Journal of Experimental Botany, Vol. 52, No. 357, pp. 725-738,
April 15, 2001
© 2001 Oxford University Press
Original Papers |
Reduced de-etiolation of hypocotyl growth in a tomato mutant is associated with hypersensitivity to, and high endogenous levels of, abscisic acid
1 Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, Saskatchewan S7N 5E2, Canada
2 Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Received 22 June 2000; Accepted 20 October 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A recessive single gene mutant, 7B-1, in tomato was originally selected for its photoperiod-dependent male sterility. The 7B-1 mutant also has some pleiotropic effects including reduced light-induced inhibition, i.e. de-etiolation, of the hypocotyl in long days (LD), increased seed size and weight, and reduced transpiration rate. These traits led us to investigate the sensitivity of 7B-1 to exogenous hormones and the interaction of these responses with daylength. In LD, but not in short days (SD), 7B-1 was more sensitive than wild-type (WT) to exogenous abscisic acid (ABA) for inhibition of seed germination, root elongation and transpiration rate. 7B-1 mutant also exhibited reduced responses to exogenous gibberellin (GA3) for hypocotyl elongation, and to inhibitors of GA biosynthesis for seed germination and root and hypocotyl elongation. 7B-1 hypocotyls contained a higher level of endogenous ABA than WT in both photoperiods, although ABA levels were higher in LD than in SD. In contrast, growth-active GAs, i.e. GA1, GA3 and GA4, and IAA were low in the mutant hypocotyls. The 7B-1 mutant appears to be an ABA-overproducer, and the photoperiod-regulated ABA levels may be responsible for the hypersensitivity of the mutant to exogenous ABA.
Key words: Abscisic acid, elongated mutant, fluridone, gibberellins, tetcyclacis, tomato.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant hormones are important internal factors which control developmental processes in interaction with various environmental stimuli, such as light and temperature (Davies, 1995
). A genetic approach, such as the isolation and characterization of mutants affected in response to hormones, is a powerful tool for the study of the role of plant hormones in gene-controlled growth and development. Many of the mutants shown to be defective in the biosynthesis or action of hormones were originally selected on the basis of changes in shoot (hypocotyl, stem) growth. For example, a number of dwarf mutants have been isolated that are affected either in endogenous GA content, or in GA-action (for review, see Ross et al., 1997). Also, mutants affected in auxin responses are known which display alterations in elongation capacity, including dwarf stature (reviewed in Reid and Howell, 1995). Recently, several dwarf mutants have been reported which have a defect in different steps of brassinosteroid biosynthesis and action (Clouse et al., 1996; Altmann, 1999; Ephritikhine et al., 1999). However, fewer hormone biosynthesis or action mutants are known which exhibit greater than normal growth (Reid and Howell, 1995; Ross et al., 1997).
Mutants exhibiting extended growth have been shown to be either GA-biosynthesis/metabolism or GA-response mutants. For example, the mutant slender (sln) in pea has a lesion in GA20 catabolism resulting in increased levels of GA1, which causes seedlings to develop exceptionally long internodes (Reid et al., 1992; Ross et al., 1997). Another elongated mutant in Brassica (ein) (Rood et al., 1990) is a GA-overproducer and it has a phytochrome lesion (Devlin et al., 1992). Other elongated mutants, such as lacrys in pea, slender (sln) in barley, spindly (spy) in Arabidopsis, and procera (pro) in tomato (Chandler, 1988; Lanahan and Ho, 1988; Jacobsen and Olszewski, 1993; Potts et al., 1985; Jones, 1987; Jupe et al., 1988) have been classified as constitutive GA response mutants. Finally, elongated mutants have been reported which have an enhanced response to applied GA, such as the phyB-deficient long hypocotyl mutants in cucumber (lh), pea (lv), and Arabidopsis (phyB) (Ross et al., 1997).
A single gene, male sterile mutant 7B-1 in tomato (Lycopersicon esculentum) is photoperiod sensitive (Sawhney, 1997). In LD it produces male sterile flowers, but in SD most of the flowers are male fertile. The 7B-1 mutant also has some pleiotropic effects, including reduced light-induced inhibition, or de-etiolation, of hypocotyls in LD, and increased seed size and weight. The objectives of this study were (1) to examine the sensitivity of the 7B-1 mutant to exogenous hormones, i.e. ABA and GA, and their biosynthetic inhibitors, (2) to determine whether endogenous levels of ABA, GAs and IAA, are affected in the mutant, and (3) to determine whether the photoperiod-dependent mutant responses are related to changes in endogenous hormones. The differential sensitivity of the mutant to exogenous hormones and its relationship with photoperiod is discussed.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and growth conditions
The spontaneous recessive male sterile mutant 7B-1 in tomato (Lycopersicon esculentum Mill.) was isolated for its photoperiod sensitivity (Sawhney, 1997
). 7B-1 plants are male sterile under LD (12–16 h), but under SD (8–10 h) most of the flowers are male fertile. Thus, for all experiments, 7B-1 and WT seeds were obtained from plants grown in SD.
For seed germination and sterile culture of plants, seeds were sterilized in 50% (v/v) Javex-5 solution (3% hypochlorite sodium) for 20 min, rinsed extensively with sterile distilled water, and were sown on 0.7% (w/v) agar medium in Petri dishes (100x15 mm). The basal medium (BM) consisted of Murashige and Skoog salts (Murashige and Skoog, 1962), 1% (w/v) sucrose and 1 mM MES (2-[N-morpholino]ethanesulphonic acid) buffer (pH adjusted to 6.1 by KOH). Petri dishes with germinated seeds were placed in an incubator at 25/23 °C, light/dark under either a 16/8 h, light/dark (LD), or an 8/16 h, light/dark (SD) photoperiod. Illumination was provided by white fluorescent tubes (F20T12/CW, Sylvania, USA) with the photon fluence rate of 25–40 µmol m-2 s-1. Older plants were grown in soil (Sunshine No. 1, Sun Gro Horticulture, USA) at a temperature regime of 25/18 °C, light/dark in a growth chamber with LD or SD photoperiod under high-intensity fluorescent tubes (F72T12/CW/VHO, Sylvania, USA) and incandescent bulbs (Long Life 7500 h, Litemor, Canada) at an illumination of 90–200 µmol m-2 s-1.
Germination tests
7B-1 and WT seeds were germinated, 40–60 seeds per Petri dish, in the BM supplemented with or without different concentrations of ABA (±)-cis, trans, GA3 (Sigma, St Louis, MO), tetcyclacis (BASF Aktiengesellschaft, Germany), uniconazole (Chevron Chemical Company, Benseville, IL) or fluridone (Eli Lilly, Indianapolis, IN). All plant growth regulating substances were added to the medium via sterile filtration while medium was cooling after autoclaving. Seed germination, defined as radicle protrusion, was scored from 2–6 d after sowing.
Root and hypocotyl elongation
Four-day-old seedlings grown on the BM were transferred to Petri dishes that were vertically aligned. Here, a new medium was used supplemented with different concentrations of ABA, GA3, BA, 2,4-D (Sigma, St Louis, MO), 24-epibrassinolide (a gift from Dr Miroslav Strnad, Olomouc, Czech Republic), tetcyclacis, uniconazole or fluridone. After 3 d of incubation, new growth of hypocotyls and roots was measured.
Measurements of cell and organ dimensions
Epidermal cell lengths were measured on hypocotyls excised from 7-d-old seedlings grown in vitro. Hypocotyls were stained with methyl-blue dye (0.01% w/v), gently squashed on a microscope slide and examined with a compound microscope (Optiphot, Nikon, Japan). Four hypocotyls of uniform size were chosen from each treatment, and 30 epidermal cells were measured for each hypocotyl. Root and hypocotyl lengths were measured with a graduated ruler to the nearest millimetre.
Transpiration measurements
The method of Häuser et al. (Häuser et al., 1990) was followed. 7B-1 and WT seedlings with a second leaf (14–20 d after seed sowing) were chosen. Roots were excised, and seedlings with hypocotyls, cotyledons and leaves were placed upright in glass vials (25 mm in diameterx55 mm height) with 10.0 ml of distilled water supplemented either with ABA (100 µM) or tetcyclacis (50 µM). To restrict evaporation of the water to a minimum, the vials were covered with plastic lids which had slits into which hypocotyls were fitted. The vials (six replicates per treatment) were then placed in a growth chamber (25/23 °C, light/dark; 24–40 µmol m-2 s-1) for 24 h and incubated under LD or SD. Transpiration losses were determined by weighing the vials (without seedlings) at the beginning and at the end of the experiment. All values were then corrected to account for evaporation that took place in the absence of seedlings. Transpiration was expressed as grams of water lost over 24 h g-1 of tissue dry weight.
Analysis of endogenous hormones
The tomato hypocotyls were ground and extracted with 80% MeOH (H2O:MeOH=20:80, v/v). Twenty ng each of [17,17-2H2]GA1, GA3, GA4, GA7, GA9, GA20, and 200 ng each of [2H6]ABA and [13C6]IAA were added to the 80% MeOH extract along with 8000 Bq of [1,2(n)-3H]GA1 (Amersham, Oakville, Ontario; 37.7 Ci mmol-1), [1,2(n)- 3H]GA4 (Amersham, 32.2 Ci mmol-1) and [1,2(n)-3H]GA9 (Yokota et al., 1976) as internal standards. The 80% MeOH extract was then purified with a C18 preparative column (C18-PC) (Koshioka et al., 1983a) made of a syringe barrel (inside diametre 2 cm) filled with 3 g of C18 preparative reversed-phase material (Waters Ltd). The 80% MeOH eluate was taken to dryness in vacuo at 35 °C. The 80% MeOH residue from the C18-PC was then loaded onto a SiO2 partition column (SiO2-PC) (Koshioka et al., 1983a) (column i.d.=1.5 cm) made of 5 g of Woelm SiO2 (32–100 mesh) deactivated by equilibration with 20% water by weight. The SiO2-PC was eluted with EtOAc:hexane (95:5, v/v; saturated with 0.5 M formic acid) and the eluate was taken to dryness in vacuo at 35 °C. The EtOAc:hexane residue from SiO2-PC was further purified by high performance liquid chromatography (HPLC) (Koshioka et al., 1983b; Pearce et al., 1994).
The HPLC (Waters Ltd) apparatus consisted of two pumps (model M-45), an automated gradient controller (model 680), and a Rheodyne injector (model 7125). The solvents were, pump A: 10% MeOH in 1% acetic acid [H2O:MeOH:acetic acid=89:10:1, (v/v)], pump B: 100% MeOH. A reversed phase C18 Radial-PAK (-Bondapak column (8 mmx10 cm) was used with a 10–73% linear gradient programme at a flow rate of 2 ml min-1. The manually implemented 10–73% linear gradient programme was 0–10 min (pump A, 100%; pump B, 70%). The HPLC fractions were taken to dryness in vacuo. The C18 HPLC fraction residues (fractions grouped based on elution of [3H]-labelled GAs) were further purified with an Alltech Associates Nucleosil N(CH3)2 HPLC column (4.6 mmx15 cm) eluted with 99.9% MeOH in 0.1% acetic acid (Pearce et al., 1994), 1 min fractions being taken to dryness in vacuo. The GA/ABA/IAA-containing fractions from the Nucleosil N(CH3)2 HPLC were methylated by ethereal CH2N2. The methylated sample was then silylated by BSTFA with 1% TMCS (Hedden, 1987; Gaskin and MacMillan, 1991).
The identification and quantification of GAs was carried out on a gas chromatograph (GC-MS) in -selected ion monitoring (-SIM) mode. The derivatized sample was injected onto a capillary column installed in a Hewlett-Packard 5890 GC with a capillary direct interface to a HP 5970 mass selective detector (MSD). The capillary column was a 0.25 (mm film thickness, 0.25 mm internal diametre, 15 m DB1-15N column (J&W Scientific, Inc.). The capillary head pressure was 4 psi with a He carrier gas flow rate of 1.1 ml min-1. The GC temperature programme was as follows: 0.1 min at 60 °C, then up to 200 °C at 20 °C min-1, then up to 250 °C at 4 °C min-1 and finally to 300 °C at 25 °C min-1, and stayed at 300 °C for 5 min before returning to 60 °C. The interface temperature was maintained at 300 °C and the MSD was operated with the electron multiplier at 1600 V. Three m/z ions from each deuterated GA and endogenous GA were monitored. The dwell time was 10 s. The data were processed using the HP G1034C MS ChemStation Software. Based on the capillary GC Rts of stable isotope internal standards and relative abundance of the characteristic m/z ions monitored, endogenous GAs were identified and amounts estimated based on the isotope dilution technique (Fujioka et al., 1988).
Statistical analysis
Statistical significance of the treatment differences was assessed using Student's t-test when only two components were compared. Two-way analysis of variance was utilized in all other comparisons.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Seed germination
7B-1 seeds that developed on plants under both SD and LD were larger in size, darker in color and weighed more than WT seeds (Table 1
). On the BM, WT and 7B-1 seeds showed similar germination percentages under both photoperiods. In both genotypes germination started after 2–4 d, and after 5–6 d it was 80% germination on average (from at least 40 independent experiments) in LD and in SD. Because of the variability in germination between seed batches (data not shown), germination comparisons between the two genotypes in response to growth regulators were calculated relative to control values on the basal medium.
|
Seed germination was assessed in the presence of various concentrations of ABA, GA3, tetcyclacis (a GA-biosynthesis inhibitor of the norbornanodiazetine type), uniconazole (a GA-biosynthesis inhibitor of triazole type) (Rademacher et al., 1987
; Grossmann, 1990), or fluridone, an inhibitor of ABA biosynthesis (Gamble and Mullet, 1986; Saab et al., 1990). Germination of WT seeds was inhibited by ABA from 3x10-6 M to 3x10-5 M under both LD and SD, although inhibition was more pronounced under SD than in LD (Fig. 1a). In LD seed germination in 7B-1 was 2–3 times more inhibitory to ABA than that in WT seeds (Fig. 1a). However, under SD, 7B-1 and WT showed similar response to a range of ABA concentrations (Fig. 1a). Germination of WT seeds was strongly inhibited by tetcyclacis over a range of concentrations in both photoperiods (Fig. 1b). In contrast, 7B-1 seeds showed a much reduced sensitivity to tetcyclacis inhibition, especially in SD (Fig. 1b). Uniconazole also strongly inhibited WT seed germination in both photoperiods over a wide range of concentrations, but germination of 7B-1 seeds was essentially unaffected by uniconazole (Fig. 1c).
|
) and the 7B-1 mutant (•) in LD and SD. Inhibition of seed germination by ABA (a), tetcyclasis (b) and uniconazole (c) relative to the mean germination on basal medium (BM), 6 d after seed sowing. 40 seeds of each genotype were sown for each treatment in each experiment. Each value represents mean germination ±SE of six independent experiments for ABA and TCY in LD, and of three experiments for ABA and TCY in SD and uniconazole in both LD and SD. (d, e) Effects of TCY (d) or UNI (e) on seed germination with or without GA3. Seeds were soaked for 2 d in TCY solution (100 µM; +5 °C, darkness) and then incubated in LD for another 5 d on the BM, with or without 10 µM or 100 µM of GA3. 40 seeds of each genotype were sown for each treatment for each experiment. Bars represent means ±SE of three independent experiments (d), or one representative experiment of two (e). **, Significantly different (P
0.01) from WT and 7B-1 controls, respectively. +, Significantly different (PThe question of whether exogenous GA would restore germination in tetcyclacis- or uniconazole-treated WT seeds was tested by transferring seeds that had been imbibed in the inhibitor solution for 2 d to a medium containing GA3 for 5 d. GA3 (10 or 100 µM) was unable to restore germination in tetcyclacis-treated WT seeds, but it did increase germination of 7B-1 seeds (Fig. 1d
). GA3 partly overcame the uniconazole-induced inhibition of WT seed, but it did not improve 7B-1 seed germination (Fig. 1e). GA3 alone had no promotive effect on seed germination of both genotypes. Similar results were obtained in SD conditions (data not shown).
Since tetcyclacis is also known to stimulate ABA accumulation in actively growing plants by inhibiting its degradation (Grossmann, 1990), the effect of fluridone (an inhibitor of ABA synthesis), alone or in combination with tetcyclasis, ABA, or tetcyclasis and GA3 was tested. Fluridone alone (10 µM) enhanced the rate of seed germination of both WT and 7B-1 seeds (Fig. 2a). ABA (30 µM) together with fluridone reduced germination of both WT and 7B-1 seeds to the level of ABA alone (Fig. 2b). Fluridone also partly overcame the inhibitory effect of tetcyclacis on WT seed germination and the addition of GA3 to this mixture further increased germination (Fig. 2b). In 7B-1 mutant, fluridone alone, or fluridone and GA3, restored germination of tetcyclacis-treated seeds to the control level (Fig. 2b). Similar results were obtained in SD conditions (data not shown).
|
Root elongation
Root growth on BM did not differ in 7B-1 or WT seedlings in LD or SD (Fig. 3a). Nor were there any differences between the two genotypes with regard to the formation of lateral roots or root hairs (data not shown). ABA inhibited the root growth of WT and 7B-1 seedlings in both photoperiods, but 7B-1 roots were 10 times more sensitive to ABA under LD, across a range of ABA concentrations, than were WT roots (Fig. 3b). For example, 50% inhibition of 7B-1 root elongation occurred at an ABA dose 10 times lower than that required for WT roots. In contrast, under SD, 7B-1 and WT roots showed similar responses to exogenous ABA (Fig. 3b). Fluridone applied at low concentrations (0.1–1 µM) under LD tended to stimulate WT root growth (approximately 20%), but inhibited 7B-1 root growth by 20% (data not shown). At higher doses (10–100 µM) root elongation was inhibited by about 60% for both genotypes and fluridone had similar inhibitory effects under SD (data not shown).
|
Tetcyclacis inhibited the root growth of WT and 7B-1 seedlings under both photoperiods at 10-6 M or higher. However, 7B-1 seedlings were less sensitive to tetcyclacis-induced inhibition in LD, though not in SD (Fig. 3c
). At high concentrations (10–30 µM) tetcyclasis caused root necrosis and this was more evident for WT than for 7B-1 (data not shown). GA3 (10 µM) had no significant effect on the growth of WT or 7B-1 roots in LD (Fig. 3d). However, GA3 partially overcame the inhibitory effect of tetcyclasis (10 µM) (Fig. 3d). Similar results were observed for SD-grown seedlings (data not shown). Uniconazole (100 µM) was appreciably less inhibitory, giving only a 40% reduction of root elongation for both genotypes, compared to 80–90% inhibition by 30 µM tetcyclasis (data not shown). The effects of an auxin (2,4-D), a cytokinin (6N-benzyladenine), and a brassinosteroid (24-epibrassinolide) were also examined across a wide range of concentrations, but there were no differences in root growth between the two genotypes (data not shown).
Hypocotyl elongation
Hypocotyls of both genotypes showed more light-induced inhibition of hypocotyl growth (de-etiolation) in LD than in SD. However, 7B-1 hypocotyls consistently showed less de-etiolation than WT in LD (Figs 4, 5a), but were of similar length to WT in SD (Fig. 5a). The epidermal cell lengths of 7B-1 hypocotyls were also longer than WT in LD, but were not different from WT in SD (Fig. 5a). Fully etiolated dark-grown 7B-1 and WT seedlings did not differ from WT in hypocotyl length (means ±SE; WT: 72.5±5.4 mm; 7B-1: 71.5±2.7 mm). Hypocotyl elongation was inhibited by ABA (3–30 µM) and the response was similar for 7B-1 and WT in both SD and LD (data not shown). Fluridone at 10-7 and 10-6 M promoted elongation of WT hypocotyls in LD by approximately 35% and 55%, respectively, relative to only 10% and 18%, respectively, promotion of 7B-1 hypocotyl elongation (Fig. 5b). Higher doses of fluridone (10-5 and 10-4 M) had a nil or weak inhibitory effect on WT hypocotyls elongation (Fig. 5b), but appreciably inhibited 7B-1 hypocotyl elongation under LD (Fig. 5c; data shown for 100 µM). In SD, fluridone at low concentrations (0.01–1 µM) had no effect on hypocotyl elongation for either genotype (data not shown), and at high concentrations (10–100 µM) fluridone had a similar inhibitory effect on WT and 7B-1 hypocotyl growth (Fig. 5c; shown for 100 µM).
|
|
Application of GA3 stimulated hypocotyl growth (Fig. 5d
) and tetcyclacis inhibited it (Fig. 5e) for both genotypes and in both photoperiods. However, the hypocotyl growth response to both GA3 and tetcyclacis was relatively less for 7B-1 (Fig. 5e; for absolute growth values see Fig. 5f). GA3 (10 µM) partially overcame the inhibitory effect of tetcyclacis (10 µM) on hypocotyl growth for both genotypes (Fig. 5f). A direct comparison of each genotype's response to GA3 (with or without tetcyclacis) is, however, complicated by 7B-1 mutant's reduced de-etiolation response in LD (Fig. 4). Similar results were observed for seedlings cultured in SD (data not shown). In both photoperiods, hypocotyl growth of 7B-1 and WT showed a similar sensitivity to application of an auxin, a cytokinin and a brassinosteroid (data not shown).
Seedling morphology after fluridone treatment
On the BM both the WT and 7B-1 seedlings had green cotyledons and yellow-green hypocotyls and the size and shape of cotyledons were similar for both genotypes in LD and in SD (Fig. 6, upper panel). With the addition of 0.1 µM fluridone, WT seedlings had yellow-green cotyledons and purple hypocotyls (not shown), but with 10 µM fluridone WT seedlings developed pale yellow, deformed cotyledons and light purple hypocotyls (Fig. 6, lower left). In contrast, 7B-1 seedlings had normal green cotyledons with 0.1 µM or 10 µM fluridone, although they had purple hypocotyls (Fig. 6, lower right). With 100 µM fluridone 7B-1 seedlings developed albino cotyledons and light purple hypocotyls. In SD, 7B-1 seedlings were more sensitive to fluridone than in LD, and with 10 µM fluridone they developed pale yellow and deformed cotyledons (not shown). Interestingly, fluridone plus ABA, or fluridone plus tetcyclacis, or fluridone plus tetcyclasis and GA3 permitted the development of normal pigmentation in cotyledons and hypocotyls in both genotypes, under either LD or SD (data not shown).
|
Transpiration experiments
Water loss from seedling shoots (roots excised) of both WT and 7B-1 was measured after 24 h in response to tetcyclacis (50 µM) or ABA (100 µM). The control WT shoots in LD showed greater transpiration than in SD, but the daylength had no effect on transpiration by 7B-1 shoots (Fig. 7). In LD the control WT shoots showed a greater transpiration than 7B-1 shoots, but there was no difference between the two genotypes in SD. Tetcyclacis is known to reduce transpiration of detached leaves in several plant species (Häuser et al., 1990), but it had no significant effect on water loss by shoots of either genotype in LD or in SD (Fig. 7). However, ABA inhibited transpiration of WT shoots in SD and of 7B-1 shoots in LD (Fig. 7).
|
Endogenous hormones in hypocotyls
Endogenous ABA, IAA and GAs were analysed from 7-d-old hypocotyls of 7B-1 and WT seedlings grown in vitro on BM in LD or SD. In both genotypes the relative amount of endogenous ABA was greater in LD than in SD (data from one experiment shown in Fig. 8a). Thus, LD favours ABA accumulation in both genotypes, although the absolute amounts of ABA varied between two independent experiments. The relative levels of endogenous ABA in hypocotyls differed between the mutant and WT. In LD 7B-1 hypocotyls had >2-times more endogenous ABA than WT hypocotyls (Fig. 8a). Here, it should be remembered that 7B-1 hypocotyls are relatively long under LD, i.e. they do not de-etiolate as much as WT. In SD 7B-1 hypocotyls also had more ABA than WT, but the relative proportion was less.
|
The absolute level of endogenous IAA was not influenced by daylength for either genotype (Fig. 8a
). However, WT hypocotyls had approximately 2x as much IAA in SD as the mutant, but the values were not different in LD (Fig. 8a).
In two separate experiments only the growth-active GAs, i.e. GA1, GA3, GA4 were consistently identified by GC-MS-SIM in WT and 7B-1 hypocotyls. These were quantified using isotope dilution techniques for seedlings grown under LD and SD. There was a photoperiod effect on endogenous GA levels in WT hypocotyls; growth-active GAs were drastically reduced under SD, compared to LD (Fig. 8b). However, photoperiod had no effect on growth-active GA levels in 7B-1 hypocotyls (Fig. 8b). As well, 7B-1 hypocotyls had only about 1/10 the amount of growth-active GAs as WT in LD, but the GA content did not differ between the two genotypes in SD (Fig. 8b).
The relative ratios of the various hormones for hypocotyls grown in LD and SD were as follows. The ratio of ABA:IAA in WT hypocotyls was consistently lower in SD relative to LD across two replicate experiments (Table 2). A similar trend was observed for 7B-1 hypocotyls (Table 2). The ratio of IAA:growth-active GAs was exceptionally elevated in WT hypocotyls in SD, but it was low in 7B-1 (Table 2). The ratio of ABA:growth-active GAs was enhanced 1.7–10.00-fold for WT in SD, relative to LD. In contrast, for 7B-1 the ratio of ABA:growth-active GAs tended to be reduced (0.1–0.9-fold; Table 2). Thus, relatively high IAA/GAs and ABA/GAs ratios in 7B-1 hypocotyls in LD, compared to WT, are correlated with the reduced de-etiolation (increased growth) in the mutant in LD (Fig. 4).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The recessive mutant 7B-1 in tomato was originally isolated for its photoperiod-dependent male sterility (Sawhney, 1997
). Here, it is shown that in LD the mutant has longer hypocotyl than the WT, i.e. it shows reduced de-etiolation, and increased sensitivity to exogenous ABA for inhibition of seed germination and root elongation. In contrast 7B-1 is less sensitive to tetcylacis in the inhibition of germination, root elongation and hypocotyl elongation relative to WT seedlings. The mutant hypocotyls also have relatively high levels of endogenous ABA and low levels of growth-active GAs in LD. Additionally, they have altered ratios of ABA:GAs and IAA:GAs. These results will be discussed in terms of the 7B-1 mutant being affected in ABA biosynthesis and/or metabolism.
Seed germination experiments suggest accumulation of endogenous ABA in 7B-1 mutant
Tomato seeds germinate well in both light and dark (Mancinelli et al., 1966) and applied GA3 had negligible effects on germination of either WT or 7B-1 seeds. A presoak with uniconazole, an early-stage inhibitor of GA-biosynthesis, reduced the germination of WT seeds and this inhibition could be partly overcome by GA3. This implies that active GA biosynthesis is required for tomato seed germination, although effects of uniconazole on ABA metabolism cannot be ruled out (see below). However, uniconazole did not inhibit germination of 7B-1 seeds. This suggests that levels of pre-existing GAs in the mutant were sufficient to allow for near-normal germination. Tetcyclacis, another early-stage GA biosynthesis inhibitor, also inhibited germination of WT seeds, but this inhibition could not be overcome by subsequent treatment with GA3. However, GA3 did partially overcome the inhibitory effect of tetcyclacis on 7B-1 seed germination. While these contrasting responses of the mutant and WT seeds to the two inhibitors are intriguing, they are not readily explained. That said, both tetcyclasis and uniconazole are also known to elevate ABA levels in growing plants (Zeevaart and Creelman, 1988; Zeevaart et al., 1988; Häuser et al., 1990; Grossmann, 1990) and ABA is a potent inhibitor of seed germination for both genotypes (Fig. 1a). Hence, the explanation may lie with ABA:GA balances within the imbibed seeds, ignoring the possible toxic effects of applied inhibitors.
Fluridone, an inhibitor of carotenoid and ABA biosynthesis (Bartels and Watson, 1978; Gamble and Mullet, 1986; Saab et al., 1990; Xu and Bewley, 1995), promoted seed germination in both genotypes (Fig. 2a). This response is similar to that in corn, alfalfa, mungbean, and lettuce (Hole et al., 1989; Xu et al., 1990; Thind et al., 1997; Yoshioka et al., 1998). Interestingly, fluridone also partly restored germination in tetcyclacis-treated WT seeds, and completely restored it when it was supplied together with GA3 (Fig. 2b). This strongly suggests that tetcyclacis-induced inhibition in WT seeds is caused not only by inhibition of GA biosynthesis but also by increases in ABA levels.
7B-1 seeds were more sensitive to exogenous ABA in LD than were WT seeds (Fig. 1a) and fluridone promoted, whereas tetcyclacis inhibited, germination in 7B-1 seeds. However, when fluridone was supplied together with tetcyclacis full germination occurred in 7B-1, but not in WT seeds. Full germination did occur for WT seeds, though, when GA3+fluridone+tetcyclacis was supplied (Fig. 2b). Together, these observations suggest that 7B-1 seeds have high endogenous ABA levels and that elevated ABA, in a fine balance with endogenous GAs, confers an increased sensitivity to exogenous ABA. However, this suggestion needs to be confirmed by an analysis of ABA and GAs in 7B-1 and WT seeds.
Differential sensitivity of WT and 7B-1 roots and hypocotyls to exogenous growth regulators is also related to elevated endogenous ABA in the mutant
Exogenous ABA generally inhibits elongation of most plant organs (Pilet and Barlow, 1987), although in a few cases low concentrations of ABA can have a stimulatory effect on root growth (Collet, 1970; Yamaguchi and Street, 1977). 7B-1 root growth was more sensitive to the inhibitory effects of ABA in LD than were WT roots (Fig. 3b). This differential response to exogenous ABA may be related to the possible higher levels of ABA in 7B-1 roots. Although ABA levels were not analysed in roots, the elevated ABA levels in 7B-1 hypocotyls (Fig. 8a) supports this view.
Hypocotyl growth in WT and 7B-1 showed a similar sensitivity to exogenous ABA. However, fluridone at low concentrations (0.1 and 1 µM) stimulated greater growth in WT than in 7B-1 hypocotyls (Fig. 5b). This observation is consistent with higher levels of ABA in 7B-1 hypocotyls (Fig. 8a), and suggests that high ABA levels tend to mute the (presumed) effect of fluridone in reducing the ABA content.
Exogenous GA3 partially overcame the inhibitory effect of tetcyclacis on root (Fig. 3d) and hypocotyl elongation (Fig. 5f). This suggests that GAs are possibly involved as causal factors in root and hypocotyl growth in tomato. Exogenous GA3 had no effect on root elongation, which implies that endogenous GA levels in roots may be at, or near, optimal levels, a suggestion consistent with earlier work (Tanimoto, 1991) which showed that GA-promotion of root growth was obtained only after use of a GA biosynthesis inhibitor.
Morphology of fluridone-treated seedlings supports the notion that the 7B-1 mutant has high ABA content
The morphology of 7B-1 and WT seedlings grown on a fluridone-containing medium further supports the view that 7B-1 has elevated levels of endogenous ABA. Fluridone inhibits pigment biosynthesis, mainly carotenoids, resulting in chlorosis of leaves (Bartels and Watson, 1978; Popova and Riddle, 1996). In these experiments, fluridone (10 µM) bleached the cotyledons in WT seedlings, but the 7B-1 mutant cotyledons remained green (Fig. 6), indicating that the mutant has high ABA content. However, when fluridone was administered with ABA, normal, green cotyledons developed in WT as well as in 7B-1 seedlings (data not shown), implying that optimal levels of ABA are required for normal pigment development.
Hypocotyls of 7B-1 treated with fluridone (10 µM) had an intense purple colour, relative to WT, suggesting high levels of anthocyanin. Interestingly, 7B-1 seeds were also darker in colour than WT seeds (Table 1), and this may also be due to high anthocyanin. Anthocyanin is formed in response to light, but it can also be produced in response to stress and hormones, including ABA (Mol et al., 1996). The presumed increase in anthocyanin in 7B-1 hypocotyls may thus be related to higher ABA level in the mutant.
ABA accumulation in 7B-1 seedlings results in reduced transpiration rate
Reduced transpiration under LD in 7B-1 seedlings relative to WT (Fig. 7) can also be explained on the basis of elevated ABA in 7B-1 hypocotyls (Fig. 8a), assuming that hypocotyl ABA levels also reflect ABA levels in the cotyledons and/or influence stomatal opening. The ability of ABA to induce stomatal closure and thus regulate transpiration is well documented (Jones and Mansfield, 1970; Kriedemann et al., 1972; Arteca et al., 1985a, b). Both tetcyclacis and ABA reduced transpiration in 7B-1 disproportionately to WT (Fig. 7). Tetcyclacis has also been reported to reduce transpiration in oilseed rape and this effect was associated with increased levels of endogenous ABA (Häuser et al., 1990). Hence, the already elevated levels of endogenous ABA in 7B-1 appear to sensitize the mutant to additional ABA whether applied directly, or indirectly, i.e. from tetcyclasis treatment.
A positive effect of endogenous IAA on stomatal opening (Pemadasa, 1982; M Fellner, unpublished results) may also be a factor, and an interaction between ABA and IAA in stomata function (Irving et al., 1992) is possible. Thus, the high ABA/IAA ratio in 7B-1 under LD may result in reduced transpiration by the mutant. In contrast, elevated IAA levels, relative to ABA, in SD (Fig. 8a) could be responsible for 7B-1 maintaining a relatively high level of transpiration under SD (Fig. 7).
7B-1 as an ABA-biosynthesis/metabolism mutant
The 7B-1 mutant has some phenotypic traits which resemble GA-response or GA-overproducing mutants (Ross et al., 1997). For example, bigger 7B-1 seeds relative to WT suggest increased level of endogenous GAs during seed development (Swain et al., 1995). Relative to WT, there was less de-etiolation in 7B-1 hypocotyls in LD (Figs 4, 5a) and 7B-1 greenhouse-grown plants were taller at late stages (after floral bud anthesis) of development (data not shown). These phenotype characters all suggest increased GA levels in 7B-1. However, the mutant hypocotyls had relatively low levels of growth-active GAs in LD relative to WT (Fig. 8b).
Unlike the GA mutants, ABA-biosynthesis or ABA-response mutants are not always drastically affected in elongation growth. ABA-deficient mutants in tomato, e.g. flacca, sitiens or notabilis (Tal and Nevo, 1973) show relatively small or no changes in stem elongation relative to WT. Similarly, the ABA-supersensitive era1 mutant in Arabidopsis shows relatively normal growth and development (Cutler et al., 1996), but the ABA-deficient (aba) mutant in Arabidopsis has reduced growth (Koornneef et al., 1982). The phenotypic traits of 7B-1 are consistent with the notion that it is an ABA-overproducing mutant. It is therefore hypothesized that a product of the 7B-1 gene lesion influences ABA biosynthesis or catabolism. Greater accumulation of endogenous ABA in the 7B-1 mutant, especially in LD relative to WT, may also explain the amplified responses of the mutant to applied ABA in LD.
The 7B-1 mutant shows phenotypic similarities to the sl-2 mutant in tomato (background cv. Rutgers). The sl-2 mutant contains higher ABA concentrations in vegetative and floral parts compared to WT tissues (Singh and Sawhney, 1998). However, unlike the 7B-1 mutant, sl-2 does not show the photoperiod-dependent male sterility. None the less, a genetic complementation test is needed to rule out the possibility that the two mutations are allelic.
7B-1 as photomorphogenetic mutant
The reduced de-etiolation of the hypocotyl, the tall stature of older plants (not shown) and the putative increased chlorophyll and anthocyanin content in 7B-1 raises the question of whether 7B-1 is a photomorphogenetic mutant, especially since light is known to reduce etiolation and enhance anthocyanin biosynthesis (Kerckhoffs and Kendrick, 1997).
The possible roles played by four hormones, or hormone classes, IAA, ABA, GAs, and cytokinins, in light signal transduction have been reviewed recently (Kraepiel and Miginiac, 1997). The pew1 mutant in Arabidopsis, which is deficient in the biosynthesis of the phytochrome A chromophore, contains higher levels of ABA relative to WT (Kraepiel et al., 1994). Given the reduced de-etiolation of 7B-1 hypocotyl in LD, it is possible that ABA and/or IAA are involved in light signalling. In WT hypocotyls, there was a low IAA:growth-active GAs ratio in LD, compared to SD (Table 2). This corresponds with the greater hypocotyl de-etiolation of WT in LD and suggests that hypocotyl de-etiolation induced by light is regulated by auxin, or more specifically by a lowered IAA:GA balance (Fig. 8a; Table 2). The fact that 7B-1 has higher IAA:GAs ratio in LD than in SD again suggests a stimulatory role for auxin in the increased elongation (reduced de-etiolation) of the 7B-1 hypocotyl in LD. That said, the greater de-etiolation in WT in LD, relative to SD, is also positively associated with higher ABA:IAA ratio in LD than in SD. However, in 7B-1, no such relationship exists. This suggests either that ABA does not play a dominant role in tomato hypocotyl elongation, or that 7B-1 (which already has elevated levels of ABA) is less sensitive to the inhibitory effect of elevated endogenous ABA levels on hypocotyl growth.
These results thus suggest that 7B-1 could be a mutant affected in light perception or signal transduction. If so, it would be an excellent system with which to investigate the complex interactions between hormone biosynthesis on the one hand and light and hormone signalling on the other.
|
Acknowledgments |
|---|
We thank Dennis Dyck for photography, and Diane Davis and Virginia Lehmkuhl for technical assistance. This research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada to VKS and RPP.
|
Notes |
|---|
4 Present address: University of Washington, Botany Department 355325, 407 Hitchcock Hall, Seattle, WA 98195-5325, USA.
3 To whom correspondence should be addressed. Fax: +1 306 966 4461. E-mail: sawhney{at}admin.usask.ca
|
Abbreviations |
|---|
ABA, abscisic acid; BA, 6N-benzyladenine; DW, dry weight; FLU (fluridone), 1-methyl-3-phenyl-5-[3-trifluoromethyl-(phenyl)]-4(1H)-pyridinone; FW, fresh weight; GA(s), gibberellin(s); IAA, indole-3-acetic acid; LD, long day(s); SD, short day(s); TCY (tetcyclacis), 5-(4-chlorophenyl)-3,4,5,9,10-pentaza-tetra-cyclo 4,5,1,O2,6,O8,11 dodeca-3, 9-diene; UNI (uniconazole), (E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol; WT, wild-type; 2,4-D, 2,4-dichlorophenoxyacetic acid.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Altmann T.1999. Molecular physiology of brassinosteroids revealed by the analysis of mutants. Planta 208, 1–11.[Web of Science][Medline]
Arteca RN, Holcomb EJ, Schlagnhaufer C, Tsai D-S.1985a. Effects of root applications of gibberellic acid on photosynthesis, transpiration, and growth of geranium plants. HortScience 20, 925–927.
Arteca RN, Tsai D-S, Schlagnhaufer C.1985b. Abscisic acid effects on photosynthesis and transpiration in geranium cuttings. HortScience 20, 370–372.
Bartels PG, Watson CW.1978. Inhibition of carotenoid biosynthesis by fluridone and norflurazone. Weed Science 2, 198–203.
Chandler PM.1988. Hormonal regulation of gene expression in the ‘slender’ mutant of barley (Hordeum vulgare L.). Planta 175, 115–120.
Clouse SD, Langford M, McMorris TC.1996. A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiology 111, 671–678.[Abstract]
Collet GF.1970. Action de l'acide abscisique sur la rhizogenéze. Compte Rendu de l'Academie des sciences Série D 221, 667–670.
Cutler S, Ghassemian M, Bonetta D, Cooney S, McCourt P.1996. A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis. Science 273, 1239–1241.[Abstract]
Davies PJ.1995. The plant hormones: their nature, occurrence, and functions. In: Davies PJ, ed. Plant hormones. Physiology, biochemistry and molecular biology, 2nd edn. Dordrecht: Kluwer Academic Publishers, 1–12.
Devlin PF, Rood SB, Somers DE, Quail PH, Whitelam GC.1992. Photophysiology of the elongated internode (ein) mutant in Brassica rapa. Plant Physiology 100, 1142–1147.
Ephritikhine G, Pagant S, Fujioka S, Takatsuto S, Lapous D, Caboche M, Kendrick RE, Barbier-Brygoo H.1999. The sax1 mutation defines a new locus involved in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana. The Plant Journal 18, 315–320.[Web of Science][Medline]
Fujioka S, Yamane H, Spray CR, Gaskin P, MacMillan J, Phinney BO, Takahashi N.1988. Qualitative and quantitative analysis of gibberellins in vegetative shoots of normal, dwarf-1, dwarf-2, dwarf-3 and dwarf-4 seedlings of Zea mays L. Plant Physiology 88, 1367–1372.
Gamble PE, Mullet JE.1986. Inhibition of carotenoid accumulation and abscisic acid biosynthesis in fluridone-treated dark-grown barley. European Journal of Biochemistry 160, 117–121.[Web of Science][Medline]
Gaskin P, MacMillan J.1991. GC-MS of the gibberellins and related compounds. Methodology and a library of spectra. University of Bristol (Cantock's Enterprises), Bristol, England, UK.
Grossmann K.1990. Plant growth retardants as tools in physiological research. Physiologia Plantarum 78, 640–648.
Häuser C, Kwiatkowski J, Rademacher W, Grossmann K.1990. Regulation of endogenous abscisic acid levels and transpiration in oilseed rape by plant growth retardants. Journal of Plant Physiology 137, 201–207.
Hedden P.1987. Gibberellins. In: Rivier L, Crozier A, eds. Principles and practice of plant hormone analysis, Vol. 1. London: Academic Press, 9–110.
Hole DJ, Smith JD, Cobb BG.1989. Regulation of embryo dormancy by manipulation of abscisic acid in kernels and associated cob tissue of Zea mays L. cultured in vitro. Plant Physiology 91, 101–105.
Irving HR, Gehring CA, Parish RW.1992. Changes in cytosolic pH and calcium of guard cells precede stomatal movement. Proceedings of the National Academy of Sciences, USA 89, 1790–1794.
Jacobsen JV, Olszewski NE.1993. Mutation at the SPINDLY locus of Arabidopsis alters gibberellin signal transduction. The Plant Cell 5, 887–896.
Jones MG.1987. Gibberellins and the procera mutant of tomato. Planta 172, 280–284.
Jones R, Mansfield TA.1970. Suppression of stomatal opening in leaves treated with ABA. Journal of Experimental Botany 21, 714–718.
Jupe SC, Causton DR, Scott M.1988. Cellular basis of the effect of gibberellin and the pro gene on stem growth in tomato. Planta 174, 106–111.
Kerckhoffs LHJ, Kendrick RE.1997. Photocontrol of anthocyanin biosynthesis in tomato. Journal of Plant Research 110, 141–149.
Koornneef M, Jorna ML, Brinkhorst-van der Swan DLC, Karssen CM.1982. The isolation of abscisic acid (ABA)-deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive line of Arabidopsis thaliana (L.) Heynh. Theoretical and Applied Genetics 61, 385–393.
Koshioka M, Takeno K, Beall FD, Pharis RP.1983a. Purification and separation of gibberellins from their precursors and glucosyl conjugates. Plant Physiology 73, 398–406.
Koshioka M, Harada J, Takeno K, Noma M, Sassa T, Ogiyama K, Taylor JS, Rood SB, Legge RL, Pharis RP.1983b. Reversed-phase C18 high-performance liquid chromatography of acidic and conjugated gibberellins. Journal of Chromatography 256, 101–115.
Kraepiel Y, Miginiac E.1997. Photomorphogenesis and phytohormones. Plant, Cell and Environment 20, 807–812.
Kraepiel Y, Rousselin P, Sotta B, Kerhoas L, Einhorn J, Caboche M, Miginiac E.1994. Analysis of phytochrome- and ABA-deficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia. The Plant Journal 6, 665–672.
Kriedemann PE, Loveys BR, Fuller GL, Leopold AC.1972. Abscisic acid and stomatal regulation. Plant Physiology 49, 842–847.
Lanahan MB, Ho T-HD.1988. Slender barley: a constitutive gibberellin response mutant. Planta 175, 107–114.
Mancinelli AL, Borthwick HA, Hendrics SB.1966. Phytochrome action in tomato seed germination. Botanical Gazette 127, 1–5.
Mol J, Jenkins G, Schafer E, Weiss D.1996. Signal perception, transduction, and gene expression involved in anthocyanin biosynthesis. Critical Reviews of Plant Science 15, 525–557.
Murashige T, Skoog A.1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.
Pearce DW, Koshioka M, Pharis RP.1994. Chromatography of gibberellins. Journal of Chromatography A 658, 91–122.
Pemadasa MA.1982. Differential abaxial and adaxial stomatal responses to indole-3-acetic acid in Commelina communis L. New Phytologist 90, 209–219.
Pilet P-E, Barlow PW.1987. The role of abscisic acid in root growth and gravireaction: a critical review. Plant Growth Regulation 6, 217–265.
Popova LP, Riddle KA.1996. Development and accumulation of ABA in fluridone-treated and drought-stressed Vicia faba plants under different light conditions. Physiologia Plantarum 98, 791–797.
Potts WC, Reid JB, Murfet IC.1985. Internode length in Pisum. Gibberellins and the slender phenotype. Physiologia Plantarum 63, 357–364.
Rademacher W, Fritsch H, Graebe JE, Sauter H, Jung J.1987. Tetcyclasis and triazole-type plant growth retardants: their influence on the biosynthesis of gibberellins and other metabolic processes. Pesticide Science 21, 241–252.
Reid JB, Howell SH.1995. Hormone mutants and plant development. In: Davies PJ, ed. Plant hormones. Physiology, biochemistry and molecular biology. Dordrecht: Klüwer Academic Publishers, 448–485.
Reid JB, Ross JJ, Swain SM.1992. Internode length in Pisum. A new slender mutant with elevated levels of C19 gibberellins. Planta 188, 462–467.
Rood SB, Williams PH, Pearce D, Murofushi N, Mander LN, Pharis RP.1990. A mutant that increases gibberellin production in Brassica. Plant Physiology 93, 1168–1174.
Ross JJ, Murfet IC, Reid JB.1997. Gibberellin mutants. Physiologia Plantarum 100, 550–560.
Saab IN, Sharp RE, Pritchard J, Voetberg GS.1990. Increased endogenous abscisic acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials. Plant Physiology 93, 1329–1336.
Sawhney VK.1997. Genic male sterility. In: Shivanna KR, Sawhney VK, ed. Pollen biotechnology for crop production and improvement. Cambridge, UK: Cambridge University Press, 183–198.
Singh S, Sawhney VK.1998. Abscisic acid in a male sterile tomato mutant and its regulation by low temperature. Journal of Experimental Botany 49, 199–203.
Swain SM, Ross JJ, Kamiya Y.1995. Gibberellins and pea seed development. Expression of the lhi, ls and le5839 mutations. Planta 195, 426–433.[Web of Science]
Tal M, Nevo Y.1973. Abnormal stomatal behaviour and root resistance, and hormonal imbalance in three wilty mutants of tomato. Biochemistry and Genetics 8, 291–300.
Tanimoto E.1991. Gibberellin requirement for the normal growth of roots. In: Takahashi N, Phinney BO, MacMillan J, eds. Gibberellins. New York: Springer Verlag Inc., 229–240.
Thind SK, Chanpreet, Mridula.1997. Effect of fluridone on free sugar level in heat stressed mungbean seedlings. Plant Growth Regulation 22, 19–22.
Xu N, Coulter KM, Bewley JD.1990. Abscisic acid and osmoticum prevent germination of developing alfalfa embryos, but only osmoticum maintains the synthesis of developmental proteins. Planta 182, 382–390.
Xu N, Bewley JD.1995. The role of abscisic acid in germination, storage protein synthesis and desiccation tolerance in alfalfa (Medicago sativa L.) seeds, as shown by inhibition of its synthesis by fluridone during development. Journal of Experimental Botany 46, 687–694.
Yamaguchi T, Street HE.1977. Stimulation of the growth of excised cultured roots of soya bean by abscisic acid. Annals of Botany 41, 1129–1133.
Yokota T, Reeve DR, Crozier A.1976. The synthesis of [3H]-gibberellin A9 with high specific activity. Agricultural Biological Chemistry 40, 2091–2094.
Yoshioka T, Endo T, Satoh S.1998. Restoration of seed germination at supraoptimal temperatures by fluridone, an inhibitor of abscisic acid biosynthesis. Plant Cell Physiology 39, 307–312.
Zeevaart JAD, Creelman RA.1988. Metabolism and physiology of abscisic acid. Annual Review of Plant Physiology and Molecular Biology 39, 439–473.[Web of Science]
Zeevaart JAD, Gage DA, Creelman RA.1988. Recent studies of the metabolism of abscisic acid. In: Pharis RP, Rood S, eds. Plant growth substances. New York: Springer Verlag, 323–332.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  V. Bergougnoux, V. Hlavackova, R. Plotzova, O. Novak, and M. Fellner The 7B-1 mutation in tomato (Solanum lycopersicum L.) confers a blue light-specific lower sensitivity to coronatine, a toxin produced by Pseudomonas syringae pv. tomato J. Exp. Bot., March 1, 2009; 60(4): 1219 - 1230. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. W. Bassel, R. T. Mullen, and J. D. Bewley procera is a putative DELLA mutant in tomato (Solanum lycopersicum): effects on the seed and vegetative plant J. Exp. Bot., February 3, 2008; (2008) erm354v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-N. Mwange, H.-W. Hou, Y.-Q. Wang, X.-Q. He, and K.-M. Cui Opposite patterns in the annual distribution and time-course of endogenous abscisic acid and indole-3-acetic acid in relation to the periodicity of cambial activity in Eucommia ulmoides Oliv. J. Exp. Bot., March 1, 2005; 56(413): 1017 - 1028. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||