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.     

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。     

Absence of CeCl3-detectable peroxisomal glycolate-oxidase activity in developing raphide crystal idioblasts in leaves of Psychotria punctata Vatke and roots of Yucca torreyi L.

Three peroxisomal enzymes, glycolate oxidase, urate oxidase and catalase were localized cytochemically in Psychotria punctata (Rubiaceae) leaves and Yucca torreyi (Agavaceae) seedling root tips, both of which contain developing and mature calcium-oxalate raphide crystal idioblasts. Glycolate-oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6) activities were present within leaftype peroxisomes in nonidioblastic mesophyll cells in Psychotria leaves, while urate-oxidase (EC 1.7.3.3) activity could not be conclusively demonstrated in these organelles. Unspecialized peroxisomes in cortical parenchyma of Yucca roots exhibited activities of all three enzymes. Reactionproduct deposits attributable to glycolate-oxidase activity were never observed in peroxisomes of any developing or mature crystal idioblasts of Psychotria or Yucca. Catalase localization indicates that idioblast microbodies are functional peroxisomes. The apparent absence of glycolate oxidase in crystal idioblasts of Psychotria and Yucca casts serious doubt that pathways involving this enzyme are operational in the synthesis of the oxalic acid precipitated as calcium-oxalate crystals in these cells.Abbreviations AMPD 2-amino-2-methyl-1,3-propandiol - CTEM conventional transmission electron microscopy - DAB 3,3-diaminobenzidine tetrahydrochloride - HVEM high-voltage electron microscopy     

Calcium channels are involved in calcium oxalate crystal formation in specialized cells of Pistia stratiotes L

BACKGROUND AND AIMS: Pistia stratiotes produces large amounts of calcium (Ca) oxalate crystals in specialized cells called crystal idioblasts. The potential involvement of Ca(2+) channels in Ca oxalate crystal formation by crystal idioblasts was investigated. METHODS: Anatomical, ultrastructural and physiological analyses were used on plants, fresh or fixed tissues, or protoplasts. Ca(2+) uptake by protoplasts was measured with (45)Ca(2+), and the effect of Ca(2+) channel blockers studied in intact plants. Labelled Ca(2+) channel blockers and a channel protein antibody were used to determine if Ca(2+) channels were associated with crystal idioblasts. KEY RESULTS: (45)Ca(2+) uptake was more than two orders of magnitude greater for crystal idioblast protoplasts than mesophyll protoplasts, and idioblast number increased when medium Ca was increased. Plants grown on media containing 1-50 microM of the Ca(2+) channel blockers, isradipine, nifedipine or fluspirilene, showed almost complete inhibition of crystal formation. When fresh tissue sections were treated with the fluorescent dihydropyridine-type Ca(2+) channel blocker, DM-Bodipy-DHP, crystal idioblasts were intensely labelled compared with surrounding mesophyll, and the label appeared to be associated with the plasma membrane and the endoplasmic reticulum, which is shown to be abundant in idioblasts. An antibody to a mammalian Ca(2+) channel alpha1 subunit recognized a single band in a microsomal protein fraction but not soluble protein fraction on western blots, and it selectively and heavily labelled developing crystal idioblasts in tissue sections. CONCLUSIONS: The results demonstrate that Ca oxalate crystal idioblasts are enriched, relative to mesophyll cells, in dihydropyridine-type Ca(2+) channels and that the activity of these channels is important to transport and accumulation of Ca(2+) required for crystal formation.     

Ca2+ as developmental signal in the formation of Ca-oxalate crystal spacing patterns during leaf development inCarya ovata

Changes in the spacing patterns of Ca-oxalate crystals during enlargement ofCarya ovata Mill. leaves were quantified by computerized image-analysis. Single Ca-oxalate crystals form in the vacuoles of young mesophyll cells transformed into crystal cells Crystals are very small in newly induced crystal cells and increase in size throughout leaf development. Crystal patterns thus reflect both induction and relative age of crystal cells. Shortly after the emergence of young leaves from the bud, very small crystals are formed in the mesophyll at high density. As leaves expand, these crystals grow larger and become separated by increasing distances. New small crystals appear in the gaps between the older, larger crystals. Later crystal patterns consist of widely spaced, larger crystals only. Finally, clusters of small crystals are formed again in the gaps between large crystals. No crystals were observed in young leaves expanding in a moist chamber, but large numbers of crystal cells were induced experimentally in sections of immature leaves floating on 4 mM Ca-acetate. The observations support the following mechanism of crystal-pattern formation: Ca²⁺ carried into leaves with the transpiration stream acts as the developmental signal inducing transdifferentiation of a few mesophyll cells into crystal cells when apoplastic [Ca²⁺] rises. Crystal cells precipitate absorbed Ca²⁺ as oxalate and, acting as Ca²⁺ sinks, inhibit crystal-cell induction in their vicinity by depleting apoplastic Ca²⁺. This prevents close spacing of crystal cells. New crystal cells form in the gaps between the depletion zones of older crystal cells when these move apart during leaf expansion. Later changes in crystal patterns result from increasing sink strength of crystal cells, lowered inducibility of mesophyll cells, and increased Ca²⁺ influx into leaves during intensive transpiration. Throughout leaf development, spacing of crystal cells permits rapid secretion of apoplastic Ca²⁺ as Ca-oxalate. Dedicated to Professor Erwin Bünning, University of Tübingen, Germany, who pioneered the analysis of spacing patterns     

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.     

The Role of Druse and Raphide Calcium Oxalate Crystals in Tissue Calcium Regulation in Pistia stratiotes Leaves

Abstract: Ca oxalate crystal formation was examined in Pistia stratiotes L. leaves during excess Ca and Ca-deficient conditions. Pistia produces druse crystal idioblasts in the adaxial mesophyll and raphide idioblasts in the abaxial aerenchyma. Raphide crystals were previously found to grow bidirectionally, and here we show that Ca is incorporated along the entire surfaces of developing druse crystals, which are coated with membrane-bound microprojections. Leaves formed on plants grown on 0 Ca medium have fewer and smaller druse crystals than leaves formed under 5 mM Ca ("control") conditions, while raphide crystal formation is completely inhibited. When plants were moved from 0 to 15 mM ("high") Ca, the size and number of crystals in new leaves returned to (druse) or exceeded (raphide) control levels. High Ca also induced formation of druse, but not raphide, crystals in differentiating chlorenchyma cells. When plants were transferred from 15 mM Ca to 0 Ca, young druse crystals were preferentially partially dissolved. Oxalate oxidase, an enzyme that degrades oxalate, increased during Ca deficiency and was localized to the crystal surfaces. The more dynamic nature of druse crystals is not due to hydration form as both crystal types are shown to be monohydrate. Part of the difference may be because raphide idioblasts have developmental constraints that interfere with a more flexible response to changing Ca. These studies demonstrate that excess Ca can be stored as Ca oxalate, the Ca can be remobilized under certain conditions, and different forms of Ca oxalate have different roles in bulk Ca regulation.     

ULTRASTRUCTURE OF DRUSE CRYSTAL IDIOBLASTS IN LEAVES OF CERCIDIUM FLORIDUM

The ultrastructure of druse crystal idioblasts in palo verde leaves is similar in certain aspects to other crystal-containing idioblasts, but also displays several notable differences. Although the crystal itself is dissolved during the preparative procedure, the druse idioblast is readily observable. The large crystal is contained in a tightly appressed vacuole in the center of the idioblast. Between the crystal vacuole and the cell wall there is a narrow stalk-like connection which has the same substructure and staining characteristics as cell wall material. The membrane of this crystal vacuole and the idioblast plasmalemma stain asymmetrically, while other cellular membranes in the idioblast appear symmetrical. Much of the remaining cell volume is occupied by a ramified vacuome and a peripherally displaced nucleus. The plastids of the druse idioblast are markedly different from chloroplasts in adjacent parenchyma cells. The former lack the size, starch grains, and well-developed grana of the latter. Idioblast mitochondria are similar in quantity and appearance to those of palisade cells, except for a greater number of cristae in the former. Dictyosomes, while rare in mesophyll cells, are quite common in the idioblast. These features suggest that the druse crystal idioblast is metabolically active and not dead at maturity.     

Leaf anatomy ofDombeya andNesogordonia (Sterculiaceae), emphasizing epidermal and internal idioblasts

We studied leaf anatomy, using clearings, resin sections, and scanning electron microscopy, from mostly herbarium specimens of 123 species ofDombeya and 11 species ofNesogordonia (Sterculiaceae). Species were placed in seven idioblast categories, ranging from those without any to single and bicelled epidermal forms to multicelled nodules and single mesophyll idioblasts. Idioblast contents are possibly mucilaginous, but were not identified. In these two genera the range of foliar idioblast morphology surpasses that known previously for the entire family. Leaves are dorsiventral with mostly abaxial anomocytic stomata and typical palisade and spongy layers; paraveinal mesophyll is lacking. Miniature glandular (clavates, capitates) and nonglandular (mostly stellate) trichomes occur. Prismatic crystals predominate; epidermal prismatics and mesophyll druses are rare.     

Incorporation of strontium into plant calcium oxalate crystals

Summary Lemna minor, which produces many calcium oxalate raphide crystals, was grown on media containing in addition to Ca, 200 M of one of the following divalent cations: Ba, Cd, Co, Mn or Sr. Energy dispersive X-ray analysis showed that only Sr was incorporated into the raphides at levels detectable by the analysis technique. Incorporation of Sr into other insoluble compounds, such as cell wall material, could not be detected. Plant species which form different crystal types in their leaves (Beta vulgaris, crystal sand;Arthrostema ciliatum, druse;Glycine canescens, prismatic) also incorporated Sr into their crystals when grown hydroponically on nutrient medium containing 200 M Sr.Axenic cultures ofL. minor were used to examine further the process of Sr incorporation into plant crystals. When grown on nutrient solution with 5 M Ca, increasing the Sr concentration resulted in increases of the amount of Sr incorporated into the raphide crystals. The ratio of Sr to Ca became greater as the Sr concentration was increased. This ratio change was due to both an increase in the amount of Sr incorporated and a decrease in the Ca incorporated. Analysis of the number of crystal idioblasts formed as a function of Sr concentration shows fewer idioblasts are produced as Sr became high. Competition with Ca and interference of Ca utilization by Sr is indicated.     

Cellular ultrastructure and crystal development in Amorphophallus (Araceae)

Background and Aims: Species of Araceae accumulate calcium oxalate in the form ofcharacteristically grooved needle-shaped raphide crystals andmulti-crystal druses. This study focuses on the distributionand development of raphides and druses during leaf growth inten species of Amorphophallus (Araceae) in order to determinethe crystal macropatterns and the underlying ultrastructuralfeatures associated with formation of the unusual raphide groove. Methods: Transmission electron microscopy (TEM), scanning electron microscopy(SEM) and both bright-field and polarized-light microscopy wereused to study a range of developmental stages. Key Results: Raphide crystals are initiated very early in plant development.They are consistently present in most species and have a fairlyuniform distribution within mature tissues. Individual raphidesmay be formed by calcium oxalate deposition within individualcrystal chambers in the vacuole of an idioblast. Druse crystalsform later in the true leaves, and are absent from some species.Distribution of druses within leaves is more variable. Drusesinitially develop at leaf tips and then increase basipetallyas the leaf ages. Druse development may also be initiated incrystal chambers. Conclusions: The unusual grooved raphides in Amorphophallus species probablyresult from an unusual crystal chamber morphology. There aremultiple systems of transport and biomineralization of calciuminto the vacuole of the idioblast. Differences between raphideand druse idioblasts indicate different levels of cellular regulation.The relatively early development of raphides provides a defensivefunction in soft, growing tissues, and restricts build-up ofdangerously high levels of calcium in tissues that lack theability to adequately regulate calcium. The later developmentof druses could be primarily for calcium sequestration.     

Biosynthesis of L-ascorbic acid and conversion of carbons 1 and 2 of L-ascorbic acid to oxalic acid occurs within individual calcium oxalate crystal idioblasts

L-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various (14)C-labeled compounds and examined by micro-autoradiography for incorporation of (14)C into calcium oxalate crystals. [(14)C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-(14)C]AsA also gave heavy labeling of crystals, whereas [6-(14)C]AsA gave no labeling. Labeled precursors of AsA (L-[1-(14)C]galactose; D-[1-(14)C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog, D-[1-(14)C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > L-galactose > D-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis via D-mannose and L-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments.     

THE DEVELOPMENT OF MUCILAGINOUS RAPHIDE CRYSTAL IDIOBLASTS IN YOUNG LEAVES OF TYPHA ANGUSTIFOLIA L. (TYPHACEAE)

Raphide crystal idioblast initiation occurs in the uppermost region of intercalary meristems in young leaves of Typha angustifolia L., and development proceeds acropetally. Idioblast differentiation commences with a loss of stored lipids, depletion of starch from amyloplasts, enlargement of the nucleus and nucleolus, cell elongation, and the formation of a central vacuole. Crystalloplastids are formed via dedifferentiation of amyloplasts, followed by an increase in plastid number as cell volume increases with cell elongation. Crystalloplastid membranes stain intensely with periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP). Following crystal production within the central vacuole, crystalloplastids differentiate lobed regions, dense with plastid ribosomes, thylakoids, lamellae, and plastoglobuli. Mucilage, which stains with PA-TCH-SP, appears to be formed at the tonoplast in the central vacuole and follows differentiation of crystalloplastid lobes. Crystal chambers are surrounded by lamellae during mucilage accumulation and the crystals undergo a change in shape. Lobed crystalloplastids may be involved in vacuolar mucilage formation in these types of raphide crystal idioblasts.     

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.     

DISTRIBUTION OF CALCIUM OXALATE CRYSTAL IDIOBLASTS IN LEAVES OF TARO (COLOCASIA ESCULENTA)

Cleared leaves of taro (Colocasia esculenta) were examined microscopically to determine changes in the distribution of both druse and raphide idioblasts during a late developmental process—leaf unfurling and expansion. Druse crystal idioblasts are small spherical cells found throughout the lamina, mostly in subepidermal areas. Two types of raphide idioblasts were observed in taro leaves: the nondefensive raphide idioblasts, which are elongated cells usually found embedded in tissues of the leaf margins; and the defensive raphide idioblasts, also elongated cells, but usually found suspended between mesophyll cells in leaf airspaces. The densities of both druse and raphide cells were highest at the fully furled stage and least in the mature, unfurled stage, after substantial leaf expansion. During leaf unfurling, the raphide cells showed a bilaterally symmetrical distribution during all stages from fully furled to mature, unfurled leaves. The distribution of druse cells was bilaterally symmetrical during the fully furled and unfurled stages, but, during unfurling, when one half of the lamina is unfurled and the other half is still tightly furled, up to 80% of the druse cells were found on the unfurled half of the lamina.     

On the formation of the pattern of crystal idioblasts — in Canavalia ensiformis DC

Summary Light and electron-microscope observations were made of the crystal idioblasts in the leaves of Canavalia. The crystal-containing cells occur as pairs in which the crystals, nuclei, and the majority of the chloroplasts are symmetrically arranged with regard to the common wall. The chloroplasts are found in the cytoplasm along this wall.The crystals originate in a vacuole. The space in which the young crystal develops is delimited by a membrane. One to several additional membranes surround the crystal inside the vacuole. Numerous vesicles are distributed between these vacuolar membranes. Dense groups of tubules or fibrils are oriented toward a portion of the crystal surface, suggesting that the material forming the crystal might be transported to the surface by these structures.The cytoplasm of the young idioblasts contains many mitochondria and dictyosomes with associated vesicles. Concentrations of what is assumed to be protein are present in the cytoplasm. These protein accumulations are not seen in neighboring cells, suggesting that protein synthesis is especially high in the idioblasts.In older crystal cells, layers of wall material are deposited on the wall between the two crystals of the pair and towards the cell wall adjacent to the mesophyll. Not only does the original wall become thickened but a new wall develops at the border of the crystal vacuole. Eventually this wall material becomes continuous and the crystal becomes, on two sides, directly connected with the wall.     

Calcium carbonate deposition in a cell wall sac formed in mulberry idioblasts

Summary. Although calcium carbonate is known to be a common biomineral in plants, very little attention has been given to the biological control of calcium carbonate deposition. In mulberry leaves, a subcellular structure is involved in mineral deposition and is described here by a variety of cytological techniques. Calcium carbonate was deposited in large, rounded idioblast cells located in the upper epidermal layer of mulberry leaves. Next to the outmost region (“cap”) of young idioblasts, we found that the inner cell wall layer expanded to form a peculiar outgrowth, named cell wall sac in this report. This sac grew and eventually occupied the entire apoplastic space of the idioblast. Inside the mature cell wall sac, various cellulosic membranes developed and became the major site of Ca carbonate deposition. Concentrated Ca²⁺ was pooled in the peripheral zone, where small Ca carbonate globules were present in large numbers. Large globules were tightly packed among multiple membranes in the central zone, especially in compartments formed by cellulosic membranes and in their neighboring membranes. The maximum Ca sink capacity of a single cell wall sac was quantified using enzymatically isolated idioblasts as approximately 48 ng. The newly formed outgrowth in idioblasts is not a pure calcareous body but a complex cell wall structure filled with substantial amounts of Ca carbonate crystals. Correspondence and reprints: Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.     

The plant vacuole: emitter and receiver of calcium signals

This review portrays the plant vacuole as both a source and a target of Ca²⁺ signals. In plants, the vacuole represents a Ca²⁺ store of enormous size and capacity. Total and free Ca²⁺ concentrations in the vacuole vary with plant species, cell type, and environment, which is likely to have an impact on vacuolar function and the release of vacuolar Ca²⁺. It is known that cytosolic Ca²⁺ signals are often generated by release of the ion from internal stores, but in very few cases has a role of the vacuole been directly demonstrated. Biochemical and electrophysical studies have provided evidence for the operation of ligand- and voltage-gated Ca²⁺-permeable channels in the vacuolar membrane. The underlying molecular mechanisms are largely unknown with one exception: the slow vacuolar channel, encoded by TPC1, is the only vacuolar Ca²⁺-permeable channel cloned to date. However, due to its complex regulation and its low selectivity amongst cations, the role of this channel in Ca²⁺ signalling is still debated. Many transport proteins at the vacuolar membrane are also targets of Ca²⁺ signals, both by direct binding of Ca²⁺ and by Ca²⁺-dependent phosphorylation. This enables the operation of feedback mechanisms and integrates vacuolar transport systems in the wider signalling network of the plant cell.     

Phytochrome-mediated uptake of calcium in Mougeotia cells

Ca²⁺ is proposed to function as a messenger in such phytochrome-mediated responses as localized cell growth, intracellular movements, and control of plasma membrane properties. To test this hypothesis, the uptake of Ca²⁺ in irradiated and non-irradiated regions of individual threads of the green alga Mougeotia was studied with the aid of ⁴⁵Ca²⁺ and low temperature autoradiography: 10–20 cells within 40–60 cell-long threads were irradiated for up to 1 min, transferred to darkness for 3 to 10 min, submersed in a radioactive medium for 1 min, washed in an unlabelled medium for 30 min, and then autoradiographed at-80° C for several days.The autoradiographs show that those cells which had been pre-irradiated with red light did take up 2–10 times more Ca²⁺ than the adjacent non-irradiated cells of the same thread. Cells pre-irradiated with farred light or red light followed by far-red light showed no enhanced uptake of Ca²⁺. These results might be interpreted to indicate, firstly, that phytochrome-Pfr is involved in the enhanced uptake of Ca²⁺ and secondly, that the accumulation of radioactive Ca²⁺ in red light irradiated cells is an expression of an increased intracellular concentration of Ca²⁺. This interpretation is based on the data that (i) the dark interval between irradiation and labelling precluded the involvement of photosynthesis, (ii) the effect of red light was reversible with far-red light, and (iii) the accumulation of Ca²⁺ persisted during the long wash-out period. We speculate, that the red light-enhanced accumulation of Ca²⁺ in Mougeotia cells is caused by a Pfr-mediated increase of the Ca-permeability of the plasma membrane, and perhaps by a Pfr-impeding of an active Ca²⁺-extrusion.Abbreviations APW artificial pond water - EGTA ethylene glycol-bis-(-amino ethyle ether) N,N-tetraacetic acid - R red irradiation - D darkness - FR far-red irradiation - Pfr physiologicallyactive form of phytochrome - Pr physiologically inactive form of phytochrome This paper is part of a Ph. D. Thesis submitted to the University of Erlangen-Nürnberg by E.M. Dreyer     

Calcium pools,calcium entry,and cell growth

The Ca²⁺ pump and Ca²⁺ release functions of intracellular Ca²⁺ pools have been well characterized. However, the nature and identity of Ca²⁺ pools as well as the physiological implications of Ca²⁺levels within them, have remained elusive. Ca²⁺ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca²⁺ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca²⁺ pools, the levels of Ca²⁺ within Ca²⁺ pools, and how these intraluminal Ca²⁺ levels may be physiologically related to ER function. Experiments utilizingin situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluroescent Ca²⁺ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺ pools within cells, and large changes in the levels of Ca²⁺ within pools following InsP₃-mediated Ca²⁺ release. Such changes in Ca²⁺ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca²⁺-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca²⁺ changes with respect to cell growth. Emptying of pools using Ca²⁺ pump blockers can result in cells entering a stable quiescent G₀-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca²⁺ pools return, and cells reenter the cell cycle. During the Ca²⁺ pool-depleted growth-arrested state, cells express a Ca²⁺ influx channel that is distinct from the store-operated Ca²⁺ influx channels activated after short-term depletion of Ca²⁺ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca²⁺ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.