Gibberellins and the photoperiodic control of stem elongation in the long-day plant Agrostemma githago L.

Agrostemma githago is a long-day rosette plant in which transfer from short days (SD) to long days (LD) results in rapid stem elongation, following a lag phase of 7–8 d. Application of gibberellin A₂₀ (GA₂₀) stimulated stem elongation in plants under SD, while 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride (AMO-1618, an inhibitor of GA biosynthesis) inhibited stem elongation in plants exposed to LD. This inhibition of stem elongation by AMO-1618 was overcome by simultaneous application of GA₂₀, indicating that GAs play a role in the photoperiodic control of stem elongation in this species. Endogenous GA-like substances were analyzed using reverse-phase high-performance liquid chromatography and the d-5 corn (Zea mays L.) assay. Three zones with GA-like activity were detected and designated, in order of decreasing polarity, as A, B, and C. A transient, 10-fold increase in the activity of zone B occurred after 8–10 LD, coincident with the transition from lag phase to the phase of rapid stem elongation. After 16 LD the activity in this zone had returned to a level similar to that under SD, even though the plants were elongating rapidly by this time. However, when AMO-1618 was applied to plants after 11 LD, there was a rapid reduction in the rate of stem elongation, indicating that continued GA biosynthesis was necessary following the transient increase in activity of zone B, if stem elongation was to continue under LD. It was concluded that control of stem elongation in A. githago involves more than a simple qualitative or quantitative change in the levels of endogenous GAs, and that photoperiodic induction alters both the sensitivity to GAs and the rate of turnover of endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - LD long day(s) - LDP long-day plant(s) - SD short day(s)     

Thermoperiodic stem elongation involves transcriptional regulation of gibberellin deactivation in pea

The physiological basis of thermoperiodic stem elongation is as yet poorly understood. Thermoperiodic control of gibberellin (GA) metabolism has been suggested as an underlying mechanism. We have investigated the influence of different day and night temperature combinations on GA levels, and diurnal steady-state expression of genes involved in GA biosynthesis (LS, LH, NA, PSGA20ox1, and PsGA3ox1) and GA deactivation (PsGA2ox1 and PsGA2ox2), and related this to diurnal stem elongation in pea (Pisum sativum L. cv Torsdag). The plants were grown under a 12-h light period with an average temperature of 17 degrees C. A day temperature/night temperature combination of 13 degrees C/21 degrees C reduced stem elongation after 12 d by 30% as compared to 21 degrees C/13 degrees C. This was correlated with a 55% reduction of GA1. Although plant height correlated with GA1 content, there was no correlation between diurnal growth rhythms and GA1 content. NA, PsGA20ox1, and PsGA2ox2 showed diurnal rhythms of expression. PsGA2ox2 was up-regulated in 13 degrees C/21 degrees C (compared to 21 degrees C/13 degrees C), at certain time points, by up to 19-fold. Relative to PsGA2ox2, the expression of LS, LH, NA, PSGA20ox1, PsGA3ox1, and PsGA2ox1 was not or only slightly affected by the different temperature treatments. The sln mutant having a nonfunctional PsGA2ox1 gene product showed the same relative stem elongation response to temperature as the wild type. This supports the importance of PsGA2ox2 in mediating thermoperiodic stem elongation responses in pea. We present evidence for an important role of GA catabolism in thermoperiodic effect on stem elongation and conclude that PsGA2ox2 is the main mediator of this effect in pea.     

Allene oxide cyclase is essential for theobroxide-induced jasmonic acid biosynthesis in Pharbitis nil

Theobroxide, a natural product, strongly stimulates the biosynthesis of jasmonic acid (JA) in Pharbitis nil. In this study, we investigated the accumulation of protein by the immunoblot analysis of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC), key enzymes in JA biosynthesis, and how the endogenous levels of JA in P. nil are affected by theobroxide. The effect of JA on the accumulations of these proteins was monitored simultaneously. The results show that theobroxide treatment led to a high level accumulation of JA, which is due to high accumulations of LOX, AOS, and AOC proteins induced by theobroxide treatment both under short day (SD) and long day (LD) conditions. However, under SD conditions AOS and AOC proteins are not enhanced by JA treatment. Kinetic analysis of protein levels shows that a biphasic activation of AOC protein by theobroxide is displayed and the first activation of AOC protein together with elevated JA levels is observed within 30min after treatment. Meanwhile, AOS and LOX proteins are activated by theobroxide later than AOC protein, suggesting that AOC plays an essential role in the initial JA formation induced by theobroxide. Since theobroxide-increased JA levels also show a biphasic manner similar to AOC activation and AOS, LOX proteins are activated later than AOC, and thus we propose a positive JA feedback regulation. Interestingly, AOS protein, which is also the enzyme for the biosynthesis of 9,10-ketol-octadecadienoic acid (KODA, a flowering inducing factor), accumulates markedly due to the simultaneous involvement of theobroxide and SD conditions, suggesting that AOS probably plays a role in flower bud formation in P. nil.     

ent-kaurene biosynthesis is enhanced by long photoperiods in the long-day plants Spinacia oleracea L. and Agrostemma githago L.

The effect of photoperiod on ent-kaurene biosynthesis was determined in the long-day (LD) plants spinach (Spinacia oleracea L.) and Agrostemma githago L. Further metabolism of ent-kaurene was blocked by application of the growth retardant tetcyclacis, and ent-kaurene accumulation was measured by isotopic dilution using gas chromatography-selected ion monitoring (GC-SIM) (E. Grosselindemann, J.E. Graebe, D. Stöckl, P. Hedden [1991] Plant Physiol 96: 1099-1104). In spinach, the rate of ent-kaurene accumulation in shoots grown under LD conditions was 3 times higher than in shoots grown under short-day (SD) conditions. ent-Kaurene also accumulated in fully expanded leaves, but at a lower rate than in shoots (15 and 55 pmol g-1 dry weight h-1, respectively). In Agrostemma, ent-kaurene accumulated at a rate 2.5 times higher in plants grown under LD conditions than in those grown under SD conditions. In spinach, enhanced ent-kaurene accumulation was detectable after 1 long day, and with exposure to additional long days, the rate of ent-kaurene accumulation increased further. Conversely, when plants were exposed to LD conditions and then returned to SD conditions, the rate of ent-kaurene accumulation decreased. Following tetcyclacis application, ent-kaurene accumulation was observed in all parts of spinach that were analyzed, but there were large quantitative differences between organs of different ages. As the leaves matured, ent-kaurene biosynthesis declined. Petioles accumulated more ent-kaurene than the corresponding leaf blades. It is concluded that stimulation of ent-kaurene biosynthesis by LD conditions leads to a higher rate of gibberellin biosynthesis, which is essential for stem elongation in rosette plants.     

Stem elongation and floral initiation on in vitro chicory root explants: influence of photoperiod

Chicory root explants (Cichorium intybus L.) were cultured in vitro under different photoperiods. In complete darkness, strong stem elongation, but no flowering induction was observed. We suggest that this stem elongation could be homologous to the pit growth in chicory heads in vivo. Under a photoperiod of 12 h (LI=±40 E m^–2 s^–1), only vegetative growth was observed. Photoperiods of 16 h or more light a day induced the in vitro explants to develop stems bearing flower buds. When the in vitro cultures were kept in the dark for different durations starting from the first day of culture and afterwards transferred to long-day conditions, 4 days dark were sufficient to cause a decrease in flowering induction. We suggest that during the dark culture, a flowering inhibitory process was started.     

Immunomodulation of gibberellin biosynthesis using an anti-precursor gibberellin antibody confers gibberellin-deficient phenotypes

Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. We have recently succeeded in obtaining gibberellin (GA)-deficient phenotypes in Arabidopsis thaliana by using anti-bioactive GA antibodies. In this study, a single-chain antibody (scFv) against GA(24), a precursor GA, was utilized to repress the biosynthesis of bioactive gibberellins. Stable accumulation of the scFv in endoplasmic reticulum (ER) was achieved by being produced as a fusion with GFP as well as KDEL ER-retention signal. The transgenic plants showed GFP fluorescence in the reticulate cortical ER network in epidermal cells. The GFP-scFv fusion produced in plants maintained its binding activity. The transgenic plants showed GA-deficient phenotypes, including reduced rosette leaf development, delayed flower induction and reduced stem elongation of the main culm, especially in the early stage of inflorescence growth. Contrarily, stem elongation of the main culm at a later stage, or that of lateral shoots was much less affected by scFv production. These phenotypes were different from anti-bioactive GA scFv-producing lines, whose stem elongation was continuously repressed throughout the inflorescence development. The GA-deficient phenotypes were recovered by treatment with GA(24) and bioactive GA(4), the latter being more effective. The transgenic lines contained conspicuously higher endogenous GA(24) and clearly less GA(4) than wild-type plants. The expression of GA 20-oxidase and GA 3-oxidase genes, which are feedback-regulated by GA signaling, were up-regulated in those plants. These results demonstrate that the scFv trapped GA(24) in ER and inhibited metabolism of GA(24) to bioactive GA(4).     

Influence of gibberellin biosynthesis inhibitors on stem elongation and floral initiation on in vitro chicory root explants under dark and light conditions

Root explants of chicory (Cichorium intybus L.) were cultured in vitro under continuous light or darkness. On a standard medium (no plant growth regulators added), flowering-stems were initiated under continuous light while under continuous dark, vegetative-stems were formed. Different types of GA (gibberellin) biosynthesis inhibitors were added to the culture medium. Paclobutrazol and compounds belonging to the group of cyclohexanetriones clearly reduced flowering-stem growth under light conditions and vegetative-stem growth under dark conditions. Under light conditions, flower bud initiation was not affected. These and other results suggest that GA₁ may be synthesized during the in vitro culture period and that it controls flowering-stem growth but not floral initiation.Abbreviations CCC chlormequat chloride - GA gibberellin - LAB 198 999 3,5-dioxo-4-butyryl-cyclohexane carboxylic acid ethyl ester - BAS 111..W 1-phenoxy-3-(1H-1,2,4-triazol-1-yl)-4-hydroxy-5,5-dimethylhexane     

Inhibitory role of gibberellins in theobroxide-induced flowering of Pharbitis nil

Theobroxide, a novel active compound isolated from a fungus, has been reported previously to induce potato tuberization and flower bud formation in Pharbitis nil under non-inductive long-day conditions. Up to date, the action mechanism of theobroxide on flower-bud induction of P. nil, however, is still unknown. In the present study, we observed a reduction of the stem length, along with the induction of flower buds, in theobroxide-treated and short-day-grown P. nil plants. Also, the results showed that flower bud formation was delayed markedly in P. nil seedlings with removal of cotyledons or exposure to night break. The suppression effect of night-break and cotyledon-removal, however, was abolished completely by spraying theobroxide. Endogenous gibberellin(1/3) contents in P. nil plants treated with theobroxide or grown under short-day conditions were relatively lower, suggesting that gibberellins probably are negatively involved in theobroxide- and short-day-induced flower-bud formation of P. nil.     

Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in which stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxidation and elimination of C-20 of C20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from spinach genomic DNA that has a high homology with GA 20-oxidase cDNAs from Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymerase chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protein of 374 amino acid residues. When this cDNA clone was expressed in Escherichia coli, the fusion protein catalyzed the biosynthetic sequence GA53-->GA44-->GA19-->GA20 and GA19-->GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of C-20. Transfer of spinach plants from short day (SD) to LD conditions caused an increase in the level of all GAs of the early-13-hydroxylation pathway, except GA53, with GA20, GA1, and GA8 showing the largest increases. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in LD than in SD conditions, with highest level of expression in the shoot tips and elongating stems. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in SD and LD conditions.     

Evidence against the involvement of phytochrome in UVB-induced inhibition of stem growth in green tomato plants

The effects of UVB on the kinetics of stem elongation of wild type (WT) and photomorphogenic mutants of tomato were studied by using linear voltage transducers connected to a computer. Twenty-one or twenty-six-day-old plants, grown in 12 h white light (150 μmol m⁻² s⁻¹ PAR)/12 h dark cycles, were first transferred to 200 μmol m⁻² s⁻¹ monochromatic yellow light for 12 h, then irradiated with 0.1 or 4.5 μmol m⁻² s⁻¹ UVB for 12 h and finally kept in darkness for another 24 h. The measurements of the kinetics of stem elongation started after 4 h under yellow light. Significant differences in stem growth during the irradiation with yellow light, as well as during the dark period, were found between the genotypes. In darkness, the magnitude of stem growth followed the order: tri > AC = fri > MMau > hp1. Two factors determined the large differences of growth in darkness: 1) the different stem elongation rate (SER) and 2) the different duration of the growing phase among the genotypes. In darkness the stem growth of au and hp1 mutants lasted for about 18 h, whereas it continued for the whole experimental period (36 h) in the other genotypes. UVB irradiation substantially reduced elongation growth of all genotypes (4.5 μmol m⁻² s⁻¹ being more effective than 0.1 μmol m⁻² s⁻¹). Both fluence rates of UVB induced a detectable reduction of SER already after 15 min of irradiation. Red light inhibited, while far red light promoted stem growth of all the genotypes tested. fri (phyA null), tri (phyB1 null), hp1 (exhibiting exaggerated phytochrome responses) mutants and WT tomato showed similar levels of UVB–induced inhibition of growth, while the aurea mutant showed the largest growth inhibition during the 12 h of irradiation. These results indicate that phytochrome is not directly involved in UVB control of stem elongation. The results of dichromatic irradiations UVB + red or UVB + far red indicate the presence of distinct and additive action of UVB photoreceptor and of the phytochrome system in the photoregulation of stem growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gibberellins and stem growth in Arabidopsis thaliana. Effects of photoperiod on expression of the GA4 and GA5 loci.

Arabidopsis thaliana (L.) Heynh. is a quantitative long-day (LD) rosette plant in which stem growth is mediated by gibberellins (CAs). Application of GAs to plants in short-day (SD) conditions resulted in rapid stem elongation and flower formation, with GA4 and GA9 being equally effective, and GA1 showing lower activity. The effects of photoperiod on the levels of endogenous GAs were measured by combined gas chromatography-mass spectrometry with selected ion monitoring. When plants were transferred from SD to LD conditions there was a slight decrease in the level of GA53 and an increase in the levels of C19-GAs, GA9, GA20, GA1, and GA8, indicating that GA 20-oxidase activity is stimulated in LD conditions. Expression of GA5, which encodes GA 20-oxidase, was highest in elongating stems and was correlated with the rate of stem elongation. By contrast, GA4, which encodes 3 beta-hydroxylase, showed low expression in stems and its expression was not correlated with the rate of stem elongation. We conclude that stem elongation in LD conditions is at least in part due to increased expression of GA5, whereas expression of GA4 is not under photoperiodic control.     

The ectopic overexpression of a citrus gibberellin 20-oxidase enhances the non-13-hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated phenotype in tobacco

Transgenic plants of Nicotiana tabacum overexpressing a gibberellin (GA) 20-oxidase cDNA ( CcGA20ox1 ) from citrus, under the control of the 35S promoter, were taller (up to twice) and had larger inflorescences and longer flower peduncles than those of control plants. Hypocotyls of transgenic seedlings were also longer (up to 4 times), and neither the seedlings nor the growing plants elongated further after application of GA₃. Hypocotyl and stem lengths were reduced by application of paclobutrazol, and this inhibition was reversed by exogenous GA₃. The ectopic overexpression of CcGA20ox1 enhanced the non-13-hydroxylation pathway of GA biosynthesis leading to GA₄, apparently at the expense of the early-13-hydroxylation pathway. The level of GA₄ (the active GA from the non-13-hydroxylation pathway) in the shoot of transgenic plants was 3–4 times higher than in control plants, whereas that of GA₁, formed via the early-13-hydroxylation pathway (the main GA biosynthesis pathway in tobacco), decreased or was not affected. GA₄ applied to the culture medium or to the expanding leaves was found to be at least equally active as GA₁ on stimulating hypocotyl and stem elongation of tobacco plants. The results suggest that the tall phenotype of tobacco transgenic plants was due to their higher content of GA₄, and that the GA response was saturated by the presence of the transgene.     

Effect of light quality and 5-azacytidine on genomic methylation and stem elongation in two ecotypes of Stellaria longipes

Changes in cytosine methylation are known to occur in response to various environmental stimuli, therefore, we looked at methylation changes in relation to stem elongation. More specifically, we investigated the response of genomic cytosine methylation to irradiance-mediated plasticity of stem elongation in two ecotypes of Stellaria longipes . Ramets of S. longipes were grown under high and low ratios of red/far-red light (F/FR; 3.7 and 0.7, respectively). Stem elongation and methylated cytosine content were measured over a period of 7 days. Ramets of S. longipes demonstrated the highest level of demethylation after 4 days of long-day warm (LDW) treatment, which coincides with the first day of rapid stem elongation initiation. The extent of demethylation associated with day 4 depended upon the relative ratio of R/FR light. In particular, those plants treated with low R/FR light ratios showed a lower level of methylation, and were taller than the high R/FR light grown counterparts. In addition, prairie ecotype plants demonstrated lower day 4 methylation levels, as well as longer day 7 stem lengths, than the alpine ecotype plants within the same R/FR light treatments. To investigate if the degree of methylation was a crucial factor in controlling the stem elongation response, ramets of both alpine and prairie plants were grown in MS media supplemented with 5-azacytidine (5-AzaC), and grown for 14 days under a R/FR ratio of 3.7 and two different PAR values. 5-AzaC treatments demonstrated that the prairie ecotype plants required greater doses of 5-AzaC, and thus lower methylation levels, than the alpine ecotype plants in order to promote maximal stem elongation. These observations suggest that DNA demethylation is involved in the shade-avoidance response.     

Nocturnal gibberellin biosynthesis is carbon dependent and adjusts leaf expansion rates to variable conditions

Optimal plant growth performance requires that the presence and action of growth signals, such as gibberellins (GAs), are coordinated with the availability of photo-assimilates. Here, we studied the links between GA biosynthesis and carbon availability, and the subsequent effects on growth. We established that carbon availability, light and dark cues, and the circadian clock ensure the timing and magnitude of GA biosynthesis and that disruption of these factors results in reduced GA levels and expression of downstream genes. Carbon-dependent nighttime induction of gibberellin 3-beta-dioxygenase 1 (GA3ox1) was severely hampered when preceded by reduced daytime light availability, leading specifically to reduced bioactive GA₄ levels, and coinciding with a decline in leaf expansion rate during the night. We attributed this decline in leaf expansion mostly to reduced photo-assimilates. However, plants in which GA limitation was alleviated had significantly improved leaf expansion, demonstrating the relevance of GAs in growth control under varying carbon availability. Carbon-dependent expression of upstream GA biosynthesis genes (Kaurene synthase and gibberellin 20 oxidase 1, GA20ox1) was not translated into metabolite changes within this short timeframe. We propose a model in which the extent of nighttime biosynthesis of bioactive GA₄ by GA3ox1 is determined by nighttime consumption of starch reserves, thus providing day-to-day adjustments of GA responses.

GA-sugar matching occurs specifically at night and determines day to day adjustment of GA levels and subsequent growth.