Differential effect of auxin on in vivo extensibility of cortical cylinder and epidermis in pea internodes

The effect of auxin indole-3-acetic acid (IAA) on growth and in vivo extensibility of third internode sections from red light grown pea seedlings (Pisum sativum L. cv Alaska) and the isolated tissues (cortex plus vascular tissue = cortical cylinder, and epidermis) was investigated. Living tissue was stretched at constant force (creep test) in a custom-built extensiometer. In the intact section, IAA-induced increase in total (E_tot), elastic (E_el), and plastic (E_pl) extensibility is closely related to the growth rate. The extensibility of the cortical cylinder, measured immediately after peeling of intact sections incubated for 4 hours in IAA, is not increased by IAA. Epidermal strips, peeled from growing sections incubated in IAA, show a E_pl increase, which is correlated to the growth rate of the intact segments. The isolated cortical cylinder expands in water; IAA has only a small growth-promoting effect. The extensibility of the cortical cylinder is not increased by IAA. Epidermal strips contract about 10% on isolation. When incubated in IAA, they do not elongate, but respond with an E_pl increase. The amount of expansion of the cortical cylinder and contraction of the epidermis (tissue tension), measured immediately following excision and peeling, stays constant during IAA-induced growth of intact sections. The results support the hypothesis that IAA induces growth of the intact section by causing an E_pl increase of the outer epidermal wall. The driving force comes from the expansion of the cortical cylinder which is under constant compression in the intact section.     

Growth and anthocyanin synthesis in excised Sorghum internodes

Summary Ligh-induced anthocyanin synthesis in excised dark-grown internodes of Sorghum was depressed by the addition of auxin to the incubating medium at physiological concentrations. Both IAA and the synthetic auxin, 2,4-D, reduced anthocyanin yield. Similar results were obtained with isolated internode segments and in internodes incubated with coleoptiles (the major source of endogenous auxins) attached. Auxin increased the duration of the lag phase before anthocyanin synthesis began and reduced the rate during the subsequent linear phase. Elongation continued longer with IAA than without it and anthocyanin formation did not begin until extension growth had ceased or was slowing down in both cases; the rate of anthocyanin synthesis in the IAA solution remained depressed compared with that in buffer even after extension growth had ceased in both.At low concentrations IAA stimulated elongation growth without reducing anthocyanin yield and it is unlikely that the effect of IAA on anthocyanin synthesis results from the increased utilisation in growth of substrates needed for anthocyanin formation. The results of reciprocal transfer experiments from dark to light, and vice versa, showed that the action of IAA was associated with its presence in the incubating medium during the irradiation period. If present only in darkness, before or after transfer to light, IAA did not reduce anthocyanin formation; in the former case total yield was increased by IAA as a result of the stimulation of elongation growth, the concentration of anthocyanin remaining unchanged.GA₃ also decreased anthocyanin content; the effect was greater in sections incubated with coleoptiles attached and it is possible that GA₃ acts by increasing the concentration of endogenous auxins. However, CCC, which has been reported to decrease endogenous auxin levels, also reduced anthocyanin yield.The effect of IAA was not influenced by the presence of ascorbate in the incubating medium, nor did added ascorbate result in the formation of any acylated cyanidin derivative in internodes maintained in darkness.Possible relationships between light-induced anthocyanin formation and the photo-inhibition of elongation are discussed.     

Osmotic properties of pea internodes in relation to growth and auxin action

The water transport properties of etiolated pea (Pisum sativum L.) internodes were studied using both dynamic and steady-state methods to determine (a) whether water transport through the growing tissue limits the rate of cell enlargement, and (b) whether auxin stimulates growth in part by increasing the hydraulic conductance of the growing tissue.

Measurements using the pressure probe technique showed that the hydraulic conductivity of cortical cell membranes was the same for both slowly growing and auxin-induced rapidly growing cells (membrane hydraulic conductivity, about 1.5 × 10⁻⁵ centimeters per second per bar). In a second technique which measured the rate of water movement through the entire pea internode, the half-time for radial water flow was about 60 seconds and was not altered by auxin application. These results indicate that auxin does not alter the hydraulic conductance of pea stem tissue, either at the cellular or the whole tissue level.

Measurements of the turgor pressure of cortical cells, combined with osmotic pressure measurements of expressed cell sap, show that the water potential of growing pea stems was about −3 bars. When the growth rate was altered by various treatments, including decapitation, auxin application, cold temperature, and KCN treatment, the water potential was independent of the growth rate of the stem. We attribute the depression of the water potential in young pea stems to the presence of solutes in the cell wall free space of the tissue. This interpretation is supported by the results of infiltration and perfusion experiments.

From the results of these dynamic and steady-state experiments, we conclude that the internal gradient in water potential (from the xylem to the epidermis) needed to sustain cell enlargement is small (no greater than 0.5 bar). Thus, the hydraulic conductance of the tissue is sufficiently large that it does not control or limit the rate of cell enlargement.

     

Evidence for an electrogenic ATPase in microsomal vesicles from pea internodes

The transmembrane electropotential of microsomal vesicles from pea internode segments, monitored by equilibrium distribution of the permeant anion SCN^?, is strongly hyperpolarized when ATP is present in the incubation medium.The stimulation of SCN^? uptake by ATP is rather specific with respect to the other nucleoside di- and triphosphates tested: ADP, GTP, CTP and UTP. ATP-stimulated SCN^? uptake is strongly inhibited by ATPase inhibitors such as $\text{p-}\text{chloromercuribenzenesulphonate}$ and $\text{N,N}{}^{\mathrm{\prime }}\text{-}\text{dicyclohexylcarbodiimide}$ and by 2.5% toluene/ethanol (1 : 4, v/v), the latter being a treatment which makes the vesicles permeable. On the contrary, oligomycin is almost ineffective in influencing ATP-induced SCN^? uptake. The proton conductor carbonyl cyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$ strongly inhibits ATP-stimulated SCN^? uptake. The effect of ATP on SCN^? uptake depends on the pH of the medium, the maximum being reached at about pH 7.0.These data support the view that microsomal fractions from pea internodes contain membrane vesicles endowed with a membrane-bound ATPase coupling ATP hydrolysis to electrogenic transport of ions, probably H⁺.     

Localization and partial characterization of the extracellular proteins centrifuged from pea internodes

The proteins removed from the extracellular space of dark-grown pea ( Pisum sativum L. cv. Alaska) internode sections by centrifugation were studied. A large number of proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These proteins ranged in size from 10 to 150 kdalton and their removal from the cell wall was greatly facilitated by the presence of salts of divalent and trivalent cations in the infiltration medium. Pulse-labelling experiments with [³⁵S)-methionine showed that many of the proteins extracted from the cell wall incorporated radioactivity and that treatment with indoleacetic acid (IAA) altered the pattern of radiolabel incorporation. One of the proteins centrifuged from pea internode sections possessed per-oxidase (EC 1.11.1.7) activity. The activity of this peroxidase increased less in auxin-treated internode segments than in untreated controls. Antibodies were raised to the total protein fraction extracted by centrifugation and used to localize antigens on protein blots. Most of the proteins centrifuged from pea internode sections were stained by a dye coupled to the cell wall antiserum. Light microscopic immunohistochemical studies showed that the proteins centrifuged from dark-grown pea internodes were localized almost exclusively in the cell wall and intercellular spaces of pea internode tissue. Light microscopic immunohistochemistry also showed that antibodies to extracted proteins penetrate into the apoplast of abraded pea internode segments and split pea stems. These antibodies did not influence growth of IAA-treated or control tissue.     

The role of endogenous gibberellin-like substances and inhibitors in the growth of pea internodes

Third internodes or whole stems of 7-days old etiolated pea plants were extracted and the content of gibberellin-like substances and inhibitors has been determined. Extracts were found to contain four or five different gibberellin-like substances, some of which are chromatographically similar to GA₃. The content of gibberellins has been high in young internodes and decreased along with the internodes elongation. Brief red light irradiation brings about quantitative changes in gibberellin content, depending also on the length of internodes. The extracts contain acidic and neutral inhibitors which interfere with the response to GA₃. The content of the inhibitors does not seem to be affected by the ageing of internodes or by the light treatment.     

Xyloglucan endotransglycosylase activity in pea internodes. Effects of applied gibberellic acid.

Xyloglucan endotransglycosylase (XET) activity extractable from internodes of tall and dwarf varieties of pea (Pisum sativum L.) was assayed radiochemically using tamarind seed xyloglucan as donor substrate and an oligosaccharidyl-[3H]alditol as acceptor substrate. Internodes I and II showed little elongation during the period 15 to 21 d after sowing; XET activity remained relatively constant and was unaffected by exogenous gibberellic acid (GA3). A single application of GA3 to the dwarf genotype resulted in a small enhancement of elongation in internode III between d 17 and 21 and caused a small increase in XET activity in internode III. Repeated applications of GA3 caused internode V to elongate between d 20 and 26, to the same extent as in the tall variety, and concomitantly led to greatly elevated XET activity (expressed per unit fresh weight, per unit of extractable protein, and per internode). Thus, XET activity correlated with GA3-enhanced length in pea internodes; the possibility that this represents a causal relationship is discussed.     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

Blockage by gibberellic Acid of phytochrome effects on growth, auxin responses, and flavonoid synthesis in etiolated pea internodes

Red light inhibits the growth of etiolated pea internodes, causes a shift toward higher indoleacetic acid (IAA) concentrations in the IAA dose-response curve of excised sections, and promotes the synthesis in intact internodes of kaempferol-3-triglucoside. Gibberellic acid (GA₃) prevents all 3 effects, the first effect substantially and the last 2 completely. This suggests GA₃ blockage of an early or basic event initiated by the active form of phytochrome. The red light-induced shift in the IAA dose-response curve of excised sections is consistent with a light-induced increase in the activity of an IAA destruction system, since the magnitude of the red light inhibition varied with IAA concentration. The red light and GA₃ effects on growth and on flavonoid synthesis are consistent with the view that phytochrome may control growth by regulating the synthesis of phenolic compounds which act as cofactors in an IAA-oxidase system. GA₃ reversal of the red light-induced shift in the IAA dose-response curve involves both growth promotion and inhibition by GA₃ at different IAA concentrations and this, together with the GA₃ reversal of light-induced flavonoid synthesis, supports the suggested regulatory role of phenolic compounds in growth.     

Effects of vasectomy and exercise upon aortic extensibility, cholesterol deposition, and stainable tissue lipids

The effects of vasectomy and exercise upon arterial extensibility, cholesterol deposition, and the extent of stainable tissue lipids were determined in rats. A 2 X 3 factorial design with two activity levels (treadmill exercise and sedentary) and three treatment levels (vasectomy, sham vasectomy and control) were employed. The exercise groups had significantly greater arterial extensibility measures at breaking load and stretch distance at 45% load and breaking point. The vasectomy-sedentary group had significantly less extensibility than the control-sedentary group for stiffness and stretch distance at 45% load and breaking point. Vasectomy significantly increased the amount of esterified and total cholesterol in aortic tissue. The vasectomy-sedentary group had significantly higher unesterified cholesterol levels than the three exercise groups. Total cholesterol levels were significantly higher for the sedentary groups than the exercise groups. There were no significant differences between groups in the extent of stainable tissue lipids.     

Characteristics of starch in developing pea seeds

Changes in the physical and chemical properties of pea starch in developing seeds have been investigated. Increase in the iodine-binding capacity of st     

Transport, metabolism, and redistribution of xylem-borne amino acids in developing pea shoots

Amino acid metabolism and transport was investigated in the leaves of 3-week-old nonnodulated seedlings of Pisum sativum L. Xylem sap entering the shoot contained nitrate (about 5 millimolar), and amino compounds (11 millimolar) of which 70% was asparagine plus glutamine; aspartate and homoserine were also present. Mature leaves showed stable nitrogen levels and incoming nitrogen was redistributed to growing leaves. Younger leaves, still enclosed in the stipules, showed negligible rates of transpiration, suggesting that most of their nitrogen must arrive in the phloem.     

Starch phosphorylase enzymes in developing and germinating pea seeds

Phosphorylase has been fractionated during development and germination of seeds of smooth and wrinkled-seeded peas. The total phosphorylase levels have been compared. In addition, a number of other pea tissues and other legumes have been examined. Some kinetic properties of the two enzymes present have been measured. Both enzymes have been further purified by affinity chromatography on Sepharose 4B-starch columns and by sequential gel filtration in the absence and presence of amylopectin. The MW and sub-unit structures of the two enzymes have been examined and their possible roles discussed.     

Senescence of detached leaves in pigeon pea and chick pea: Regulation by developing pods

The senescence rate of the subtending leaves in deflowered and control plants of pigeon pea ICajanus cajan (L.) Millsp. cv., Prabhat] and chick pea ( Cicer arietinum L. cv. JG 62) were examined during the course of natural and induced senescence, at several stages of pod growth. The leaves from the top 5 nodes on the main axis in pigeon pea and the top 8 nodes on the main axis in chick pea were used throughout the experiments. The natural senescence was characterized in leaves taken directly from the field-growing plants. For the study of induced senescence, the leaves were excised from both control and deflowered plants at various stages of pod growth and placed in test tubes containing water under dark conditions. Senescence was assessed in terms of peroxidase activity and contents of tola] chlorophyll, soluble amino acids and total protein. During natural ageing in the field, the leaves from deflowered plants exhibited delayed senescence in both the species. In contrast, the rate of ageing during induced senescence was higher in the leaves of deflowered plants than in the controls. Although of the same chronological age when excised for induced senescence, the leaves of deflowered plants were evideatly metabolically different from the controls, due to the fact that deflowered plants did not support the development of pods. This difference probably determined the subsequent rate of induced senescence.     

Chromatin-bound DNA-dependent RNA polymerase in developing pea cotyledons

Summary The pattern of cotyledon development in three varieties of Pisum sativum has been defined in terms of cell number, DNA and RNA content and chromatin, bound RNA polymerase activity. Variation was observed in the relative periods of growth by cell division and cell expansion between the three varieties. The mean DNA content per cotyledon cell during growth by cell expansion increased to approximately 50C in one variety, 30C in the second variety and 15C in the third variety. The pattern of chromatin-bound RNA polymerase activity during development suggested that some of the DNA above the 2C level may contribute to RNA synthesis in two of the three varieties studied. In the third variety the RNA polymerase activity decreases throughout the phase of increase in DNA per cell. The chromatin-bound RNA polymerase activity per cell was correlated with the rate of RNA increase per cell.