Effect of indole-3-acetic acid on cell wall loosening: Changes in mechanical properties and noncellulosic glucose content of Avena coleoptile cell wall

The effect of indole-3-acetic acid on cell wall loosening andchemical modifications of noncellulosic components of the cellwall in Avena coleoptile segments was studied and the followingresults were obtained. (1) Auxin decreased both the minimum stress-relaxation time(T_o) and the noncellulosic glucose content of the cell wall. (2) Decreases were observed in the absence or presence of mannitolsolution at concentrations lower than 0.20 M which osmoticallysuppressed auxin-induced extension, while at concentrationshigher than 0.25 M, there was little auxin effect, indicatingthat it is turgor-dependent. (3) The decrease in T_o of the cell wall and that in the noncellulosicglucose content caused by auxin in the presence of mannitolsolutions of various concentrations paralleled each other (thecorrelation coefficient was 0.897). (4) Both decreases in T_o and glucose content caused by auxinwere inhibited by nojirimycin (5-amino-5-deoxy-D-glucopyranose)in the presence of mannitol. The results suggest that auxin-induced cell wall loosening iscaused by the degradation of noncellulosic rß-glucanin the cell wall. (Received December 24, 1976; )     

Changes in biochemical composition of the cell wall of the cotton fiber during development

The composition of the cell wall of the cotton fiber (Gossypium hirsutum L. Acala SJ-1) has been studied from the early stages of elongation (5 days postanthesis) through the period of secondary wall formation, using cell walls derived both from fibers developing on the plant and from fibers obtained from excised, cultured ovules. The cell wall of the elongating cotton fiber was shown to be a dynamic structure. Expressed as a weight per cent of the total cell wall, cellulose, neutral sugars (rhamnose, fucose, arabinose, mannose, galactose, and noncellulosic glucose), uronic acids, and total protein undergo marked changes in content during the elongation period. As a way of analyzing absolute changes in the walls with time, data have also been expressed as grams component per millimeter of fiber length. Expressed in this way for plant-grown fibers, the data show that the thickness of the cell wall is relatively constant until about 12 days postanthesis; after this time it markedly increases until secondary wall cellulose deposition is completed. Between 12 and 16 days postanthesis increases in all components contribute to total wall increase per millimeter fiber length. The deposition of secondary wall cellulose begins at about 16 days postanthesis (at least 5 days prior to the cessation of elongation) and continues until about 32 days postanthesis. At the time of the onset of secondary wall cellulose deposition, a sharp decline in protein and uronic acid content occurs. The content of some of the individual neutral sugars changes during development, the most prominent change being a large increase in noncellulosic glucose which occurs just prior to the onset of secondary wall cellulose deposition. Methylation analyses indicate that this glucose, at least in part, is 3-linked. In contrast to the neutral sugars, no significant changes in cell wall amino acid composition are observed during fiber development.     

Turgor-dependent Changes in Avena Coleoptile Cell Wall Composition

The effects of reduced turgor pressure on growth, as measured by cell elongation, and on auxin-mediated changes in cell walls, as measured by analyses of wall composition, were examined using Avena coleoptile segments. Although moderate (1-4 bar) decreases in turgor resulted in a progressive decline in growth proportional to the decrease in turgor, the major auxin-induced change in wall composition, a decrease in noncellulosic wall glucose, was unaffected. Severe (5-8 bar) decreases, however, did inhibit this auxin effect on the wall, and with turgor decreases of 9 bars or more this auxin effect was no longer apparent. The results show that turgor pressure is required for this auxin-mediated wall modification and also that this modification of wall glucose occurs at turgor pressures less than those required for wall extension. Changes in other wall components were generally unaffected by altering turgor pressure.     

Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa)

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.     

Growth and cell wall changes in rice coleoptiles growing under different conditions I. Changes in turgor pressure and cell wall polysaccharides during intact growth

The effect of the oxygen supply on growth, water absorptionof cells and cell wall changes was studied in coleoptiles ofrice seedlings growing under three different conditions: underwater, under water with constant air bubbling and in air. Coleoptilegrowth was larger when they were grown under water than in waterwith air bubbling and in air. Coleoptile growth under waterwas limited by the suction force of their cells rather thanby mechanical properties of the cell wall, while that of thecoleoptiles growing under the other two conditions was limitedby the cell wall rigidity. A decrease in the relative amountof noncellulosic glucose of the cell wall, and an increase inthe noncellulosic xylose during coleoptile growth were foundfor all three culture conditions. ¹ Present address: Departamento de Fisiologia Vegetal, Facultadde Ciencias, Universidad, Salamanca, Spain. (Received May 21, 1979; )     

Partial chemical characterization of corn root cell walls

The present study reports on chemical changes which occur in the cell wall of Zea mays during early phases of growth. Roots of seedling corn plants were divided into a meristematic zone, the zone of elongation, and the maturation zone, and the cell wall isolated from each of these zones. The wall preparations were then extracted sequentially to obtain pectin, hemicellulose, cellulose, and lignin fractions. Each of these, except for the lignin fraction, was hydrolyzed and the resultant sugars isolated, identified, and estimated quantitatively. Quantitative analysis of the products of hydrolysis of these fractions demonstrated that the classical scheme of fractionation is a valuable indicator of the changes in solubility properties which the various polysaccharide components for the wall undergo. It does not however yield definite chemical entities. For example, the “pectin” fraction contains only about 3% galacturonic acid; the bulk of it being composed of glucose, xylose, and galactose. By summation of analysis of these various fractions, it was found that substances yielding glucose and xylose upon hydrolysis increase with advancing age of the tissue. Galactose- and arabinose-yielding compounds decrease and mannose appears during maturation. Anhydrouronic acids first decrease, then increase. Most interestingly, of the total dry weight of the cell wall, only 24, 45, and 50% of the meristematic, elongation, and maturation zones respectively are accounted for as simple sugars in the acid hydrolysates. Oligosaccharides were not encountered in large amounts so that the 50 to 75% of the wall weight unaccounted for would consist of polysaccharides or oligosaccharides not precipitated by ethanol from the extracting solutions employed and by polysaccharides in the hemicellulose fraction which are resistant to acid hydrolysis.     

Phosphorylated and nucleotide sugar metabolism in relation to cell wall production in Avena coleoptiles treated with fluroride and peroxyacetyl nitrate

Coleoptile sections of Avena sativa L. were pretreated with sodium fluoride or peroxyacetyl nitrate at levels which inhibit auxin-induced growth but did not affect glucose uptake or CO₂ production when postincubated for 30 minutes in a ¹⁴C-glucose medium without auxin. Labeling of metabolites involved in cell wall synthesis was measured. Peroxyacetyl nitrate decreased labeling, and it was concluded that the pool size of uridine di-phosphoglucose, sucrose, and cell wall polysaccharides decreased compared to control. The changes suggest that peroxyacetyl nitrate inactivated sucrose and cell wall synthesizing enzymes including cellulose synthetase and decreased cell growth by inhibiting production of cell wall constituents. Fluoride treatment had no effect on production of cell wall polysaccharides, with or without indoleacetic acid stimulation of growth. The only change after fluoride treatment was a decrease in uridine diphosphoglucose during incubation without indoleacetic acid, a decrease that disappeared when indoleacetic acid was present. It was concluded that some other aspect of cell wall metabolism, not determined here, was involved in fluoride-induced inhibition of growth.     

Effect of 2,4-D on Cell Wall Metabolism of Peroxyacetyl Nitrate Pretreated Avena Coleoptile Sections

Avena coleoptile sections were exposed to nonlethal concentrations of peroxyacetyl nitrate (PAN). The sections were then incubated in solutions of 50 mM glucose plus 2.5 mM poassium phosphate with various concentrations of 2,4-dichlorophenoxycetic acid (2,4-D). Growth after 4 hours was measured. A corresponding series of experiments was carried out with glucose-¹⁴C (U) in the subsequent incubation medium and the effect of the 2,4-D treatments on ¹⁴C incorporation into various cell wall components was determined. Growth in the PAN-treated sections, although still partially inhibited, was greater at auxin levels normally superoptimal for growth than at the former optimum. Incorporation into all cell wall fractions was similar to growth in the case of control treated tissue. Most of the cell wall constituents, but particularly cellulose and less soluble noncellulosic polysaccharides, tended to show higher incorporation at the levels where PAN-treated growth was also higher. It was concluded that effects by PAN on cell wall metabolism in growing tissue are similar to the effects on growth and that the mechanism of alleviation of growth inhibition is probably through decreased inhibition of wall metabolism.     

Changes in cell wall composition of rice mesocotyls in relation to growth and treatment with 4-ethoxy-1-(p-tolyl)-s-triazine-2,6(1H, 3H)-dione

A synthetic substance, 4-ethoxy-l-(p-tolyl)-s-triazine-2,6(1H,3H)-dione [TA] dramatically promoted mesocotyl growth in dark-grownrice (Oryza sativa L. cv. Nato) seedlings, the optimal concentrationbeing 0.1–0.2 mM. Changes in the cell wall compositionof the rice mesocotyls were examined in relation to growth andtreatment with 0.1 mM TA. The amount of the cell wall increasedduring the elongation in control and treated mesocotyls. Particularly,TA caused a large increase in the amount of the cell wall permesocotyl but a decrease per unit length of mesocotyl. Hydrolysisof the cell wall with trifluoroacetic acid liberated xylose,glucose, arabinose, galactose, and trace amounts of rhamnose,fucose and mannose. An increase in the relative amount of xyloseand a decrease in that of glucose in the noncellulosic fractionduring growth were found in control and treated mesocotyl walls.On the 2nd day after planting when the mesocotyl emerged, TAsignificantly affected the cell wall composition; TA decreasedthe relative amount of -cellulose in the wall, and caused anincrease in the relative amount of glucose and decreases inthose of xylose and arabinose in the noncellulosic fraction. ¹This paper is Part 7 in the series "Plant Growth-regulatingActivities of Isourea Derivatives and Related Compounds." (Received March 18, 1980; )     

Growth capacity in response to auxin of coleoptile segments of normal and dwarf rice strains

Auxin-induced growth, epidermal cell length, cellular osmotic potential, and cell wall composition of coleoptile segments excised from one normal and two dwarf rice strains were studied 2, 3, 4, and 5 days after soaking. The auxin-induced growth was higher at the early stages of coleoptile growth and decreased with age, being always higher in normal than in the two dwarf strains. A good correlation between auxin-induced growth and auxin-induced decrease in the minimum stress-relaxation time has been found, suggesting that the different growth capacity in response to auxin among the three different strains is due to differences in the structure of their cell walls. In fact, cell wall analysis revealed that (1) the relative α-cellulose content of the cell walls was higher in the two dwarf strains than in the normal one, and (2) the auxin-induced decrease in noncellulosic glucose was high, compared with dwarf strains, in the normal strain, which showed the higher auxin-induced growth, showing a highly significant correlation between the decrease in noncellulosic glucose and the growth in response to auxin. Thus, the different growth between normal and dwarf strains might be attributed to their different capacity to degrade β-glucan of their cell walls.     

Auxin-induced extension, cell wall loosening and changes in the wall polysaccharide content of barley coleoptile segments

Contents of the cell wall and sugar pool and the response toexogenously applied auxin (cell extension and cell wall loosening)were investigated with barley coleoptile segments excised from4-, 5- and 6-day-old seedlings. The first two groups exhibiteda high capacity to grow in terms of the intact growth rate andwere responsive to auxin, while those excised from 6-day-oldseedlings had a low growth capacity. The cell wall of 4- and5-day-old coleoptile segments contained almost the same amountof noncellulosic wall components per unit length while the 6-day-oldones had a lesser amount. The sugar pool and -cellulose contentper unit length decreased as the coleoptile aged. Auxin-stimulatedextension was most marked in the 4-day-old coleoptile segments.Auxin caused quantitative changes in the cell wall componentsof 4-day-old coleoptiles and, to a lesser extent, of 5-day-oldcoleoptiles, i.e., an increase in the contents of xylose andarabinose, both of which are constituents of noncellulosic polysaccharidesof the cell wall, and of -cellulose and a decrease in the noncellulosicglucose content. Auxin caused very little change in the noncellulosicsugar content and -cellulose content of the cell wall from 6-day-oldcoleoptile segments. The auxin-induced change in mechanicalproperties of the cell wall was significant in 4- and 5-day-oldcoleoptiles but very small in 6-day-old ones. The results suggestedthat the content of noncellulosic wall components is closelyrelated to the intact growth and auxin responsiveness of barleycoleoptiles. (Received April 20, 1978; )     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring     

Ultrastructural and biochemical aspects of cell wall reconstitution in recalcitrant (grapevine) and regenerating (tobacco) leaf protoplasts

Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study was undertaken on [U-¹⁴C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher [U-¹⁴C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts. Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl sulfoxide to the culture medium resulted in a dramatic increase in [U-¹⁴C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic and noncellulosic cell wall fractions.     

Growth capacity in response to auxin of coleoptile segments of normal and dwarf rice strains

Auxin-induced growth, epidermal cell length, cellular osmotic potential, and cell wall composition of coleoptile segments excised from one normal and two dwarf rice strains were studied 2, 3, 4, and 5 days after soaking. The auxin-induced growth was higher at the early stages of coleoptile growth and decreased with age, being always higher in normal than in the two dwarf strains. A good correlation between auxin-induced growth and auxin-induced decrease in the minimum stress-relaxation time has been found, suggesting that the different growth capacity in response to auxin among the three different strains is due to differences in the structure of their cell walls. In fact, cell wall analysis revealed that (1) the relative -cellulose content of the cell walls was higher in the two dwarf strains than in the normal one, and (2) the auxin-induced decrease in noncellulosic glucose was high, compared with dwarf strains, in the normal strain, which showed the higher auxin-induced growth, showing a highly significant correlation between the decrease in noncellulosic glucose and the growth in response to auxin. Thus, the different growth between normal and dwarf strains might be attributed to their different capacity to degrade -glucan of their cell walls.     

Comparison of Cell Wall Polysaccharide Hydrolysis by a Dilute Acid/Enzymatic Saccharification Process and Rumen Microorganisms

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72?h conversion assay (CONV) with in vitro rumen microflora incubation for 72?h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r?=?0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.     

RADIOAUTOGRAPHIC STUDY OF CELL WALL DEPOSITION IN GROWING PLANT CELLS

Segments cut from growing oat coleoptiles and pea stems were fed glucose-³H in presence and absence of the growth hormone indoleacetic acid (IAA). By means of electron microscope radioautography it was demonstrated that new cell wall material is deposited both at the wall surface (apposition) and within the preexisting wall structure (internally). Quantitative profiles for the distribution of incorporation with position through the thickness of the wall were obtained for the thick outer wall of epidermal cells. With both oat coleoptile and pea stem epidermal outer walls, it was found that a larger proportion of the newly synthesized wall material appeared to become incorporated within the wall in the presence of IAA. Extraction experiments on coleoptile tissue showed that activity that had been incorporated into the cell wall interior represented noncellulosic constituents, mainly hemicelluloses, whereas cellulose was deposited largely or entirely by apposition. It seems possible that internal incorporation of hemicelluloses plays a role in the cell wall expansion process that is involved in cell growth.     

Changes in expansin activity and cell wall susceptibility to expansin action during cessation of internodal elongation in floating rice

We investigated the involvement of expansin action in determining the growth rate of internodes of floating rice (Oryza sativa L.). Floating rice stem segments in which rapid internodal elongation had been induced by submergence for 2 days were exposed to air or kept in submergence for 2 more days. Both treatments reduced the elongation rate of the internodes, and the degree of reduction was much greater in air-exposed stem segments than in continually submerged segments. These rates of internodal elongation were correlated with acid-induced cell wall extensibility and cell wall susceptibility to expansins in the cell elongation zone of the internodes, but not with extractable expansin activity. These results suggest that the reduced growth rate of internodes must be due, at least in part, to the decrease in acid-induced cell wall extensibility, which can be modulated through changes in the cell wall susceptibility to expansins rather than through expansin activity. Analysis of the cell wall composition of the internodes showed that the cellulosic and noncellulosic polysaccharide contents increased in response to exposure to air, but they remained almost constant under continued submergence although the cell wall susceptibility to expansins gradually declined even under continued submergence. The content of xylose in noncellulosic neutral sugars in the cell walls of internodes was closely and negatively correlated with changes in the susceptibility of the walls to expansins. These results suggest that the deposition of xylose-rich polysaccharides into the cell walls may be related to a decrease in acid-induced cell wall extensibility in floating rice internodes through the modulation of cell wall susceptibility to expansins.     

Distribution of Cellular Carbohydrates during Development of the Mycelium and Fruitbodies of Flammulina velutipes

Flammulina velutipes (Curt. ex Fr.) Sing. was grown on potato-glucose solution freed of most starch. Glucose uptake and dry weight changes in the colony indicated that the large fruitbodies derived their substrates partly from glucose remaining in the medium and partly from cellular constituents stored in the mycelium and small fruitbodies. Changes in the amounts of low molecular weight carbohydrates, glycogen, and four cell wall polysaccharide fractions were followed in the mycelium and fruitbodies. Trehalose, arabitol, and smaller amounts of mannitol were the main stored low molecular weight carbohydrates. A large net loss of these compounds occurred in the mycelium and small fruitbodies after their growth ceased. The carbohydrates accumulated in the large fruitbodies, but were also partly metabolized in the colony. Reducing sugars were minor components, and included about 30 to 50% glucose and a small undetermined quantity of fructose. Glycogen was the main storage carbohydrate in the mycelium, and was also stored in the small fruitbodies. It was broken down in both structures during growth of the large fruitbodies which accumulated only small amounts. During the same period, almost 45% of the maximum amount of cell wall polysaccharides were degraded in the small fruitbodies, but not in the mycelium.     

Changes in cell wall polysaccharides associated with growth

Changes in the polysaccharide composition of Phaseolus vulgaris, P. aureus, and Zea mays cell walls were studied during the first 28 days of seedling development using a gas chromatographic method for the analysis of neutral sugars. Acid hydrolysis of cell wall material from young tissues liberates rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose which collectively can account for as much as 70% of the dry weight of the wall. Mature walls in fully expanded tissues of these same plants contain less of these constituents (10%-20% of dry wt). Gross differences are observed between developmental patterns of the cell wall in the various parts of a seedling, such as root, stem, and leaf. The general patterns of wall polysaccharide composition change, however, are similar for analogous organs among the varieties of a species. Small but significant differences in the rates of change in sugar composition were detected between varieties of the same species which exhibited different growth patterns. The cell walls of species which are further removed phylogenetically exhibit even more dissimilar developmental patterns. The results demonstrate the dynamic nature of the cell wall during growth as well as the quantitative and qualitative exactness with which the biosynthesis of plant cell walls is regulated.     

Exogenously applied ascorbic acid ameliorates detrimental effects of NaCl and mannitol stress in Vicia faba seedlings

The adverse effects of either NaCl or mannitol on growth, nitrogen content, and antioxidant system in Vicia faba seedlings were investigated. The role of exogenous ascorbic acid in increasing resistance to these stressors was also evaluated. Thus, with an increase in concentration of either NaCl or mannitol in culture media, a progressively greater significant decrease in percentage germination, in growth parameters, and in nitrogen constituents of the germinating beans, was observed. On the other hand, amide-, nitrate-, and total soluble-N contents appeared to show a progressive significant increase. Exogenous addition of ascorbic acid (4 mM) to the stressful media induced a pronounced significantly increased percentage germination and the growth attributes, whereas nitrogen constituents were variably changed in relation to values maintained in beans treated with either NaCl or mannitol. Furthermore, exogenous addition of ascorbic acid to NaCl or mannitol media induced a significant increase in the contents of ascorbate and glutathione and enzymatic antioxidant activities, in particular, in beans treated with the three highest concentrations of NaCl or mannitol, throughout the period of the experiments (12 days). Thus, ascorbic acid ameliorates the adverse effects of the stressful media; the magnitude of amelioration being a function of the type and the concentration of the stressful agent as well as of the duration of treatment. The importance of the above-mentioned changes in growth and metabolism to stress tolerance in broad bean is discussed.