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.     

Endogenous abscisic acid during the germination of chick-pea seeds

Over the past twenty years many studies have been undertaken to elucidate the regulation of seed germination. Abscisic acid (ABA) and the gibberellins (GAs) are the hormones proposed to control this process, the first by inhibiting and the second by inducing germination. It has been proposed that a high water potential increases the growth potential of the embryo, presumably permitting the production or activation by GA of the cell wall hydrolases and thus decreasing the yield threshold of the endosperm close to the radicle tip. A low water potential, e.g., imbibition in an osmoticum. imposes a stress on cell metabolism, by reducing the turgor of the radicle cells, and there is a decrease in growth potential. Exogenous ABA also causes a decline in growth potential of the radicle: however, the actions of low water potential in preventing germination are not mediated through an increase in ABA in the seeds. In the present paper an attempt is made to asses the role of ABA and polyethylene glycol (PEG) in the germination of chick-pea (Cicer arietinum L.) seeds. The endogenous ABA of chick-pea seeds was purified by reversed-phase HPLC and quantified by GC-ECD. The variations in the ABA levels in the embryonic axes and the cotyledons were studied during 120 h. of imbibition. The highest ABA level in the embryome axes was found at 18 h. coinciding with an increase in fresh weight and a high germination percentage. ABA was not detected in the cotyledons during incubation which probably indicates that the hormone is more involved in the active growth of the embryonic axes itself than in the mobilization process of the reserves. When seeds were treated with different PEG-cycles. PEG delayed germination, reduced the fresh weight of embryonic axes, and retarded the onset of ABA synthesis. It is concluded that endogenous ABA is related to the onset of germination and the growth of the embryonic axis. In addition, there is no correlation among the different PEG-cycles and the level of ABA and germination. Germination was related more to the water conditions inside the embryo's cells than to ABA levels.     

Diamine oxidase of Lathyrus sativus seedlings

A diamine oxidase has been purified (ca 40-fold) from 5-day old etiolated seedlings of L. sativus by MnCl₂ treatment, (NH₄)₂SO₄ and Me₂CO fractionations, positive adsorption on alumina Cγ-gel followed by column chromatography on DEAE-Sephadex. This cytosol enzyme oxidatively deaminates a number of aliphatic and aryl alkylamines but not histamine. NSD-1055, semicarbazide and other carbonyl reagents, α,α'bipyridyl, 1,10-phenanthroline and 8-hydroxyquinoline inhibit the enzyme. Pargyline, SKF trans-385, atabrine were without effect on the enzyme.     

Abscisic acid and temperature modify the levels of calmodulin in embryonic axes of Cicer arietinum

Changes in calmodulin (CaM) levels in embryonic axes of Cicer arietinum L. cv. Castellana germinated under three different conditions were measured. Abscisic acid (ABA) and a temperature of 30°C, which delay chick-pea germination, respectively decreased and increased the concentration of CaM compared to the values obtained under normal germinating conditions (H₂O-25°C). The CaM concentration was higher in those zones of the axes undergoing an active cell division.
The compartmentalization of CaM in 36-h-old embryonic axes grown under these three conditions was also measured. Cytosolic and mitochondrial CaM was higher in axes where a delayed germination occurred as well as in the cell walls of normally grown ones. On the other hand, CaM was higher in nuclear and microsomal fractions extracted from H₂-O-25°C-treated axes. From these data we postulate that delayed germination could be an effect of altered CaM distribution in chick-pea embryonic axes.     

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.     

Regulation of cadaverine and putrescine levels in different organs of chick-pea seed and seedlings during germination

In chick-pea ( Cicer arietinum L.) seed germinated in the presence of ¹⁴C-lysine, the latter is taken up and partly metabolised to cadaverine and TCA-precipitable molecules. Labelled cadaverine is detectable in seedlings only after 3 days, on a labelled lysine-containing medium, as confirmed also by the presence of lysine decarboxylase (LDC) activity, measured in the embryo axis and cotyledons of the seed and in the epicotyl, cotyledons, hypocotyl and roots of the seedling on the basis of ¹⁴CO₂ evolution from the labelled precursor. Putrescine biosynthesis occurred only via arginine decarboxylase (ADC) activities in soaked seeds and via both ADC and ornithine decarboxylase (ODC) activities in seedlings. Both putrescine and cadaverine were present in soaked seed, and accumulated in very large amounts in the different portions of both 3- and 8-day-old seedlings, while spermidine and spermine titers were maintained at similar levels with respect to the seed. Diamine oxidase activity, measured by evaluating oxygen consumption in the presence of putrescine, was absent in ungerminated seed and appeared in 3- and 8-day-old seedlings. In order to clarify the metabolic relationships between cadaverine and the more common polyamines, gradients of biosynthesis, accumulation and degradation of putrescine and cadaverine along the seedling axis were compared, indicating that the two diamines behave similarly during seed germination and seedling development. Their conspicuous accumulation (up to 6 m M for putrescine) seems to be regulated mainly via oxidation rather than biosynthesis.     

Calcium dependence of the effects of abscisic acid on RNA synthesis during germination of Cicer arietinum seeds

The effect of abscisic acid (ABA) and CaCl₂ in the medium on RNA synthesis in embryonic axes of seeds of chick-pea (Cicer arietinum L. cv. Castellana) during the first hours of germination has been studied. ABA decreases the incorporation of ³H-uridine in the embryonic axes and modifies the mRNA populations by preventing the disappearance of three polypeptides of 25, 22 and 21.6 kDa and inducing the appearance of two polypeptides of 35 and 27 kDa absent in the control. Calcium increases the effect of ABA on the synthesis of these mRNAs and seems to be involved in the synthesis of at least the polypeptide of 25 kDa, as this polypeptide disappears in the treatments with EGTA and Verapamil. Moreover, cytosolic calcium seems to be necessary for the accumulation of these messengers since treatment with TMB-8 (chelating agent for endogenous calcium) decreases the intensity of the bands corresponding to these 5 polypeptides. The possible synergistic action of ABA and calcium in this process is discussed.     

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.     

Abscisic acid increases the content of free polyamines and delays mitotic activity induced by spermine in isolated embryonic axes of chick-pea seeds

The effects of abscisic acid (ABA) on the free polyamine: spermine (Spm), spermidine (Spd), putrescine (Put) and cadaverine (Cad) content, mitotic index (MI) and DNA synthesis have been studied in embryonic axes isolated from chick-pea ( Cicer ariennum L.) seeds. Spermine and spermidine decreased in controls during the first 24 h of germination, the former by 70% and the latter by 20%. This decrease was slowed down by ABA (5 μ M or 25 μ M ) in response to its concentration; at 24 h the values for Spm and Spd were 3-fold and 1.5-fold those of the controls, respectively. Exogenous ABA induced the synthesis of Put from 0 to 24 h, by which time the content of this polyamine had doubled. The controls at 24 h registered the same Put content as those with 25 μ M ABA, suggesting that ABA had ceased to induce Put synthesis. The pattern for cadaverine ABA-induced synthesis was similar to that of Put. From 18 to 24 h, however, the level of Cad in the controls was 3 times higher than in the treatments. The mitotic index in meristem cells reached a maximum at 30–36 h, when cell elongation stopped in the sub-apical region. This maximum was lowered by exogenous ABA in response to the concentration and occurred 12 h earlier in the presence of Spm (0.1–1.0 m M ), which induced high MI levels during the period studied. With 10 m M Spm, two complete waves of mitosis were observed, but when Spm and ABA were added together, the tetramine had no observable effect. Spermine stimulated ³H-thymidine uptake as well as DNA synthesis, with a maximum at 12 h, but did not counteract the inhibitory effect of ABA. Our results suggest that ABA causes changes in mitotic activity that Spm cannot overcome.