Hormonal regulation of barley nuclease: investigation using a monoclonal antibody

Abstract. A monoclonal antibody prepared against barley ( Hordeum vulgare L., cv. Himalaya) nuclease (EC 3.1.30.2) was characterized with solid-state enzyme-linked immunosorbent assays and immuno-blotting. The antibody was specific for intracellular and secreted nuclease. Hormonal regulation of the synthesis and secretion of nuclease in isolated aleurone layers was investigated by immunoprecipitation of biosynthetically-labelled nuclease using polyclonal antibodies and by immunoblot analyses using the monoclonal antibody, respectively. Gibberellic acid (GA₃) induced the de novo synthesis and secretion of nuclease in a time-and concentration-dependent manner. Nuclease was detected in aleurone layers incubated in 1 mmol m⁻³ GA₃, after 24 h. The maximum rates of nuclease synthesis and secretion occurred 36–48 h after hormone treatment. A minimum concentration of 10⁻⁶ mol m⁻³ GA₃ was required for nuclease synthesis and secretion, whereas the maximum rate of nuclease secretion occurred at concentrations of 10⁻⁵ mol m⁻³ and higher. In the presence of abscisic acid, the synthesis and secretion of nuclease from GA₃-treated aleurone layers was almost completely inhibited. Based on these findings, the authors conclude that all nuclease within and secreted from aleurone layers treated with GA₃ is the result of its de novo synthesis.     

Regulation of alcohol dehydrogenase gene expression in barley aleurone by gibberellin and abscisic acid

The expression of the Adh1 gene (alcohol dehydrogenase, EC 1.1.1.1) was studied in the aleurone layer of barley ( Hordeum vulgare cv. Himalaya). Expression increased markedly during grain development at the levels of activity, enzyme protein and mRNA. mRNA content, but not enzyme activity, could be increased further by exogenous abscisic acid (ABA) when isolated, de-embryonated developing grains were pre-treated with gibberellic acid (GA₃) or fluridone. In isolated mature aleurone layers incubated with exogenous hormones, ADH mRNA was strongly up-regulated by ABA and down-regulated by GA₃ within 6 h. With ABA, this increase in mRNA was followed by an increase in ADH protein and activity, peaking at 18 h. With GA₃, the decrease in mRNA was accompanied by simultaneous decreases in protein and activity. In general, GA₃ counteracted the effect of ABA and vice versa. In the aleurone of germinating grain, ADH activity decayed in a distal direction from the embryo, consistent with down-regulation by gibberellin(s) diffusing from it. It was concluded that ADH gene expression in the aleurone of the intact grain is regulated by an ABA/gibberellin interaction.     

Metabolism of C19- and C20-gibberellins by cell-free preparations from immature Phaseolus coccineus seed

Gibberellin biosynthesis pathways were investigated using isotopically-labelled C₁₉- and C₂₀-gibberellins and cell-free preparations from immature seed of Phaseous coccineus cv. Prizewinner. The initial steps in an early 13-hydroxylation pathway involved the conversion gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to GA₁₂ which was 13-hydroxylated to yield GA₅₃, Metabolism of GA₅₃ yielded GA₄₄. In contrast to other cell-free systems, GA₄₄ was not further converted, either as a δ-lactone or an open-lactone structure, to the C-20 aldehyde GA₁₉. GA₁₉ was, however, metabolised to GA₂₀, GA₅ and GA₁. GA₂₀ represented a branch point in the pathway as it was converted both to GA₁, which was an end product, and GA₅ which was further converted to GA₆. Like GA₁, GA₆ was also an end-product of the early 13-hydroxylation pathway.
A non-13-hydroxylation pathway involving GA₄, GA₁₅, GA₂₄ GA₃₇ and GA₃₆ also originated from GA₁₂. The terminal product of this pathway was the 3β-hydroxy C₁₉-gibberellin, GA₄.     

Role of endogenous gibberellins in germination of melon (Cucumis melo) seeds

The effect of the plant growth retardants ancymidol. mefluidide and uniconazole on germination of two melon accessions differing in their ability to germinate at 14°C was examined. The accessions were the cold sensitive Noy Yizre'el and the cold tolerant Persia 202. The three growth retardants were able to delay the germination of intact Noy Yizre'el seeds, but did not affect that of intact Persia 202 seeds. On the other hand germination of decoated seeds of both accessions was unaffected by these inhibitors at normal oxygen concentration, but was inhibited at 5% oxygen. When gibberellin-like activity was measured by a dwarf rice biological assay following HPLC fractionation, it was found that seeds of Persia 202 contained much more gibberellin-like activity than Noy Yizre'el seeds. Among the extracted compounds several endogenous gibberellins were identified by combined gas chromatography-mass spectrometry (GC-MS). They included GA₄, GA₂₀, GA₁ and GA₃ in Noy Yizre'el and GA₃₄, GA₂₀, GA₁ and GA₈ in Persia 202. It is suggested that the better germination of intact Persia 202 seeds, compared to Noy Yizre'el seeds at low temperature and low oxygen concentration, is due to a higher endogenous level of GA and a better seed coat permeability to oxygen.     

Effects of temperature and drying rate during dehydration of celery seeds on germination, leakage and response to gibberellin and cytokinin

A high temperature treatment of 32°C which prevents dehydration injury in celery seeds imbibed for 3 days at 17°C and then dried at 20°C, reduced leakage during rehydration, compared with seeds not given the high temperature treatment. Treatments which would normally release celery seeds from dormancy, such as low temperature imbibition or gibberellin (GA_4/7) and benzyladenine (BA) applications had little effect on the germination of seeds exhibiting desiccation injury. However, GA_4/7 did induce splitting of the seed coat and swelling of the endosperm, and this effect was enhanced by BA. It is suggested that in celery seeds high temperature prevents irreversible embryo damage, including membrane damage, caused by drying.     

Ethylene-dependent action of gibberellin in seed germination of Amaranthus caudatus

The role of gibberellins in the germination of seeds of Amaranthus caudatus L. was examined. Tetcyclacis (BAS 106), an inhibitor of gibberellin biosynthesis, inhibited germination of the seeds. The inhibition caused by BAS 106 was antagonised by gibberellin A₄₊₇ (GA₄₊₇). Ethephon (2-chloroethylphosphonic acid) and 1-aminocyclopropane-1-carboxylic acid (ACC) could replace GA₄₊₇. Ethephon and ACC counteracted also the side effects of BAS 106 that are not reversible by GA₄₊₇. The rate of seed germination was not increased by gibberellin in the presence of aminoethoxyvinylglycine (AVG). AVG increased the effect of BAS 106. GA₄₊₇ could not reverse the effect of BAS 106 when AVG was simultaneously applied. The results indicate that the biosynthesis of endogenous gibberellins may be required for germination of A. caudatus seeds and that main physiological effects of GA₄₊₇ on seed germination may depend on ethylene biosynthesis.     

Factors affecting the induction and release of secondary dormancy in Kalanchoë seeds

Abstract. Several short daily R irradiations are required from the first day of incubation on water to induce germination of Kalanchoë seeds. When the same light treatment is given after a prolonged dark incubation period at 20°C, secondary dormancy prevents germination. Factors controlling the induction and breaking of secondary dormancy have been investigated. The induction of secondary dormancy is very temperature dependent. Locally puncturing the seed coat strongly delays it. Secondary dormancy is not induced in the presence of GA₃ during the first 10 d of dark incubation, although this growth substance cannot induce dark germination. Prolonged or cyclic daily R irradiations can relieve secondary dormancy of seeds kept on water, even after a dark period of 20 d. A 24 h treatment at 4°C restores responsiveness to short R exposures of slightly secondarily dormant seeds. The synergism between GA₃ and Pfr in non-dormant Kalanchoë seeds, leading to high effectiveness of even one short FR irradiation, still occurs in seeds made secondarily dormant before transfer to GA₃, but more R or FR irradiations, in combination with GA₃, are required for the release of secondary dormancy. A combination of red light and 6-benzyl-aminopurine is ineffective in removing dormancy.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

Comparison of endogenous gibberellins in roots and shoots of elongating Salix pentandra seedlings

Gibberellins GA₁, GA₈. GA₁₉. GA₂₉. GA₂₀ and GA₅₆ (2-epi-GA₈). were identified by combined gas chromatography-mass spectrometry in root extracts of elongating Salix pentandra L. seedlings. The presence of GA₈ was also demonstrated for the first time in S. pentandra shoots. The levels of GA₁, GA₈, GA₁₉, GA₂₀ in shoot tissue and in roots were estimated by selected ion monitoring. While the amounts of GA₈ and GA₁₉ were similar in both plant parts. the levels of the biologically active GA₁ and its immediate precursor GA₂₀. were found to be much lower in roots than in shoots.     

Identification and quantitation of gibberellins in fruits of Lycopersicon esculentum, and their relationship to fruit size in L. esculentum and L. pimpinellfolium

GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀ and GA₂₉ were identified by combined gas chromatography-mass spectrometgry (GC-MS) in immature seeds and pericarp of Lycopersicon esculentum Mill. (tomato). Higher levels of these GAs were present in the seeds than in the pericarp; seeds in addition contained GA₁₅, GA₂₄, GA₂₅, and GA₄₄. Fruits of the Lycopersicon pimpinellifolium Mill. mutant I were smaller and contained lower GA₁ concentrations, but higher GA₂₀ concentrations, than those of mutants III and IV. In contrast, differences in fruit size in L. esculentum due to position on the truss did not correlate with GA₁ concentration in either the pericarp or seeds.     

Photoperiodic control of endogenous gibberellins in seedlings of Salix pentandra

In the temperate-zone woody species Salix pentandra elongation growth is regulated by the photoperiod. Long days sustain active growth, whereas short days induce cessation of apical growth, which is a prerequisite for winter hardening. It is shown that this is correlated to quantitative changes in levels of endogenous GA₁₉ GA₂₀, and GA₁. Within two short days the amount of the active GA₁ and its immediate precursor GA₂₀, decreased markedly in young leaves us well as in stem tissue. Also, the amount of GA¹⁹, declined, but the decrease was delayed relative to that of GA₁ and GA₂₀. The ability of S. pentandra seedlings to respond to exogenous GA₁₉, decreased with increasing numbers of short days. Observations that support the hypothesis that the level of GA₁ in S. pentandra is regulated by the photoperiod in a quantitative mode with conversion of GA₁₉, to GA₂₀, being one target for control.
Different distribution of GAs in various plant parts was observed. The level of GA was higher in young leaves than in other plant parts, and the amount of GA₁₉ was 5–10 times higher in stem tissue than in leaves and roots. The ratios of GA₈ to GA¹ and GA₂₀, were higher in roots as compared with other parts, as rods contained very low levels of GA₁ and GA₂₀, but amounts of GA₂₀ comparable with other parts.     

The role of hormones in regulating the germination of polymorphic seeds and early seedling growth of Atriplex triangularis under saline conditions

Germination of the polymorphic seeds and seedling growth of Atriplex triangularis under various salinity, gibberellic acid and kinetin treatments were determined. Gibberellic acid (GA₃; 2.9 m M ) promoted germination and growth at high NaCl concentrations (345 m M ). Kinetin (4.7 μ M ) stimulated germination at all salinities and seed sizes tested. GA₃ and kinetin generally increased seedling growth at all concentrations of salinity studied. Higher concentrations of kinetin were found to be inhibitory.     

Gibberellins and photoperiodic control of shoot elongation in Salix

Effects of exogenous gibberellins GA₅₃, GA₄₄, GA₁₉, GA₂₀ and GA₁ on photoperiodically controlled shoot elongation in seedlings of Salix pentandra L. were studied. Gibberellins GA₂₀ and GA₁ induced shoot elongation under short days (SD) and could substitute for a transfer to long day (LD), while gibberellins A₅₃, A₄₄ and A₁₉ were inactive. In seedlings exposed to a prolonged SD-treatment (30 days) there was a significant positive interaction between a transfer to LD and a treatment with GA₂₀ and GA₁ on shoot elongation. In addition, GA₁₉ enhanced the growth promotive effect of LD in these seedlings. The results are compatible with the suggestion that conversion of GA₁₉ to GA₂₀ is blocked under SD. This effect is supposed to be an early process leading to the cessation of shoot elongation under SD. Responsiveness of the seedlings to LD and to a GA-treatment gradually decreased with an increasing length of exposure to SD.     

Effects of different gibberellins on elongation growth under short day conditions in seedlings of Salix pentandra

Fifteen different gibberellins (GA's) were tested for their ability to induce elongation growth under short day conditions in seedlings of Salix pentandra L. GA's were applied either to the apex or they were injected into a mature leaf. GA₃ was highly active and also GA₄₊₇ and GA₄ showed high activity. GA₁, GA₂, GA₅, GA₉, GA₁₃, GA₂₀, GA₃₆ and GA₄₇ showed moderate activity. GA₁₆, GA₁₇, GA₂₇ and GA₄₁ exhibited low or no activity in doses up to 10 μg per plant. In general, a better growth response was obtained with an application to the apex than with an injection into the leaf.