Intracellular distribution of mitochondria after geotropic stimulation of the oat coleoptile

The number of mitochondria is greater in the bottom than in the top of cells in geotropically stimulated oat (Avena sativa) coleoptiles. In the avascular tip and outer epidermis of subapical regions this difference occurs only in the lower tissues. These inequalities are found both in the KMnO₄⁻ and in the glutaraldehyde-fixed tissues; however, they are significant only in the former. Also, the number of mitochondria scored is consistently lower when the tissues were fixed in KMnO₄. These results suggest that mitochondria undergo a small degree of sedimentation after geostimulation, a redistribution reduced by the slower fixation with glutaraldehyde. Differences in mitochondrial number begin later than those in the amyloplast and the Golgi apparatus after geotropic stimulation. The cells in the avascular-tip region (a region having an important role in geotropism) have two to three times more mitochondria than the subapical cells.     

Electrical properties of parenchymal cell membranes in the oat coleoptile

Summary Parenchymal cells of oat (Avena sativa) coleoptiles had an osmotic concentration of 410 mM (determined by plasmolysis); of this only 22 mM was K⁺ and 1 mM Na⁺ (flame photometry). Cells were impaled with micropipette electrodes. Iontophoretic injection of the dye Niagara sky-blue from the micropipette showed that the tip of the electrode penetrated the vacuole. When sections of tissue were immersed in a solution of 22 mM KCl, 1 mM CaCl₂, and 50 mM glucose, average membrane potential was found to be 38.5 mV inside negative specific membrane resistance was 510 cm², and specific membrane capacitance, 2 f cm^-2. The cell membranes showed <25% retification and no electrical excitability. Electrotonic coupling of adjacent cells could not be demonstrated.     

Matrix polysaccharides of oat coleoptile cell walls

Separation of component polysaccharides in extractable fractions of the noncellulosic matrix of Avena sativa coleoptile cell walls shows that the principal classes of polymers present are glucuronoarabinoxylans (GAX) and iodine-negative hemicellulosic β-glucans. Rhamnogalacturonan is a minor component. GAX contains about 5–10% glucuronic acid and its 4-O-methyl ether, arabinose in amount almost equal to xylose, and a small amount of galactose; some subfractions contained appreciable amounts of glucose and galacturonic acid but these may derive from separate, contaminating polysaccharides. From the sedimentation and diffusion coefficients and intrinsic viscosities of one subfraction each of the GAX and of the hemicellulosic glucan that had been purified to apparent homogeneity by criteria of sedimentation and borate electrophoresis, MWs of about 200 000 were calculated by two methods. The viscosity characteristics and gel-forming ability of the hemicellulosic glucan give evidence of appreciable molecular interactions which suggest that this polymer is an important structural component of the cell wall.     

Brefeldin A: a specific inhibitor of cell wall polysaccharide biosynthesis in oat coleoptile segments

The effect of brefeldin A (BFA) on the synthesis and incorporation of polysaccharides, proteins and glycoproteins into the cell wall of subapical coleoptile segments isolated from etiolated oat seedlings (Avena sativa L. cv. Angelica) has been investigated. In the presence of D-[U-¹⁴C]-glucose, the incorporation of radioactive glycosyl residues into buffer-soluble, membrane (matrix polysaccharides) and cell wall polysaccharides was drastically inhibited by increasing concentrations of BFA up to 10 μ·mL⁻¹. BFA also altered the pattern of these polysaccharides suggesting a different sensitivity of glycosyltransferases toward the action of the drug. The incorporation of [U-¹⁴C]-glycine or L-[U-¹⁴C]-leucine into non-covalently- and covalently-bound cell wall proteins as well as the incorporation of radioactive N-acetylglucosamine residues into the newly synthesised oligosaccharidic chains of cytosolic, membrane and cell wall glycoproteins remained unchanged in the presence of 10 μg·mL⁻¹ BFA. The data demonstrate that, in oat coleoptile segments, BFA specifically inhibits the synthesis of cellulose and matrix polysaccharides without altering the synthesis and incorporation of proteins and glycoproteins into the cell wall. In addition, it is demonstrated that BFA does not affect the in vivo activity of glycosyltransferases involved in the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the oligosaccharidic chains of glycoproteins.     

Proton efflux from oat coleoptile cells and exchange with wall calcium after IAA or fusicoccin treatment

Elongation growth of plant cells occurs by stretching of cell walls under turgor pressure when intermolecular bonds in the walls are temporarily loosened. The acid-growth theory predicts that wall loosening is the result of wall acidification because treatments (including IAA and fusicoccin) that cause lowered wall pH cause elongation. However, conclusive evidence that IAA primarily reduces wall pH has been lacking. Calcium has been reported to stiffen the cell walls. We have used a microelectrode ion-flux measuring technique to observe directly, and non-invasively, the net fluxes of protons and calcium from split coleoptiles of oats (Avena sativa L.) in unbuffered solution. Normal net fluxes are 10 nmol · m⁻² · s⁻¹ proton efflux and zero calcium flux. The toxin fusicoccin (1 μM) causes immediate efflux from tissue not only of protons, but also of calcium, about 110 nmol · m⁻² · s⁻¹ in each case. The data fit the “weak acid Donnan Manning” model for ion exchange in the cell wall. Thus we associate the known “acid-growth” effect of fusicoccin with the displacement of calcium from the wall by exchange for protons extruded from the cytoplasm. Application of 10 μM IAA causes proton efflux to increase transiently by about 15 nmol · m⁻² · s⁻¹ with a lag of about 10 min. The calcium influx decreases immediately to an efflux of about 20 nmol · m⁻² · s⁻¹. It appears that auxin too causes an “acid-growth” effect, with extruded protons exchanging for calcium in the cell walls. I. Arif is currently recieving an AIDAB scholarship. This work was supported by an Australian Research Council grant to I.A. Newman.     

Galactose inhibition of auxin-induced cell elongation in oat coleoptile segments

Auxin-induced cell elongation in oat coleoptile segments was inhibited by galactose; removal of galactose restored growth. Galactose did not appear to affect the following factors which modify cell elongation: auxin uptake, auxin metabolism, osmotic concentration of cell sap, uptake of tritium-labeled water, auxin-induced wall loosening as measured by a decrease in the minimum stress-relaxation time and auxininduced glucan degradation. Galactose markedly prevented incorporation of [¹⁴C]-glucose into cellulosic and non-cellulosic fractions of the cell wall. It was concluded that galactose inhibited auxin-induced long-term elongation of oat coleoptile segments by interfering with cell wall synthesis.     

Auxin-indnced changes in barley coleoptile cell wall composition

Auxin induces extension growth of barley coleoptile segments,causing cell extension and cell wall loosening represented bya change in mechanical properties of the cell wall. This responsedecreased after the segments were starved for more than 12 hrin buffer solution. Auxin decreased the noncellulosic glucosecontent of the cell wall of the segments starved for 0 and 6hr, but very little that of segments starved for 12 and 18 hr.The contents of arabinose, xylose and galactose, among noncellulosicpolysaccharides, and -cellulose of the cell wall increased duringthe starvation, but auxin did not affect them. The auxin-induceddecrease in glucose content was inhibited by nojirimycin, apotent inhibitor of ß-glucanase, which inhibited auxin-inducedextension and changes in mechanical properties of the cell wall,suggesting that cell wall loosening, and thus cell extension,resulted from partial degradation of ß-glucan of thecell wall. (Received April 20, 1978; )     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring     

Stress relaxation properties of the cell wall of growing intact plants

The cell wall of dark-grown Avena coleoptiles and the epidermisof light-grown mungbean hypocotyls was subjected to stress-relaxationanalysis and the following results were obtained. 1. Actively growing apical regions of the organs, either coleoptilesor hypocotyls, had certain threshold values of minimum stress-relaxationtime, T_O, 0.04 sec for coleoptile cell wall and 0.03 sec forthe epidermal cell wall of hypocotyls. The cell wall of thebasal region of the organs, which were mature and not growing,had a higher value of To. 2. When the apical regions of the organs, either coleoptilesor hypocotyls, ceased to grow, their cell walls showed T_O valuesabove these thresholds. 3. The relaxation rate, b, was small in the cell wall of activelygrowing regions of the organs, compared with that of non-growingregions. 4. The maximum relaxation time, T_m, was variable and no significantrelationship with growth capacity was found. 5. The extensibility, mm/gr, was large not only in activelygrowing regions of the organs but also in fully grown regions,suggesting that the value represents complex properties of thecell wall including the history of cell wall extension. From these results, we concluded that biochemical modificationsoccur in the cell wall matrix of actively growing organs ofeither monocots or dicots, and these are the bases of the capacityof the cell wall to extend and are represented chiefly by T_oand possibly by b. (Received August 12, 1974; )     

Effect of auxin on changes in the oriented structure of wall polysaccharides in response to mechanical extension in oat coleoptile cell walls

The increase in the dichroism of the ester-C=O and COO^–bands in response to mechanical extension in oat coleoptilecell walls was much enhanced by IAA pretreatment while the decreasein that of the C-O-C band was not enhanced or even suppressed.The results indicate the importance of the ultrastructure ofnon-cellulosic polysaccharides in controlling auxin-inducedcell elongation. ¹ Present adress: Department of Agricultural Chemistry, KyotoPrefectural University, Shimogamo, Kyoto 606, Japan. (Received May 16, 1978; )     

Effect of galactose on auxin-induced cell elongation in oat coleoptile segments in mannitol solutions

Oat coleoptile segments were treated with or without 10 mM galactose in the presence or absence of 10 μM IAA and various concentrations of mannitol (pre-incubation). Auxin-induced growth was inhibited by galactose. Segments were then transferred to buffer solutions containing or not containing 10 mM galactose (post-incubation). Expansion growth due to rapid water absorption was observed. The expansion growth during the post-incubation was inhibited by galactose when galactose was applied during the post-incubation period or all through the pre- and post-incubation but was not affected by galactose when it was applied only during the pre-incubation. This result indicates that the galactose effect on the expansion growth is due to its inhibitory action during the post-incubation period. Galactose has been reported to be a specific inhibitor for cell wall synthesis. Thus, it is suggested that the expansion growth during post-incubation requires cell wall synthesis and is not just the process of passive water absorption. The primary action of auxin does not seem to require new synthesis of polysaccharides.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

Wounding-induced cell wall pH shifts in coleoptile segments of various Poaceae

Wounding-induced extracellular pH shifts were characterized previously in excised segments of maize (Zea mays L.) coleoptiles. In the present study it is demonstrated that similar pH shifts also occur in Triticum aestivum L., Secale cereale L., Hordeum vulgare L., Avena sativa L., Sorghum durra (Forsk.) Stapf, and Setaria italica (L.) Beauv., with characteristic quantitative differences between the species. Indole-acetic acid induces pronounced drops of the medium pH in all species except Setaria italica. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of indole-3-acetic acid on cell wall loosening: Changes in mechanical properties and noncellulosic glucose content of Avena coleoptile cell wall

The effect of indole-3-acetic acid on cell wall loosening andchemical modifications of noncellulosic components of the cellwall in Avena coleoptile segments was studied and the followingresults were obtained. (1) Auxin decreased both the minimum stress-relaxation time(T_o) and the noncellulosic glucose content of the cell wall. (2) Decreases were observed in the absence or presence of mannitolsolution at concentrations lower than 0.20 M which osmoticallysuppressed auxin-induced extension, while at concentrationshigher than 0.25 M, there was little auxin effect, indicatingthat it is turgor-dependent. (3) The decrease in T_o of the cell wall and that in the noncellulosicglucose content caused by auxin in the presence of mannitolsolutions of various concentrations paralleled each other (thecorrelation coefficient was 0.897). (4) Both decreases in T_o and glucose content caused by auxinwere inhibited by nojirimycin (5-amino-5-deoxy-D-glucopyranose)in the presence of mannitol. The results suggest that auxin-induced cell wall loosening iscaused by the degradation of noncellulosic rß-glucanin the cell wall. (Received December 24, 1976; )     

Second positive phototropic response patterns of the oat coleoptile

Summary 1.During second positive irradiation, bending increases steadily with time. Under optimal conditions, the lag between onset of illumination and beginning of parabolic bending behavior is about 3 min. — 2. Shortly after irradiation ceases, bending becomes linear with time. On a clinostat, bending continues for about 2.5 hr. Auxanometric measurements show that the ultimate cessation of bending is not due to failing growth rate. — 3. The second positive response shows a striking dependence on intensity of irradiation. Inactivation occurs when irradiation approaches the intensity of full daylight. — 4. Induction is linear with duration of illumination, both at purely activating intensities and at partially inactivating intensities. — 5. Induction at 2°, while somewhat slower than at 25°, retains linear dependence on exposure duration. This suggests that the reactions immediately following light reception are slowed but not stopped at low temperature. — 6. Growth, which drops to about 0.5 /min at 2°, resumes at about 18 min^-1 as soon as plants are warmed to 25°. Curvature does not seem to begin for about 10 min. Combined with information about lag time for primary auxin action, this suggests that lateral auxin transport, as well as growth, is strongly inhibited at near-freezing temperatures. — 7. The induced transport system is highly stable at 2°. — 8. Under optimal conditions, the lag between onset of irradiation and induction of capacity to produce measurable curvature is only a few seconds. The length of the lag is dependent on the rate of induction. The lag is thought to be due to the requirement that enough induction be accumulated to overcome resistance of the coleoptile. — 9. Induction is dependent on the gradient of light across the coleoptile, whether measured for purely activating or partially inactivating intensities. The light received is probably integrated either across individual cells or across the entire width of the coleoptile.     

Cell wall yield properties of growing tissue : evaluation by in vivo stress relaxation

Growing pea stem tissue, when isolated from an external supply of water, undegoes stress relaxation because of continued loosening of the cell wall. A theoretical analysis is presented to show that such stress relaxation should result in an exponential decrease in turgor pressure down to the yield threshold (Y), with a rate constant given by ε where is the metabolically maintained irreversible extensibility of the cell wall and ε is the volumetric elastic modulus of the cell. This theory represents a new method to determine in growing tissues.

Stress relaxation was measured in pea (Pisum sativus L.) stem segments using the pressure microprobe technique. From the rate of stress relaxation, of segments pretreated with water was calculated to be 0.08 per megapascal per hour while that of auxin-pretreated tissue was 0.24 per megapascal per hour. These values agreed closely with estimates of made by a steady-state technique. The yield threshold (0.29 megapascal) was not affected by auxin. Technical difficulties with measuring by stress relaxation may arise due to an internal water reserve or due to changes in subsequent to excision. These difficulties are discussed and evaluated.

A theoretical analysis is also presented to show that the tissue hydraulic conductance may be estimated from the T_½ of tissue swelling. Experimentally, pea stems had a swelling T_½ of 2.0 minutes, corresponding to a relative hydraulic conductance of about 2.0 per megapascal per hour. This value is at least 8 times larger than . From these data and from computer modeling, it appears that the radial gradient in water potential which sustains water uptake in growing pea segments is small (0.04 megapascal). This means that hydraulic conductance does not substantially restrict growth. The results also demonstrate that the stimulation of growth by auxin can be entirely accounted for by the change in .

     

Activation of Avena coleoptile cell wall glycosidases by hydrogen ions and auxin

Several cell wall-bound glycosidases present in Avena sativa coleoptiles were assayed by following the hydrolysis of p-nitrophenyl-glycosides. Particular emphasis was placed on characterizing some parameters affecting the activity of β-galactosidase. The pH optimum of this enzyme is 4.5 to 5.5; it is sensitive to copper ions and p-chloromercuribenzoate treatment and apparently has an exceptionally low turnover rate. Indoleacetic acid treatment enhanced in vivo β-galactosidase activity of coleoptile segments by 36% over control after 60 minutes. This enhancement was prevented by abscisic acid and cycloheximide. High buffer strengths and low pH reduced the indoleacetic acid-enhanced increase in enzyme activity. These data lend support to the following proposed model of indoleacetic acid action. Indoleacetic acid enhances the release of hydrogen ions into the cell wall which promote the activities of cell wall glycosidases, some of which may participate in the cell extension process.     

Infrared analysis of oat coleoptile cell walls and oriented structure of matrix polysaccharides in the walls

Infrared absorption spectra of film specimens of oat coleoptilecell walls, before and after protease treatment and after treatmentfor removal of lipid materials, pectic substances and hemicellulose,were recorded, and the characteristic bands in the spectrumof the wall assigned. Polarization spectrum measurements onthe wall provided evidence indicating that the non-cellulosicpolysaccharide matrix as well as cellulose micronbrils has anoriented structure in the wall and that the oriented structurechanges during extension growth as well as upon mechanical extensionof the walls. (Received July 22, 1977; )     

An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls

Plant cell walls expand considerably during cell enlargement, but the biochemical reactions leading to wall expansion are unknown. McQueen-Mason et al. (1992, Plant Cell 4, 1425) recently identified two proteins from cucumber (Cucumis sativus L.) that induced extension in walls isolated from dicotyledons, but were relatively ineffective on grass coleoptile walls. Here we report the identification and partial characterization of an oat (Avena sativa L.) coleoptile wall protein with similar properties. The oat protein has an apparent molecular mass of 29 kDa as revealed by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Activity was optimal between pH 4.5 and 5.0, which makes it a suitable candidate for acid growth responses of plant cell walls. The oat protein induced extension in walls from oat coleoptiles, cucumber hypocotyls and pea (Pisum sativum L.) epicotyls and was specifically recognized by an antibody raised against the 29-kDa wall-extension-inducing protein from cucumber hypocotyls. Contrary to the situation in cucumber walls, the acid-extension response in heat-inactivated oat walls was only partially restored by oat or cucumber wall-extension proteins. Our results show that an antigenically conserved protein in the walls of cucumber and oat seedlings is able to mediate a form of acid-induced wall extension. This implies that dicotyledons and grasses share a common biochemical mechanism for at least part of acid-induced wall extensions, despite the significant differences in wall composition between these two classes of plants.Abbreviations ConA concanavalin A - CM carboxymethyl - DEAE diethylaminoethyl - DTT dithiothreitol - Ex29 29-kDa expansin