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 osmotic stress on the growth of epicotyls of Cicer arietinum in relation to changes in cell wall composition

The inhibition of growth by polyethlene glycol (PEG)-induced osmotic stress led to modifications in the changes taking place in cell wall composition during normal growth of epicotyls of Cicer arietinum L. cv. Castellana. Epicotyls growing under normal conditions showed a decrease in the amount of pectic fractions and an increase in the hemicellulosic fractions and α-cellulose that led to an increase in the rigidity and a loss in growth capacity. Among the hemicellulosic fractions, the KI-2 fraction (insoluble fraction of 10% KOH-extracted hemicelluloses) seemed to be the only one related to the elongation process and subsequent rigidity. During normal growth a decrease was observed in the total amount of galactose, mainly from the pectic fractions. The inhibition of elongation led to an increase in the amount of the cell walls, due to inhibition of cellular elongation. PEG prevented the increase in the hemicelluloses and the α-cellulose, indicating that these changes were related to elongation. Thus, during the inhibition of elongation there is probably an inhibition of new synthesis that prevents cell wall rigidity and maintains cell wall growth capacity. Changes in the pectic fractions during growth were not affected by the inhibition of elongation, showing that these fractions are related to cell wall loosening rather than to elongation. Study of the cell wall composition confirms the idea that the autolytic process is regulated by changes in the cell wall structure during epicotyl growth     

Cell wall localization of the natural substrate of a β-galactosidase, the main enzyme responsible for the autolytic process of Cicer arietinum epicotyl cell walls

The Hw pectic fraction, extracted with hot water, is the major component of 4 days old epicotyl cell walls of Cicer arietinum L. cv. Castellana and is formed of arabinose and galactose, with smaller amounts of rhamnose, xylose, glucose and mannose. The cell wall 2βIII enzymatic fraction, with β-galactosidase activity (EC 3.2.1.23) and the main enzyme responsible for the autolytic process, essentially acts on the Hw fraction, and is able to hydrolyze 560 μg of this fraction per g of epicotyls, releasing mainly galactose as monosaccharide.
The 2βIII fraction acts very weakly on the other polysaccharide fractions of the cell wall, both pectic and hemicellulosic, releasing 80, 60 and 14 μg per g of epicotyls from the fractions extracted with oxalate (Ox), KOH 10% (KI) and KOH 24% (KII), respectively. It can be concluded that the natural substrate of this enzyme is the Hw pectic fraction, probably an arabinogalactan that is found in the cell wall in isolated form or as side chains of the rhamnogalacturonan I.     

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.     

Autolysis of the cell wall. Its possible role in endogenous and IAA-induced growth in epicotyls of Cicer arietinum

Indoleacetic acid (IAA), a factor that induces growth in epicotyls of cicer arietinum L. cv. Castellana, increases the autolytic capacity of the cell walls by 50%, suggesting that autolysis is related to the processes of cell wall loosening that accompany growth. IAA promotes an increase in the specific activities of the enzymes involved in autolysis, mainly α-galactosidase (EC 3.2.1.22). This relationship autolysis-growth. was also observed in a study of the autolytic capacity of cell walls from regions of the epicotyl with different growth capacity. The sugars released and the level of enzymatic protein were higher in the subapical region that towards the base.     

Characterization of a cell wall β-galactosidase of Cicer arietinum epicotyls involved in cell wall autolysis

β-Galactosidase (EC 3.2.1.23) has been established as the main enzyme involved in the autolytic process. The enzyme extracted from cell walls of epicotyls of Cicer arietinum L. cv. Castellana with 3 M LiCl is a 45 kDa protein composed of a single subunit, having an optimum pH of 4; an optimum temperature of 45°C and K_m and V_max of 1.72 m M and 18.5 nkat (mg protein)^–1 respectively, as evaluated against p -nitrophenyl-β- d -galactopyranoside. The enzyme is inhibited by Hg²⁺, d -galactono-1,4-lactone and galactose, substances that also inhibit the autolytic process. Ca²⁺ and EDTA, which do not affect the activity of the β-gaiactosidase, do however modify the hydrolysis of the cell wall mediated by the enzyme, and they also inhibit the autolytic process. Ca²⁺ decreased both processes, whereas EDTA increased them; and when both substances were added together, their individual effects were neutralized. The effects of both agents is probably due to modifications in the cell wall that prevent access of the enzyme to its substrate.     

Cell wall structure regulates the autolytic process throughout growth of Cicer arietinum epicotyls

The autolytic process in epicotyl cell walls of Cicer arietinum L. cv. Castellana, and also the hydrolysis of heat-inactivated cell walls as mediated by a cell wall β-galactosidase (EC 3.2.1.23) (named βIII and previously characterized as responsible for the autolysis), are maximal on the fourth day of germination and coincide with the maximal growth capacity. They decrease during the following days, in which the growth rate diminishes. In both cases, no differences were observed in the percentages of the different sugars released, galactose being the principal one. The βIII fraction from aged epicotyl cell walls hydrolyzed young walls in proportion to its specific activity, and more efficient than when cell walls from aged material were used as the substrate. The βIII fraction from 4 day-old epicotyls (the time for maximal autolysis) was incapable of hydrolyzing aged epicotyl cell walls to the same extent as young ones. These results, together with the levels and activity of the enzyme throughout growth, allow the assumption that the variations in the autolysis and hydrolysis caused by βIII during growth processes are due to structural modifications in the cells walls, modifications that would limit access of the enzyme to its substrate, thus impeding the release of galactose, even though the enzyme is present.     

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

Two protein fractions with activity as α-galactosidase (EC 3.2.1.22) and α-arabinosidase (EC 3.2.1.55), respectively, were identified in the proteins of cell wall of Cicer arietinum L. cv. Castellana extracted with 3 M LiCl. These fractions were partially purified by gel filtration chromatography (Bio Gel P-150), increasing the specific arabinosidase activity 57-fold and the α-galactosidase activity 6-fold. Other protein fractions with glucosidase (EC 3.2.1.21) and glucanase (EC 3.2.1.6) activity also appeared. According to earlier authors, α-arabinosidases and α-galactosidases are related to alterations in linkages occurring in cell walls, since the enzymes are able to hydrolyze isolated wall polymers. However, our preparations hydrolyze intact cell walls only to a very limited extent, such that their participation in the autolytic processes of cell walls can be ruled out.     

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.     

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.     

Effect of anti-wall protein antibodies on auxin-induced elongation, cell wall loosening, and β-D-glucan degradation in maize coleoptile segments

Antiserum raised against the LiCl extract of maize shoot cell walls suppresses auxin-induced elongation of maize coleoptile segments. A series of polyclonal antibodies were raised against protein fractions separated from the LiCl extract of maize ( Zea mays L. cv. B73 x Mo17) coleoptiles by SP-Sephadex and Bio-Gel P-150 chromatography. To understand the role of cell wall proteins in growth regulation, the effect of these antibodies on auxin-induced elongation and changes in the cell walls of maize coleoptiles was examined. Four of the fractions prepared reacted with the antiserum raised against the total LiCl extract and effectively suppressed its growth-inhibiting activity. Only these fractions contained the proteins responsible for eliciting growthinhibiting antibodies. The antibodies capable of growth inhibition of auxin-induced elongation of segments also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. The antibodies raised against one of the protein fractions separated by SP-Sephadex inhibited the autolytic reactions of isolated cell walls and the auxin-induced decrease in (1→3), (1→4)-β-D-glucans in the cell walls. Thus, the degradation of β-D-glucans by cell wall enzymes may be associated with the cell wall loosening that is responsible for cell elongation. Because the other antibodies did not influence the auxin-induced degradation of (1→3), (1→4)-β-D-glucanses, β-D-glucanases and other cell wall enzymes may cooperate in regulation of cell elongation in maize coleoptiles.     

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.     

Increased expression of two cDNAs encoding metallothionein‐like proteins during growth of Cicer arietinum epicotyls

The present study was undertaken to identify and characterize clones whose expression increase during Cicer arietinum epicotyl growth. Two cDNAs encoding two different plant metallothionein (MT)‐like proteins have been isolated from a cDNA library from epicotyls of Cicer arietinum L. cv. Castellana. The CanMT‐1 deduced protein appears to have the typical structure of type 1 MT where all Cys residues are in Cys‐X‐Cys motifs, while the CanMT‐2 has the typical structure of type 2 MT having Cys‐Cys and Cys‐X‐X‐Cys motifs within the N‐terminal domain. Both chickpea CanMTs are up‐regulated during epicotyl growth, showing increased expression in mature tissues, mostly CanMT‐1, which is undetectable in young epicotyls. Accordingly, stem of chickpea plants displayed the highest level of CanMT‐1 expression in the basal internode, with reduced growth, decreasing towards the apex. Osmotic stress by PEG, which inhibited growth, and ABA treatment induced the expression of MT‐like genes, which points to a relationship between chickpea MTs and ABA‐mediated stress response. Unlike CanMT‐2, CanMT‐1 is induced in chickpea epicotyls by cadmium indicating a different function for both clones. We conclude that these MT‐like proteins, in particular CanMT‐1, are regulated by the developmental stage and may participate in cell maturation process.     

The immunolocation of a xyloglucan endotransglucosylase/hydrolase specific to elongating tissues in Cicer arietinum suggests a role in the elongation of vascular cells

In a previous work, a Cicer arietinum cDNA clone (CaXTH1) encoding a xyloglucan endotransglucosylase/hydrolase (XTH1) protein was isolated and characterized. CaXTH1 showed an expression pattern specific to growing tissue: mostly epicotyls and the upper growing internodes of adult stems. CaXTH1 mRNA was not detected in any other organs of either seedlings or adult plants, suggesting an involvement of the putative XTH encoded by CaXTH1 in the chickpea cell expansion process. After the generation of polyclonal antibodies by using the XTH1 recombinant protein and the analysis of the specificity of the antibodies for XTH proteins, here the specific location of the chickpea XTH1-cross-reacting protein in cell walls of epicotyls, radicles, and stems is reported, evaluated by western blot and immunocytochemical studies. The results indicate a function for this protein in the elongation of parenchyma cells of epicotyls and also in developing vascular tissue, suggesting a role in the elongation of vascular cells.