Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.     

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.     

Effect of water stress on functioning and structure ofCicer arietinum L. nodules

Chickpea (Cicer arietinum L. cv. 235) plants were grown in sand culture at moisture equal to 45–50% of sand saturation capacity under greenhouse conditions. 60 d after sowing, pots were divided into four lots, leaving one as control and sand moisture content of others was brought to 25–30% (S₁), 12–15% (S₂) and 5–6% (S₃) of sand saturation capacity, by withholding the water supply and then maintaining the required levels gravimetrically till the harvest. Relative water content of leaves and nodule water content were measured as indices of water stress. With increase in the severity and duration of water stress nitrogenase activity and nitrogen and leghemoglobin content of the nodules decreased and the ratio of leghemoglobin components I and II were changed. Nodules developed under limited water availability showed decreased branching, breakdown of the endodermis, greater compactness and decreased vacuolation of cells in the central symbiotic tissue as compared to the control.     

Effect of applied nitrate on enzymes of ammonia assimilation in nodules ofCicer arietinum L.

Summary The relationship between N₂-fixation, nitrate reductase and various enzymes of ammonia assimilation was studied in the nodules and leaves ofC. arietinum. In the nodules of the plants growing on atmospheric nitrogen, maximum activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aspartate aminotransferase (AAT) were recorded just prior to maximum activity of nitrogenase. In nitrate fed plants, the first major peak of GDH and AS coincided with that of nitrate reductase in the nodules. With the exception of AS, application of nitrate decreased the activities of all these enzymes in nodules but not in leaves. Activities of GS, GOGAT and AAT were affected to much greater extent than that of GDH. On comparing the plants grown without nitrate and those with nitrate, the ratios of the activities of GDH/GS and GDH/GOGAT in nitrate given plants, increased by 4 and 12 fold, respectively. The results presented in this paper suggest that in nodules of nitrate fed plants, assimilation of ammonia via GDH assumes much greater importance.     

Hemicellulose fractions and associated protein of lupin hypocotyl cell walls

The alkali extraction of polysaccharide fractions from depectinated primary cell walls of lupin hypocotyls was studied using sequential extractions at 0° and 18–22°. Aqueous 10% KOH at 0° removed hemicellulose-A (95%) heteroglycan-B (80%) and linear 1–4 linked hemicellulose-B (60%). Arabinose accounts for 88% of the monosaccharides of the linear 1–4 linked hemicellulose-B extracted between 2 and 5 h at 18–22°. Extraction of the 0° and 18–22° alkali-soluble fractions by denaturants, was also examined. 6M guanidine thiocyanate removed about 60% of the 0° 10% KOH soluble polysaccharide but little of the 18–22° soluble material. Although rapidly extracted by 10% KOH at 0° the hemicellulose-A is not extracted by this reagent. Analyses of cell walls and extracted fractions showed that there is little change in amino acid composition and little extraction of wall protein upon removal of about 60% of total wall hemicellulose with 10% KOH at 0°. It is therefore not bound to the wall through galactosylserine links. 10% KOH at 18–22° caused a marked change in composition and extracted most of the wall protein. An alkali resistant fraction high in hydroxyproline and low in serine was not extracted by 24% KOH at 18–22° in 24 hr.     

Hormonal regulation of growth of Cicer arietinum L. epicotyls. changes in the chemical composition of the cell wall

The growth capacity of the epicotyls of Cicer arietinum L. decreased from the sub-apical region to the basal region. The pectic fraction content falls from the upper to the lower part of the epicotyls. Contrary to this, the -cellulose content was lower in the upper part and higher in the basal one. The hemicellulosic content was almost constant over the whole epicotyl. Treatment of the epicotyls with IAA or GA₃, compounds which induce growth in epicotyls, induced a decrease in galactose content in the hemicellulosic fraction as well as in the pectic fraction. On the other hand, epicotyls treated with kinetin, a compound which does not stimulate growth, did not show a decrease, though they did show a considerable increase in -cellulose content.Abreviations GLC gas liquid chromatography - GA₃ gibberellic acid - IAA indole-3-acetic-acid - TFA trifluoroactic acid     

The level of proteins and leakage of solutes in germinating fresh and stored seeds ofCicer arietinum L.

One-year-old seeds of chickpea (Cicer arietinum L. cv. C-235) lost about 23 % germinability and leaked larger quantities of N, P, K, saccharides and proteins into the soaking medium in the first 48 h, as compared with fresh seeds. The protein content in stored seeds decreased more than in fresh seeds, as the soaking progressed.     

Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.     

Dimeric calcium complexes of arabinan-rich pectic polysaccharides from Olea europaea L. cell walls

The study carried out in this work concerns the pectic polysaccharides of olive cell walls as present in olive pulp and that remained entrapped in the cellulosic residue after sequential extraction of the cell wall material (CWM) with imidazole, carbonate and KOH aqueous solutions. These polymers, obtained after neutralisation and dialysis of an aqueous suspension of the residue (sn-CR fraction), extracted with 4 M KOH, were arabinan-rich pectic polysaccharides. They accounted for 11–19% of the total pectic polysaccharides found in the olive pulp cell walls of fruits collected in two years and in three stages of ripening (green, cherry and black). The analysis by powder X-ray diffraction highlighted the existence, in all sn-CR fractions, of crystalline phases related with the presence of calcium-pectic polysaccharide complexes (CPPC) occurring in an amorphous carbohydrate network. The relative crystallinity of the CPPC varied linearly with the Ca²⁺/GalA molar ratio until a maximum of 0.57. Size-exclusion chromatography showed that sn-CR fractions possessed a bimodal molecular weight distribution. The lower molecular weight fraction of sn-CR (M_w = 70–135 kDa) was independent on the ripening stage of olive fruit, whereas the higher molecular weight fraction showed values of 1.1, 0.6–0.9 and 0.5–0.7 MDa, respectively, for green, cherry and black olives. Treatment of the sn-CR pectic polysaccharides with a 2 M imidazole solution disrupted the CPPC crystalline network showing the loss of low molecular weight galacturonan-rich material during dialysis (12–14 kDa cut off) and the decrease of molecular weight of the polymers to roughly half. These results allowed to infer the presence of oligogalacturonides held within cell walls by calcium ions and that the pectic polysaccharides of sn-CR fraction occurred in olive pulp cell walls as calcium bridged macrodimers.     

Partial purification of cell wall β-galactosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

The protein extracted from the cell wall of the epicotyls of Cicer arietinum L. cv. Castellana was separated by ion exchange chromatography in four different fractions with β-D-galactosidase (EC 3.2.1.23) activity. These were called βI, βII, βIII and βIV, according to their order of elution. βII was associated with a particularly high β-D-glucosidase (EC 3.2.1.21) activity. Gel filtration chromatography of each of the fractions gave further subdivision of fractions βI and βIII. Subfractions 1 βI, 1 βII and 1 βIV have glucosidase activity and subfractions 2 βI and 2 βIII have galactosidase activity.
The studies on the hydrolytic capacity of these fractions and its relationship with the autolytic process seem to show that subfraction 2 βIII is responsible for autolysis. The release of total and reducing sugars is very similar for autolysis and hydrolysis by 2 βIII. The sugars released are mainly galactose and, to a lesser extent arabinose and glucose. Galactose is released as a monosaccharide, while arabinose remains associated to a polysaccharide component together with glucose and small amounts of galactose.     

Relation of cell wall peroxidase activity with growth in epicotyls of Cicer arietinum. Effects of calmodulin inhibitors

Sánchez, O.J., Pan, A., Nicolás, G. and Labrador, E. 1989. Relation of cell wall peroxidase activity with growth in epicotyls of Cicer arietinum. Effects of calmodulin inhibitors.
Peroxidases are bound ionically to cell walls in epicotyls of Cicer arietinum L. cv. Castellana. The cell wall peroxidase activity increases during the growth of epicotyls, being the lowest in 3-day-old epicotyls with high growth capacity. The cell wall phenolic compounds, postulated natural substrates of cell wall peroxidases, also increase during growth.
The calmodulin inhibitors chlorpromazine and trifluoperazine decrease the elongation rate of epicotyls of Cicer arietinum. These inhibitors also cause an increase in the cell wall peroxidase activity and in the level of phenolic compounds. A possible regulatory effect of calmodulin on peroxidase activity is postulated.     

Altered matrix polysaccharides in cell walls of pocket galls formed by an aphid on Distylium racemosum leaves

The present work reports the results of a study on the isolation and characterization of matrix polysaccharides in the cell walls of galls formed by an aphid (Neothoracaphis yanonis) on Distylium racemosum leaves. Cell walls were isolated from both healthy Distylium leaf and gall tissues and then extracted sequentially with cyclohexane‐trans‐1,2‐diaminetetra‐acetate (CDTA), Na₂CO₃, 1 m KOH, and 4 m KOH. The amount of pectin solubilized from gall cell walls was approximately 2.6‐fold higher than the pectin solubilized from leaf cell walls, whereas the amount of hemicellulose solubilized from gall cell walls was 1.4‐fold higher than that from normal leaf cell walls. When the polysaccharides were fractionated by anion‐exchange chromatography, considerable increases in arabinose and galactose were observed in CDTA‐soluble pectic polymer (fraction PI‐1) from gall cell walls, whereas the gall cell walls had less xylose in 1 m KOH‐soluble hemicellulosic polymers (fractions HI‐2, HI‐3, and HI‐4) than did the cell walls from the healthy leaf. The hemicellulosic polymers of the gall cell walls exhibited distinctly different patterns of molecular mass, compared with the healthy leaf cell walls. These results suggest that an extensive change occurs in the matrix polysaccharide structure of the cell walls of Distylium galls formed by an aphid. In addition, many glycosylhydrolase activities were detected in the protein fraction solubilized with strong saline solution from the gall cell walls, and the activities of β‐galactosidase, β‐xylosidase and α‐l ‐arabinofuranosidase were considerably increased under gall formation.     

Enzymatic degradation of cell walls of apples and characterization of solubilized products

Partially depectinated apple cell walls were digested by pectin lyase or endoglucanase or a combination. By combining these commercial enzymes, a higher yield of 22.2% of carbohydrate material was obtained compared with only 13.9% and 5.7%, respectively, when using them singly. Only small amounts of carbohydrates were extracted by buffer (0.8%). The solubilized extracts were fractionated using a combination of ion-exchange chromatography and gel filtration. The indidividual subfractions were analysed for neutral sugar and uronic acid content. The results indicated the existence of a synergistic effect between pectin lyase and endoglucanase based on the percentage of material extracted.     

Effect of auxin on cell wall glycanhydrolytic enzymes in epicotyls of Cicer arietinum

Cell wall glycanhydrolytic enzymes have been related to cell wall loosening and cell growth, although the mechanism of this relationship has not been clarified. Since auxins are plant hormones that stimulate growth in elongating organs, in the present work we studied the effect of auxin on cell wall glycanhydrolytic enzymes, which were extracted with LiCl. Our results show that incubation of sections of Cicer arietinum epicotyls with indoleacetic acid elicit some minor changes in electrophoretic patterns of cell wall proteins when compared with control sections. This indicates that there is no appearance of a specific polypeptide synthesized de novo in response to the hormone, although there are increases in the intensity of some of the polypeptides, which could indicate an enhancement of wall protein biosynthesis. Brief incubation with IAA led to a general increase in the specific activities of these different cell wall enzyme fractions separated by chromatography, with the exception of the α-fraction, with α-galactosidase activity. Longer incubation resulted in an increase in the amount of protein associated with some of the enzyme fractions. In particular, it induced a large increase in the amount of protein associated with the β111-galactosidase fraction that is involved in the autolytic process of cell walls of chick-pea epicotyls. Our results indicate that auxin-enhanced growth could be the result of the action of the hormone al the level of the cell wall glycanhydrolytic proteins that have been related to the wall-loosening process.     

Effect of calcium and other metal ions on pectic activities from autolysed cultures of Alternaria alternata

Abstract The effect of increasing concentrations of Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Na⁺ and EDTA on the pectic enzymic activities (polymethylgalacturonase, endopectinase and pectin-lyase) present in the autolytic complex from Alternaria alternata has been studied. In all cases the divalent metal ions and EDTA produced an increased inhibition correlated with increasing concentration of each ion. An opposite effect was shown by the Na⁺ ion, which produced an increase in pectic enzymic activities, principally at low concentrations.     

Behaviour of the cell walls of Aspergillus niger during the autolytic phase of growth

Abstract Autolysis of Aspergillus niger cultures has been studied. The stability of the cell wall fraction was followed throughout a 57-day period of autolysis. The results indicated that both a persistence of existing walls and an extra wall deposition due to continued synthesis of walls or wall components during the first 12 days of autolysis seem to take place.     

Self-assembly of plant cell walls

The object of this paper is to define criteria for distinguishing between self-assembly and template-based assembly in plant cell walls. The example of cellulose shows that cell wall polymers biosynthesized at a membrane may retain parallel chain packing arrangements that are thermodynamically unstable and cannot be reproduced in vitro, making the experimental testing of the self-assembly hypothesis difficult. Also, natural cellulose is ordered on a number of scales of pattern, each of which may be constructed by either self- or template-based assembly independently of the rest. These conceptual problems apply equally to the self-assembly of complete cell walls and other cell wall polymers. It is suggested that the self-assembly concept should be applied only to one stage or level in the synthesis of a cell wall, and that an additional concept of parallel assembly may be useful for understanding the synthesis of some polysaccharides.     

Pectic polysaccharides of rice endosperm cell walls

Two pectic polysaccharide fractions were purified from rice endosperm cell walls. Methylation analysis including carboxyl-reduction and also selective     

Matrix polysaccharides of oat coleoptile cell walls

Separation of component polysaccharides in extractable fractions of the noncellulosic matrix of Avena sativa coleoptile cell walls shows that the principal classes of polymers present are glucuronoarabinoxylans (GAX) and iodine-negative hemicellulosic β-glucans. Rhamnogalacturonan is a minor component. GAX contains about 5–10% glucuronic acid and its 4-O-methyl ether, arabinose in amount almost equal to xylose, and a small amount of galactose; some subfractions contained appreciable amounts of glucose and galacturonic acid but these may derive from separate, contaminating polysaccharides. From the sedimentation and diffusion coefficients and intrinsic viscosities of one subfraction each of the GAX and of the hemicellulosic glucan that had been purified to apparent homogeneity by criteria of sedimentation and borate electrophoresis, MWs of about 200 000 were calculated by two methods. The viscosity characteristics and gel-forming ability of the hemicellulosic glucan give evidence of appreciable molecular interactions which suggest that this polymer is an important structural component of the cell wall.