Response of Arabidopsis to iron deficiency stress as revealed by microarray analysis.

Gene expression in response to Fe deficiency was analyzed in Arabidopsis roots and shoots through the use of a cDNA collection representing at least 6,000 individual gene sequences. Arabidopsis seedlings were grown 1, 3, and 7 d in the absence of Fe, and gene expression in roots and shoots was investigated. Following confirmation of data and normalization methods, expression of several sequences encoding enzymes known to be affected by Fe deficiency was investigated by microarray analysis. Confirmation of literature reports, particularly for changes in enzyme activity, was not always possible, but changes in gene expression could be confirmed. An expression analysis of genes in glycolysis, the tricarboxylic acid cycle, and oxidative pentose phosphate pathway revealed an induction of several enzymes within 3 d of Fe-deficient growth, indicating an increase in respiration in response to Fe deficiency. In roots, transcription of sequences corresponding to enzymes of anaerobic respiration was also induced, whereas in shoots, the induction of several genes in gluconeogenesis, starch degradation, and phloem loading was observed. Thus, it seemed likely that the energy demand in roots required for the Fe deficiency response exceeded the capacity of oxidative phosphorylation, and an increase in carbon import and anaerobic respiration were required to maintain metabolism.

Two iron-regulated cation transporters from tomato complement metal uptake-deficient yeast mutants.

Although iron deficiency poses severe nutritional problems to crop plants, to date iron transporters have only been characterized from the model plant Arabidopsis thaliana. To extend our molecular knowledge of Fe transport in crop plants, we have isolated two cDNAs (LeIRT1 and LeIRT2) from a library constructed from roots of iron-deficient tomato (Lycopersicon esculentum) plants, using the Arabidopsis iron transporter cDNA, IRTI, as a probe. Their deduced polypeptides display 64% and 62% identical amino acid residues to the IRT1 protein, respectively. Transcript level analyses revealed that both genes were predominantly expressed in roots. Transcription of LeIRT2 was unaffected by the iron status of the plant, while expression of LeIRT1 was strongly enhanced by iron limitation. The growth defect of an iron uptake-deficient yeast (Saccharomyces cerevisiae) mutant was complemented by LeIRT1 and LeIRT2 when ligated to a yeast expression plasmid. Transport assays revealed that iron uptake was restored in the transformed yeast cells. This uptake was temperature-dependent and saturable, and Fe2+ rather than Fe3+ was the preferred substrate. A number of divalent metal ions inhibited Fe2+ uptake when supplied at 100-fold or 10-fold excess. Manganese, zinc and copper uptake-deficient yeast mutants were also rescued by the two tomato cDNAs, suggesting that their gene products have a broad substrate range. The gene structure was determined by polymerase chain reaction experiments and, surprisingly, both genes are arranged in tandem with a tail-to-tail orientation.

Kinetics Analysis of the Plasma Membrane Sucrose-H+ Symporter from Sugar Beet (Beta vulgaris L.) Leaves.

The kinetics behavior of the H+-sucrose (Suc) symporter was investigated in plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves by analyzing the effect of external and internal pH (pHo and pHi, respectively) on Suc uptake. The apparent Km for Suc uptake increased 18-fold as the pHo increased from 5.5 to 7.5. Over this same pHo range, the apparent Vmax for Suc uptake remained constant. The effects of pHi in the presence or absence of internal Suc were exclusively restricted to changes in Vmax. Thus, proton concentration on the inside of the membrane vesicles ([H+]i) behaved as a noncompetitive inhibitor of Suc uptake. The Km for the proton concentration on the outside of the membrane vesicles was estimated to be pH 6.3, which would indicate that at physiological apoplastic pH Suc transport might be sensitive to changes in pHo. On the other hand, the [H+]i for half-maximal inhibition of Suc uptake was approximately pH 5.4, making regulation of Suc transport through changes in [H+]i unlikely. These results were interpreted in the framework of the kinetics models for co-transport systems developed by D. Sanders, U.-P. Hansen, D. Gradmann, and C. L. Slayman (J Membr Biol [1984] 77: 123-152). Based on their analysis, the behavior of the Suc symporter with respect to the [H+]i is interpreted as an ordered binding mechanism by which the binding of Suc on the apoplastic side of the membrane and its release on the symplastic side precedes that of H+ (i.e. a first-on, first-off model).

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.).

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (DeltapH), alkaline inside, and approximately fourfold by a negative membrane potential (DeltaPsi), generated as a K(+)-diffusion potential, negative inside. The -fold stimulation was directly related to the relative DeltapH or DeltaPsi gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. DeltaPsi- and DeltapH-dependent glucose uptake showed saturation kinetics with a K(m) of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a DeltapH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H(+)-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H(+)-sucrose symporter.

Substrate Specificity of the H-Sucrose Symporter on the Plasma Membrane of Sugar Beets (Beta vulgaris L.) : Transport of Phenylglucopyranosides.

Previous results (TJ Buckhout, Planta [1989] 178: 393-399) indicated that the structural specificity of the H(+)-sucrose symporter on the plasma membrane from sugar beet leaves (Beta vulgaris L.) was specific for the sucrose molecule. To better understand the structural features of the sucrose molecule involved in its recognition by the symport carrier, the inhibitory activity of a variety of phenylhexopyranosides on sucrose uptake was tested. Three competitive inhibitors of sucrose uptake were found, phenyl-alpha-d-glucopyranoside, phenyl-alpha-d-thioglucopyranoside, and phenyl-alpha-d-4-deoxythioglucopyranoside (PDTGP; K(i) = 67, 180, and 327 micromolar, respectively). The K(m) for sucrose uptake was approximately 500 micromolar. Like sucrose, phenyl-alpha-d-thioglucopyranoside and to a lesser extent, PDTGP induced alkalization of the external medium, which indicated that these derivatives bound to and were transported by the sucrose symporter. Phenyl-alpha-d-3-deoxy-3-fluorothioglucopyranoside, phenyl-alpha-d-4-deoxy-4-fluorothioglucopyranoside, and phenyl-alpha-d-thioallopyranoside only weakly but competively inhibited sucrose uptake with K(i) values ranging from 600 to 800 micromolar, and phenyl-alpha-d-thiomannopyranoside, phenyl-beta-d-glucopyranoside, and phenylethyl-beta-d-thiogalactopyranoside did not inhibit sucrose uptake. Thus, the hydroxyl groups of the fructose portion of sucrose were not involved in a specific interaction with the carrier protein because phenyl and thiophenyl derivatives of glucose inhibited sucrose uptake and, in the case of phenyl-alpha-d-thioglucopyranoside and PDTGP, were transported.

Fe-Chelate Reductase Activity of Plasma Membranes Isolated from Tomato (Lycopersicon esculentum Mill.) Roots : Comparison of Enzymes from Fe-Deficient and Fe-Sufficient Roots.

Reduction of Fe(3+) to Fe(2+) is a prerequisite for Fe uptake by tomato roots. Ferric chelate reductase activity in plasma membranes (PM) isolated from roots of both iron-sufficient (+Fe) and iron-deficient (-Fe) tomatoes (Lycopersicon esculentum Mill.) was measured as NADH-dependent ferric citrate reductase and exhibited simple Michaelis-Menten kinetics for the substrates, NADH and Fe(3+)(citrate(3-))(2). NADH and Fe(3+)(citrate(3-))(2)K(m) values for reductase in PM from +Fe and -Fe tomato roots were similar, whereas V(max) values were two- to threefold higher for reductase from -Fe tomatoes. The pH optimum for Fe-chelate reductase was 6.5. Fe-chelate reductases from -Fe and +Fe tomato roots were equally sensitive to several triazine dyes. Reductase was solubilized with n-octyl beta-d-glucopyranoside and electrophoresed in nondenaturing isoelectric focusing gels. Three bands, with isoelectric points of 5.5 to 6.2, were resolved by enzyme activity staining of electrofocused PM proteins isolated from +Fe and -Fe tomato roots. Activity staining was particularly enhanced in the isoelectric point 5.5 and 6.2 bands solubilized from -Fe PM. We conclude that PM from roots of +Fe and -Fe plants contain Fe-chelate reductases with similar characteristics. The response to iron deficiency stress likely involves increased expression of constitutive Fe-chelate reductase isoforms in expanding epidermal root PM.

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves.

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K(+). The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (DeltapH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K(+)-selective ionophore, valinomycin, stimulated uptake in the presence of a DeltapH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited DeltapH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H(+)/sucrose symporter in PM vesicles isolated from spinach leaves.

Sucrose-dependent H(+) transport in plasma-membrane vesicles isolated from sugarbeet leaves (Beta vulgaris L.) : Evidence in support of the H(+)-symport model for sucrose transport.

The mechanism of sucrose transport across the plasma membrane (PM) was investigated in membrane vesicles isolated from sugarbeet (Beta vulgaris L.) leaves. In the presence of a membrane potential (Δψ) generated as a K(+)-diffusion potential, negative inside, sucrose induced a rapid and transient alkalization of the medium. Alkalization was inhibited by carbonyl cyanide m-chlorophenylhydrazone, was specific for the sucrose sugar and was dependent on the sucrose concentration with a Km of approx. 1 mM. Sucrose-induced alkalization and sucrose transport were inhibited by the sulfhydryl-reactive reagent, p-chloromercuribenzene sulfonic acid, and by the histidine-reactive reagent, diethyl pyrocarbonate. Parallel analysis of sucrose uptake and alkalization indicated that the stoichiometry of sucrose uptake to proton consumed was 1∶1. These results provide clear evidence that the saturable mechanism of sucrose transport across the PM in plants is a coupled H(+)-sucrose symport.

Apparent processing of a soybean oil body protein accompanies the onset of oil mobilization.

The membrane surrounding the oil body contains several different specific polypeptides. To study the biosynthesis and posttranslational modification of these polypeptides we have prepared monoclonal antibodies against purified oil bodies of soybean (Glycine max). Three of the five monoclonals selected recognize a molecular mass 34 kilodalton protein (P34). Epitope mapping of CNBr and proteolytic fragments of P34 indicates that two of the anti-P34 monoclonal antibodies are directed at different epitopes. P34 is accumulated during seed maturation at the same time as the reserve proteins and oil. SDS/PAGE-immunoblots of germinating soybean seed cotyledons indicate that the protein is initially present as a molecular mass 34 kilodalton polypeptide and is processed to molecular mass 32 kilodalton on the fourth through sixth days of seedling growth simultaneously with the onset of oil mobilization. A comparison of reduced and carboxymethylated oil body proteins with nonreduced proteins by SDS/PAGE indicates that P34 exists in vivo as a dimer of molecular mass 58 kilodalton. Comparing the amino terminal sequences of P34 and P32 indicates that their difference is at least in part due to the removal of the amino terminus of P34. The amino terminal sequences of P34 and P32 were aligned to show that the transition of P34 to P32 was accompanied by the removal of a hydrophilic decapeptide (KKMKKEQYSC) at the amino terminus of P34. Hopp-Woods hydrophilicity analysis of the deleted amino terminus of P34 shows that it is more hydrophilic and charged than the sequence of the protein which immediately follows.

Purification and Identification of a Plasma Membrane Associated Electron Transport Protein from Maize (Zea mays L.) Roots.

Plasma membranes isolated from three-day-old maize (Zea mays L.) roots by aqueous two-phase partitioning were used as starting material for the purification of a novel electron transport enzyme. The detergent-solubilized enzyme was purified by dyeligand affinity chromatography on Cibacron blue 3G-A-agarose. Elution was achieved with a gradient of 0 to 30 micromolar NADH. The purified protein fraction exhibited a single 27 kilodalton silver nitrate-stained band on sodium dodecyl sulfate polyacrylamide gel electrophoretograms. Staining intensity correlated with the enzyme activity profile when analyzed in affinity chromatography column fractions. The enzyme was capable of accepting electrons from NADPH or NADH to reduce either ferricyanide, juglone, duroquinone, or cytochrome c, but did not transfer electrons to ascorbate free-radical or nitrate. The high degree of purity of plasma membranes used as starting material as well as the demonstrated insensitivity to mitochondrial electron transport inhibitors confirmed the plasma membrane origin of this enzyme. The purified reductase was stimulated upon prolonged incubation with flavin mononucleotide suggesting that the enzyme may be a flavoprotein. Established effectors of plasma membrane electron transport systems had little effect on the purified enzyme, with the exception of the sulfhydryl inhibitor p-chloromercuriphenyl-sulfonate, which was a strong inhibitor of ferricyanide reducing activity.

Iron-Stress Induced Redox Activity in Tomato (Lycopersicum esculentum Mill.) Is Localized on the Plasma Membrane.

Tomato plants (Lycopersicum esculentum Mill.) were grown for 21-days in a complete hydroponic nutrient solution including Fe(3+)-ethylenediamine-di(o-hydroxyphenylacetate) and subsequently switched to nutrient solution withholding Fe for 8 days to induce Fe stress. The roots of Fe-stressed plants reduced chelated Fe at rates sevenfold higher than roots of plants grown under Fe-sufficient conditions. The response in intact Fe-deficient roots was localized to root hairs, which developed on secondary roots during the period of Fe stress. Plasma membranes (PM) isolated by aqueous two-phase partitioning from tomato roots grown under Fe stress exhibited a 94% increase in rates of NADH-dependent Fe(3+)-citrate reduction compared to PM isolated from roots of Fe-sufficient plants. Optimal detection of the reductase activity required the presence of detergent indicating structural latency. In contrast, NADPH-dependent Fe(3+)-citrate reduction was not significantly different in root PM isolated from Fe-deficient versus Fe-sufficient plants and proceeded at substantially lower rates than NADH-dependent reduction. Mg(2+)-ATPase activity was increased 22% in PM from roots of Fe-deficient plants compared to PM isolated from roots of Fe-sufficient plants. The results localized the increase in Fe reductase activity in roots grown under Fe stress to the PM.

Localization of a protein, immunologically similar to a sucrose-binding protein from developing soybean cotyledons, on the plasma membrane of sieve-tube members of spinach leaves.

Immunocytochemical studies using antibodies raised against a 62-kDa membrane protein isolated from developing soybean (Glycine max (L.) Merr.) cotyledons were performed on leaf tissue of spinach (Spinacia oleracea L.). This 62-kDa protein was labeled by 6'-deoxy-6'-(4-azido-2-hydroxy)-benzamidosucrose (HABS), a photoaffinity sucrose analogue (K. G. Ripp et al., 1988, Plant Physiol.88, 1435-1445). Western-blot analysis of spinach plasma-membrane proteins indicated a cross-reactive polypeptide identical in molecular mass to that found in soybean. Indirect immunogold labeling of resin-embedded sections of fully expanded leaf tissue resulted in specific localization of colloidal gold on the sieve-tube plasma membrane. The label was uniform and, except for a few non-specific gold particles over the cell wall, all other cellular organelles and membrane systems were free of label. With the exception of occasional gold particles associated with the companion-cell plasma membrane, all other cell types of the leaf contained little or no label. Control sections treated with non-immune rabbit immunoglobulin-G were also essentially free of label. Immunogold labeling of young leaves, in which the phloem contained no mature sieve-tube members, were free of label for the 62-kDa protein. However, young leaf tissue in which mature or nearly mature sieve tubes could be identified, contained immunolabel associated with the sieve-tube plasma membranes. Similar results were obtained with mature leaf tissue of sugar beet (Beta vulgaris L.). The results of the immunocytochemical studies are consistent with the suggestion that the concentrating step in the phloem-loading process in this species may occur across the sieve-tube plasma membrane.

Surcose transport in isolated plasma-membrane vesicles from sugar beet (Beta vulgaris L.) Evidence for an electrogenic sucrose-proton symport.

An analysis of the molecular mechanism of sucrose transport across the plasmalemma was conducted with isolated plasma-membrane (PM) vesicles. Plasma membrane was isolated by aqueous two-phase partitioning from fully expanded sugar beet (Beta vulgaris L.) leaves. The isolated fraction was predominantly PM vesicles as determined by marker-enzyme analysis, and the vesicles were oriented right-side-out as determined by structurally linked latency of the PM enzyme, vanadate-sensitive Mg(2+)-ATPase. Sucrose uptake was investigated by equilibrating PM vesicles in pH 7.6 buffer and diluting them 20-fold into pH 6.0 buffer. Using this pH-jump technique, vesicles accumulated acetate in a pH-dependent, protonophore-sensitive manner, which demonstrated the presence of a pH gradient (ΔpH) across the vesicle membrane. Addition of sucrose to pH-jumped PM vesicles resulted in a pH-dependent, protonophoresensitive uptake of sucrose into the vesicles. Uptake was sucrose-specific in that a 10-fold excess of mannose, glucose, fructose, mannitol, melibiose, lactose or maltose did not inhibit sucrose accumulation. The rate of pH-dependent uptake was saturable with respect of sucrose concentration and had an apparent K m, of 0.45 mM. Sucrose uptake was stimulated approximately twofold by the addition of valinomycin and K(+), which indicated an electrogenic sucrose-H(+) symport. Membrane potentials (ΔΨ) were imposed across the vesicle membrane using valinomycin and K(+). A membrane potential, negative inside, stimulated pH-dependent sucrose uptake while a ΔΨ, positive inside, inhibited uptake. Conditions that produce a negative ΔΨ in the absence of a pH gradient supported, although weakly, sucrose uptake. These data support an electrogenic sucrose-H(+) symport as the mechanism of sucrose transport across the PM in Beta leaves.

Characterization of Ca Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots.

The characteristics of Ca(2+) transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca(2+) and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0.The Ca(2+)-dependent modulation protein, calmodulin, was tested for its effect on Ca(2+) transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca(2+) transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca(2+) into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca(2+) transport activity. The inhibition of Ca(2+) transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca(2+) uptake into root endoplasmic reticulum.

Can a Ca2+ pump in the endoplasmic reticulum of the Lepidium root be the trigger for rapid changes in membrane potential after gravistimulation?

Since gravistimulation is followed by alterations in the external current symmetry (Behrens et al., 1982), the effect of gravistimulation on cellular membrane potential was investigated using conventional glass microelectrode techniques. The resting potential of statocytes in a vertically oriented root is approx. -118 mV. Upon gravistimulation, the membrane potential is temporarily depolarized (lag time = 2 s) to a potential of approx. -93 mV. This depolarization is only observed in statocytes located on the physically lower root flank while those on the corresponding upper flank become weakly hyperpolarized (approx. -13 mV). These results reflect altered ion fluxes across the plasma membrane. The perception of gravistimulus was suggested to result from a pressure of the amyloplasts on the distal endoplasmic reticulum (ER) of the statocytes (Sievers and Volkmann, 1972). A causal relationship between changes in ER-amyloplast interactions and the rapid alterations in plasma membrane potential described above is not known. A candidate for such an intracellular messenger is Ca2+. As a first step in establishing the validity of such an assumption, we have isolated ER membranes from roots. When incubated with micromolar concentrations of Ca2+, the vesicular membrane fraction accumulates Ca2+. The accumulation is ATP-dependent and -specific and is directly coupled to ATP hydrolysis since a protonophore shows no inhibitory effect. Thus, in analogy to the sarcoplasmic reticulum of muscle, regulation of an ER-localized Ca2+ compartment might be an important step in such complex processes as stimulus-transduction in gravitropism.

ATP-dependent CA(2+) transport in endoplasmic reticulum isolated from roots ofLepidium sativum L.

Endoplasmic reticulum membranes were isolated from roots of garden cress (Lepidium sativum L. cv Krause) using differential and discontinuous sucrose gradient centrifugation. The endoplasmic reticulum fraction was 80% rough endoplasmic reticulum oriented with the cytoplasmic surface directed outward and contaminated with 12% unidentified smooth membranes and 8% mitochondria. Marker enzyme analysis showed that the activity for endoplasmic reticulum was enriched 2.4-fold over total membrane activity while no other organelle activity showed an enrichment. All evidence indicated that the fraction was composed of highly enriched endoplasmic reticulum membranes. Ca(2+) uptake activity was measured using the filter technique described by Gross and Marmé (1978). The results of these experiments showed an ATP-dependent, oxalate-stimulated Ca(2+) uptake into vesicles of the endoplasmic reticulum fraction. The majority of the transport activity was microsomal since specific inhibitors of mitochondrial Ca(2+) transport (ruthenium red, LaCl3 and oligomycin) inhibited the activity by only 25%. Sodium azide showed no inhibition. The transport was likely directly coupled to ATP hydrolysis since there was no inhibition with carbonylcyanidem-chlorophenylhydrazone. The transport activity was specific for ATP showing only 36% and 29% of the activity with inosine diphosphate and guanosine 5'-triphosphate, respectively. The results indicate a Ca(2+) transport function located on the endoplasmic reciculum of garden cress roots.

Fractionation and characterization of cellular membranes from root tips of garden cress (Lepidium sativum L.).

The first step in the gravitropic reaction chain, i.e. perception, is known to occur in the statenchyma of the root cap. Because of the importance of the root tip in graviperception, a procedure has been developed to isolate root tips from garden cress (Lepidium sativum L.). The root tip fraction contains the tissues of the root cap plus the lower half of the meristem zone, but is clearly separated from the tissues of the elongation zone, the zone of gravitropic response. Membranes from the root tip and root base fractions have been centrifuged on sucrose density gradients and the marker enzyme profiles analyzed. These results show that the marker enzyme profiles for vacuoles, dictyosomes, mitochondria, and plasma membranes are similar in the root tip or root base fractions. The endoplasmic reticulum (ER) has a shoulder of cytochrome c reductase activity at a density of 1.16 g cm(-3) which is distinct from the other enzyme activities and is only observed in root tip preparations. The specific enzyme activity for ER, cytochrome c reductase, was enriched in root tip membranes 1.7 fold. This latter increase is interpreted as at least in part an increased ER content in the root tip.

Endoplasmic Reticulum Formation during Germination of Wheat Seeds : A QUANTITATIVE ELECTRON MICROSCOPE STUDY.

This study demonstrates germination-induced ultrastructural changes in wheat (Triticum aestivum L. cv Arthur) aleurone cells. Seeds imbided for 4 hours in water contained endoplasmic reticulum (ER) or ER-like membranes as vesicles or as short segments of membrane associated with the spherosomes on the periphery of aleurone grains. Aleurone cells incubated between 8 and 10 hours contained abundant ER membranes mainly associated with the nuclear envelope and, to a lesser extent, with the spherosomes surrounding the aleurone grain. The membranes located on the periphery of the nucleus occurred as regions of stacked cisternae. When aleurone cells were analyzed by morphometry, the increase in ER during incubation was found to be greater than 2-fold. During the same incubation period, other organelles did not change significantly. The early increase in ER was not affected by gibberellin incubation. Thus, the rapid proliferation of ER observed during the early stages of germination in aleurone cells of wheat is not likely to be controlled directly by gibberellin.

In vitro promotion by auxins of divalent ion release from soybean membranes.

Release of divalent ions from membrane pellets of soybean hypocotyls was promoted by the natural auxin, indole-3-acetic acid, and the synthetic auxin, 2,4-dichlorophenoxyacetic acid. The calcium release occurred at auxin concentrations as low as 1 nanomolar, and maximum release was observed at 1 micromolar. Hormone concentrations greater than 1 micromolar showed reduced effectiveness in releasing membrane-associated calcium. 2,3-Dichlorophenoxyacetic acid, a weak-auxin analog of 2,4-dichlorophenoxyacetic acid, did not promote calcium release. In some experiments, the analog actually promoted calcium association with the membranes. Red blood cells treated in a similar manner to soybean hypocotyl membranes did not release calcium in response to indole-3-acetic acid. The release phenomenon was hormone specific but not ion specific. Auxin released manganese from membranes in a manner similar to that of calcium. The calcium release, following auxin treatment, is accompanied by a decrease in membrane-associated sites for calcium binding.