camphora)叶片为材料,分别于春夏秋冬四个季节露地取样,制作超薄切片,用透射电子显微镜(TEM)观察叶肉含晶细胞超微结构的变化。结果表明:春季时香樟叶肉中只有少数细胞有草酸钙晶体,数量较少,晶体结构多为柱状晶、方晶; 夏季时香樟叶肉细胞中随机分布于液泡的草酸钙晶体明显比春季的数量多、体积大、形态丰富,晶体多为柱状晶、方晶、针晶、簇晶; 秋季时香樟叶肉细胞草酸钙晶体和夏季的类似,数量较多,形态多样,以方晶和柱状晶针晶为主,伴有晶簇; 冬季时香樟叶肉含晶细胞晶体形态为柱状晶、方晶、针晶,数量比夏季和秋季的数量略有减少。该研究结果表明在一年四季中香樟叶肉细胞液泡中均有草酸钙晶体结构存在。     

Specific and constitutive expression of oxalate oxidase during the ageing of leaf sheaths of ryegrass stubble

Changes in the activity of oxalate oxidase (OxO) and of the concentrations of oxalate and H₂O₂ were investigated during the ageing of leaf sheaths of ryegrass (Lolium perenne L.) stubble. The accumulation of H₂O₂ during ageing coincides with the increases of both oxalate level and OxO activity. Western and Northern blot analyses using protein and RNA extracts of the different categories of leaf sheaths suggested that OxO gene expression, as well as Ca-oxalate synthesis, are crucial events of ageing for leaf sheaths. Immunocytochemistry experiments have revealed that OxO, which is an extracellular enzyme, is nearly always present in the parenchymatous cells surrounding the vascular bundles and in the cells of the lower epidermis. Overall, results suggest that in ryegrass that synthesizes both Ca-oxalate and OxO, the production of H₂O₂ and Ca²⁺ during ageing of stubble might be involved in the constitutive defences against pathogens, thus allowing the phloem mobilization of nutrient reserves from the leaf sheaths towards elongating leaf bases of ryegrass.     

Degradation of chloroplast DNA in second leaves of rice (Oryza sativa) before leaf yellowing

Summary The second leaf ofOryza sativa develops, grows and ages within the 10 days that follow imbibition under our controlled continuous-light conditions. Proplastids in the leaf cells develop, mature to become chloroplasts and then age and disintegrate. In an examination of this life process, we studied first the behavior and the number of copies of plastid DNA and levels of chlorophyll by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified microscope photon-counting system (VIMPCS). The results indicated that the number of copies of the plastid DNA per plastid increased and reached to plateau value of approximately 100 at the time when the elongation of the mesophyll cells and the enlargement of chloroplasts ceased 96 h after imbibition. However, 24 h later, the number of copies of plastid DNA per chloroplast began to decrease and fell rapidly to approximately 30 copies within 168 h after imbibition. Our examination of the number of chloroplasts per mesophyll cell indicated that no division of chloroplasts occurred more than 72 h after imbibition. The results suggest that the decrease in number of copies of plastid DNA per chloroplast was not due to an increase in the number of chloroplasts, but that this decrease was caused by degradation by unidentified enzymes. Since visible senescence of leaves, which was characterized by development of a yellowish color, began 168 h after imbibition, the degradation of plastid DNA seemed to occur 48 h before the visible leaf senescence. When we tested the nucleolytic activities in the second leaves after imbibition by digestion of plasmids in vitro and DNA-SDS polyacrylamide gel electrophoresis, five Ca²⁺–, four Zn²⁺–, and four Mn²⁺–dependent nucleases were detected in the leaf blades, and one of the Ca²⁺–, two of the Zn²⁺–, and two of the Mn²⁺–dependent nucleases were also identified in a purified preparation of intact chloroplasts. When the activity of the Zn²⁺–dependent nucleases (51 kDa and 13 kDa) increased markedly, degradation of the plastid DNA occurred. These results suggest that the destruction of chloroplast DNA, which occurs approximately 48 h before leaf yellowing, could be due to the activation of some metallo-nucleases and, furthermore, this enzymatic degradation propels the leaf towards senescence.