Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )     

A novel {beta}-galactosidase gene isolated from the bacterium Xanthomonas manihotis exhibits strong homology to several eukaryotic {beta}-galactosidases

The gene encoding a ß-galactosidase from Xanthomonasmanihotis was cloned into Escherichia coli. The gene resideson a 2.4 kb DNA fragment which was isolated from a partial Sau3Alibrary in the cloning vector pUC19 using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as the selection. The enzyme produced by the clone hasa specificity for ß1-3->ß1-4-linked galactose.The nucleotide sequence of the gene was determined. The deducedprotein sequence contained 597 amino acids yielding a monomericmolecular mass of 66 kDa. The cloned ß-galactosidaseshowed no similarity to any known prokaryotic ß-galactosidase.However, extensive similarity was observed with eukaryotic ß-galactosidasesfrom animals, plants and fungi. The strongest similarity waswith the ß-galactosidases found hi the human and mouselysosomes (42 and 41% identity, respectively). Alignment ofthe X.manihotis and eukaryotic ß-galactosidase sequencesrevealed seven highly conserved domains common to each protein.Additionally, Domain 1 in X.manihotis showed similarity to regionswithin catalytic domains from seven xylanases and cellulasesbelonging to family 10 of glucosyl hydrolases. A region spanningDomain 2 showed similarity to the catalytic domain of endo ß1-3glucanases from tobacco and barley. cellulase ß-galactosidase G_M$$$gangliosidosis Morquio B syndrome Xanthomonas     

Changes in composition of the outer epidermal cell wall of pea stems during auxin-induced growth

The gross composition of the outer epidermal cell wall from third internodes of Pisum sativum L. cv. Alaska grown in dim red light, and the effect of auxin on that composition, was investigated using interference microscopy. Pea outer epidermal walls contain as much cellulose as typical secondary walls, but the proportion of pectin to hemicellulose resembles that found in primary walls. The pectin and hemicellulose fractions from epidermal peels, which are enriched for outer epidermal wall but contain internal tissue as well, are composed of a much higher percentage of glucose and glucose-related sugars than has been found previously for pea primary walls, similar to non-cellulosic carbohydrate fractions of secondary walls. The epidermal outer wall thus has a composition rather like that of secondary walls, while still being capable of elongation. Auxin induces a massive breakdown of hemicellulose in the outer epidermal wall; nearly half the hemicellulose present is lost during 4 h of growth in the absence of exogenous sugar. The percentage breakdown is much greater than has been seen previously for whole pea stems. It has been proposed that a breakdown of xyloglucan could be the basis for the mechanical loosening of the outer wall. This study provides the first evidence that such a breakdown could be occurring in the outer wall.M.S. Bret-Harte would like to thank Dr. Peter M. Ray, of Stanford University, for helpful discussions and for technical and editorial assistance, Dr. Winslow R. Briggs, of the Camegie Institude of Washington, for the use of experimental facilities and for helpful discussions, Dr. Wendy K. Silk, of the University of California, Davis, for helpful discussions and financial support, Dr. Paul B. Green for financial support, and Drs. John M. Labavitch and L.C. Greve, of the University of California, Davis, for performing the -cellulose analysis on short notice, in response to a request by an anonymous reviewer. This work was supported by a National Science Foundation Graduate Fellowship to M.S. B.-H., National Science Foundation Grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk (Department of Land, Air, and Water Resources, University of California, Davis) during the final writing.     

Production and secretion of {alpha}-galactosidase and endo-{beta}-mannanase by carob (Ceratonia siliqua L.) endosperm protoplasts

Viable protoplasts were isolated for the first time from maturecarob (Ceratonia siliqua L.) endosperm tissue. After 5 d ofincubation 75% of the protoplasts were viable. During incubationthey underwent vacuolation and produced the carob endospermhydrolases, agalactosidase and endo-ß-mannanase, whichwere secreted in the incubation medium. The secretion of bothenzymes were under Ca²⁺ control. Many characteristics of -galactosidaseand endo-ß-mannanase production by protoplasts werethe same as those of whole endosperms: their production didnot require any hormonal signal and was inhibited in the presenceof ABA or the leachate from the carob endosperm/seed coat. Moderatewater stress (—2.0 MPa) neither affected the activityof these hydrolases nor their secretion by endosperm protoplast.However, when the osmoticum of protoplast incubation mediumwas higher, the production and secretion of both hydrolaseswere reduced. Comparison of the hydrolases activities in theincubation media of leached carob endosperms, which were incubatedunder normal and water stress (—1.5 MPa) conditions, withthe activities of the protoplast-secreted hydrolases indicatedthat (i) carob endosperm cell wall acts as a barrier for thesecreted enzymes and (ii) that water stress reduces the cellwall porosity of the carob endosperm cells, and thus the releaseof the secreted -galactosidase and endo-ß-mannanaseis inhibited. The isolation of carob endosperm protoplasts offersa potent experimental system for the study of aspects of endospermcell physiology, such as enzyme secretion Key words: Abscisic acid, carob endosperm, Ceratonia siliqua L, endo-ß-mannanase, -galactosidase, leachate, protoplasts, water stress     

The effect of nojirimycin on plant growth and its implications concerning a role for exo-{beta}-glucanases in auxin-induced cell expansion

Nojirimycin (5-amino 5-deoxy-D-glucopyranose), at concentrationsof 0.1 to 3.0 nM, is a potent inhibitor of IAA-induced growthof excised Avena coleoptile and pea stem segments. Both therapid initial responses to IAA as well as sustained growth areaffected, however growth of sections not treated with IAA isrelatively unperturbed for incubation periods up to 9 hr inthe presence of the inhibitor. There is no evidence for competitiveeffects involving nojirimycin and IAA, as increasing the auxinconcentration does not reverse the inhibition. Analysis of cellwall components of sections treated with nojirimycin shows thata parallel relationship exists between the extent to which glucoseis removed from the noncellulosic polysaccharides and the amountof tissue growth. Since nojirimycin is an inhibitor of exo-ß-glucanases,these results implicate a role for this enzyme in IAA-inducedmodification of noncellulosic wall glucan and a requirementfor such enzymes in IAA-induced cell extension. (Received December 27, 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; )     

Regulation of ethylene biosynthesis after short-term heat treatment in etiolated pea stems

Incubation of plant tissues at a constant elevated temperature greatly inhibits both basal and wound ethylene production. However, recovery from heat treatment is relatively rapid and is followed by stimulated ethylene production. The present investigation examines the kinetics of ethylene production after short-term heal treatment and the regulation of heat-altered ethylene production. Subapical stem segments of 7-day-old etiolated pea L. cv. Alaska) seedlings were analyzed for ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) oxidation, and ACC and l-(malonylamino)cyclopropane-l-carboxylic acid (MACC) content after a 2-min 40°C heat pulse. The short-term heat pulse transiently inhibited ethylene production and ACC oxidation accompanied by a slight ACC accumulation within a 30-min time period. Conjugation to MACC did not appear to play an integral role in heat-regulated ethylene production. It was concluded that the major factor affecting ethylene production after heat treatment is the temporary inactivation of ACC oxidation. The possible roles of ACC synthase, ACC oxidase and lipoxygenase in regulating ethylene production after heat treatment are discussed.     

Ethylene-induced lateral expansion in etiolated pea stems : kinetics, cell wall synthesis, and osmotic potential

Treatment of etiolated pea (Pisum sativum L.) internode tissue with ethylene gas inhibits elongation and induces lateral expansion. Precise kinetics of the induction of this altered mode of growth of excised internode segments were recorded using a double laser optical monitoring device. Inhibition of elongation and promotion of lateral expansion began after about 1 hour of treatment and achieved a maximum by 3 hours. Similar induction kinetics were observed after treating internodes with colchicine and 2,6-dichlorobenzonitrile, an inhibitor of cellulose synthesis. In sealed flask experiments, ethylene had no detectable effect on incorporation of label from [¹⁴C]glucose into any of the classical pectin, hemicellulose, or cellulose wall fractions. Ethylene inhibited fresh weight increase (total cell expansion) of both excised internode segments (in sealed flasks) and intact seedlings. Ethylene treatment resulted in an increase in cell sap osmolality in those tissues (intact and excised) which are inhibited by the gas. A model for ethylene-induced inhibition of elongation and induction of lateral expansion is presented.     

Ethylene-induced lateral expansion in etiolated pea stems : the role of Acid secretion

Ethylene-induced inhibition of elongation and promotion of lateral expansion in the stems of etiolated pea (Pisum sativum L. var Alaska) seedlings is not associated with any alteration of auxin-stimulated proton extrusion. Indeed, lateral expansion in response to ethylene apparently requires an acidified wall since it is prevented by strong neutral buffers and by the ATPase inhibitor orthovanadate. Ethylene treatment reduces the capacity of live and frozen-thawed sections to extend in the longitudinal direction in response to acid. The effect of ethylene on lateral acid growth capacity is more complicated. Ethylene-treated internodes do not exhibit acid-induced lateral expansion. Ethylene-treated segments which have been frozen-thawed do show an enhanced capacity to extend in the transverse direction at acid pH, but only when the inner tissues have been removed by coring. We conclude that two of the factors which control the directionality of expansion during ethylene treatment are a decrease in the sensitivity of the walls to acid longitudinally and an increase in the sensitivity of the outer cortical parenchyma walls to acid in the transverse direction.     

Transforming growth factor {beta} (TGF-{beta})-Smad target gene protein tyrosine phosphatase receptor type kappa is required for TGF-{beta} function

Transforming growth factor beta (TGF-beta) inhibits proliferation and promotes cell migration. In TGF-beta-treated MCF10A mammary epithelial cells overexpressing HER2 and by chromatin immunoprecipitation, we identified novel Smad targets including protein tyrosine phosphatase receptor type kappa (PTPRK). TGF-beta up-regulated PTPRK mRNA and RPTPkappa (receptor type protein tyrosine phosphatase kappa, the protein product encoded by the PTPRK gene) protein in tumor and nontumor mammary cells; HER2 overexpression down-regulated its expression. RNA interference (RNAi) of PTPRK accelerated cell cycle progression, enhanced response to epidermal growth factor (EGF), and abrogated TGF-beta-mediated antimitogenesis. Endogenous RPTPkappa associated with EGF receptor and HER2, resulting in suppression of basal and ErbB ligand-induced proliferation and receptor phosphorylation. In MCF10A/HER2 cells, TGF-beta enhanced cell motility, FAK phosphorylation, F-actin assembly, and focal adhesion formation and inhibited RhoA activity. These responses were abolished when RPTPkappa was eliminated by RNA interference (RNAi). In cells expressing RPTPkappa RNAi, phosphorylation of Src at Tyr527 was increased and (activating) phosphorylation of Src at Tyr416 was reduced. These data suggest that (i) RPTPkappa positively regulates Src; (ii) HER2 signaling and TGF-beta-induced RPTPkappa converge at Src, providing an adequate input for activation of FAK and increased cell motility and adhesion; and (iii) RPTPkappa is required for both the antiproliferative and the promigratory effects of TGF-beta.     

Orientation of cell growth in the etiolated pea stem

Summary Both ethylene and IAA induce swelling in the sub-apical region of etiolated pea plants. The modified cells of these two types of swellings differ both morphologically and in their enzyme composition. In ethylene the cell walls become thickened within 24 h and the level of peroxidase is enhanced; ethylene does not affect cellulase levels. IAA induced swellings are not accompanied by early thickening of cell walls or enhanced peroxidase activity, but IAA greatly increases the level of cellulase. It is proposed that the retardation of extension growth by ethylene treatment results from the deposition of longitudinal microfibrils in the walls and that cross linking bonds in the polysaccharide matrix prevent their separation. Lateral expansion can occur, however, in the presence of auxin-induced cellulase which breaks or prevents the formation of these bonds.     

Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments

Branca, C, De Lorenzo, G. and Cervone, F. 1988. Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments. - Physiol. Plant. 72: 499–504.
α-D-galacturonide oligomers (OG) were prepared by partial hydrolysis of sodium polypectate with an homogeneous Aspergillus niger endopolygalacturonase (EC 3.2.1.15). OG, obtained after digestion for 10, 20, 30, 60, 120 min and 24 h, were assayed for their ability to interfere with the IAA-induced elongation of pea ( Pisum sativum L. cv. Alaska) stems. Maximum inhibiting activity was exhibited by oligomers with an approximate degree of polymerization higher than 8. Inhibition by longer OG was much lower, and the products of the 24 h digestion and the unhydrolysed polypectate were ineffective. The addition of OG to pea stems caused a parallel shift to the right of the IAA dose-effect curve. The shift depended on the amount of OG used, showing that oligogalacturonides behave as competitive antagonists of IAA. The presence of OG caused the disappearance of the second maximum of the elongation rate and reduced the first maximum. OG were also tested for their ability to inhibit IAA-induced ethylene evolution of pea stem segments. Maximal inhibition was obtained with OG of the same size as those that interfered with IAA-induced elongation. Inhibition of the auxin action seemed to be specific as OG did not interfere with the activity of gibberellic acid (GA₃) or kinetin. It was concluded that oligogalacturonides strongly interfere with the activity of IAA, although they are by themselves incapable to influence the elongation of pea stem segments directly.     

Inhibition of cell wall autolysis and auxin-induced elongation of Cicer arietinum epicotyls by β-galactosidase antibodies

Polyclonal antibodies were raised in response to βIII-galactosidase purified from cell wall of Cicer arietinum epicotyls. The antibody preparation generated, bound to βIII protein giving a major protein band in the zone corresponding to M_r 45 000, the molecular mass previously estimated for βIII-galactosidase. These antibodies clearly suppress autolytic reactions in isolated walls of Cicer arietinum epicotyl segments, while the preimmune serum had no effect on autolytic reaction. The results strongly support the idea that the autolytic degradation of the cell wall is carried out by the βIII-galactosidase.
The antibodies against β-galactosidase were also able to inhibit cell wall hydrolysis mediated by both total cell wall protein extracted by LiCl and cell wall hydrolysis mediated by βIII-galactosidase.
Since autolysis is thought to be related to the process of cell wall loosening, the effects of the antibodies against the autolytic enzyme was also tested on epicotyl growth. β-galactosidase antibodies consistently inhibited IAA-induced elongation.     

Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl

The occurrence and chemical nature of the cross-links between cellulose microfibrils in outer epidermal cell walls in Pisum sativum cv. Alaska was investigated by rapid-freezing and deep-etching techniques coupled with chemical and enzymatic treatments. The cell wall in the elongating region of epidermal cells was characterized by the absence of the cross-links, while in the elongated region, the cell wall was characterized by the presence of cross-links. The cross-links remained in the cell wall of the elongated region after treatment with SDS electrophoresis sample buffer and treatment with 4% potassium hydroxide. After treatment with endo-1,4-beta-glucanase, which fragments xyloglucan, the cross-links were remarkably reduced from the cell wall of the elongated region. The endoglucanase treatment also reduced immunogold labeling of xyloglucan in the cell wall. The endoglucanase hydrolysate from the cell wall fraction of the elongated region gave spots of oligosaccharides in thin layer chromatography, which were identical to the spots of xyloglucan oligosaccharides produced by xyloglucanase from both the cell wall fraction and tamarind xyloglucan. These results indicate that the cross-links are made of xyloglucan. We discussed the possibility of cross-links involved in the control of mechanical properties of the cell wall.     

Smad4 dependency defines two classes of transforming growth factor {beta} (TGF-{beta}) target genes and distinguishes TGF-{beta}-induced epithelial-mesenchymal transition from its antiproliferative and migratory responses

In response to transforming growth factor beta (TGF-beta), Smad4 forms complexes with activated Smad2 and Smad3, which accumulate in the nucleus, where they both positively and negatively regulate TGF-beta target genes. Mutation or deletion of Smad4 is found in about 50% of pancreatic tumors and in about 15% of colorectal tumors. As Smad4 is a central component of the TGF-beta/Smad pathway, we have determined whether Smad4 is absolutely required for all TGF-beta responses, to evaluate the effect of its loss during human tumor development. We have generated cell lines from the immortalized human keratinocyte cell line HaCaT or the pancreatic tumor cell line Colo-357, which stably express a tetracyline-inducible small interfering RNA targeted against Smad4. In response to tetracycline, Smad4 expression is effectively silenced. Large-scale microarray analysis identifies two populations of TGF-beta target genes that are distinguished by their dependency on Smad4. Some genes absolutely require Smad4 for their regulation, while others do not. Functional analysis also indicates a differential Smad4 requirement for TGF-beta-induced functions; TGF-beta-induced cell cycle arrest and migration, but not epithelial-mesenchymal transition, are abolished after silencing of Smad4. Altogether our results suggest that loss of Smad4 might promote TGF-beta-mediated tumorigenesis by abolishing tumor-suppressive functions of TGF-beta while maintaining some tumor-promoting TGF-beta responses.     

Vascular endothelial growth factor A (VEGF-A) induces endothelial and cancer cell migration through direct binding to integrin {alpha}9{beta}1: identification of a specific {alpha}9{beta}1 binding site

Integrin α9β1 mediates accelerated cell adhesion and migration through interactions with a number of diverse extracellular ligands. We have shown previously that it directly binds the vascular endothelial growth factors (VEGF) A, C, and D and contributes to VEGF-induced angiogenesis and lymphangiogenesis. Until now, the α9β1 binding site in VEGF has not been identified. Here, we report that the three-amino acid sequence, EYP, encoded by exon 3 of VEGF-A is essential for binding of VEGF to integrin α9β1 and induces adhesion and migration of endothelial and cancer cells. EYP is specific for α9β1 binding and neither requires nor activates VEGFR-2, the cognate receptor for VEGF-A. Following binding to EYP, integrin α9β1 transduces cell migration through direct activation of the integrin signaling intermediates Src and focal adhesion kinase. This interaction is biologically important because it mediates in vitro endothelial cell tube formation, wound healing, and cancer cell invasion. These novel findings identify EYP as a potential site for directed pharmacotherapy.