Effect of Auxin on Autolysis of Cell Walls in Azuki Bean Epicotyls

Native cell walls of azuki bean epicotyls incubated in bufferautolytically released neutral sugars, abundant in galactose,and uronic acids. Treatment with 10^–5 M IAA of subapicalor basal epicotyl segments for 3 h did not influence the amountof total neutral sugars released from the cell walls duringautolysis. However, the amount of glucose and xylose releasedfrom subapical cell walls was increased by IAA. Pretreatmentwith IAA of subapical epicotyl segments enhanced the solubilizationof neutral sugars from pectinase-treated cell walls during incubationin buffer at pH 5 to 6. The amount of fucose, xylose, and glucosereleased was specifically increased by IAA. Of the sugar fractionsreleased from pectinase-treated cell walls during autolysisand subsequently separated by gel filtration on a ToyopearlHW-40S column, IAA promoted the release of oligosaccharides,consisting mainly of glucose and xylose. These results suggestthat autolytic degradation of xyloglucans is closely relatedto IAA-induced growth of azuki bean epicotyls. (Received May 19, 1989; Accepted January 5, 1990)     

Effect of Xyloglucan Nonasaccharide on Cell Elongation Induced by 2,4-Dichlorophenoxyacetic Acid and Indole-3-acetic Acid

Xyloglucan nonasaccharide (XG9) is recognized as an inhibitorof 2,4-D-induced long-term growth of segments of pea stems.In the presence of 10^–5 M 2,4-D, inhibition by 10^–9M XG9 of elongation of third internode segments of pea seedlingswas detected within 2 h after the start of incubation, in someexperiments. Analysis by double-reciprocal (Lineweaver-Burk)plots of elongation in the presence of various concentrationsof 2,4-D, with or without XG9, gave parallel lines, indicatingthat XG9 inhibited 2,4-D-induced elongation in an uncompetitivemanner. XG9 did not influence the 2,4-D-induced cell wall loosening.Thus, XG9 does not fulfill the proposed definition of an "antiauxin". XG9 at 10^–11 to 10^–6 M did not influence IAA-inducedelongation of segments from pea third internodes, azuki beanepicotyls, cucumber hypocotyls, or oat coleoptiles. Inhibitionof IAA-induced elongation by XG9 was not observed even whenthe segments from pea or azuki bean were abraded. Furthermore,fucosyl-lactose at 10^–11 to 10^–4 M did not affectthe IAA-induced elongation of segments of pea internodes orof azuki bean epicotyls. XG9 may be incapable of inhibitingthe IAA-induced cell elongation (especially in oat) or, alternatively,the endogenous levels of XG9 may be so high that exogenouslyapplied XG9 has no inhibitory effect on IAA-induced elongation. (Received February 28, 1991; Accepted May 25, 1991)     

Graviperception in growth inhibition of plant shoots under hypergravity conditions produced by centrifugation is independent of that in gravitropism and may involve mechanoreceptors

Hypergravity caused by centrifugation inhibits elongation growth of shoots by decreasing the cell wall extensibility via suppression of xyloglucan breakdown as well as by the thickening of cell walls. The mechanism of graviperception in hypergravity-induced growth inhibition was investigated in Arabidopsis [A. thaliana (L.) Heynh.] hypocotyls and azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. Hypergravity caused growth suppression in both sgr1-1 and pgm1, which are Arabidopsis mutants deprived of gravitropism, as in wild-type plants, suggesting that the graviperception in hypergravity-induced growth inhibition of shoots is independent of that in gravitropism. Hypergravity had no effects on growth of azuki bean epicotyls or Arabidopsis hypocotyls in the presence of lanthanum or gadolinium, which are blockers of mechanoreceptors. Moreover, lanthanum or gadolinium at the same concentration had no influence on gravitropism of azuki bean epicotyls and Arabidopsis hypocotyls. Hypergravity had no effects on cell wall extensibility and affected neither xyloglucan metabolism nor the thickness of cell walls in the lanthanum- or gadolinium-treated azuki bean epicotyls. Lanthanum or gadolinium inhibited the hypergravity-induced increase in the pH of the apoplastic fluid in the epicotyls, which is involved in the processes of the suppression of xyloglucan breakdown due to hypergravity. These findings suggest that plants perceive the hypergravity stimuli by mechanoreceptors in the plasma membrane, and utilize the perceived signal to regulate the growth rate of their shoots.Abbreviations HC-I Hemicellulose-I - HC-II Hemicellulose-II     

Growth and cell wall changes in azuki bean epicotyls II. Changes in wall polysaccharides during auxininduced growth of excised segments

Changes in cell wall polysaccharides and mechanical propertiesof the cell wall were examined during IAA-induced elongationgrowth of excised azuki bean epicotyl segments under differentgrowth conditions. Sucrose promoted IAA-induced cell elongation,but had very little effect on IAA-induced cell wall loosening.In the absence of sucrose, the amount of galactose in the cellwall decreased during the incubation period. IAA enhanced thedecrease in the galactose level. In the presence of sucrose,on the other hand, IAA induced increases in the amounts of cellulose,galactose and xylose in noncellulosic polysaccharides. TheseIAA-induced increases were not observed in the presence of mannitolat concentrations higher than 0.1 M, although cell wall looseningwas induced by IAA even in the presence of 0.2 M mannitol. (Received November 21, 1978; )     

Hypergravity induces reorientation of cortical microtubules and modifies growth anisotropy in azuki bean epicotyls

We examined the changes in the orientation of cortical microtubules during the hypergravity-induced modification of growth anisotropy (inhibition of elongation growth and promotion of lateral growth) in azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. The percentage of cells with transverse microtubules was decreased, while that with longitudinal microtubules was increased, in proportion to the logarithm of the magnitude of gravity. The percentage of cells with longitudinal microtubules showed an increase within 0.5 h of transfer of the 1g-grown seedlings to a 300g-hypergravity condition. Lanthanum and gadolinium, blockers of calcium channels, nullified the modification of growth anisotropy and reorientation of microtubules by hypergravity. Horizontal and acropetal hypergravity modified growth anisotropy and reorientation of microtubules, as did basipetal hypergravity, and these changes were not seen in the presence of lanthanum or gadolinium. These results suggest that hypergravity changes activities of lanthanum- and gadolinium-sensitive calcium channels independently of its direction, which may lead to reorientation of cortical microtubules and modification of growth anisotropy in azuki bean epicotyls.     

Effects of 2-Deoxygalactose on Auxin-Induced Growth and Levels of UDP-Sugars in Higher Plants

Galactose inhibited the IAA-induced elongation of cells in segmentsof oat coleoptiles but not in segments of azuki bean and cucumberstems. In contrast, its analogs, 2-deoxygalactose and 2-deoxyglucose,inhibited the IAA-induced elongation of cells in these planttissues. In segments of cucumber stems, 2-deoxygalactose inhibitedformation of cell-wall polysaccharides with little effect onrespiration in terms of either reduced consumption of O₂ orlevels of ATP. 2-Deoxygalactose and 2-deoxyglucose caused arapid decrease in the levels of UTP and UDP-sugars with a concomitantincrease in levels of UDP in cucumber segments. These resultssuggest that 2-deoxysugars inhibit synthesis of cell-wall polysaccharidesby decreasing the levels of UDP-sugars, thereby inhibiting IAA-inducedelongation of cells in cucumber segments. 2-Deoxysugars can,therefore, be used as potential inhibitors with which to assessthe role of UDP-sugars in the IAA-induced elongation of cellsin dicotyledonous plants. (Received September 27, 1990; Accepted February 5, 1991)     

Roles of auxin and gibberellic acid in growth and maturation of epicotyls of Vigna angularis: Cell wall changes

The effects of auxin and gibberellic acid on cell wall composition in various regions of epicotyls of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) were investigated with the following results. (1) Young segments excised from apical regions of the epicotyl elongated in response to added 10⁻⁴ M indole-3-acetic acid (IAA). When the segments were supplied with 50 m M sucrose, the IAA-induced segment growth was accompanied by enhanced overall synthesis of cell wall polysaccharides, such as xyloglucans, polyuronides and cellulose. This IAA effect on the cell wall synthesis is a consequence of extension growth induced by IAA. Gibberellic acid (GA) at 10⁻⁴ M synergistically enhanced the IAA-induced cell wall synthesis as well as IAA-induced extension growth, although GA by itself neither stimulated the cell wall synthesis nor extension growth. In the absence of sucrose, cell wall synthesis was not induced by IAA or GA. (2) In mature segments excised from basal regions of the epicotyl, no extension growth was induced by IAA or GA. GA enhanced the synthesis of xylans and cellulose when the segments were supplied with 50 m M sucrose. IAA had no effect on the cell wall synthesis. These findings indicate that synthesis of polyuronides, xyloglucans and cellulose, which occurs during extension growth of the apical region of the epicotyl, is regulated chiefly by auxin whereas synthesis of xylans and cellulose during cell maturation in the basal region of the epicotyl is regulated by GA.     

Effect of cycloheximide on IAA-induced proton excretion and IAA-induced growth in abraded Avena coleoptiles

IAA-induced proton excretion in peeled or abraded oat ( Avena saliva L. cv. Victory) coleoptiles is closely associated with IAA-induced growth. It was attempted to separate these two processes by using cycloheximide to inhibit them differentially. Growth of abraded coleoptile segments was measured by a shadow graphic method, and their IAA-induced acidification of the external solution was monitored with a pH meter. IAA stimulated proton excretion in abraded Avena coleoptile segments after a 13 min lag. IAA-induced proton excretion was inhibited within 5 min by cycloheximide at concentrations of 1.8 × 10⁻⁶, 3.6 × 10 or 3.6 × 10⁻⁵ M. Cycloheximide at these concentrations, added within 4 min of IAA, prevented IAA-induced acidification of the medium for at least 60 min. However, it did not prevent IAA-induced growth during this time. It is concluded that some of the initial IAA-induced growth seen in Avena coleoptiles is independent of detectable IAA-induced proton excretion.     

Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl₁Glc₁), xyloglucan heptasaccharides (Xyl₃Glc₄), and octasaccharides (Gal₁Xyl₃Glc₄). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.     

Effects of temperature on the cell wall and osmotic properties in dark-grown rice and azuki bean seedlings

The mechanism inducing the difference in growth rate under various temperature (10–50 °C) conditions was analyzed using rice and azuki bean seedlings. The growth rate of rice coleoptiles and azuki bean epicotyls increased as temperature increased up to 40 and 30 °C, respectively, and the elongation was retarded at a higher temperature. The cell wall extensibility of rice coleoptiles and azuki bean epicotyls also showed the highest value at 40 and 30 °C, respectively, and became smaller as the temperature rose or dropped from the optimum. The opposite tendency was observed in the minimum stress-relaxation time of the cell wall. On the other hand, the cellular osmotic concentration of rice coleoptiles and azuki bean epicotyls was lower at the temperature optimum for growth at 40 and 30 °C, respectively. When rice and azuki bean seedlings grown at 10, 20, 40, or 50 °C were transferred to the initial temperature (30 °C), the growth rate of coleoptiles and epicotyls was mostly elevated, concomitant with an increase in the cell wall extensibility. The growth rate was correlated with the cell wall mechanical parameters in both materials. These results suggest that the environmental temperature modulates the growth rate of plant shoots by affecting mainly the mechanical properties of the cell wall. Electronic Publication     

Involvement of wall microtubules in gibberllin promotion and kinetin inhibition of stem elongation

The elongation of light-grown azuki bean (Azukia angularis =Vigna angularis) epicotyl segments was promoted by indoleaceticacid (IAA) and this IAA-induced elongation was inhibited byboth kinetin and benzimidazole (BIA). Increased stem thickeningwas observed with kinetin- or BIA-treated segments, but thiswas not accompanied by incresed cell number in the transversedirection, suggesting that both kinetin and BIA promoted lateralcell expansion. Colchicine at a concentration with no effecton IAA-induced elongation reversed both the kinetic- and BIA-inducedinhibition. Electron-microscopic examination revealed that wall microtubulesin cells treated with kinetin together with IAA ran parallelto the cell axis, while wall microtubules in cells treated withonly IAA were randomly oriented. In the cell treated with gibberellintogether with IAA, wall microtubules ran tranverse to the cellaxis. (Received July 13, 1973; )     

Gibberellin A3 Causes a Decrease in the Accumulation of mRNA for ACC Oxidase and in the Activity of the Enzyme in Azuki Bean (Vigna angularis) Epicotyls

Differential screening, aimed at the isolation of cDNA clonesof mRNAs whose accumulation is influenced by GA₃, resulted inthe isolation of a cDNA clone of an mRNA whose level was decreasedby GA₃ in segments of epicotyls of Vigna angularis. The putativeprotein encoded by this cDNA resembled the 1-aminocyclopropane-l-carbox-ylateoxidases (ACC oxidases) identified in other plant species (about80% homology at the amino acid level). Thus, the correspondinggene was designated AB-ACO1 (azuki bean ACC oxidase). GA₃ alsodecreased the activity of ACC oxidase in azuki bean epicotyls,but it did not decrease the rate of ethylene evolution. In fact,GA₃ increased the rate of ethylene evolution and the level ofACC. Thus, GA₃ seemed to increase the production of ethyleneby promoting the synthesis of ACC. (Received January 10, 1997; Accepted July 31, 1997)     

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.     

Increase in the gibberellin response by low temperature pretreatment of azuki bean epicotyls

Epicotyl segments cut from azuki bean (Vigna angularis) seedlings grown at 27 C for 5 days and then treated at 15 C for 2 days were compared with those cut from seedlings not receiving the 15-C treatment, for their response to auxin and auxin plus gibberellin. The segments cut from 15C-treated seedlings showed greater elongation than the segments cut from untreated seedlings both in the absence and in the presence of gibberellin. The 15-C treatment increased the elongation caused by auxin plus gibberellin to a greater extent than it did the elongation caused by auxin alone; the gibberellin response of epicotyl was increased by the treatment. Diffusates from leaves and buds inhibited both the elongation caused by auxin and that caused by auxin plus gibberellin. The diffusates inhibited the latter more strongly than the former; the gibberellin response was decreased by the diffusates. Leaves or buds appear to supply epicotyls with a substance which decreases the gibberellin response. The supply of this substance was found to be temperature dependent. The diffusates obtained at 15 C caused no inhibition, while those obtained at 27 C decreased the gibberellin response. The lack of the supply of this substance at 15 C may account for the increase in the gibberellin response by the 15-C treatment.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.     

Occurrence of osmiophilic particles is correlated to elongation growth of higher plants

Summary The occurrence of elongation growth-related osmiophilic particles (OPs) was investigated in hypocotyls of sunflower, bean, and spruce as well as in pea epicotyls and in cress roots of intact seedlings. In all analyzed species, OPs were found to occur specifically within the periplasmic space between plasma membrane and the outer epidermal cell walls of elongating parts of hypocotyls, epicotyls, and roots, whereas cells of nonelongating parts were devoid of OPs. Auxin (IAA) markedly increased the number of OPs in epicotyl and hypocotyl segments. Treatment of pea epicotyl segments with the lectin concanavalin A inhibited their elongation growth in the presence of IAA. At a subcellular level this effect was characterized by the occurrence of a pronounced osmiophilic layer in the periplasmic space of the outer periclinal and the outer part of the anticlinal epidermal cell walls. Treatment of IAA-incubated segments with the secretion inhibitor brefeldin A inhibited both elongation growth and periplasmic occurrence of OPs. This effect was accompanied by complementary accumulation of OPs in the peripheral cytoplasm of epidermal cells. Together the results indicate that IAA-induced epidermis-specific secretion of OPs is closely related to cell elongation growth not only in organs of monocotyledonous species, but also in dicotyledonous angiosperms as well as in gymnosperms.Abbreviations OPs osmiophilic particles - ConA concanavalin A - BFA brefeldin A - IAA -indolyl acetic acid     

Action of xyloglucan hydrolase within the native cell wall architecture and its effect on cell wall extensibility in azuki bean epicotyls

Xyloglucan hydrolase (XGH) has recently been purified from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls as a new type of xyloglucan-degrading enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154]. In the present study, the effects of XGH on the mechanical properties of the cell wall and on the level and the molecular size of xyloglucans within the native wall architecture were examined in azuki bean epicotyls. When the epidermal tissue strips from the growing regions of azuki bean epicotyls were incubated with XGH, the mechanical extensibility of the cell wall dramatically increased. XGH exogenously applied to cell wall materials (homogenates) or epidermal tissue strips decreased the amount of xyloglucans via the solubilization of the polysaccharides. Also, XGH substantially decreased the molecular mass of xyloglucans in both materials. These results indicate that XGH is capable of hydrolyzing xyloglucans within the native cell wall architecture and thereby increasing the cell wall extensibility in azuki bean epicotyls.     

Role of chloride ions in the promotion of auxin-induced growth of maize coleoptile segments

Background and Aims

The mechanism of auxin action on ion transport in growing cells has not been determined in detail. In particular, little is known about the role of chloride in the auxin-induced growth of coleoptile cells. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were carried out with maize (Zea mays) coleoptile segments, this being a classical model system for studies of plant cell elongation growth.

Methods

Growth kinetics or growth and pH changes were recorded in maize coleoptiles using two independent measuring systems. The growth rate of the segments was measured simultaneously with medium pH changes. Membrane potential changes in parenchymal cells of the segments were also determined for chosen variants. The question of whether anion transport is involved in auxin-induced growth of maize coleoptile segments was primarily studied using anion channel blockers [anthracene-9-carboxylic acid (A-9-C) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS)]. In addition, experiments in which KCl was replaced by KNO₃ were also performed.

Key Results

Both anion channel blockers, added at 0·1 mm, diminished indole-3-acetic acid (IAA)-induced elongation growth by ∼30 %. Medium pH changes measured simultaneously with growth indicated that while DIDS stopped IAA-induced proton extrusion, A-9-C diminished it by only 50 %. Addition of A-9-C to medium containing 1 mm KCl did not affect the characteristic kinetics of IAA-induced membrane potential changes, while in the presence of 10 mm KCl the channel blocker stopped IAA-induced membrane hyperpolarization. Replacement of KCl with KNO₃ significantly decreased IAA-induced growth and inhibited proton extrusion. In contrast to the KCl concentration, the concentration of KNO₃ did not affect the growth-stimulatory effect of IAA. For comparison, the effects of the cation channel blocker tetraethylammonium chloride (TEA-Cl) on IAA-induced growth and proton extrusion were also determined. TEA-Cl, added 1 h before IAA, caused reduction of growth by 49·9 % and inhibition of proton extrusion.

Conclusions

These results suggest that Cl^– plays a role in the IAA-induced growth of maize coleoptile segments. A possible mechanism for Cl^– uptake during IAA-induced growth is proposed in which uptake of K⁺ and Cl^– ions in concert with IAA-induced plasma membrane H⁺-ATPase activity changes the membrane potential to a value needed for turgor adjustment during the growth of maize coleoptile cells.     

Changes in the apoplastic pH are involved in regulation of xyloglucan breakdown of azuki bean epicotyls under hypergravity conditions

Hypergravity inhibited elongation growth of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls by decreasing the mechanical extensibility of cell walls via the increase in the molecular mass of xyloglucans [Soga et al. (1999) Plant Cell Physiol. 40: 581]. Here, we report that the pH value of the apoplastic fluid in epicotyls increased from 5.8 to 6.6 by hypergravity (300 x g) treatment. When the xyloglucan-degrading enzymes extracted from cell walls of the 1 x g control epicotyls were assayed in buffer at pH 6.6 and 5.8, the activity at pH 6.6 was almost half of that at pH 5.8. In addition, when enzymically active cell wall preparations obtained from 1 x g control epicotyls were autolyzed in buffer at pH 5.8 and 6.6 and then xyloglucans were extracted from the autolyzed cell walls, the molecular mass of xyloglucans incubated at pH 5.8 decreased during the autolysis, while that at pH 6.6 did not change. Thus, the xyloglucans were not depolymerized by autolysis at the pH value (6.6) observed in the hypergravity-treated epicotyls. These findings suggest that in azuki bean epicotyls, hypergravity decreases the activities of xyloglucan-degrading enzymes by increasing the pH in the apoplastic fluid, which may be involved in the processes of the increase in the molecular mass of xyloglucans, leading to the decrease in the cell wall extensibility.