Auxin and hydrogen ion actions on light-grown pea epicotyl segments II. Effect of hydrogen ions on extension of the isolated epidermis

Isolated epidermis treated differently, i.e. fresh, frozen-thawedand methanol-treated, was subjected to extension tests undera tension of 2 ? 10⁷ dyne/cm² in buffer solution at differentpH values. (1) Fresh and frozen-thawed epidermis extended in response tobuffer solution at pH values lower than 5.5. Half of the maximumextension was achieved at pH 4.5. (2) Epidermis boiled in methanol or treated with pronase didnot extend in response to pH 4.5. Low temperature reduced therate of extension of fresh or frozen-thawed epidermis inducedby pH 4.5. (3) Pretreatment of the epidermis with 0.1% deoxycholate for30 min did not inhibit acid-induced extension. (4) Nojirimycin, 3 ? 10^-3M, added to the buffer solution inhibitedacid-induced extension. (5) Epidermis peeled off from segments which had been treatedwith cycloheximide for 90 min, extended in response to pH 4.5. (6) Methanol-treated epidermis did not extend at pH 4.5, butextended somewhat at pH 3.0. These results suggest that hydrogen ions induce cell wall loosening,possibly through activation of wall-bound enzymes. (Received May 17, 1974; )     

Role of the epidermis in auxin-induced elongation of light-grown pea stem segments

1. Effects of auxin on elongation and cell wall properties werestudied using 5th internode segments of light-grown pea epicotyl.The results were:
2. The optimum concentration of 2,4-D for elongationinductionwas about 1 µg/ml, both for unpeeled and peeledsegments.
3. Using stress-relaxation analysis, mechanical propertiesof thecell wall were expressed by the parameters 1/₁, To andT_m. Unpeeledsegments were first treated with 2,4-D, then theepidermis waspeeled off. Parameters of the epidermal cell wallwere conspicuouslychanged by 2,4-D but those of the inner tissuewere not.
4. Actinomycin D and cycloheximide inhibited 2,4-D-inducedchangesin cell wall parameters, as well as in elongation, ofunpeeledsegments apd of the epidermis.
5. 2,4-D did not induceelongation of the isolated epidermis butpromoted that of peeledsegments. This promotion was smalleras compared with unpeeledsegments. 2,4-D did not significantlyinfluence the diffusionpressure deficit of peeled segmentsbut did increase their elasticand plastic extensibilities.
6. We conclude that auxin primarilyinduces cell wall looseningof the epidermis, most likely throughnucleic acid and proteinsynthesis.

¹ Present address: Biological Institute, Department of GeneralEducation, Nagoya City University, Mizuho-ku, Mizuho-cho, Nagoya467, Japan. (Received April 22, 1971; )     

Activities of growth inhibitors isolated from light-grown dwarf pea shoots

The growth inhibitory activities of 6 endogenous growth inhibitors isolated from light-grown dwarf peas (Pisum sativum cv. Progress No. 9) were examined in the epicotyl of dark-grown seedlings of the same cultivar in the dark in order to examine the possible contribution of these compounds to the growth inhibition brought about by red light. The activities of these natural inhibitors, including two A-2 and A-2 of as yet undetermined structure, were compared with those of synthetic growth retardants and benzyladenine. Samples were applied directly into the epicotyls via a glass capillary tube. In 24-h tests doses for a 25% inhibition (I₂₅) were: A-2, 4.3 × 10^-2: cis-xanthoxin, 1.2 × 10^-1 ; A-2, 1.6 × 10^-1; trans-xanthoxin, 1.2; R,S-dihydromaleimide, 3.5 × 10² and pisatin, 4.0 × 10² nmol plant^-1 . In 72-h tests, I₂₅'s were: benzyladenine, 1.5; AMO-1618 (ammonium-(5-hydroxycarvacryl)-trimethylchloride piperidine carboxylate), 2.4; R,S-dihydromaleimide, 4.0 × 10² and CCC (chlorocholine chloride), 1.1 × 10³ nmol plant^-1. -D-Glucosyl-R-dihydromaleimide had no activity at all. Benzyladenine caused the thickening as well as elongation inhibition of the epicotyls of intact plants. The possible involvement of A-2 and in the red light growth inhibition of dwarf peas is discussed.Abbreviations AMO-1618 ammonium-(5-hydroxycarvacryl)-trimethylchloride piperidine carboxylate - CCC chlorocholine chloride - G-DHMD -D-glucosyl-R-dihydromaleimide - I₂₅ dose required for a 25% growth inhibition - R red light author for correspondence     

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.     

Auxin-induced synthesis of TB-RNA in pea stems

The effect of 2,4-dichlorophenoxyacetic acid on RNA synthesisin etiolated pea internode segments was studied. Auxin stimulatedsynthesis of TB-RNA only in short period incubation, suggestingits primary importance in auxin action. The nucleotide compositionof newly synthesized TB-RNA seems to be modified under auxininfluence. (Received February 8, 1969; )     

Activity of mitochondria isolated and purified from dwarf and tall,light-grown pea seedlings

Mitochondria from dwarf and tall light-grown peas have been separated from contaminating chloroplasts and other organelles on discontinuous sucrose gradients. A comparison between activities of the mitochondria from the two cultivars of light-grown pea seedlings was made. It was observed that both respiratory control (R.C.) indices and phosphate to oxygen (P : O) ratios were superior in mitochondria from the tall cultivar than the dwarf cultivar. The results are discussed in relation to energy status and possible gibberellic acid content in the two cultivars.     

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.     

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.     

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.     

Auxin-induced longitudinal-to-transverse reorientation of cortical microtubules in nonelongating epidermal cells of azuki bean epicotyls

Summary In epidermal cells of azuki bean (Vigna angularis) epicotyl segments, that were sequentially treated with an auxin-free solution and an auxin solution, cortical microtubules changed their orientation from longitudinal to transverse. Auxin caused the reorientation of microtubules from longitudinal to transverse in segments that were kept under anaerobic conditions and, therefore, showed no elongation, indicating that auxin can regulate the orientation of microtubules by a mechanism that does not involve auxin-induced change in the rate of cell elongation.Abbreviations DMSO dimethylsulfoxide - GA₃ gibberellic acid - IAA indoleacetic acid - MT microtubule - PBS phosphate-buffered saline     

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.     

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.     

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     

Effect of red light on geotropism in pea epicotyls

Dose response curves were determined for phytochrome phototransformation and for a phytochrome-controlled decrease in geotropic curvature in epicotyls of dark-grown Pisum sativum L. cv. Alaska. Ten times as much light was required to produce a spectrophotometrically detectable transformation of phytochrome as was required to produce a significant change in the geotropic response. The red light energy required for a 50% phytochrome transformation caused a 90% change in the physiological response.     

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.     

Phytochrome control of hypocotyl extension in light-grown Chenopodium rubrum

The regulation of hypocotyl extension in light-grown Chenopodium rubrum L. seedlings by light analogous to dense vegetation canopy shade has been monitored. Hypocotyl extension was controlled by both the quantity and quality of the actinic light. At the higher of the two background photon fluence rates which were used (10.0 μmol m⁻²s⁻¹ in the 400–700 nm waveband), increasing the proportion of phytochrome calculated to exist as Pfr resulted in greater inhibition of growth. At the lower photon fluence rate (1.0 μmol m⁻²s⁻¹ in the 400–700 nm waveband), a biphasic response was observed in which minimum inhibition was observed at intermediate photoequilibria. Although photosynthesis was not directly involved in the photomorphogenetic responses, it did play an indirect quantitative role in determining the response.