Etiolated Maize Coleoptiles Research Articles

Ca(2+)-Transport through Plasma Membrane as a Test of Auxin Sensitivity.

Auxin is one of the crucial regulators of plant growth and development. The discovered auxin cytosolic receptor (TIR1) is not involved in the perception of the hormone signal at the plasma membrane. Instead, another receptor, related to the ABP1, auxin binding protein1, is supposed to be responsible for the perception at the plasma membrane. One of the fast and sensitive auxin-induced reactions is an increase of Ca2+ cytosolic concentration, which is suggested to be dependent on the activation of Ca2+ influx through the plasma membrane. This investigation was carried out with a plasmalemma enriched vesicle fraction, obtained from etiolated maize coleoptiles. The magnitude of Ca2+ efflux through the membrane vesicles was estimated according to the shift of potential dependent fluorescent dye diS-C3-(5). The obtained results showed that during coleoptiles ageing (3rd, 4th and 5th days of seedling etiolated growth) the magnitude of Ca2+ efflux from inside-out vesicles was decreased. Addition of ABP1 led to a recovery of Ca2+ efflux to the level of the youngest and most sensitive cells. Moreover, the efflux was more sensitive, responding from 10−8 to 10−6 M 1-NAA, in vesicles containing ABP1, whereas native vesicles showed the highest efflux at 10−6 M 1-NAA. We suggest that auxin increases plasma membrane permeability to Ca2+ and that ABP1 is involved in modulation of this reaction.

Zea mays CCaMK: autophosphorylation-dependent substrate phosphorylation and down-regulation by red light.

The role of protein kinases has been extensively studied in various signal transduction pathways and they are one of the most important components that link the signal perception to the final response. However, not many studies have been reported, especially from the plant systems, that show the regulation pattern of the kinase itself under different conditions. A calcium/calmodulin-dependent protein kinase has already been purified and characterized from etiolated maize coleoptiles. In this paper a detailed study of how the kinase itself is regulated at the autophosphorylation level is provided. Evidence is also given that the autophosphorylation of kinase effects its activity towards substrate phosphorylation. It is further shown that the kinase is an important component of the light signalling pathway as the level of the kinase itself decreases by red light irradiation.

Suitable experimental design for determination of auxin polar transport in space using a spacecraft.

It is necessary to establish a suitable experimental design for the determination of auxin (indole-3-acetic acid: IAA) polar transport in space using a spacecraft in concerning with the role of gravity. Problems in space experiments are as follows: I) Selection of suitable plant species; II) Preservation of integrity of plant segments for activities of auxin polar transport; III) Stop of auxin polar transport of the segments after the transport experiment in space. Segments of etiolated pea epicotyls and etiolated maize coleoptiles showed relatively high activities of auxin polar transport among dicotyledonous and monocotyledonous plants tested, respectively. The activities decreased dramatically when the segments were pre-stored at 25 degrees C only for 1 day. On the other hand, the storage at low temperature (5 degrees C) in the presence of antioxidants or chelating agents, especially EGTA, maintained relatively high activities of auxin polar transport in pea epicotyl segments. Low temperature (5 degrees C) substantially inhibited the activity of auxin polar transport. Based on the results in this study, a suitable experimental design for the space experiment of auxin polar transport using a spacecraft is also proposed.

Biochemical evidence for a calmodulin-stimulated calcium-dependent protein kinase in maize.

We provide biochemical evidence for the presence of a Ca2+-dependent calmodulin (CaM)-stimulated protein kinase (CCaMK) from etiolated maize coleoptiles. The kinase, with a molecular mass of 72.3 kDa, was purified to homogeneity by means of ammonium sulphate precipitation, DEAE-Sephacel chromatography. CaM-Sepharose chromatography and gel purification. The purified kinase required 5 mM Mg2+ for activity and had an optimum pH of 7.5. The kinase is a Ca2+-binding protein, as was evident by 45Ca2+-binding and Ca2+ mobility-gel-shift assays. 1 microM Ca2+ stimulated the kinase activity about 12-fold and was further stimulated by the addition of exogenous CaM (approximately 100 nM). Addition of Ca2+ and CaM antagonists decreased the kinase activity. Under in vitro assay conditions the kinase phosphorylated preferentially syntide-2, histone IIIS and casein. Syntide-2 and histone IIIS were phosphorylated at serine residues, showing that the kinase belongs to the serine/threonine family of protein kinases. Autophosphorylation of CCaMK occurred on threonine residue(s) and was Ca2+ dependent. Addition of exogenous CaM had no effect on autophosphorylation. The properties of the maize kinase suggests that it is a CCaMK that shows dual stimulation with Ca2+ and CaM for substrate phosphorylation and only Ca2+ requirement for autophosphorylation. Antibodies raised against the kinase cross-reacted with maize total proteins to give a single band of 72 kDa and precipitated substrate (syntide-2 and histone IIIS)-phosphorylation and autophosphorylation activities in a specific manner. Localisation studies with antibodies showed that the kinase is ubiquitous.

Immunocytochemical localisation of auxin-binding proteins in coleoptiles and embryos ofZea mays L.

The auxin-binding protein ABP-1 was localised immunocytochemically in coleoptiles and immature embryos ofZea mays. Two primary polyclonal antibodies raised against ABP-1 and secondary antibodies were either labelled with FITC or 10 nm gold particles for light microscopy, and with 10 nm gold particles for transmission electron microscopy. Light microscopy revealed that ABP-1 was localised in the epidermal cells of etiolated maize coleoptiles, in subepidermal parenchymatic mesophyll cells of the coleoptile and in the companion cells of the vascular bundles. Most labelling was found in the cytoplasm, less in nuclei and vacuoles and cell walls appeared negative. The region of the plasma membrane exhibited prominent labelling. Embryos showed low labelling throughout their tissues just after excision, but after culture for 7 days intensive labelling was found in the epidermis of the scutellum. Quantitative electron microscopy confirmed that ABP-1 was present in the cytoplasm of epidermal, mesophyll, and companion cells of coleoptiles. Gold particles were neither found in cell walls nor in the cuticle. Areas with ER and dictyosomes within epidermal and mesophyll cells of coleoptiles had a denser labelling with gold particles than elsewhere. Labelling at the plasma membrane, being the site where the auxin binds to the ABP, was observed at low levels in all cells examined, which is due to the method applied. Epidermal cells of embryos cultured for 5 days exhibited high levels of gold particles in ER and nuclei, and lower levels in the cytoplasm. The distribution is only partly in accordance with the model in which ABP is thought to cycle through the plant cell from the ER via the Golgi system towards the plasma membrane.

Evidence That Maize Acetyl-Coenzyme A Carboxylase Does Not Function Solely as a Homodimer

Acetyl-coenzyme A carboxylase (EC 6.4.1.2; ACCase) was purified from etiolated maize coleoptiles using cyclohexanedione affinity and DyeMatrex gel orange A, followed by anion-exchange chromatography. Purification yielded ACCase240, which was bound to orange A dye (purified 68x), and ACCase220, having no affinity for orange A dye (purified 79x). ACCase220 contained two biotinylated polypeptides with molecular mass of 220 and 85 kDa. The 85-kDa protein was separated from the 220-kDa protein and had ACCase activity. ACCase220 was composed of seven proteins as determined by native PAGE. Two-dimensional electrophoresis (native/SDS PAGE) of ACCase220 revealed that these seven native proteins share constituent subunits. These subunits of ACCase220 were cross-linked using dithiobis(succinimidylpropionate), and unassociated polypeptides were removed by YM100 ultrafiltration. Reduction of cross-linked ACCase resulted in the reappearance of multiple polypeptides originally observed by SDS PAGE. On the basis of these results, we provide the first evidence that gramineae ACCase does not function solely as a homodimer.

UDP-glucose sterol ß- d-glucosyltransferase, a plasma membrane-bound enzyme of plants: Enzymatic properties and lipid dependence

UPD-glucose sterol ß- d-glucosyltransferase (UDPG-SGTase) catalyzes the glucosylation of plant sterols. This enzyme has been shown to be membrane-bound, most of its activity being associated with plasma membrane in etiolated maize coleoptiles. After solubilization with detergents, total delipidation and purification, kinetic studies performed with a purified enzyme preparation in the presence of detergent and soybean phosphatidylcholine (PC) strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. A reduced sulfhydryl group and an arginyl residue were shown to be essential for activity. Lipid dependence studies have been performed on the delipidated enzyme in two systems: a micellar one composed of a mixture of enzyme, detergent and phospholipids and another one where the enzymatic activity was reconstituted in unilamellar lipid vesicles. In both systems it was shown that the UDPG-SGTase activity was stimulated to a large extent by negatively charged phospholipids. Enzymatic assays were performed with membrane fractions originating from plants whose sterol content was profoundly modified following treatment with a sterol biosynthesis inhibitor. Results showed that the sterol glucosylating activity was strongly inhibited in these fractions in accordance with sterol substrate specificity studies. All these results show that the UDPG-SGTase is exquisitely sensitive to its lipid environment. Physiological implications of these data are discussed in the light of the putative role of sterols in the plant cell.

Direct Observation of the Effects of 3,5-Diisopropylsalicylate and Light on Maize Coleoptiles using 31P NMR

Summary Metabolic effects caused by 3,5-diisopropylsalicylate (DIPS) and light were observed in etiolated maize coleoptiles using in vivo31P NMR spectroscopy. A decrease in the nucleotide triphosphate (NTP) resonances and an increase in nucleotide diphosphate hexose (NDP-hexose) signals were induced by 200 μM DIPS, with an acidification of the cytoplasm occurring in a fraction of the cells. Increased hexose phosphate and NDP-hexose signals, accompanied by a decreased cytoplasmic Pi resonance, were observed under intermittent blue light, while in red light only the increase of the hexose-P and decrease in cytoplasmic Pi signals occurred. These results provide evidence for an analogy between the action of DIPS and light at the molecular level, while the similar effects of red and blue light are compatible with phytochrome as the principal photoreceptor responsible for the metabolic changes.

Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family.

We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family. Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40%. Most amino acid residues showing identity are clustered in the GTP/GDP binding domain. In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well. Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs. The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells. The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers. The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes.

THE BLUE LIGHT GRADIENT IN UNILATERALLY IRRADIATED MAIZE COLEOPTILES: MEASUREMENT WITH A FIBER OPTIC PROBE

Abstract— The blue light (450 nm) gradient was measured with a fiber optic microsensor in etiolated maize coleoptiles that were irradiated unilaterally. Patterns of transmitted and scattered light across the shoot were related to the morphology of the coleoptile and varied greatly between the coleoptile base, mid‐region and tip. In the coleoptile base, light was scattered equatorially around the coleoptile sheath so that there was more light on the shaded side of the sheath than in the shaded side of the primary leaves. In the hollow mid‐region there were strong reflecting boundaries at the air‐coleoptile interfaces, which resulted in step like transitions in the light gradient. In the coleoptile tip, there was a steep, near‐linear gradient of blue light, with the greatest amount of light on the irradiated side. There was no evidence that the coleoptile tip acts as a lens or that there are anomalies that result in more light on the shaded than irradiated side. Immediately beneath the irradiated surface, the space irradiance was about twice the fluence rate of the light beam. The magnitude of the light gradient was 4:1 between the irradiated and shaded side of the mid‐region and tip and 8:1 for the coleoptile base.

Phospholipid-dependence of plant UDP-glucose-sterol-β-D-glucosyltransferase. II. Acetone-mediated delipidation and kinetic studies

Etiolated maize coleoptiles microsomes were delipidated by acetone. The resulting acetone powder, when resuspended in buffer and tested with radioactive UDP-glucose has lost the UDP-glucose:sterol β-D-glucosyltransferase (UDPG-SGTase) activity present in the microsomes. Addition of Triton X-100 at solubilizing concentration reveals only a residual activity (1% of the original solubilized microsomal activity). Simultaneous addition of Triton and sterols restores a low activity (20% of the solubilized microsomal activity). The subsequent addition of the coarse acetonic extract, or phospholipids purified from it, produces the restoration of 100% of the original activity. These results, together with those of the preceding paper, P. Bouvier-Navé et al., Plant Sci. Lett., 36 (1984) 19, point toward the phospholipid dependence of plant UDPG-SGTase. Unlike the microsomal or plasma membrane preparations of UDPG-SGTase activity, the acetone powder is a sterol-depleted preparation and thus, allowed a bisubstrate kinetic analysis of UDPG-SGTase mechanism. Reciprocal plots of initial rates of activity indicate clearly that the kinetics follow a sequential mechanism. The sterolic substrate specificity of the enzyme was also studied and is discussed.

Electron microscope localization of concanavalin a-binding sites in plasma membrane- and endoplasmic reticulum-rich fractions from maize coleoptiles

The binding of concanavalin A (Con A) to plasma membrane(PM)-and endoplasmic reticulum(ER)-rich fractions from etiolated maize coleoptiles has been investigated using sequential treatment with Con A and mannosyl-ferritin (Man-Fer). PM vesicles were shown to be extensively labelled at their outer surface, indicating the presence of available mannosyl and/or glucosyl residues in this fraction. Ferritin particles presented a clustered distribution. No label was observed on control vesicles stained in the presence α-methyl-D-mannoside (αMM). Membranes of the ER-rich fraction appeared largely devoid of ferritin particles. Combining this electron microscope study with procedures which render the interior of the vesicles accessible to the lactin, it was found that whereas no modification in the label of PM vesicles occurred, ER membranes were extensively labelled on their cisternal face. The results are totally in agreement with biochemical data reported in the preceding paper (Hartmann et al., Plant Sci. Lett., 30 (1983) 227) and suggest that PM and ER vesicles retain their orientation after grinding of coleoptile tissue.

Concanavalin a binding as a tool in determining the sidedness of membrane vesicles from maize coleoptiles

Plasma membrane(PM)-and endoplasmic reticulum(ER)-rich fractions from etiolated maize coleoptiles were assayed for their ability to bind concanavalin A (Con A). Evidence is presented for lectin binding sites in both fractions, proving the presence of available mannosyl and /or glucosyl residues on the membrane surface of vesicles. Results from quantitative binding studies using [ 3H] Con A showed that Con A binding is specific, of high affinity and saturable. Compared to ER vesicles, PM vesicles bound three times as much Con A. Con A binding by both fractions was also assessed after disruption of the vesicle membrane by Triton X-100 or low pH. While a dramatic increase in binding capacity of Con A to ER vesicles was achieved after treatment with low concentrations of detergent or at pH 5.0, the level of Con A binding to PM vesicles was not altered, indicating that these treatments did not expose additional lectin binding sites. Results are discussed in relation to the sidedness of PM and ER vesicles.

Activation of etiolated maize coleoptiles plasma membrane-bound uridine-diphosphate-glucose-sterol-gb- d-glucosyltransferase by Triton X-100, hydroxyl ions and phospholipase A 2

The plasma membrane-bound uridine-diphosphate-glucose-sterol-β- d-glucosyltransferase (UDPGSGT) of etiolated maize coleoptiles is activated by low concentrations (close to the critical micellar concentration (CMC)) of Triton X-100. Treatment of plasma membrane (PM) vesicles with hydroxyl ions or with phospholipase A 2 resulted in a stimulation of the enzymatic activity and in an elimination of the capacity of the enzyme to be activated by Triton X-100. The action of the phospholipase A 2 resulted also in a strong inhibition of the incorporation of uridine diphosphate (UDP) glucose into 6′-acyl steryl-β- d-glucosides (ASG). The results obtained lead to the conclusion that hydroxyl ions and phospholipase A 2 can be used in place of detergents to reveal latent enzymatic sites. They bring some further insights into the knowledge of the topology of the UDPGSGT in the transverse plane of the PM vesicles.

Biosynthesis of Δ 5.23 sterols in etiolated coleoptiles from Zea mays

In addition to the previously found ergosta-5, E-23-dien-3β-ol and 5α-ergosta-7, E-23-dien-3β-ol, the following Δ 23 sterols have been identified in etiolated maize coleoptiles: cyclosadol, 4α, 14α-dimethyl-5α-ergosta-8, E-23-dien-3β-ol, 4α, 14α-dimethyl-9β, 19-cyclo-5α-ergosta-8, E-23-dien-3β-ol and 4α-methyl-5α-ergosta-7, E-23-dien-3β-ol. The incubation of maize coleoptile microsomes in the presence of cycloartenol and of [ 14C-methyl] S-adenosyl methionine gave a mixture of labelled 24-methylene cycloartanol and cyclosadol. No trace of cyclolaudenol could be detected in these conditions. It is suggested that Δ 23 sterols are products of the C-24 methyltransferase reaction and they probably do not arise from a Δ 24 → Δ 23 isomerization occurring at a later stage of the biosynthesis. The Δ 13-sterols may play an intermediary role in the biosynthesis of 24-methyl sterols in this plant material.

Very long chain fatty acids: Occurrence and biosynthesis in membrane fractions from etiolated maize coleoptiles

The endoplasmic reticulum from maize coleoptiles elongates stearoyl-CoA more effectively than the plasmalemma-enriched fraction. The alkane and very lo

Latency of uridine diphosphate glucose-sterol-β-D-glucosyltransferase, a plasma membrane-bound enzyme of etiolated maize coleoptiles

Triton X-100 at low concentrations stimulates the activity of uridine diphosphate glucose-sterol-β-D-glucosyltransferase, a plasma membrane-bound enzyme of etiolated maize coleoptiles. Enzyme inactivation experiments using trypsin in the presence or absence of Triton X-100 suggested that the stimulatory effect of the detergent could be due to the existence of enzymatic sites which are not accessible to the substrate: uridine diphosphate glucose (UDP-glucose) and exposed on the inner surface of plasma membrane vesicles.

Sterol biosynthetic capability of purified membrane fractions from maize coleoptiles

Membrane fractions were isolated from etiolated maize coleoptiles by differential and sucrose density gradient centrifugation. Specific membrane components were identified by using marker enzyme activities. Fractions were also tested for α-naphthylacetic acid (NAA).and N-naphthylphthalamic acid (NPA) binding activities. Evidence is presented for the isolation of a plasma-membrane fraction containing specific binding sites for NPA, a high concentration of sterols, and most of the total UDP-glucose sterol glucosyltransferase activity. A fraction rich in endoplasmic reticulum is shown to contain most of the binding sites for NAA and all of the activity of both S-adeno-Syl- L-methionine-Δ24 cycloartenol methyltransferase and cycloeucalenol-obtusifoliol isomerase.