Heavy metal stress reduces the deposition of calcium oxalate crystals in leaves of Phaseolus vulgaris

Calcium oxalate (CaOx) crystals in plants may serve as a sink for the absorption of excess calcium, and they could play an important role in heavy metal detoxification. In this study, the effect of heavy metals and different calcium concentrations on the growth of calcium oxalate crystals in leaves of Phaseolus vulgaris was investigated. Different analytical techniques were used to determine the influence of exogenous lead and zinc on CaOx deposition and to detect a presence of these metals in CaOx crystals. We found a positive correlation between the calcium concentration in the nutrient medium and the production of calcium oxalate crystals in leaves of hydroponically grown plants. On the other hand, addition of the heavy metals to the nutrient medium decreased the number of crystals. Energy dispersive X-ray spectrometry did not detect the inclusion of heavy metals inside the CaOx crystals. Our investigation suggests that CaOx crystals do not play a major role in heavy metal detoxification in P. vulgaris but do play an important role in bulk calcium regulation.     

Calcium oxalate formation is a rapid and reversible process inLemna minor L.

Summary Lemna minor root tips form raphide Ca oxalate crystals in both the root cap and root proper. An in vivo system was developed to examine raphide crystal bundle formation in the root of intact plants. By increasing the exogenous Ca concentration, crystal bundle formation could be induced. Entire new crystal bundles could be formed within 30 minutes of an inductive stimulus. The process was reversible with recently formed crystal bundles being dissolved over a period of about 3 hours. Older, previously existing bundles were more resistant to dissolution. The calmodulin antagonists, chlorpromazine and trifluoperazine (300 M), prevented crystal formation and caused dissolution of some crystal bundles, even in the presence of exogenous Ca. When the antagonists were flushed out and replaced with fresh medium, crystals were formed in cells where dissolution had occurred under the influence of the antagonists. The Ca ionophore A 23187 (20 M) caused slow dissolution of crystal bundles, even in the presence of exogenous Ca. A model describing the control of and physiological significance of Ca oxalate formation in plants is presented and discussed with respect to the results obtained in this study.     

Calcium and oxalate content of the leaves of Phaseolus vulgaris at different calcium supply in relation to calcium oxalate crystal formation

Soluble and insoluble oxalate and insoluble calcium were measured in the leaves of Phaseolus vulgaris. The plants were grown in nutrient solutions with two different concentrations of calcium. Two developmental stages of the leaves were studied. Although the content of insoluble calcium differs widely according to leaf age and growth conditions, the percentage bound in crystals is nearly the same in all cases. In the growing leaves, concentrations of total oxalate are independent of calcium supply, thus, showing that the known rise in numbers of crystals, and of cells containing them, is not induced via oxalate biosynthesis. Fully expanded leaves contain more oxalate when grown in a nutrient solution with higher calcium concentration. Amounts of oxalate in percent of dry weight are similar to those given in the literature for other legume leaves.     

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     

Calcium oxalate crystals and crystal cells in the leaves ofRhynchosia caribaea (Leguminosae: Papilionoideae)

Summary The development and distribution of calcium oxalate crystals, stomates and hairs were studied in the first trifoliolate leaf ofRhynchosia caribaea (Leguminosae: Papilionoideae, Phaseoleae). Using light and transmission electron microscopy, the crystals were shown to occur in both bundle sheath and mesophyll cells. Crystal distribution and shapes are characteristic forRhynchosia. Crystals develop late in leaf development in contrast to the stomates and hairs. As these latter two structures decrease in number per unit area with leaf age, crystal number increases.     

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - PPi inorganic pyrophosphate - PGA 3-phosphoglycerate - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(aminoethyl ether) - N,N tetraacetic acid     

Origin of gynogenetic embryos of Beta vulgaris L.

Haploid plants of Beta vulgaris were obtained by gynogenesis from ovules isolated from male-fertile and annual and biennial male-sterile plants. We show that on a N6 basal medium, supplemented with 2.85 M IAA and 0.94 M Kinetin or 0.88 M BAP, haploids originate directly through embryogenesis. In order to determine the optimal developmental stage of the ovule of Beta vulgaris for gynogenesis, we carried out a histological study of whole ovules from open male-sterile flowers (collected 1 to 5 days after flowering) and unopened male-fertile flowers (collected 1 to 3 days before anthesis). In all cases, the gametophyte appeared completely differentiated. These results suggest that maturity of the gametophyte is reached a few days before anthesis and therefore ovules from unopened flowers are already suitable for plating. A developmental study of the haploid cells of the sugarbeet embryo sac during the first week of in vitro culture showed that the viable gynogenetic embryo originated only from the egg cell.     

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.     

Transport of ascorbate into plasma membrane vesicles ofPhaseolus vulgaris L.

Summary Incubation of bean hook plasma membrane vesicles in the presence of L-[¹⁴C]ascorbate (ASC) resulted in a specific recovery of significant levels of the ligand with the vesicles. The strong decrease in radioactive ASC detected after hypotonic disruption of the vesicles or after an assay at 4 °C indicated that ASC was probably transported from the medium into the lumen of the membrane vesicles. The concentration kinetics of this presumptive transport process revealed a saturation curve which best fitted a biphasic model. Each phase in this model showed Michaelis-Menten type kinetics. The kinetic parameters for the different phases were calculated to be 14 and 79 M (K _m1 andK _m2) and 26 and 53 pmol/min · mg protein (V _max1 andV _max2). High concentrations of iso-ascorbate, dehydroascorbate (DHA) or non-labelled ASC significantly reduced the uptake of the radioactive vitamin. It was demonstrated that sugar or amino acid carriers are not involved in the ASC transport reaction. Generation of transmembrane cation gradients (H⁺, K⁺, Ca²⁺, Na⁺) or addition of sulfhydryl reagents (pCMBS or NEM) did not affect the ASC uptake in any way. It is suggested that ASC is taken up by a facilitated diffusion mechanism.Abbreviations ASC ascorbate - DHA dehydroascorbate - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid     

Calcium oxalate crystals in the red algaAntithamnion kylinii (Ceramiales): cytoplasmic and limited to indeterminate axes

Summary The red alga,Antithamnion kylinii Gardner, was found to have needle-shaped inclusions about 10 m long and less than 0.4 m thick. They ranged in abundance from one or a few in young cells to hundreds in fully enlarged cells. Under polarized light, the inclusions were birefringent, indicating crystallinity. Solubility tests suggested that the inclusions were composed of calcium oxalate: they dissolved in 1 N hydrochloric acid and in a saturated solution of aqueous cupric acetate, but they were not soluble in 10 N acetic acid or 5.25% sodium hypochlorite. X-ray microanalysis confirmed the presence of calcium. Calcium oxalate crystals were present in cells of indeterminate axes, but cells of determinate lateral filaments lacked them. Light and electron microscopic study demonstrated that the crystals were associated with the parietal cytoplasm. Calcium oxalate crystals were also present inA. defectum Kylin, but they were not found in ten more distantly related taxa.     

Short-term effects of Al3+ on the osmotic behavior of red beet (Beta vulgaris L.) protoplasts

The short-term (less than 10 min) effects of Al³⁺ on the biophysical properties of plasma membranes were investigated by time-series image analysis of osmotically-induced volumetric and morphologic changes of red beet (Beta vulgaris L.) protoplasts. Exposure to Al³⁺ under hypotonic conditions reduced the volumetric expansion of protoplasts and their resultant burst: i.e. lysis of protoplasts in a concentration-dependent manner. Under hypertonic conditions, protoplasts exposed to Al³⁺ underwent an enhanced volumetric contraction in cross-sectional area, while maintaining higher protoplast roundness. The residual effects of Al³⁺ pre-treatment on subsequent osmotic behavior were also examined, and protoplasts pre-treated with Al³⁺ also exhibited less lysis during subsequent exposure to hypotonic conditions and enhanced volumetric contractions and higher roundness under subsequent hypertonic conditions. Under our experimental conditions, Al³⁺ consistently minimized protoplast surface area by inhibiting osmotic expansion or by enhancing osmotic contraction, as well as by maintaining higher protoplast roundness. These results suggested that the electrostatic property of Al³⁺ might have induced the binding and possible cross-linking of negatively-charged sites on the plasma membrane surface. This may be an important factor in understanding the mechanism of Al³⁺ phytotoxicity.     

A proline-rich chitinase from Beta vulgaris

A gene (Chl) encoding a novel type of chitinase was isolated from Beta vulgaris. The Ch1 protein consists of an N-terminal hydrophobic prepeptide of 25 amino acids followed by a hevein-like domain of 22 amino acid residues, an unusually long proline-rich domain of 131 amino acid residues with 90 prolines, and finally a catalytic domain of 261 amino acid residues. Proteins with similar proline-rich domains are present in some other plants. The Chl gene shows a transient expression in response to fungal infection.     

Genetical control and linkage relationships of isozyme markers in sugar beet (B. vulgaris L.)

Summary Five isozyme systems were genetically investigated. The different separation techniques, the developmental expression and the use as marker system in sugar beet genetics and breeding is discussed. Isocitrate dehydrogenase was controlled by two genes. The gene products form inter- as well as intralocus dimers, even with the gene products of the Icd gene in B. procumbens and B. patellaris. Adenylate kinase was controlled by one gene. Three different allelic forms were detected, which were active as monomeric proteins. Glucose phosphate isomerase showed two zones of activity. One zone was polymorphic. Three allelic variants, active as dimers, were found. Phosphoglucomutase also showed two major zones of activity. One zone was polymorphic and coded for monomeric enzymes. Two allelic forms were found in the accessions studied. The cathodal peroxidase system was controlled by two independent genes, of which only one was polymorphic. The gene products are active as monomers. Linkage was found between red hypocotyl color (R) and Icd ₂. Pgm ₁, Gpi ₂, Ak ₁ and the Icd ₂-R linkage group segregated independently.     

Influence of long-term drought stress on osmolyte accumulation in sugar beet (Beta vulgaris L.) plants

Accumulation of various osmolytes was examined in plants of sugar beet cv. Janus grown under two soil water treatments: control (60% of the field water capacity; FWC) and drought (30–35% FWC). The water shortage started on the 61st day after emergence (DAE), at the stage of the beginning of tap-roots development and was imposed for 35 days. Osmotic potential of sugar beet plant organs, particularly tap-roots, was decreased significantly as a consequence of a long-term drought. Water shortage reduced univalent (K⁺, Na⁺) cations concentrations in the petioles and divalent (Ca²⁺, Mg²⁺) ions level in the mature and old leaves. Cation concentrations in the tap-roots were not affected by water shortage. The ratio of univalent to divalent cations was significantly increased in young leaves and petioles as a consequence of drought. Long-term water deficit caused a significant reduction of inorganic phosphorus (P_i) concentration in young and old leaves. Under the water stress condition, the concentration of proline was increased in all individual plant organs, except proline concentration in the youngest leaves. Drought treatment caused a significant increase of glycine betaine content in shoot without any change in tap-roots. Glucose concentrations were significantly increased only in tap-roots as the effect of drought. In response to water shortage the accumulation of sucrose was observed in all the examined leaves and tap-roots. Overall, a long-term drought activated an effective mechanism for osmotic adjustment both in the shoot and in the root tissues which may be critical to survival rather than to maintain plant growth but sugar beet organs accumulate different solutes as a response to water cessation.     

Implications of calcium nutrition on the response of Phaseolus vulgaris L. to salinity

The effect of Ca²⁺ level in the growth medium on the response of germination and early seedling growth of Phaseolus vulgaris to NaCl salinity was investigated. When NaCl concentration was increased germination and early seedling growth was decreased. The addition of Ca²⁺ to the media increased both germination percentage and seedling growth. Chloride concentrations were not affected by the level of Ca²⁺. Potassium and Ca²⁺ concentrations and transport from roots to shoots were decreased by NaCl, but were restored by increasing Ca²⁺ in the medium. The opposite was true for Na⁺. Leakage of NO₃ ^- and H₂PO₄ ^- was increased by salinity and reduced by high Ca²⁺ in the medium. The results are discussed in terms of the beneficial effects of calcium for plant growth under saline conditions.     

Rapid hybridization-based assays for identification by DNA probes of male-sterile and male-fertile cytoplasms of the sugar beet Beta vulgaris L.

Summary Methods are described whereby hybridization of mitochondrial (mt) DNA with different DNA probes can definitely distinguish male-fertile and and male-sterile (cms) cytoplasms of sugar beet Beta vulgaris L. We have developed two types of miniassays. (1) Comparative methods requiring the isolation and restriction of total cellular DNA, hybridization with cloned mtDNA fragments from either fertile or male-sterile cytoplasms, and comparison of the hybridization patterns to the fertile-and sterile-specific patterns of mtDNA of sugar beet for the given mtDNA probe. For these analyses, we routinely used 1 g of plant material to determine the type of cytoplasm. (2) Noncomparative (plus-minus) methods requiring neither the isolation of pure DNA nor restriction, electrophoresis, or Southern blotting. Instead, alkaline-SDS plant extracts from as little as 50 mg of plant material were dot-blotted and hybridized with fertile-specific (mitochondrial minicircular DNA) and/or cms-specific probes (consisting of a 2.3-kb mtDNA sequence exclusively occurring in the cms cytoplasm). The assays are simple to perform, give definitive results, are nonde-structive to the plants, and may be used in mass screening of sugar beet populations for hybrid production or in in vitro culture processes.     

Immunodetection of pectin and arabinogalactan protein epitopes during pollen exine formation of Beta vulgaris L.

Summary. We present the results of ultrastructural and immunocytochemical studies of sugar beet microsporocytes during the developmental phase that begins with the first meiotic metaphase and ends with the formation of young tetrads. The most prominent feature noted during this period of microsporogenesis was the presence of numerous cisternae of endoplasmic reticulum which frequently lie perpendicular to the surface of the plasma membrane and eventually fuse to it. Microscopic observations have been combined with the detection of several carbohydrate epitopes representing pectins and arabinogalactan proteins in the primexine and incipient exine. Pectin domains that possess both low and highly methylesterified epitopes, as well as pectin side chains enriched in (1→4)-β-D-galactose residues, are deposited in this young microspore wall. The epitopes of arabinogalactan protein that bind to JIM13, JIM8, and LM2 antibodies are localised within the callose wall surrounding posttelophase tetrads. The possibility of endoplasmic-reticulum involvement in the synthesis, transport, or metabolism of several microspore wall compounds is discussed. Correspondence and reprints: Institute of Plant Breeding and Acclimatization, Powstańców Wielkopolskich 10, 85-090 Bydgoszcz, Poland.     

Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel

The patch clamp technique has been used to investigate ion permeation and Ca²⁺-dependent gating of a voltage-sensitive Ca²⁺ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca²⁺ Sr²⁺ Ba²⁺ > Mg²⁺ K⁺ which is inversely related to the size of the unitary conductances K⁺ Mg²⁺ Ba²⁺ > Sr²⁺ Ca²⁺, suggesting that ion movement is not independent. In the presence of Ca²⁺, the unitary K⁺ current is reduced in a concentration- and voltage-dependent manner by Ca²⁺ binding at a high affinity site (K _0.5 = 0.29 mm at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca²⁺ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca²⁺ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca²⁺ at –41 mV predicts that two Ca²⁺ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.We thank Ian Jennings for writing and implementing some of the software used in this study and Anna J. Bate for technical assistance. The work was supported by grants from the Biotechnology and Biological Sciences Research Council to E.J. (PDF/14) and DS (PG87/529).     

A linkage map of sugar beet (Beta vulgaris L.)

Summary We have established a first linkage map for beets based on RFLP, isozyme and morphological markers. The population studied consisted of 96 F₂ individuals derived from an intraspecific cross. As was expected for outbreeding species, a relatively high degree of polymorphism was found within sugar beet; 47% of the DNA markers were polymorphic for the chosen population. The map consists of 115 independent chromosomal loci designated by 108 genomic DNA probes, 6 isozyme and one morphological marker. The loci cover 789 cM with an average spacing of 6.9 cM. They are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Eighteen markers (15.4%) showed distorted segregation which, in most instances, can be explained by gametic selection of linked lethal loci. The application of the linkage map in sugar beet breeding is discussed.     

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.