No effect of plant growth retarding compounds and growth stimulators on indolo-3-acetic acid oxidase activity in greening cucumber cotyledons

Cotyledons dissected from 5-day-old etiolated cucumber seedlings were incubated in solutions on AMO-1618, B-Nine, CCC and Phosfon D for 48 h in light. In some tests the retardants were applied in mixed solutions with GA₃ or BAP. IAA oxidase was extracted and purified by means of molecular sieving through a bed of Sephadex G-25. The retardants inhibited chlorophyll synthesis by 50 % or more, and had essentially no effect on IAA oxidase activity per cotyledon basis. GA₃ and BAP also had no effect on enzyme activity in spite of a fact that the compounds stimulated growth of the cotyledons. The crude enzyme extract from B-Nine treated cotyledons showed lower IAA oxidase activity in comparison with the water treated control, the effect being due to a longer lag-phase preceding the initiation of IAA oxidation. KNO₃ strikingly stimulated expansional growth of the cotyledons, the effect being correlated with the accelerated chlorophyll accumulation. KNO₃ had no effect on IAA oxidase activity per cotyledon and decreased it per gram fr wt. It is concluded that [1] the growth rate of cucumber cotyledons is not correlated with IAA oxidase activity, and ;[2] the growth retarding compounds do not affect IAA oxidase system is this tissue.

Effects of growth retarding compounds on chlorophyll accumulation and nitrate reductase activity in nitrate induced cucumber cotyledons

Cotyledons were excised from 5-day old etiolated cucumber seedlings and .grown for 24 or 48 h in solutions of plant growth retardants: AMO-1618,B-Nine, CCC and phosfon D, supplemented with KNO₃ (10^-2M) in light. Nitrate reductase (NR) activity was determined <i>in vivo</i>. CCC and Phosfon D at high concentrations had no effect on nitrate reductase activity in 24 h tests. CCC at 5xl0^-2 M enhanced NR activity in longer 48 h tests; Phosfon D was inhibitory in that case. AMO-1618 markedly decreased NR activity. B-Nine strikingly enhanced NR activity in KNO₃ induced cytoledons; the effect was positively correlated with the concentration of B-Nine. Ali the compounds inhibited chlorophyll synthesis.

Effects of gibberellic acid and AMO-1618 on the development of vegetative systems in generatively matured thalli of Chara vulgaris L.

Effects of GA₃ (10^-11-10^-4 M) and AMO-1618 (10^-6-10^-4 M) on the development of generatively matured thalli of <em>Chara vulgaris</em> were investigated during 21-day culture of plants in axenic conditions. It has been found that in the main bud the divisions of apical cells of the thalli are not stimulated by GA₃, whereas in the lateral buds the cell divisions are stimulated by higher GA₃ concentrations. Subsequent mitotic activity of the apical cells in the branches of the main axis is not stimulated by GA₃, whereas the lateral buds of these branches are activated. The development of rhizoids in younger nodes is accelerated by high GA₃ concentrations. The elongation of the polynuclear, internodal cells of the main axis and that of pleuridia are inhibited proportionally to the GA₃ concentration. AMO-1618 stimulates the development of new nodes, elongation of internodes and delays the activation of lateral buds as well as the formation of rhizoids. These results suggest that the GA₃-induced inhibition of elongation of the thalli and diminution of the apical domination is connected with a high level of endogenous gibberellins in the generatively matured thallus.

The effect of the plant growth retardants AMO-1618 and CCC on the synthesis of ribonucleic acids and proteins in triticale embryos during the initial phase of germination

Triticale var. Grado caryopses were subjected to imbibition and germination in the presence of the growth retardants, AMO-1618 (2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride) and CCC (2-chloroethyl)-trimethylammonium chloride) at the following concentrations, 3 x 10^-4 M and 10^-3 M. These compounds exerted a very strong inhibitory effect on the initiation of germination processes, growth of embryos and the germination capacity of the caryopses. At the concentration of 10^-3 M, AMO-1618 showed an especially strong effect, lowering the germination capacity of the caryopses to about 50%. It was also shown that both retardants are decidedly more effective on the germination of whole, intact caryopses than on that of isolated embryos. During the very earliest hours of germination, these retardants already inhibited RNA synthesis. The participation of the polyribosome fraction in the total ribosome fraction of embryos in the control sample after 24 hrs of germination of caryopses equalled about 70%, while in the samples treated with CCC (10^-3 M)- about 57%, in the samples treated with AMO-1618 (10^-3 M) about 35%,. The inhibition of incorporation of ¹⁴C-amino acids into ribosomal proteins in the polyribosome fraction was in the case of CCC about 13%, while in the samples treated with AMO-1618, about 55%. In the monosome fraction (80S), the inhibition by CCC was about 23%, whereas in the samples treated with AMO-1618 it reached around 73%. From this data it is evident that the studied retardants have a significant influence on the synthesis of ribonucleic acids as well as on ribosome proteins. These results also suggest the existance of another mechanism, aside from that of inhibition of gibberellin biosynthesis, inhibiting the growth and development of cells. The high percentage of ribosome subunits in the samples treated with CCC, in comparison with controls and samples treated with AMO-1618, points to different mechanisms by which these two compounds affect protein biosynthesis.

The effect of ABA and AMO-1618 on the preharvest sprouting of triticale caryopses

The experiments were conducted on developing triticale var. Grado caryopses. Treatment of freshly gathered, unripe triticale caryopses with abscisic acid (ABA) decreased their precocious germination throughout the entire period of development and ripening. The more mature the embryos that were germinated, the lower the inhibition by ABA. This indicates that the sensitivity of the embryo to ABA decreases during the course of caryopsis development and that the role that this hormone can play in the prevention of spouting in ears during the final stages of maturation, is limited. A known inhibitor of gibberellin synthesis, the retardant AMO-1618, was also tested in these experiments. This compound caused the reduction of germination capability only in the initial stages of development of triticale caryopses. Its most visible effect was noted during the germination of caryopses collected 30 days after flowering. The effect of this compound on the precocious germination of isolated embryos also decreased as the degree of maturity of these embryos increased. In the opinion of this author, this is connected with the fact that intense gibberellin synthesis and accumulation occurs only in the early stages of caryopsis development.

Studies on the role of gibberellins in the regulation of spermatogenesis in Chara vulgaris L.

Antheridia from isolated nodes of Chara vulgaris, developing in the presence of either AMO-1618 or GA₃, were studied. AMO-1618 which lowers the level of endogenous gibberellins causes a significant, proportional to the concentration, reduction in: 1) the number of antheridial filaments formed in antheridia, 2) spermatid number within a filament, as a result of eliminating one mitotic division at the first stage of spermatogenesis. Exogenous GA₃ at the concentration 10^-5 M evokes opposite effect i.e. increase in the number of antheridial filaments and increase in the number of spermatids within filament. Total number of spermatids within an antheridium decreases under the influence of 10^-4M AMO-1618 three times in comparison with the control, whereas it increases twice following 10^-5M GA₃ treatment. It has been suggested that the normal course of spermatogenesis requires precisely determined level of endogenous gibberellins.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: search for the enzyme’s repressor derived from mevalonate

Three inhibitors of squalene 2, 3-oxide–lanosterol cyclase (AMO 1618, 4, 4,10β-trimethyl- trans -decal-3β-ol (TMD) and 2, 3-iminosqualene (ISq)) were used to study effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and on sterol and polyprenyl synthesis from [ 14 C]acetate and [ 14 C]mevalonate in cultured rat hepatoma (H4) cells. After a 4 h exposure of cultures to AMO 1618 or TMD, followed by removal of the inhibitors, the utilization of [ 14 C]acetate for synthesis of digitonin-precipitable sterols increased about twofold, an increase parallelled by the rise in HMG-CoA reductase. Mevalonate at 2.3 mM counteracted the effects of these inhibitors on the reductase. When ( R )-[2- 14 C]mevalonate at 2.3 mM was included with the two inhibitors in the culture media, the cells were still able to synthesize cholesterol although in lesser amounts than the controls. In the presence of TMD the H4 cells also accumulated [ 14 C]squalene 2, 3-oxide and [ 14 C]squalene 2, 3-22, 23-dioxide. ISq added to cells kept in full-growth medium (10 μg ml -1 ) caused an almost complete and irreversible inactivation of the squalene oxide–lanosterol cyclase but did not inhibit polyprenyl synthesis, as the amount of [ 14 C]mevalonate converted into squalene, squalene 2, 3-oxide, squalene 2, 3-22, 23-dioxide plus a little cholesterol was equal to the amount converted by control cells into cholesterol plus squalene. After a 24 h exposure of cells kept in full-growth medium to ISq (10 μg ml -1 ), the levels of HMG-CoA reductase rose about twofold. ISq completely abolished the suppressive effect of 2.3 mM ( R )-mevalonate on the reductase. Chromatin isolated from cell nuclei contains cholesterol, which is renewed biosynthetically. It is argued that the suppressor of HMG-CoA reductase, derived from mevalonate, is a sterol and not a non-steroidal product of mevalonate metabolism.