Auxin and hydrogen ion actions on light-grown pea epicotyl segments I. Tissue specificity of auxin and hydrogen ion actions

Growth responses to added auxin and hydrogen ions of differentlyprepared tissue segments excised from 5th internodes of light-grownAlaska peas were studied. Unpeeled segments extended in responseto auxin but not to hydrogen ions. Peeled segments elongatedwell in response to hydrogen ions but much less when exposedto auxin. Hollow-cylinder (central part removed) and infiltrated(by reduced pressure) unpeeled segments significantly elongatedin response to hydrogen ions as well as to auxin. Outward curvatureof split segments was enhanced by buffer solution at pH lowerthan 5.0. Peeled segments secreted hydrogen ions into an incubationmedium containing auxin or a fungal toxin, FC. Unpeeled segmentsand the isolated epimermis secreted much less hydrogen ionsin response to these agents. Hydrogen ions generated by theaction of auxin thus seem to accumulate in the region adjacentto the cuticle, that is, epidermal cells. The significance ofthe epidermis in auxin and hydrogen ion actions is discussed. (Received May 17, 1974; )     

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; )     

Red light and auxin effects on rubidium uptake by oat coleoptile and pea epicotyl segments

Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [⁸⁶Rb⁺] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10⁻⁷ molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light-enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [⁸⁶Rb⁺] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [⁸⁶Rb⁺] flux is not a general feature of phytochrome action.     

Counter-flow of3H-IAA and14C-ABA in long pea epicotyl segments

The counter-flow of³H-IAA and¹⁴C-ABA was studied in both aeropetal and basipetal arrangement in 11-cm long segments of pea epicotyls. A significant acropetal flow of¹⁴C-ABA was promoted by exogenous application of³H-IAA in the apical part of the segment together with a simultaneous promotion of its elongation growth. In the case of limited basipetal flow of¹⁴C-ABA neither the accumulation of this growth regulator on site of³H-IAA application nor a growth promotion were observed. Part I. Influence of Auxin-like Substances upon the Transport of¹⁴CABA in Long Pea Epicotyl Segments.     

Effect of galactoglucomannan-derived oligosaccharides on elongation growth of pea and spruce stem segments stimulated by auxin

Galactoglucomannan-derived oligosaccharides (GGMOs) (degree of polymerization 4–8) isolated from the wood of poplar (Populus monilifera Ait.) were shown to be inhibitors of the 2,4-dichlorophenoxyacetic acid-stimulated elongation growth of pea (Pisum sativum L. cv. Tyrkys) and spruce [Picea abies (L.) Karst] stem segments. A dependence on the concentration of GGMOs (between 10^-5-10^-10M) as well as plant species was ascertained. Pea stem segments were much more sensitive (10^-10M) than spruce (10^-8M). The GGMOs did not exhibit toxicity even at high concentrations and during long-term bioassays. The timing of the action of GGMOs and auxin in the growth process was also studied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - d.p degree of polymerization - GGMOs galactoglucomannan-derived oligosaccharides This research was supported by the Slovak Grant Agency for Science.     

Apparent Pfr killer reaction and P* denaturation in segments of etiolated pea epicotyl

When totally etiolated pea epicotyls were cut into segments and incubated with potassium phosphate buffer, pH 6.0, in the dark at 25 C, an instantaneous loss of photoreversible absorbance change, Δ (ΔA) between 660 and 730 nm, was observed after the first irradiation with actinic red light in the spectrophotometric measurement of phytochromein vivo. The shorter the epicotyl segments, and the longer the period of dark incubation, the greater was the loss detected in the measurement. A remarkable decline of Δ(ΔA) in the far-red region was seen inin vivo difference spectra for phytochrome, after the epicotyl segments were incubated in the dark at 25 C. As the period of dark incubation was prolonged, the ratio of the maximal change of Δ(ΔA) in the far-red region to that in the red region was reduced. It decreased to ca. one third of the initial value after incubation for 8 hr. The evidence indicates that Pfr killer activity and P^* denaturation, both of which have so far been known onlyin vitro, can also occur in segments of etiolated pea epicotyls.     

Auxin and the response of pea roots to auxin transport inhibitors: morphactin

The auxin transport inhibitor methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (CFM), a morphactin, inhibits negative geotropism, causes cellular swelling, and induces root hair formation in roots of intact Pisum sativum L. seedlings. In excised pea root tips, CFM inhibits elongation more than increase in fresh weight (swell ratio = 1.3 at 20 mum CFM). CFM growth inhibition was expressed in the presence of ethylene. Indoleacetic acid (IAA) prevented the expression of CFM growth inhibition possibly because IAA inhibited the accumulation of CFM into the tissue sections. CFM inhibited the accumulation of IAA and 2,4-dichlorophenoxyacetic acid into excised root tips. Applying Leopold's (1963. Brookhaven Symp. Biol. 16: 218-234) model for polar auxin transport, this result suggests a possible explanation for CFM inhibition of geotropism in pea roots, i.e. disruption of auxin transport by interfering with auxin binding.     

The effect of cycloheximide on IAA-stimulated transport of 14C-ABA and 14C-sucrose in long pea epicotyl segments

The effect of cycloheximide (CH) on the indol-3yl-acetic acid (IAA)-stimulated transport of ¹⁴C-labelled abscisic acid (ABA) and ¹⁴C-labelled sucrose was studied in 110 mm long pea epicotyl segments. IAA application resulted in elongation growth of the segments. This effect was decreased by CH treatment which also reduced [¹⁴C] ABA and [¹⁴C] sucrose accumulation in the growing apical part of the segments. A reduction in [¹⁴C] IAA uptake and in protein synthesis in this part of the segments was also observed. The simultaneous inhibition of protein synthesis and reduction of [¹⁴C] ABA and [¹⁴C] sucrose transport suggests that IAA can stimulate the transport of ABA and sucrose through a protein synthesis-based elongation growth.     

Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

Solubilization and properties of GDP-fucose: xyloglucan 1,2-alpha-L-fucosyltransferase from pea epicotyl membranes

GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase from pea (Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% (w/v) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona- and decasaccharide subunits. The apparent Km for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 microM GDP-fucose. This was 10 times the apparent Km for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at -70 degrees C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% (w/v) tamarind XG, and its activity located by incubation in GDP-[14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.     

Effects of gibberellic acid and indole-3-acetic acid on stress-relaxation properties of pea hook cell wall

The effects of GA, IAA and PCIB on the cell wall propertiesof Alaska pea hooks were examined using stress-relaxation analysis.The results were:

1. GA caused a decrease in the stress-relaxation parameter To ofplumular hook sections after the first 30 min of incubation,long before it induced elongation.
2. PCIB increased To, andIAA tended to negate the PCIB effecton To in GA-treated sectionsafter 90 min of incubation, whenthe effect of PCIB and IAAon the elongation was not yet found.In this case, IAA couldnot be substituted by an extra amountof GA.
3. GA decreasedTo in the middle part of the sections after 24hr of incubation,and then stimulated elongation.
4. In any case, the effect ofGA, IAA or PCIB on To was recognizedin both epidermis and innertissue of plumular hook sections.
5. The stress-relaxation parameterTo appears to represent thecapacity of the cell wall to extend;we thus concluded thatboth gibberellin and auxin increase theextensibility of thecell wall, when they stimulate the elongationof plumular hooksections.

(Received October 4, 1974; )     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.     

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.

     

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.     

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.     

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 M found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.