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.     

Abscisic acid and gibberellic acid-regulated responses of embryos and aleurone layers isolated from dormant and nondormant barley grains

Dormant and nondormant isogenic barley grains were obtained by maturing grains under short day (SD) or long day (LD) growth conditions, respectively. Hormonal responses of isolated embryos and aleurone layers from these grains were studied. Addition of abscisic acid (ABA) reduced germination rate and percentage of embryos, and induced Rab (ABA-responsive) mRNA in aleurone layers from both types of grain. Embryos and aleurone layers from dormant grains responded stronger to ABA than those from nondormant grains. Gibberellic acid (GA₃) increased the germination rate and percentage of embryos from dormant grains and counteracted the ABA-induced inhibition of embryo germination. GA₃ did not affect the amount of Rab mRNA in aleurone layers, suggesting that expression of the Rab gene has no direct correlation with germination. The stronger response of embryos and aleurone layers from dormant grains to ABA may not be explained by higher endogenous ABA levels, but might be due to differences in hormone signal transduction. Aleurone protoplasts from dormant grains had a higher cytosolic pH than those from nondormant grains. To inhibit the ABA-induced Rab mRNA, a much higher concentration of weak acid was required for aleurone layers from dormant grains than for those from nondormant grains. A possible difference in ABA signal transduction between dormant and nondormant grains is discussed.     

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.     

Temporal, spatial and hormonal regulation of the S-adenosylmethionine synthetase gene in petunia

Gibberellic acid (GA₃) promotes corolla elongation and pigmentation in petunia flowers. We have previously shown that G.A₃ induces pigmentation by activating specific genes of the anthocyanin biosynthetic pathway. The aim of the present work was to examine whether GA₃ induces also the expression of genes from other metabolic pathways in petunia corollas that may be associated with growth. Recently we reported the cloning of the petunia sam gene coding for S -adenosylmethionine synthetase (SAM-S). In the present work we show that sam expression is induced by GA₃ in both corollas and stems. The expression of the gene was correlated with corolla elongation. GA₃ and the cylokinin, N -6-benzyladenine (BA) promoted corolla growth and sam expression, whereas abscisic acid (ABA) inhibited corolla elongation and repressed sam mRNA accumulation. An analysis of sam expression in stems indicated a high level in young, elongating internodes and a very low level in the mature, non-elongating stem zone. The results of the present study show that the effect of GA₃ on gene expression in the corolla of petunia, is not restricted to the anthocyanin biosynthetic pathway, they also suggest a possible role for sam in GA₃-induced corolla and stem elongation.     

Effects of the triazole plant growth retardant BAS 111¨W on gibberellin levels in oilseed rape, Brassica napus

Three-week-old shoots of the spring oilseed rape cv. Petranova ( Brassica napus L. ssp. napus ) were found by combined gas chromatography-mass spectrometry to contain GA₁, GA₈, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₂₉, 3-epi-GA₁ and a previously uncharacterised C₁₉ dicarboxylic acid that is probably structurally related to GA₂₄. Shoots of the winter cultivar Belinda, harvested at the early flowering stage, contained the same GAs with the exception of the C₁₉ dicarboxylic acid and, in addition, GA₃₄ and GA₅₁ were identified. All material contained higher levels of GA₂₀ than of GA₁; the ratio of GA₁ to GA₂₀ was highest in shoots containing the largest proportion of young immature tissues. Soil treatment of cv. Petranova seedlings with the growth retardant BAS 111¨W [1-phenoxy-5,5-dimethyl-3-(1,2,4-triazol-1-yl)-hexan-4-ol] caused 80% reduction in height 18 days after treatment and the levels of all GAs were 20% or less that of control plants. Foliar treatment at the same dosage reduced height by 50% and caused an 85% or greater reduction in the concentrations of the GA₁ precursors GA₂₀, GA₁₉ and GA₄₄. However, the levels of GA₁, GA₈ and GA₂₉ were affected to a much smaller extent. Foliar application of BAS 111¨W to cv. Belinda 1 month after sowing resulted in only a 20% height reduction at flowering, but no uniform decrease in the concentrations of endogenous GAs at this stage.     

Effect of gibberellic acid on growth and carbohydrate metabolism of developing tubers of potato (Solanum tuberosum)

Potato plants (Solanum tuberosum L. cv. Ostara) were grown in aerated water culture in a controlled environment. When the tubers had reached a diameter of 1–3 cm. ¹⁴C-labelled or unlabelled gibberellic acid (GA₃) was applied to the surface of the stolons at points approximately 1 crn from the developing tubers, and treatment continued for 10 days. - Significant quantities of GA₃ moved into tuber tissue within 2–4 days of hormone application. This influx of GA₃ was accompanied by a marked reduction in both the activity of ADPG-pyrophospharylase and the ratio ADPG-pyrophosphorylase/starch phosphorylase and an increase in the activity of UDPG-pyrophosphorylase. Starch phosphorylase activity initially increased slightly but then fell, whereas the activity of starch synthase remained constant throughout the experiment. The soluble sugar composition of the tubers changed qualitatively towards a pattern characteristic of growing stolon tips prior to tuber initiation, but there was no clear evidence of net starch degradation. Changes in the activities of the enzymes were observed prior to noticeable effects of the hormone on tuber growth rate or the development of new stolons at the tuber eyes. - GA₃- treated tubers imported more ¹⁴C from labelled photosynthate than expected on the basis of growth rate. However, the capacity to convert solub#e-¹⁴C to ethaTiol-insoluble-¹⁴C (predominantly starch) was reduced in comparison with non-treated tubers. - The observed changes in carbohydrate composition and enzyme activities indicate that GA₃ induces a drastic change in potato tuber metabolism towards a pattern characteristic for the termination of the storage process.     

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.     

Ca2+ and AtCaM3 are involved in the expression of heat shock protein gene in Arabidopsis

The involvement of calcium and different calmodulin isoforms (Ca²⁺-CaM) in heat shock (HS) signal transduction in Arabidopsis ( Arabidopsis thaliana ) was investigated. Using transgenic Arabidopsis plants which have the AtHsp18.2 promoter/GUS fusion gene, it was found that the level of β -glucuronidase (GUS) activity was up-regulated by the addition of CaCl₂ and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl₃ and verapamil, or the CaM antagonists N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), chlorpromazine (CPZ) and trifluoperazine (TFP). CaCl₂ not only increased the GUS activity after HS, but also up-regulated the GUS activity under non-HS conditions. These results provide additional support for the involvement of the Ca²⁺-CaM signalling system in HSP gene expression. The expression of nine CaM genes (AtCaM1–9) from Arabidopsis was differentially regulated by HS at 37 °C. The expression of AtCaM3 and AtCaM7 genes increased during HS. The temporal expression of the AtCaM3, AtCaM7 and hsp18.2 genes demonstrated that up-regulation of AtCaM3 expression occurred earlier than that of AtCaM7 or hsp18.2 .     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Effects of hypochlorite disinfection on the response of barley aleurone layers to gibberellic acid

Although the use of hypochlorite to disinfect seeds is widespread, the effects on tissues isolated from them have been largely ignored. Disinfection of barley ( Hordeum vulgare L. cv. Himalaya) half-seeds with hypochlorite solutions of ≥1.0% (w/v) available chlorine caused the pericarp to separate from the underlying tissues. Aleurone layers isolated from these grains had lower rates of oxygen consumption and released significantly less protein, PO₄³⁻ Mg²⁺, K⁺ and amylase (EC 3.2.1.1) into the medium in response to gibberellic acid (GA₃) than layers isolated from grains disinfected with a 0.1% hypochlorite solution. Disinfection with 1.0% hypochlorite also quantitatively altered the spectrum of proteins released into the incubation medium by the layers in response to GA₃.     

Effect of plant growth hormones and polyamines on ornithine decarboxylase activity during the germination of barley seeds

Gibberellic acid (GA₃) and β-indolylacetic acid (IAA), two of the well known growth hormones, induce four fold the activity of ornithine decarboxylase (ODC) during the germination of barley seeds ( Hordeum vulgare L. var. Beca). The optimal concentration for induction of ODC was 10^–5 M for GA₃ and 10^–3 M for IAA. When 10^–3 M of a polyamine, putrescine or spermidine, is added to the growth medium, ODC activity is significantly inhibited. This inhibition is due to the induction of a protein inhibitor of ODC (antizyme), whose apparent molecular weight is 16 000 ± 2 000 daltons. Addition of GA₃ to cultures which have been grown for 50 or 98 h in the presence of polyamines, abolishes the observed inhibition of ODC activity, while in the reverse experiment, addition of polyamines at 50 or 98 h does not affect the ODC activity induced by GA₃. Cadaverine, a physiological plant diamine, enhances ODC activity; whereas 1,8-diaminooctane (the alkyl analogue of spermidine) does not have any effect.     

Gibberellin-induced elongation and osmoregulation in internodes of floating rice

Internodal elongation in floating rice ( Oryza sativa L. cv. Habiganj Aman II) is known to be enhanced by treatment with ethylene or gibberellic acid (GA₃) at high relative humidity (RH). However, ethylene-induced internodal elongation is inhibited at low RH. while GA₃-induced internodal elongation is hardly affected by humidity. We examined the possible involvement of osmoregulation in the stimulation by GA₃ of the elongation of internodes at low RH. Submergence and treatment with ethylene or GA3₃ at 100% RH increased the osmotic potential in internodes of excised stem segments, while GA₃ at 20% RH maintained the osmotic potential at a low level. In internodes of stem segments that had been treated with GA₃ at 20% RH, the activity of invertase and the level of soluble sugars were almost 2- and 1.5-fold higher, respectively, than those in internodes that had been treated with GA₃ at 100% RH. These results indicate that one of the possible mechanisms by which GA₃ promotes elongation of internodes at low RH involves the osmoregulation that is achieved by promotion of the synthesis of invertase.