The Casparian strip in pea epicotyls: Effects of light on its development

The Casparian strip, which is specific to roots, was studied in the epicotyls of dark-grown seedlings of pea (Pisum sativum L.) where it was found to have the same morphology and properties as the strip in roots. In dark-grown seedlings, the distance between the upper-most position of the Casparian strip and the bending point of the hook (about 37 mm) did not change during growth of the seedlings. In the uppermost 0.5-mm region of the region in which the Casparian strip could be detected by fluorescence microscopy, the plasma membrane was not firmly attached to the cell wall. The development of the Casparian strip continued for about 42 h after dark-grown seedlings were transferred to the light, indicating that (i) the cells that have been determined to form the Casparian strip in darkness form the strip in the light, and that (ii) it takes about 42 h for the cells to complete formation of the strip. Cells in the hook of dark-grown seedlings did not form a Casparian strip when such seedlings were transferred to the light. The Casparian strip was formed in rapidly elongating internodes of light-grown seedlings when the seedlings were transferred to darkness. Light did not control the formation of the Casparian strip in roots.Abbreviation PBS phosphate-buffered saline     

Electropotential in excised pea epicotyls

In contrast to intact etiolated pea seedling tissue (Pisum sativum L.), excised segments immersed in a complete nutrient solution show marked increases in ion content, largely of K⁺ and NO₃⁻, over a 72-hour period. During this time there is increase in cell electropotential difference, PD. During the initial 6 to 8 hours there is a lag in ion uptake; cell PD, however, increases rapidly from approximately −50 to −100 mv then increases more slowly. The increase in PD precedes and thus may be a prerequisite for the rapid ion accumulation phase. Cell PD increases in either water or nutrient solution but eventually reaches higher levels in the latter. Following water pretreatment of sufficient duration K⁺ accumulation shows no lag period. The lag phase noted here appears dissimilar to that of storage tissues.     

Effect of red light on ribulose 1,5-bisphosphate carboxylase activity in pea leaves

The ribulose 1,5-bisphosphate activity and its relative content in pea (Pisum sativum L., cv. Bordi) seedlings grown either under white or red light were investigated. Plants grown under red light had a lower ribulose 1,5- bisphosphate carboxylase (RuBPCO) activity as compared to plants grown under white light, if expressed on a fresh mass. These activities were very similar under both lights, as calculated on protein basis, although the relative content of RuBPCO was higher in the red one. The activity of RuBPCO under red light corresponds to the lower rate of net photosynthesis. The results are discussed in respect to possible presence of RuBPCO inhibitor in pea plants growth under red light.     

Soluble auxin-binding proteins in pea epicotyls

Auxin-binding proteins, have been identified in the soluble cytoplasrnic protein fraction of etiolated pea epicotyls, Pisum sativum L., cv. "Dippes Gelbe Victoria". The binding is specific for the auxins NAA, IAA and 2,4-D with a K_D in the range of 0.1–0.4 μ M . Moreover, the binding is competitive, sensitive to digestion by proteinase and shows linearity with the protein content of the assay mixture. The binding proteins appear to be very labile, since repeated freezing and thawing destroys specific binding. No clear pH-optimum could be detected in the physiological pH-range 5.5–8.0, but the binding was doubled at pH 8.0 compared to pH 5.5–7.0.     

Gibberellic-acid-induced cell elongation in pea epicotyls: Effect on polyploidy and DNA content

In gibberellic-acid(GA₃)-treated epicotyls of dwarf peas (Pisum sativum L.) grown in the light, DNA (per cell and per epicotyl) is followed. Histofluorometric DNA determinations show that GA₃-promoted cell elongation is not accompanied by increased endomitosis, but chemical estimations show an increased DNA content per epicotyl. This difference must therefore be the result of increased mitotic activity in the GA₃-treated tissue. Epicotyls of seedlings grown with or without cotyledons under continuous light with GA₃ are tetraploid, as are those of ecotylized embryos grown in darkness. These epicotyls reach no more than half the length of octaploid epicotyls of seedlings grown in darkness. This result provides evidence for a relationship between polyploidy and final possible cell length.     

Genotype-specific soluble auxin-binding in etiolated pea epicotyls

Using different independent procedures for assaying soluble auxin-binding in etiolated pea epicotyls, wo could prove the reliability of the (XH₄)₂SO₄-pelleting assay both for crude cytosols as well as for specific protein fractions obtained after chromatofocusing. Three distinct genotypes (two parent lines, one tall recombinant) investigated so far exhibit characteristic differences with respect to soluble auxin-binding kinetics in their cytosols.     

Auxin-regulated changes in protein phosphorylation in pea epicotyls

Auxins regulate various aspects of plant growth and development. However, the mechanism by which these hormones elicit diverse physiological processes is not clear. We present evidence for the role of auxin in protein phosphorylation and the possible involvement of calmodulin in auxin-induced changes. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides markedly decreased whereas phosphorylation of 19,000, 24,000 and 28,000 molecular weight polypeptides increased. These results open up a new experimental approach in understanding the molecular mechanism by which auxins regulate various physiological processes in plants.     

Amyloplast development in etiolated and ethylene-treated pea epicotyls

The amyloplasts found in the apical hook cells of etiolated pea (Pisum sativum L.) epicotyls were randomly distributed. Sedimentation of endodermal amyloplasts in the direction of gravity became apparent in the transition from the hook to the top of the main axis of the epicotyl. Cortical amyloplasts in this region were not, however, sedimented. These patterns of sedimentation could not be related to changes in amyloplast size, and it is proposed that cytoplasmic properties determine amyloplast behaviour.The differentiation of plastids in the hook differed between the amyloplast-containing endodermal cells and the cortical cells, in which amoeboid plastids predominated over amyloplasts. Amyloplasts disappeared from the cortical cells in the main axis of the epicotyl, but in the endodermal cells sedimented amyloplasts were found throughout the upper epicotyl.Etiolated epicotyls induced to grow horizontally by treatment with ethylene had a normal content of amyloplasts, sedimented in the direction of gravity.     

Occurrence of diamine oxidase in the apoplast of pea epicotyls

Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epicotyl sections previously infiltrated under vacuum with a buffer solution. No detectable amount of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase is present in this fluid, showing that there is very little contamination by cell contents. Polyacrylamide-gel electrophoresis and specific-activity data indicate that diamine oxidase is the most plentiful protein in the extracellular solution obtained from pea epicotyl sections and that an active process is involved in the selective transfer of the enzyme outside the cell. The possible involvement of diamine oxidase in the supply of H₂O₂ to peroxidase-catalyzed reactions occurring inside the cell wall is discussed.Abbreviations DAO diamine oxidase - Glc6P glucose-6-phosphate     

Action potentials resulting from mechanical stimulation of pea epicotyls

Summary Non-propagating, asynchronous action potentials may be detected with an extracellular electrode placed on the hook of the pea epicotyl following mechanical stimulation. These may well mediate the release of ethylene which has previously been shown to control the diameter of mechanically stressed shoots.     

Purification and properties of diamine oxidase from pea epicotyls

Diamine oxidase (DAO) (EC 1.4.3.6) was purified from pea epicotyls to homogeneity by the criterion of polyacrylamide gel electrophoresis (PAGE). The pu     

Auxin-induced extension of the isolated epidermis of light-grown pea epicotyls

1. The effect of IAA and FC on the extension of isolated epidermisof light-grown Alaska pea epicotyls was studied under differentconditions with an extension apparatus. The following resultswere obtained.
2. The epidermis extended in response to low pHbuffer solutionof 1–10 mM, maximum extension being achievedat pH below5.5.
3. IAA, 5 mg/liter, caused, although not consistently,an extensionof epidermal strips in 1 mM buffer, but not at10 mM.
4. Consistent extension of the isolated epidermis dueto IAA wasobtained by addition of GTP, ATP, ITP or UTP (sodiumsalts),but not nucleosides, nitrogen bases or sugars.
5. A fungaltoxin, FC, at 10^–5 M induced extension of theepidermiswithout addition of the nucleoside triphosphates.
6. IAA andFC caused H⁺ extrusion in peeled epicotyl segments bothin thepresence and absence of GTP. IAA caused appreciable H⁺extrusionin the isolated epidermis only in the presence ofGTP, whereasH⁺ extrusion by the epidermis was induced by FCeven in theabsence of GTP.

From these results, we concluded that IAA induces extensionof the isolated epidermis under the above conditions throughthe mediation of H⁺ ions. (Received July 12, 1976; )     

Extraction and partial characterization of cellulases from expanding pea epicotyls

Etiolated pea (Pisum sativum) epicotyls synthesize a buffer-soluble cellulase (cellulase A) and a salt-soluble cellulase (cellulase B) (EC 3.2.1.4) after treatment with high (0.5%) auxin levels. Only cellulase A increased in activity after treatment with low (0.005%) auxin. Cellulase A was released into the supernatant after homogenization of tissue in dilute buffer (buffer-soluble), had a pH optimum at 5.5, was relatively thermostable, and its activity was inhibited by NaCl. Cellulase B was released by 1 m NaCl (salt-soluble) from excised tissue segments or from the insoluble residue remaining after removal of the buffer-soluble form. It had a pH optimum at 7.0, was thermolabile, and required salt for maximum activity. When subjected to polyacrylamide gel electrophoresis, the cellulase fraction released by NaCl from excised segments showed two bands of cellulase activity compared to several for the buffer-soluble fraction. Electrophoretic analysis of the buffer and salt-soluble fractions for marker enzymes indicated the presence of malate dehydrogenase activity in all fractions and glutamate dehydrogenase activity in the buffer-soluble fraction only.     

Glucomannan synthesis in pea epicotyls: The mannose and glucose transferases

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a -1,4-[su14C]mannan from GDP-d-[U-¹⁴C]-mannose, a mixed -1,3- and -1,4-[¹⁴C]glucan from GDP-d-[U-¹⁴C]-glucose and a -1,4-[¹⁴C]-glucomannan from both GDP-d-[U-¹⁴C]mannose and GDP-d-[U-¹⁴C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The -glucan synthase had different properties from other preparations which bring about the synthesis of -1,3-glucans (callose) and mixed -1,3- and -1,4-glucans and which use UDP-d-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-d-xylose in addition to GDP-d-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-d-glucose acted competitively in the presence of GDP-d-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-d-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-d-glucose and GDP-d-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-d-mannose and GDP-d-glucose to bring about the synthesis of the heteropolysaccharide.Abbreviations CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate - CHAPSO 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate - CHD 1,2-cyclohexanedione - CDP cytidine 5-diphosphate - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - GDP guanosine 5-diphosphate - NAI N-acetyl-imidazole - NEM N-ethylmaleimide - PGO phenylglyoxal This work has been made possible by grants of M.A.F. and M.U.R.S.T. 40% of Italy. Dr. A. Zuppa wishes to thank the C.N.R. of Italy for his research scolarship.     

Effects of brefeldin A on the development of the Casparian strip in pea epicotyls

Summary The Casparian strip, a structure that is present in roots, is also present in epicotyls of dark-grown pea seedlings. In a dark-grown epicotyl, the cells in each stage of the development of the Casparian strip have been suggested to be lined up basipetally in the region 3 to 37 mm below the bending point of the hook, in order of the developmental stage. Brefeldin A (BFA), a specific inhibitor of secretory transport, was administrated at 200 M. to dark-grown pea epicotyls for 2 h via a thread passed through the epicotyl 40 mm below the bending point. The basipetal sequence of development of the modification of the cell wall at the Casparian strip, as judged by fluorescence microscopy, stopped 5 h after the start of 2 h treatment with BFA and resumed after 30 h. This basipetal sequence of development did not stop in control seedlings. Electron micrographs of endodermal cells in epicotyls treated with BFA showed striking morphological changes in the Golgi stacks and the ER. Histological examination made 20 h after the start of the experiment revealed that the basipetal sequence of development of the cell wall modification stopped at a point which was present at 25.2 ± 1.6 mm (mean with SD, n=5) from the bending point of the hook at the start while the basipetal sequence of development of the tight adhesion of the plasma membrane to the cell wall at the Casparian strip stopped 0.9 ± 0.5 mm (mean with SD, n=5) below this point. These results indicate the involvement of secretory transport not only in the introduction of the modification of the cell wall but also in the completion of the tight adhesion of the plasma membrane.Abbreviations BFA brefeldin A - PBS phosphate-buffered saline - ER endoplasmic reticulum     

Ethylene effect on extensin and peroxidase distribution in the subapical region of pea epicotyls

In dark grown pea (Pisum sativum) seedlings ethylene causes the triple response in which elongation growth is inhibited, radial growth is promoted, and orientation of shoots to gravity is altered. The distribution of extensin and peroxidase activity in pea epicotyls upon ethylene treatment was studied by tissue printing on nitrocellulose paper. It was found that the localization of extensin and peroxidase activity changes after 72 and 96 hours of ethylene treatment. In untreated plants, peroxidase activity is detected only in the vascular bundles. Nonetheless, after 72 and 96 hours of ethylene treatment peroxidase activity is hardly detected in the vascular system but present in the epidermal and cortical cells. Extensin increases in the epidermal and cortical cells upon ethylene treatment but it also appears in the vascular system when peroxidase activity is no longer detected.     

Active and passive transport of potassium in cells of excised pea epicotyls

The Ussing-Theorell equation, which provides a fundamental test for the independent passive movement of ions under conditions of nonequilibrium, has been used to assess the active and passive components of K⁺ uptake by segments of pea epicotyl (Pisum sativum L. cultivar Alaska), incubated for 24 hours in both 1-fold and 10-fold concentrations of a complete nutrient solution. Measurements of the rates at which ⁴²K diffused out of the segments provided data from which were estimated the K⁺ content of, and the fluxes to and from, the nonfree space compartments, interpreted as being cytoplasm and vacuole. For this analysis the serial model of MacRobbie and Dainty and Pitman for the spatial arrangement of cell compartments was used. On the basis of these values, and measurements of electrical potential across the cell membranes, the vacuolar K⁺ concentration was found to be fairly close to that expected as a result of passive diffusion between the cytoplasm and vacuole provided that no potential exists across the tonoplast. Cytoplasmic K⁺ concentration, however, was much too high in both treatments to be accounted for in passive terms. It was concluded, therefore, that, on the basis of the model, the high ratio of influx to efflux was maintained in the cells by an active K⁺ pump located at the plasmalemma. There is some reason to question the applicability of this model for flux analysis to the conditions of high net influx as encountered here; nonetheless, it provides a first approach to an over-all flux analysis in pea stem tissue.