Effects of Gibberellic Acid on Primary Terpenoids and △9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration,and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP)pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA3) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA3 caused a decrease in DXS activity in both sexes that it wasaccompanied by a decrease in chlorophylls, carotenoids and △9-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA3 showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants.The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA3 can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

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.     

Geibberllin and temperature influence carbohydrate content and flowering in Phalaenopsis

When Phalaenopsis amabilis is grown under high temperature (30/25°C, day/night), flowering is blocked, and this can be reversed by gibberellin A₃ (GA₃) treatment. Associated with GA₃ treatment under high temperature are increases in sucrose, glucose and fructose as compared with warm-treated plants. Spraying with sucrose solution alone caused leaf epinasty in plants grown under high temperature. Epinasty was released by about 9 days of GA₃ treatment. In GA₃-treated plants under high temperatures, sucrose application to the source leaves led to an increase in sugar content in both leaves and inflorescence. In contrast, although in warm-treated plants sucrose application to the source leaves increased sugar content in the leaves, it did not increase sucrose content in the inflorescence. These results corroborate our hypothesis that in Phalaenopsis GA₃ stimulates sink activity in the apical meristem and promotes the translocation of sucrose from source leaves to the apex of the inflorescence, where it accumulates. GA₃ treatment led to an increase in sucrose synthase activity and had no effect on invertase activity.     

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.     

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 Lathyrus odoratus. Effects of mutants l and lb on gibberellin metabolism and levels

After the application of [¹³C³H]-gibberellin A₂₀ to wild-type (tall) sweet peas ( Lathyrus odoratus L.) labelled gibberellin A₁ (GA₁), GA₈, GA₂₉ and 2-epiGA₂₉ were identified as major metabolities by gas chromatography-mass spectrometry after high performance liquid chromatography. By contrast in genetically comparable dwarf ( II ) plants only labelled GA₂₉ and 2-epiGA₂₉ were produced in significant amounts, although evidence was obtained for trace amounts of labelled GA₁ and GA₈. The apical portions of dwarf plants contained 8–10 times less GA₁ than those of tall plants but at least as much GA₂₀ (measured using di-deuterated internal standards). In conjunction with previous data these results strongly indicate that in genotype ll internode length is reduced and leaf growth altered by a reduction in GA1 levels attributable to a partial block in the 3β-hydroxylation of GA₂₀ to GA₁.
In contrast to dwarf plants, semidwarf plants (genotype lblb ) contained more GA₁ in the apical portion than wild-type counterparts. This is consistent with the suggestion that lb alters some aspect of GA sensitivity.     

Internode length in Lathyrus odoratus. The involvement of gibberellins

Evidence was obtained by gas chromatography-mass spectrometry and gas chromatography-selected ion monitoring for the presence of gibberellin A₂₀), GA₁, GA₂₉, GA₈ and 2-epiGA₂₉ in vegetative shoots of tall sweet pea, Lathyrus odoratus L. Both tall (genotype L –) and dwarf (genotype II ) sweet peas elongated markedly in response to exogenous GA₁ attaining similar internode lengths at the highest dose levels. Likewise internode length in both genotypes was reduced by application of the GA biosynthesis inhibitor, PP333. The ratio of leaflet length to width was reduced by application of PP333 to tall plants and this effect was reversed by GA₁. When applied to plants previously treated with PP333, GA₂₀ promoted internode elongation of L – plants as effectively as GA₁, but GA₂₉ was not as effective as GA₁ when applied to II plants. In contrast, GA₂₀ and GA₁ were equally effective when applied to the semidwarf lb mutant but GA-treated lblb plants did not attain the same internode length as comparable GA-treated Lb – plants. The difference in stature between the tall and dwarf types persisted in dark-grown plants. It is concluded that GA₁ may be important for internode elongation and leaf growth in sweet pea. Mutant l may influence GA₁ synthesis by reducing 3β-hydroxylation of GA₂₀ whereas mutant lb appears to affect GA sensitivity.     

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.     

Effect of gibberellic acid on K+ (Rb+) uptake and transport in sunflower roots

Four-week-old sunflower plants ( Helianthus annuus L. cv. Halcón), grown in different nutrient solutions, were used to study the effects of gibberellic acid (GA₃) on K⁺ (Rb⁺) uptake by roots or transport to the shoot. Gibberellic acid application to the nutrient solution did not affect the exudation process of excised roots. When GA₃ was sprayed on leaves 2 to 6 days before excising the roots, the rate of exudation and the K⁺ flux increased. When the exudation study was done keeping the roots in a nutrient solution in which Rb⁺ replaced K⁺, the GA₃ effects were evident also on Rb⁺ uptake and transport. In intact plants, GA₃ increased the Rb⁺ transported to the shoot but did not affect Rb⁺ accumulation in the root. It is suggested that these GA₃ effects can be explained if it is assumed that GA₃ acts on the transport of ions to the xylem vessels.     

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.     

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 substitution for long day secondary induction of flowering in Poa pratensis

Plants of Poa pratensis cv. Holt initiate inflorescence primordia when exposed to short days (SD) and low temperature, but require a secondary induction by at least 4 long days (LD) for further inflorescence development and stem elongation. Single or double applications of 10 µg per plant of gibberellins A₁, A₃, A₅ and 16,17‐dihydro A₅ (DHGA₅) induced inflorescence development in a high proportion of plants in SD, but only if the plants were detillered to a single stem. Exposure to 2 LD cycles did not cause heading and flowering alone but enhanced the effect of exogenous gibberellins (GAs), bringing flowering to 100%. GA₅ and DHGA₅ were less effective than GA₁ and GA₃ in SD, especially with double applications, but were more effective than GA₁ and GA₃ when given together with 2 LD. The GAs had differential effects on vegetative growth and flowering, GA₅ and DHGA₅ causing much less leaf and stem growth than the other two GAs. Marginal induction, whether by LD or GA application, resulted in a high proportion of spikelets with viviparous proliferation. Thus, whereas GAs are inhibitory to the primary induction by SD, they can replace secondary induction by LD when vegetative growth is limited.     

Effects of gibberellic acid on patterns of carbohydrate distribution and acid invertase activity in Phaseolus vulgaris

The application of gibberellic acid (GA₃,10 μ M ) as a root drench to 16-day-old plants of Phaseolus vulgaris L. cv. Masterpiece stimulated growth of the stem internodes and reduced root growth. GA₃ treatment did not affect the export of ¹⁴C from a primary leaf to which [¹⁴C]-sucrose was applied, but greatly increased upward translocation to the elongation region of the stem at the expense of transport to the hypocotyl and root system. The observed changes in the patterns of growth and [¹⁴C]-labelled assimilate distribution were correlated with an increase in the specific activity of acid invertase in the elongating stem internodes and a decrease in invertase activity in the hypocotyl and root. Sucrose concentration in the elongating internodes fell substantially after treatment with GA₃ while the concentration of hexose sugars increased. We suggest that by stimulating acid invertase synthesis in the elongating internodes, GA₃ acts to establish a more favourable sucrose gradient between these sinks and source leaves. Under source-limiting conditions this, in turn, will lead to a reduced rate of assimilate translocation to competing sinks in the root system.     

Effects of ABA on primary terpenoids and Δ<Superscript>9</Superscript>-tetrahydrocannabinol in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. at flowering stage

This work examined the effects of exogenously applied abscisic acid (ABA) on the content of chlorophyll, carotenoids, α-tocopherol, squalene, phytosterols, Δ⁹-tetrahydrocannabinol (THC) concentration, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS) activity in Cannabis sativa L. at flowering stage. Treatment with 1 and 10 mg l⁻¹ ABA significantly decreased the contents of chlorophyll, carotenoids, squalene, stigmasterol, sitosterol, and HMGR activity in female cannabis plants. ABA caused an increase in α-tocopherol content and DXS activity in leaves and THC concentration in leaves and flowers of female plants. Chlorophyll content decreased with 10 mg l⁻¹ ABA in male plants. Treatment with 1 and 10 mg l⁻¹ ABA showed a decrease in HMGR activity, squalene, stigmasterol, and sitosterol contents in leaves but an increase in THC content of leaves and flowers in male plants. The results suggest that ABA can induce biosynthesis of 2-methyl-d-erythritol-4-phosphate (MEP) pathway secondary metabolites accumulation (α-tocopherol and THC) and down regulated biosynthesis of terpenoid primary metabolites from MEP and mevalonate (MVA) pathways (chlorophyll, carotenoids, and phytosterols) in Cannabis sativa.     

Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia

Spike lavender ( Lavandula latifolia ) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T₀ plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression of HMGR1S also increased the amount of the end-product sterols, β-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not affect the accumulation of carotenoids or chlorophylls. We also analysed T₁ plants derived from self-pollinated seeds of T₀ lines that flowered after growing for 2 years in the greenhouse. The increased levels of essential oil and sterols observed in the transgenic T₀ plants were maintained in the progeny that inherited the HMG1 transgene. Our results demonstrate that genetic manipulation of the MVA pathway increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species.     

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.     

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 .