Low temperature enhances petunia flower pigmentation and induces chalcone synthase gene expression

Flower coloration is controlled by internal and external factors, including temperature. The aim of the present work was to examine the effect of temperature on anthocyanin synthesis and chalcone synthase gene ( chs ) expression in petunia flowers. A moderate-low temperature enhanced both anthocyanin accumulation and chs expression in the corollas. However, the effect on chs expression was not always correlated with that on anthocyanin content, suggesting a post-translational effect. The effect was local and required the exposure of corollas, but not the whole plant, to the ambient temperature. The response of chs to moderate-low temperatures did not coincide with its expression during flower development. Moderate-low temperatures only slightly affected gibberellic acid (GA₃)-induced chs expression in the light, but activated chs expression under non-inducing conditions, i.e. in the absence of GA₃ in the dark. The results of this study suggest that moderate-low temperatures do not simply enhance the developmental regulation of anthocyanin biosynthetic gene expression; they act as a specific and separate signal.     

Leaf-Mediated Light Responses in Petunia Flowers

In the present work we studied the role of light in the regulation of flavonoid gene expression and anthocyanin synthesis in petunia (Petunia hybrida) corollas. We found that light is required for chalcone synthase gene (chs) expression, anthocyanin synthesis, and growth of detached and attached petunia corollas. Although direct illumination induced chs expression, pigmentation, and elongation of the detached corollas, irradiation of green leaves or sepals played the main role in the attached corollas. The duration, intensity, and spectrum of the light reaction suggest that phytochrome-mediated high-irradiance reactions are involved in the regulation of corolla development. Using the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, we showed that photosynthesis does not significantly contribute to the leaf-mediated light responses. When sepals were removed or covered. [14C]sucrose up-take by the corolla of detached intact flowers was inhibited. The results of this study suggest that light is perceived by leaves and sepals and enhances corolla sink activity, elongation, pigmentation, and chs expression. The role of leaves and sepals in the light regulation of petunia corolla development is discussed.     

Sugars enhance the expression of gibberellin-induced genes in developing petunia flowers

Sugar is essential for the development of detached Petunia hybrida flowers. We have shown that sucrose (Suc) and gibberellic acid (GA₃) are required for anthocyanin accumulation and the expression of various genes in developing petunia corollas. The effect of GA₃ on the expression of the gibberellin-induced gene and chalcone synthase gene, in detached corollas, was promoted by metabolic sugars such as Suc, glucose (Glc) and fructose, but not by the nonmetabolized 3- O -methylglucose and the sugar alcohol, mannitol. Several pieces of evidence support sugars' signaling role in the corollas and the possible involvement of hexokinase as the sugar sensor. Mannose, which is inefficiently metabolized but is phosphorylated by hexokinase at efficiency similar to Glc, was as effective as Glc in promoting gene expression and pigmentation. 2-Deoxyglucose, which is a substrate for hexokinase but is not metabolized in glycolysis, also promoted gene expression. On the other hand, mannoheptulose, a competitive inhibitor of hexokinase, completely abolished the promotive effect of Glc. We suggest that sugar-phosphorylation-related signal transduction interacts with the gibberellin signal to induce gene expression and anthocyanin accumulation in developing petunia corollas.     

Ca2+, calmodulin, and protein dephosphorylation are required for GA-induced gene expression in petunia corolla

Gibberellic acid (GA₃) induces the expression of different genes, including chalcone synthase ( chs ) and gip , in detached petunia corollas. To initiate a study on gibberellin (GA)-signal transduction in this tissue, we examined the effect of agents that inhibit or promote specific steps in signal-transduction pathways. The calcium chelator 1,2- bis ( o -aminophenoxy)ethane N,N,N ' ,N '-tetraacetic acid (BAPTA) had no effect on GA-induced gene expression, while the calcium-channel blocker, ruthenium red (RR), inhibited the activation of the genes. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) inhibited the induction of chs and gip by the hormone, and its analog, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5), had lower effect. The activation of chs and gip by GA₃ was completely blocked by the protein phosphatase inhibitor, okadaic acid (OA), and partially inhibited by the protein kinase inhibitor, 1-(5-isoquinoline-sulfonyl)- 2-methylpiperazine dihydrochloride (H-7). We suggest that Ca²⁺ from intracellular sources, calmodulin and protein dephosphorylation and phosphorylation are involved in GA-induced gene expression in petunia corollas.     

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.     

The germination characteristics of phytochrome-deficient aurea mutant tomato seeds

The role of light reactions in anthocyanin synthesis was studied in both attached and detached corollas of Petunia hybrida (cv. Hit Parade Rosa), the latter grown in vitro in media containing 150 m M sucrose and 50 μ M gibberellic acid (GA). Light was essential for the synthesis of anthocyanin in detached corollas, whereas in intact corollas its effect was only to enhance anthocyanin synthesis. Continuous white light at a fluence rate of at least 20 μmol m⁻² s⁻¹ was needed for anthocyanin synthesis in detached corollas. Blue light was more effective than red or green, and far-red was ineffective. Pigmentation of detached corollas exposed to light was inhibited by the photosynthetic inhibitor 3-(4-dichlorophenyl)-1,1-dimethylurea (DCMU). The chloroplast uncoupler NH₄Cl did not affect anthocyanin synthesis, which was, however, inhibited by the blocking of ATP synthesis in both the chloroplast and the mitochondria by dicyclohexylcarbodiimide (DCCD). Sucrose uptake in vitro was inhibited by DCMU and by darkness, and was promoted equally by blue and red light. The activity of phenylalanine ammonialyase (EC 4.3.1.5) was inhibited in detached corollas grown in the dark or in the light in the presence of DCMU. The activity of chalcone isomerase (EC 5.5.1.6) was not affected by light. These findings suggest that at least two different light reactions are involved in the regulation of anthocyanin synthesis in petunia corollas, namely the high irradiance reaction (HIR) and photosynthesis.     

Gibberellic acid decreases anthocyanin accumulation in wild carrot cell suspension cultures but does not alter 3'-nucleotidase activity

Gibberellic acid (GA₃) applied at different times during the growth of wild carrot ( Daucus carota ssp. Carota ) cell suspension cultures inhibited anthocyanin accumulation. Application of 3 × 10^–6 M GA₃ to cultures on day 0 or day 4 gave, respectively, 10 or 35% of anthocyanin accumulation relative to levels occurring when GA₃ was applied at the end of the growth period. Endogenous GAs were separated by high pressure liquid chromatography, and identified and quantified by gas chromatography-selected ion monitoring. Gibberellins GA₁, GA₃ and traces of GA₈. GA₁₉ and GA₂₀ were identified in carrot cell suspension cultures of both high and low anthocyanin-accumulating clones. The concentrations of GA₁. GA₃ and GA₈ in the two clones were similar and were not significantly different after the application of uniconazole which promoted anthocyanin accumulation. This suggests that these endogenous GAs are not the sole factors controlling the accumulation of anthocyanin in these different clones. Exogenous GA₃ and uniconazole had no effect on 3'-nucleotidase and 5'-nucleotidase activity in the carrot cell suspension cultures. Thus 3'-nucleotidase does not appear to play a role in the inhibition of anthocyanin accumulation by exogenous GA₃.     

Sugar-Dependent Gibberellin-Induced Chalcone Synthase Gene Expression in Petunia Corollas

The induction of anthocyanin synthesis and anthocyanin biosynthetic gene expression in detached petunia (Petunia hybrida) corollas by gibberellic acid (GA3) requires sucrose. Neither sucrose nor GA3 alone can induce these processes. We found that GA3 enhances sucrose uptake by 20 to 30%, and we tested whether this is the mechanism by which the hormone induces gene expression. Changing the intracellular level of sucrose with the inhibitors p-chloromercuribenzenesulfonic acid and vanadate did not inhibit the induction of chalcone synthase gene (chs) expression by GA3. Growing detached corollas in various sucrose concentrations did not affect the induction of the gene but did affect its level of expression and the level of anthocyanin accumulated. Only metabolic sugars promoted GA3-induced anthocyanin accumulation. Mannitol and sorbitol had no effect and 3-O-methylglucose only slightly promoted chs expression and anthocyanin accumulation. Our results do not support the suggestion that sugars act as specific signals in the activation of anthocyanin biosynthetic gene expression during petunia corolla development. We suggest that sugars are essential as general sources of carbohydrates for carbon metabolism, upon which the induction of pigmentation is dependent.     

Effect of chlorocholine chloride and gibberellic acid on the anthocyanin synthesis in radish seedlings

Anthocyanin synthesis in radish ( Raphanus sativus L. cv. Scarlet Globe) seedlings after treatment with chlorocholine chloride (CCC) and gibberellic acid (GA) has been investigated. CCC promotes and GA₃ inhibits the synthesis. When both substances are given together, CCC reverses the inhibition caused by GA₃. Simultaneous external feeding of anthocyanin precursors (sucrose and phenylalanine) reverses the GA₃ inhibition. A higher amount of total free amino acids, in particular phenylalanine, was present in CCC-treated seedlings compared to controls grown on distilled water. The amount of phenylalanine was lower in seedlings treated with both CCC and GA₃ as compared to seedlings treated with CCC alone, and total free sugars (reducing plus non-reducing) was lower in CCC treated seedlings than in controls grown on distilled water. We conclude that CCC and GA3 control the anthocyanin synthesis at the level of precursors.     

Methyl Jasmonate Induces Gums and Stimulates Anthocyanin Accumulation in Peach Shoots

The effect of methyl jasmonate (JA-Me) on the induction of gum was studied in relation to the action of ethylene in peach (Prunus persica Batsch cv. Benishimizu) shoots. JA-Me applied at concentrations of 0.1–2.5% (w/w) in lanolin paste to current growing or older shoots substantially induced gums 3 days after treatment. The amount of gums exuded increased depending on the dose of JA-Me. Ethephon (2-chloroethyl- phosphonic acid) at 1 or 2% (w/w) in lanolin induced gum and strongly enhanced the promoting effect of JA-Me on gum formation. JA-Me also induced anthocyanin accumulation in current growing shoots, but ethephon did not. Anthocyanin accumulation in response to JA-Me at a concentration of 10 mg/liter or higher was observed also in the cut shoots of peach. Ethephon (100 mg/liter) substantially inhibited anthocyanin accumulation induced by JA-Me. These facts suggest that JA-Me plays an important role in gum formation as well as ethylene and in anthocyanin accumulation and that these processes are not necessarily accompanied by each other in peach shoots. Received January 26, 1998; accepted March 4, 1998     

The end-of-day far-red irradiation increases gibberellin A1 content in cowpea (Vigna sinensis) epicotyls by reducing its inactivation

It has been shown previously that gibberellins (GAs) mediate the phytochrome (Phy) control of cowpea ( Vigna sinensis L.) epicotyl elongation induced by end-of-day (EOD)-far-red light (FR). In the present work, the EOD-FR effect on GA metabolism and GA levels in cowpea has been investigated. GA₁, GA₈, GA₁₉ and GA₂₀ were identified in epicotyls, and GA₁, GA₁₉, GA₂₀ and GA₂₉-catabolite in leaves of 6-day-old cowpea seedlings. The content of GA₁ in the epicotyl paralleled the decrease of its growth rate, supporting the hypothesis that this is the GA bioactive in controlling cowpea epicotyl elongation. FR enhanced both the amount of [3H]GA₁ in the epicotyl produced from applied [3H]GA₂₀, and that of applied [3H]GA₁ that remained unmetabolized in epicotyl explants, suggesting that Phy may regulate the inactivation of GA₁. In agreement with this effect of light on GA₁ metabolism, the contents of GA₁ in the epicotyl remained higher in FR-treated than in R-treated explants. Moreover, in intact seedlings EOD-FR treatment increased both epicotyl elongation and GA₁ content in the responsive epicotyl, whereas it was not altered in the leaves. These results show, for the first time, that photostable Phys modulate the stem elongation in light-grown plants by locally controlling the GA₁ levels through regulation of its inactivation.     

An acylcyclohexadione retardant inhibits gibberellin A1 metabolism, thereby nullifying phytochrome-modulation of cowpea epicotyl explants

The regulation by phytochrome of stem elongation in light-grown plants depends on gibberellins (GAs). To investigate whether this is mediated by a change in GA metabolism, the effect of the GA biosynthesis inhibitor LAB 198 999 (an acylcyclohexadione derivative) on the end-of-day far-red (FR) response in cowpea ( Vigna sinensis L.) epicotyl explants has been investigated. Growth of epicotyl explants of light-grown seedlings was enhanced when treated with far-red light before incubation in the dark (end-of-day FR effect). Low doses of LAB 198 999 (0.05 and 0.5 μg explant⁻¹) reduced the effect of FR, whereas 5 to 50 μg explant⁻¹ stimulated elongation of both red light (R)- and FR-treated epicotyl explants while nullifying the differences between R and FR treatments. In paclobutrazol-treated epicotyl explants, FR enhanced the response to applied GA₁ and GA₂₀, whereas LAB 198 999 increased the activity of GA₁ and decreased that of GA₂₀, [³H]Gibberellin A₁, injected into the basal part of the epicotyl, was transported and metabolized mainly to [³H]GA₈ in the apical 20 mm of the epicotyl. The conversion of [³H]GA₁ to [³H]GA₈ was dramatically reduced by both end-of-day FR treatments and LAB 198 999 applications. In addition, both treatments enhanced epicotyl elongation. It is proposed that the regulation of cowpea epicotyl growth by phytocrome is mediated, at least partially, by modifying GA₁ degradation.     

Internode length in Pisum. IV. The effect of the Le gene on gibberellin metabolism

The highly active, polar gibberellin-like substance found in the apical region of shoots of tall (genotype Le ) peas ( Pisum sativum L.) is shown by combined gas chromatography-mass spectrometry (GC/MS) to be GA₁. This substance is either absent or present at only low levels in dwarf ( le ) plants. Multiple ion monitoring (MIM) tentatively suggests that GA₈ may also be present in shoot tissue of tall peas. Gibberellin A₁ is the first 3 β-hydroxylated gibberellin positively identified in peas, and its presence in shoot tissue demonstrates the organ specificity of gibberellin production since GA₁ has not been detected in developing seeds. Application of GA₁ can mask the Le/le gene difference. However, whilst Le plants respond equally to GA₂₀ and GA₁, le plants respond only weakly to GA₂₀, the major biologically active gibberellin found in dwarf peas. These results suggest that the Le gene controls the production of a 3 β-hydroxylase capable of converting GA₂₀ to GA₁. Further support for this view comes from feeds of [³H] GA₂₀ to Le and le plants. Plants with Le metabolise [³H] GA₂₀ to three major products whilst le plants produce only one major product after the same time. The metabolite common to Le and le plants co-chromatographs with GA₂₉. The additional two metabolites in Le peas co-chromatograph with GA₁ and GA₈.     

Gibberellins in relation to flowering in Polianthes tuberosa

Endogenous gibberellins (GAs) in corms of Polianthes tuberosa L. (cv. Double) were isolated and identified by high performance liquid chromatography, bioassay and combined capillary gas chromatography-mass spectrometry (GC-MS). Gibberellins A₁, A₁₉, A₂₀ and A₅₃ were quantified at the vegetative, early floral initiation and flower development stages. The identification of 13-hydroxylated GAs indicates the presence of the early 13-hydroxylation pathway in P. tuberosa corms. An increase in GA₁ and GA₂₀, and a decrease in GA₁₉ levels, coincided with the transition from the vegetative phase to the stages of early floral initiation and flower development. GA₅₃ stayed at constant levels at the 3 different growth stages. The absence of GA₁ in vegetative corms and its presence in corms at early floral initiation and flower development stages suggest that GA₁ is a causal factor in inducing floral initiation in P. tuberosa . When GA₁, GA₃, GA₄, GA₂₀ and GA₃₂ were applied to corms at the vegetative stage (plants about 5 cm in height), floral initiation was promoted by all of the GAs used, GA₃₂ being the most active. In contrast with the other GAs, GA₃₂ had no effect on stem elongation. Therefore, it is suggested that hydroxylated C-19 GAs play an important role in flower induction in P. tuberosa .     

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.     

Effects of Methyl Jasmonate on Anthocyanin Accumulation, Ethylene Production, and CO2 Evolution in Uncooled and Cooled Tulip Bulbs

Effects of methyl jasmonate (JA-Me) on anthocyanin accumulation, ethylene production, and CO₂ evolution in uncooled and cooled tulips (Tulipa gesneriana L. cvs. Apeldoorn and Gudoshnik) were studied. JA-Me stimulated anthocyanin accumulation in stems and leaves from uncooled and cooled bulbs of both cultivars. The highest level of anthocyanin accumulation was observed in leaves from cooled bulbs treated with 200 μL/liter JA-Me. In sprouting bulbs treated with 100 μL/liter and higher concentrations of JA-Me, the ethylene production began to increase at 3 days after treatment, being extremely greater in uncooled bulbs than in cooled ones. JA-Me also stimulated CO₂ evolution in both cultivars, depending on its concentrations. CO₂ evolution in sprouting bulbs was not affected by cooling treatment. These results suggest that anthocyanin accumulation by JA-Me in tulip leaves is not related to ethylene production stimulated by JA-Me. Received October 10, 1997; accepted November 17, 1997