Phytochrome Control of Cell Wall-bound Hydroxyproline Content in Etiolated Pea Epicotyls

The red light inhibition of growth of the intact pea (Pisum sativum L. cv. Alaska) third internode was correlated with an increase in the content of cell wall-bound hydroxyproline. These changes were detected 3 hours after irradiation, and possibly at 1 hour. Far red light reversed the effects of red light. The iron chelator α,α′-dipyridyl reversed the red light effects on both growth and hydroxyproline content. Using segments incubated in vitro, no phytochrome-mediated change in hydroxyproline content could be observed, perhaps because of an overwhelming wounding response. If plants were irradiated in situ and grown for 8 hours before excision and incubation of segments, some enhancement of hydroxylation by red light was detectable both colorimetrically and radioisotopically. The red light inhibition of segment growth was reversed by α,α′-dipyridyl. These results are examined in reference to the role of extensin in normal and induced growth cessation.     

On the Nature and Origin of the Calcium Asymmetry Arising during Gravitropic Response in Etiolated Pea Epicotyls

Seven day old etiolated pea epicotyls were loaded symmetrically with ³H-indole 3-acetic acid (IAA) or ⁴⁵Ca²⁺, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca²⁺ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca²⁺ asymmetries, but substances known to interfere with normal Ca²⁺ transport (nitrendipine, nisoldipine, Bay K 8644, A 23187) do not significantly alter either IAA or Ca²⁺ asymmetries. These substances, however, are active in modifying both Ca²⁺ uptake and efflux through oat and pea leaf protoplast membranes. We conclude that the ⁴⁵Ca²⁺ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca²⁺ movement secondary in gravitropism. We hypothesize that apoplastic Ca²⁺ changes during graviresponse because it is displaced by H⁺ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increases calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H⁺ efflux, increase Ca²⁺ release from pea epicotyl segments, whereas cycloheximide, which inhibits H⁺ efflux, has the reverse effect. We suggest that Ca²⁺ does not redistribute actively during gravitropism: the asymmetry arises because of its release from the wall adjacent to the region of high IAA concentration, proton secretion, and growth. Thus, the asymmetric distribution of Ca²⁺ appears to be a consequence of growth stimulation, not a critical step in the early phase of the graviresponse.     

Phytochrome-dependent Reduction of Nicotinamide Nucleotides in the Mitochondrial Fraction Isolated from Etiolated Pea Epicotyls

When mitochondria isolated from etiolated pea (Pisum sativum cv. Alaska) epicotyls were exposed briefly to red light, their ability to reduce exogenous NADP was enhanced. The red light effect was reversed by far red light. Photoreversible absorbance changes between 730 nm and 800 nm were spectrophotometrically detected in the purified mitochondria and its membrane fraction. The dehydrogenase activity in the mitochondria was heat-labile and was dependent on the presence of magnesium ion and appropriate substrates such as glucose 6-phosphate, isocitrate, pyruvate, 6-phosphogluconate, and succinate. The photoreversible effect was seen only for a few minutes after the irradiation, and was cancelled by hypotonic treatment or addition of Triton X-100. A similar but lesser effect was observed in the pea microsome fraction, whereas no photoreversible response was seen with a supernatant fraction resulting from centrifugation at 10⁵g for 30 minutes.     

Binding Proteins to Phytochrome A in Etiolated Pea Seedlings

In order to detect and characterize a putative receptor(s) fora signal from PhyA, proteins that bind to purified pea PhyAwere searched for in the crude extract of etiolated pea seedlingswith affinity chromatography. PhyA was coupled to the columnsubstrate either in P_R form (P_R column) or in red-irradiatedform (P_FR column). The coupled PhyA of both columns retainsits spectral reversibility between P_R and P_FR, although theirpeptide mapping by trypsin digestion suggests that the C-terminalhalf of PhyA in the P_FR column is partially fixed in P_FR structure.15 polypeptides were detected reproducibly in the elution fromthe P_FR column by silver-staining of SDS-PAGE. These 15 polypeptidesmay form two complexes judging from their elution profiles.Of the 15 polypeptides, the 6 major polypeptides have approximatemol wt of 80, 55, 53, 46, 40 and 35 kDa. On the other hand,only a trace amount of protein, which mainly consists of the46 kDa species, was eluted from P_R column, indicating the presenceof P_FR-specific BPs in the crude extract of etiolated pea seedlings.Of the 6 major polypeptides, the 40 kDa species binds to thePhyA in a photoreversible manner. (Received June 19, 1998; Accepted December 19, 1998)     

Effects of Extreme Acceleration on the Germination, Growth and Cell Wall Composition of Pea Epicotyls

Pea (Pisum sativum) seedlings were germinated and grown in darknessfor 5 d whilst being subjected to continuous accelerations ofbetween 1 ? g and 1054 ? g. Increased acceleration retardedthe elongation of both epicotyls and roots, and inhibited lateralroot growth. Changes in the cell wall carbohydrate and lignincomponents were also recognized. Nevertheless, increasing accelerationfrom 1 ? g to 1054 ? g had no effect on germination and peaswere able to germinate and grow successfully at up to approximately10 000 ? g. Key words: Acceleration, cell walls, Pisum     

Calcium Antagonist TMB-8 Inhibits Cell Wall Formation and Growth in Pea

The effects on auxin-stimulated growth and cell wall formationof 8-(N, N-diethylamino)-octyl-3, 4, 5-trimethoxybenzoate.HCI(TMB-8), an intracellular Ca²⁺ antagonist, were investigatedin abraded stem segments from aetiolated seedlings of Pisumsativum L. cv. Alaska. Incubation of segments at pH 6.0 with200 mmol m^–3 TMB-8 resulted in a 50% inhibition of auxin-stimulatedgrowth. Added Ca²⁺ did not restore normal auxin-stimulated growth,presumably because of its well-known stiffening effect on thecell wall. In segments incubated at a pH (7–2) which preventedelongation, auxin promoted the incorporation of [³H]glucoseinto the cell wall relative to total uptake of label. TMB-8abolished about 60% of the total incorporation of label intocell walls in the presence of auxin, but was not effective inthe absence of auxin. Exogenous CaCl₂ reversed the inhibitoryeffect of TMB-8 on relative cell wall incorporation in a parabolicmanner, with a 50% reversal at about 100 mmol m^–3 andcomplete reversal at 1.0 mol m^–3 Ca²⁺. Other ions tested(Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺) were without substantial effect atconcentrations of 0.5 mol m^–3. Both apparent uptake ofCa²⁺ and consequent reversal of TMB-8 inhibition of cell wallincorporation were blocked by the Ca²⁺ channel blockers verapamiland La³⁺. The data provide further evidence that auxin-stimulatedgrowth is dependent upon continued cell wall incorporation,and suggest that a Ca²⁺ messenger system may be involved inthe promotory actions of auxin on cell wall synthesis and long-termgrowth. Key words: Auxin, calcium, cell wall synthesis     

In vivo Binding of Gibberellin A(1) in Dwarf Pea Epicotyls

Binding of [(3)H]gibberellin A(1) (GA(1)) to extracts of dwarf pea epicotyls was investigated using sliced pea epicotyls (0.5-1.0 millimeter thick) that had been incubated in a solution containing [(3)H]GA(1) at 0 C for 3 days. Gel filtration of a 100,000g supernatant indicated binding to a high (HMW) and an intermediate molecular weight (IMW) fraction with estimated molecular weights of 6 x 10(5) daltons and 4 to 7 x 10(4) daltons, respectively. The bound (3)H-activity was [(3)H]GA(1) and not a metabolite as deduced by thin layer chromatography. The bound label did not sediment during centrifugation at 100,000g for 2 hours; also, binding was not disrupted after treatment of a combined HMW and IMW fraction with DNase, RNase, or phospholipase A or C, but it was disrupted by protease or heat treatment. These facts suggest that binding of [(3)H]GA(1) was occurring to a soluble protein(s). [(3)H]GA(1) bound to a combined HMW and IMW fraction was not susceptible to changes in pH, nor could it be exchanged with a variety of GAs tested under in vitro conditions. Under in vivo equilibrium conditions, biologically active GAs, such as GA(1), GA(3), GA(4), GA(5), GA(7), and keto GA(1), could reduce the level of [(3)H]GA(1) binding, whereas inactive GAs, such as iodo GA(1) methyl ester, GA(8), GA(13), GA(26), and non-GAs, such as (+/-)abscisic acid, had no effect. By varying the concentration of [(3)H]GA(1) in the incubation medium, the specific binding of [(3)H]GA(1) appeared to be due to two classes of binding sites having estimated K(d) of 6 x 10(-8) molar and 1.4 x 10(-6) molar. The concentrations of the two sites were estimated to be 0.45 picomole per gram and 4.04 picomoles per gram on a fresh weight and 0.1 picomole per milligram and 0.9 picomole per milligram on a soluble protein basis, respectively.     

Role of Cell Wall Calcium in Red Light- inhibited Elongation of Hypocotyls of Etiolated Phaseolus radiatus

When the hypocotyl segments of Phaseolus radiatus L. were incubated in CaC12 (1 mmol/L) medium, the cell wall calcium was increased over threefold more than those incubated in a Ca2+ -free medium. However, red light inhibited elongation of the hypocotyl was 20% to 25% both in the medium with or without Ca2 + . The amount of calcium removed from the wall by ethylene glycol-bis (2-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) ( 1 to 10 mmol/L) was 58.13 % to 75.33%, which offset the red light-inhibited elongation of the hypocotyl by 61.29% to 87.1%. Moreover, treatment with the channel blocker, verapamil ( 10 to 100 μ mol/L), wall calcium was the same as that of the darkness control, by which the red light-inhibited growth was also offset. La3 + ( 100 to 1 000 μmol/L) had no effect on wall calcium as compared to hypocotyl segments treated with red light alone, but eliminated the inhibitory effect of red light. Treatment with the calcium ionophore, A23187 (10 to 100 μmol/L), red light-inhibited elongation was abolished by 66.67% to 142.45% while wall calcium was reduced by 24.53% to 42.81%. In addition, calmodulin antagonist chlorpromazine (1 to 10 μmol/L) also counter acted the red light-induced elongation inhibition. These data indicated that exogenous Ca2+ was involved in the red light-inhibition effect, but that' did not mean that Ca2 + was not required. Perhaps Ca2 + in the wall itself was sufficient for red light-induced inhibition of hypocotyl elongation. The role of wall calcium might be quite complex, it not only acted as a signal of influx Ca2 + from the Ca2 + pool, but also played a regulatory role in the cell wall.     

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [³H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [³H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K_d) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [³H]NPA binding.     

Increase in Cell Wall-Associated Phosphatase Activity in Cucumber Roots during Calcium Starvation: Binding Nature and Properties of the Phosphatase and Cell Wall Analysis

In Ca^2$-starved cucumber roots, about 23% of phosphatase assayedat pH 9.0 (ALPase) in the crude cell walls was solubilized witheither 2 M NaCl or purified endo type polygalacturonase (endo-PG)from yeast culture broth. Coexistence of NaCl and endo-PG hadlittle effect on further release of ALPase, and a small amountof the activity was solubilized from the NaCl-pretreated cellwalls by incubation with endo-PG. Ionically bound ALPase, therefore,seemed to be localized in the fraction which was hydrolyzedby endo-PG in the crude cell walls of Ca^2$-starved cucumberroots. In the control roots, however, ALPase was not effectivelysolubilized by the treatment with endoPG. Ca^2$ starvation reducedthe contents of rhamnose, uronic acids and galactose among non-cellulosicsugars in the cell walls, suggesting that the structure of pecticsubstances, possibly rhamnogalacturonan, is altered during thestarvation. Activities of both ionically and covalently bound ALPases greatlyincreased during Ca^2$ starvation. The increased ALPase in theNaCl-solubilized fraction hydrolyzed most phosphate esters tested,whereas the enzyme from control roots only cleaved nucleoside2'(3')-monophosphates and p-nitrophenylphosphate. Differencesin the properties between both types of roots were also foundwhen the effects of various inhibitors were tested. Profilesof ALPase-isozymes after polyacrylamide gel electrophoresiswere also altered by Ca^2$ starvation. (Received June 2, 1982; Accepted July 20, 1982)     

Differential effect of Calcium and Magnesium on Mechanical Properties of Pea Stem Cell Walls

Replacement of calcium ion with magnesium ion in the cell wallof the pea epicotyl makes the wall more extensible. A possiblerole of this differential effect of Ca²⁺ and Mg^2– in regulatingcell elongation in pea epicotyl is discussed. The ratio of the content of calcium ion to that of magnesiumion (Ca²⁺/Mg²⁺) in the walls decreased markedly in the orderof the first > the second > the third internodes of thepea epicotyl. The capacity of the walls for cation exchangeincreased in the same order, whereas the calcium-magnesium ionexchange selectivity of the walls was virtually constant. Ourresults indicate that the changes in the Ca²⁺/Mg²⁺ ratio amongthe internodes is not attributable to the ion exchange propertiesof the walk per se, but is due to other physiological conditionswhich regulate the activities of free calcium and magnesiumions in the environment with which the walls are in equilibrium. ¹ Present address: Wakayama Research Laboratories, Kao SoapCo., Ltd., Wakayama 640-91, Japan ² Present address: Foold Development Laboratories, Meiji SeikaKaisha Ltd., Kawasaki 210, Japan (Received May 1, 1981; Accepted August 26, 1981)     

The Effect of Calcium Antagonists on Auxin-induced Elongation and on the Expression of Two Auxin-Regulated Genes in Pea Epicotyls

Calcium has been implicated in various regulatory roles in plantcells including auxin-induced cell elongation. Treatment ofpea epicotyl segments with the calcium chelators, EGTA and chlorotetracycline(CTC), the calcium ionophore, A23187 [GenBank] , and channel blocker, D-600,inhibits auxin-induced cell elongation. Depletion of tissuecalcium either by EGTA or EGTA and a calcium ionophore doesnot interfere with the induction of the early auxin induciblemRNAs pIAA4/5 and pIAA6. Similarly, an increase in cytosoliccalcium with calcium and calcium ionophore neither induces thehormonally regulated mRNAs nor interferes with their inductionby auxin. The calcium channel blocker, D-600, is without effecton the auxin-regulated mRNA induction. The results indicatethat calcium is not involved in the rapid induction of IAA4/5and IAA6 genes in pea tissue. However, a possible role for calciumin the translation of these mRNAs, or in the expression of otherauxin-regulated genes, is not excluded. ³Present Address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo, Japan. (Received April 8, 1988; Accepted July 30, 1988)     

Stimulation of Endomitotic DNA Synthesis and Cell Elongation by Gibberellic Acid in Epicotyls Grown from Gamma-irradiated Pea Seeds

Large doses of γ-irradiation, given to air-dried pea seeds, inhibit the endomitotic DNA synthesis in pea epicotyls during germination in darkness. The cortex cells of the etiolated epicotyls reach only the 4 C DNA level, whereas cortex cells of unirradiated seeds reach the 8 C DNA level. Epicotyl elongation and cell elongation are also reduced.     

Ethylene-forming Systems in Etiolated Pea Seedling and Apple Tissue

Auxin-induced ethylene formation in etiolated pea (Pisum sativum L. var. Alaska) stem segments was inhibited by inhibitors of RNA and protein synthesis. Kinetics of the inhibitions is described for actinomycin D, cordycepin, α-amanitin, and cycloheximide. α-Amanitin was the most potent and fast-acting inhibitor, when added before induction or 6 hours after induction of the ethylene-forming system. The ethylene-forming system of postclimacteric apple (Malus sylvestris L.) tissue, which is already massively induced, was not further stimulated by auxin. Ethylene production in apples was inhibited least by α-amanitin and most by actinomycin D. The relative responses of the ethylene system in apples to RNA inhibitors were different from the ethylene system of pea stems. However, the protein synthesis inhibitor, cycloheximide, appeared to act equally in both tissue systems. The effect of cycloheximide on ethylene production in postclimacteric apple tissue, already producing large quantities of ethylene, suggests a dynamic regulating system for the synthesis and degradation of the ethylene-forming system.     

小麦黄化胚芽鞘的细胞壁钙调素和钙调素结合蛋白

小麦黄化胚芽鞘经苯基琼脂糖亲和层析提取和纯化,其细胞壁CaM在有钙和缺钙时SDS电泳呈现不同的迁移率;依赖Ca~(2 )与苯基疏水结合;在紫外吸收光谱上具有五个特征峰;对PDE的激活剂量反应曲线和从非活性状态向活性状态转变时所需的Ca~(2 )浓度均和胞内CaM相同,说明细胞壁CaM和胞内CaM具有相同的基本理化特性。采用CaM琼脂糖亲和层析,发现在小麦细胞壁中存在CaM结合蛋白,其中以分子量为40.7 kD的 CaM结合多肽为主。细胞壁CaM结合蛋白不具有过氧化物酶、ATP酶或酸性磷酸酯酶的活性。     

pH-Dependent Interactions between Pea Cell Wall Polymers Possibly Involved in Wall Deposition and Growth

In an effort to detect a pH-dependent release of polymers such as xyloglucans, thought to be involved in auxin-induced cell wall expansion during growth, radioactively labeled cell walls from pea stem tissue were incubated at different pH values, and changes in water-soluble, ethanol- or trichloroacetic acid-insoluble components were determined. This revealed the occurrence, at neutral pH, of a time- and pH-dependent binding of soluble pectin, in the walls, to a heat-labile, presumably protein, wall component, yielding a trichloroacetic acid-insoluble pectin-protein complex. This reaction, which can also be observed between polymers in water extracts of cell walls, is inhibited at low pH and by Ca²⁺, and appears to be of a physical, possibly lectin-like, nature. Progressive binding of pectin or of the pectin-protein complex to the insoluble wall structure is also observed. These reactions may be involved in wall assembly during its deposition, and may participate in, or be analogous to pH-dependent physical interactions that participate in, wall extension during cell growth.     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH