Salinity affects intracellular calcium in corn root protoplasts

Previous work with the fluorescent Ca probe chlorotetracycline (CTC) showed that salinity displaces Ca from membranes of root cells. Using a variety of indirect approaches, we studied whether salinity displaces Ca from the cell surface or from internal membranes of corn (Zea mays L. cv Pioneer 3377) root protoplasts. Preloading the cells with supplemental Ca counteracted subsequent NaCl effects on CTC fluorescence. CTC quenching by exogenous EGTA was not competitive with CTC quenching by NaCl. The Ca channel reagent (+)-202-791 had significant interactions with the effect of NaCl on CTC fluorescence. The effect of NaCl on CTC fluorescence was attenuated by pretreatment with Li, but was restored by inositol. Salinity increased Na influx, decreased Ca influx, and increased Ca efflux from the cells. Fluorescence anisotropy indicated that NaCl decreased the fluidity of the external face of the plasmalemma but increased the fluidity of cell membranes in general. Our results suggest that salinity displaces Ca associated with intracellular membranes through activation of the phosphoinositide system and depletion of intracellular Ca pools.     

Salinity reduces membrane-associated calcium in corn root protoplasts

Mesocotyl elongation in 4 day old etiolated seedlings immediately following 3 hours of white light (3 h W) is reversibly controlled by phytochrome. Time-lapse video measurements were made of the 5 millimeter zone just below the coleoptile which is the main growth region of the mesocotyl. The growth kinetics were determined for five contiguous 1 millimeter zones subtending the coleoptile node for nonirradiated seedlings, for seedlings given 3 h W, and 3 h W followed by terminal far-red (FR) or red subsequent to the far-red (FR/R) irradiation. Each zone in nonirradiated seedlings exhibits exponential elongation kinetics during the early stages of elongation. This finding suggests that during elongation, a growth limiting factor is also exponentially increasing. Following 3 h W differences in the kinetic responses were found for each zone. In all zones, the inhibitory effect following the 3 h W is totally FR reversible. The effect of FR is reversed by R. The upper zone exhibits the fastest response and is the most plastic in its growth response. The three upper zones all exhibit spontaneous and sharp recoveries with time. It is suggested that the control by phytochrome is not inductive but rather continuous, the controlling factor being either the level of the far red-absorbing form of phytochrome (Pfr) or the ratio Pfr to total phytochrome.     

Mechanical release and lectin labeling of maize root protoplasts

Summary An improved method for the mechanical release of protoplasts from plant tissues is described. The historically-low yield of mechanically-released protoplasts is greatly increased by use of a simple electrically-driven tissue sheer and by optimization of various other steps in the procedure. As counted by light microscopy of a purified preparation, the number of mechanically-released protoplasts obtained is about 6×10⁴ per gram fresh weight of cortical tissue from the primary root of maize (Zea mays L. WF9×Mo 17) seedlings. Nuclear staining of the preparation, however, shows that about half of these protoplasts lack a nucleus and thus are actually subprotoplasts. Comparison of lectin binding to the plasma membranes of mechanically-and enzymatically-released protoplasts shows that both types contain binding sites forRicinus communis agglutinin. Binding sites for peanut (Arachis hypogaea) agglutinin are not naturally present on mechanically-released protoplasts but are generated by exposure to a mixture of Cellulysin and Pectolyase Y-23, the cell wall-degrading enzymes used to prepare enzymatically-released protoplasts.Abbreviations BSA bovine serum albumin - DDT dithiothreitol - gfw gram fresh weight - Mes 2-(N-morpholino) ethanesulfonic acid - PNA peanut (Arachis hypogaea) agglutinin - RCA Ricinus communis agglutinin - Tris tris(hydroxymethyl)aminomethane     

Light-affected ca fluxes in protoplasts from vallisneria mesophyll cells

In Vallisneria gigantea Graebner mesophyll cells, red light irradiation induces cytoplasmic streaming by decreasing the Ca²⁺ concentration in the cytoplasm, while far-red light irradiation inhibits it by increasing the concentration (S Takagi, R Nagai 1985 Plant Cell Physiol 26: 941-951). To examine the effects of light irradiation on Ca²⁺ fluxes across the cell membrane, protoplasts are isolated from the mesophyll cells. Changes in Ca²⁺ concentration in a solution bathing the protoplasts are monitored by spectrophotometry, using the Ca²⁺ -sensitive dye murexide. Red light irradiation induces an increase in Ca²⁺ concentration, which means an efflux of Ca²⁺ from the protoplasts. Subsequent far-red light irradiation produces a rapid decrease in Ca²⁺ concentration down to the dark control level; however, this is not observed in the presence of the Ca²⁺ -channel blocker nifedipine. Vanadate inhibits both the streaming and the Ca²⁺ efflux induced by red light irradiation. The results suggest that red light and far-red light control Ca²⁺ movements across the cell membrane, which in turn regulate the streaming.     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

Hydroquinone peroxidase activity of maize root mitochondria

Summary. The oxidation of hydroquinone with H₂O₂ in the presence of mitochondria isolated from maize (Zea mays L.) roots was studied. The results indicate that a reduced form of quinone may be a substrate of mitochondrial peroxidases. Specific activities in different mitochondrial isolates, the apparent K _m for hydrogen peroxide and hydroquinone, and the influence of some known peroxidase inhibitors or effectors are presented. Zymographic assays revealed that all mitochondrial peroxidases, which were stained with 4-chloro-1-naphthol, were capable of oxidizing hydroquinone. A possible antioxidative role of hydroquinone peroxidase in H₂O₂ scavenging within the mitochondria, in cooperation with ascorbate or coupled with mitochondrial NAD(P)H dehydrogenases, is proposed. Correspondence: M. Vuletić, Laboratory of Plant Physiology, Maize Research Zemun Polje, P.O. Box 89, 11185 Belgrade, Serbia.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Ca2+, annexins, and GTP modulate exocytosis from maize root cap protoplasts

Protoplasts isolated from root cap cells of maize were shown to secrete fucose-rich polysaccharides and were used in a patch-clamp study to monitor changes in whole-cell capacitance. Ca2+ was required for exocytosis, which was measured as an increase in cell capacitance during intracellular dialysis with Ca2+ buffers via the patch pipette. Exocytosis was stimulated significantly by small increases above normal resting [Ca2+]. In the absence of Ca2+, protoplasts decreased in size. In situ hybridization showed significant expression of the maize annexin p35 in root cap cells, differ-entiating vascular tissue, and elongating cells. Dialysis of protoplasts with maize annexins stimulated exocytosis at physiological [Ca2+], and this could be blocked by dialysis with antibodies specific to maize annexins. Dialysis with milli-molar concentrations of GTP strongly inhibited exocytosis, causing protoplasts to decrease in size. GTPgammaS and GDPbetaS both caused only a slight inhibition of exocytosis at physiological Ca2+. Protoplasts were shown to internalize plasma membrane actively. The results are discussed in relation to the regulation of exocytosis in what is usually considered to be a constitutively secreting system; they provide direct evidence for a role of annexins in exocytosis in plant cells.     

Mitotic activity in the maize root apex after freeze-decapping

Mitosis and nuclear DNA synthesis have been examined in root apices of maize whose caps were removed by a freezing technique. These processes are not impaired by this technique even though cells at the surface of the decapped apex experience a temperature close to 0°±1.5° C for a brief period. We conclude that freeze-decapping is without significant deleterious effects to the apex and therefore the technique is a useful adjunct in studies of the role of the cap on root growth.     

Hexokinase activity alters sugar-nucleotide formation in maize root homogenates

Two pools of hexokinase activities differing in sensitivity to ADP inhibition were characterised in maize roots. In order to evaluate how glucose utilisation could be affected by these hexokinases, glucose-6-P and NDP-5'-sugar levels were measured after a D-[U-14C]glucose pulse in root extracts in the presence of 0 or 1 mM ADP. Analysis of radio-labelled activated sugars by paper chromatography revealed that: (1) without ADP, nearly 20% of the 14C appeared in NDP-5'-sugars; (2) 0.1 mM ADP inhibited 14C-NDP-5'-sugar formation by 85%; and (3) with 1 mM ADP, 14C-NDP-5'-sugars were undetectable, but substantial (14%) 14C accumulated as glucose-6-P. Mannoheptulose, a hexokinase inhibitor, blocked the NDP-5'-sugar formation, but did not modify the amount of 14C-glucose-6-P in root extracts either with or without ADP. The analysis of the hexokinase activities with 0.8 mM glucose in maize root extracts showed that: (1) mitochondrial hexokinase activity was totally inhibited by 30 mM mannoheptulose; and (2) the cytosolic hexokinase was inhibited by only 30%. These data suggest that NDP-5'-sugar synthesis is sensitive to ADP fluctuations and that mannoheptulose affects preferentially the mitochondrial-bound hexokinase, but the cytosolic form is less sensitive. We propose that the mitochondrial hexokinase is the main energy charge sensor in this pathway in maize.     

Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants

ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed strength. The Escherichia coli mutant glgC gene (glgC16), which encodes a highly active and allosterically insensitive AGPase, was introduced into maize (Zea mays L.) under the control of an endosperm-specific promoter. Developing seeds from transgenic maize plants showed up to 2–4-fold higher levels of AGPase activity in the presence of 5 mM inorganic phosphate (Pi). Transgenic plants with higher cytoplasmic AGPase activity under Pi-inhibitory conditions showed increases (13–25%) in seed weight over the untransformed control. In addition, in all transgenic maize plants, the seeds were fully filled, and the seed number of transgenic plants had no significant difference compared with that of untransformed control. These results indicate that increasing cytoplasmic AGPase activity has a marked effect on sink activity and, in turn, seed weight in transgenic maize plants.     

Cytochemical localization of adenyl cyclase activity in maize root tips

Adenyl cyclase activity has been investigated in the cells of maize root tips and other tissues using a cytochemical staining procedure in which adenylyl-imidodiphosphate (AMP-PNP) is utilized as substrate. Heavy deposits of the reaction product were found in the plasma membrane, endoplasmic reticulum and nuclear membrane; no clear staining was associated with other cellular membranes. These findings are discussed in relation to the reported localization of adenyl cyclase in animal cells and to the possible role of this enzyme in plants.     

Subcellular localization of a carboxypeptidase activity in maize root homogenates

Maize root homogenates were prepared and centrifuged to sediment the mitochondria. The supernatants (6 KS) and pellets (6 KP) were collected and fractionated on linear sucrose density gradients. The distribution of carboxypeptidase was similar to that of α-mannosidase (a soluble vacuolar enzyme) and glucose-6-phosphate dehydrogenase (a soluble cytoplasmic enzyme). Carboxypeptidase was therefore a soluble in maize root cells and cannot be used as a marker for the tonoplast.     

Characterization of energy-dependent ca transport in maize mitochondria

Experimental conditions which optimize both substrate- and ATP-dependent Ca²⁺ transport in corn (Zea mays) mitochondria have been determined. It has been found that a substrate (pyruvate + succinate) dependent, Pi independent, binding of Ca²⁺ occurs. This reaction is very rapid and complete in less than 30 seconds. For massive accumulation of calcium, Pi is essential. Phosphate is accumulated along with the calcium and the ratio of Ca:Pi accumulated is about 1.6:1 indicating the precipitation of hydroxyapatite inside the mitochondria.

The activation energies and Michaelis constants for both the substrate- and ATP-driven reactions have been determined. It has also been shown that the substrate-driven system is more efficient in Ca²⁺ accumulation than the ATP-driven system. This is partially due to the fact that Mg²⁺ is essential for the ATP-driven system but not for the substrate-driven system and that Mg²⁺ acts as a strong competitor of Ca²⁺ transport. The effect of other inorganic ions on Ca²⁺ transport energized by both substrate and ATP were examined.

The results lend support to the hypothesis that high energy intermediates of oxidative phosphorylation participate directly in Ca²⁺ binding and transport in plant mitochondria.

     

Root cap removal increases root penetration resistance in maize (Zea mays L)

The root cap assists the passage of the root through soil by means of its slimy mucilage secretion and by the sloughing of its outer cells. The root penetration resistance of decapped primary roots of maize (Zea mays L. cv. Mephisto) was compared with that of intact roots in loose (dry bulk density 1.0 g cm-3; penetration resistance 0.06 MPa) and compact soil (1.4 g cm-3; penetration resistance 1.0 MPa), to evaluate the contribution of the cap to decreasing the impedance to root growth. Root elongation rate and diameter were the same for decapped and intact roots when the plants were grown in loose soil. In compacted soil, however, the elongation rate of decapped roots was only about half that of intact roots, whilst the diameter was 30% larger. Root penetration resistances of intact and decapped seminal axis were 0.31 and 0.52 MPa, respectively, when the roots were grown in compacted soil. These results indicated that the presence of a root cap alleviates much of the mechanical impedance to root penetration, and enables roots to grow faster in compacted soils.     

Sugar transport in isolated corn root protoplasts

Isolated corn (Zea mays L.) root protoplasts were used to study sucrose and hexose uptake. It is found that glucose was preferentially taken up by the protoplasts over sucrose and other hexoses. Glucose uptake showed a biphasic dependence on external glucose concentration with saturable (K_m of 7 millimolar) and linear components. In contrast, sucrose uptake only showed a linear kinetic curve. Sucrose and glucose uptake were linear over a minimum of 1 hour at pH 6.0 and 1 millimolar exogenous sugar concentration. Glucose uptake showed a sharp 42°C temperature optimum, while sucrose uptake showed a lower temperature sensitivity which did not reach a maximum below 50°C. Uptake of both sugars was sensitive to several metabolic inhibitors and external pH. Differences between sucrose and glucose uptake in two different sink tissue (i.e. protoplasts from corn roots and soybean cotyledons) are discussed.     

The keratin intermediate filament—like system in maize protoplasts

The application of Penman‘s method of cell fractionation to plant protoplasts leads to our finding of keratin intermediate filament(IF)-like system in maize protoplasts,which was identified by using immunogold labelling with monoclonal antibody of cytokeratin from animal cells.Many gold particles were found to be bound on filaments,linked by 3 nm filaments.After further digestion and extraction with DNase I and ammonium sulphate.IF-like framework-lamina-nuclear matrix system was shown under electron microscope.That IF system exists in plant protoplasts just like in animal cells,and their main component is keratin-like protein.     

Localization of acid phosphatase activity in maize root under phosphorus deficiency

The effect of phosphorus deficiency on acid phosphatase activity in the apical, middle and basal parts of the root of maize plants was followed. The supernatant obtained by centrifuging the homogenate of plant tissue at 1500 ×g was further centrifuged at 18 000 ×g, the sediment being marked as fraction II and the supernatant as fraction III. The results obtained document the fact that acid phosphatase activity of the two fractions of all analyzed root segments was higher in plants cultured in nutrient medium without phosphate than in those containing phosphorus in nutrient medium. In most cases this difference was significant to highly significant. The results of experiments proved unambiguously a higher enzymatic activity in all root segments in fraction III than in fraction II. In fraction III the highest acid phosphatase activity was found in the apical part, in fraction II in the basal part of the root.     

Ultrastructure of cortical cells of maize root under water stress conditions

Following moderate water stress in the cortical cells of theZea mays primary roots, the condensation of the nuclear chromatin, a higher density of free ribosomes and a reduction of polyribosomes, the reduction of mitochondrial cristae, elongation of ER elements, less compact dictyosomes and inhibited production of the Golgi vesicles were observed. Severe water stress would cause more severe structural damage in the cortical cells. The more differentiated cortical cells showed more expressive ultrastructural damage when compared with the meristematic nonvacuolated cells. Similarly, the cells of the peripheral layers of the cortex suffered more from water deficit than the cells of the layers situated closer to the central cylinder.