Crop Science 40:1702-1705 (2000)
© 2000 Crop Science Society of America
SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY
Tetcyclacis and Abscisic Acid Differentially Affect Growth of Wheat (Triticum aestivum L.) Seedlings Isogenic for Reduced-Height Genes
Joshua D. Kleina and
Yonit Hebbeb
a Dep. of Field Crops, ARO-Volcani Center, Bet Dagan, Israel
b Institute for Agriculture according to the Torah, Yad Binyamin, Israel
vcjosh{at}agri.gov.il
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ABSTRACT
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Wheat (Triticum aestivum L.) varieties with the dwarfing Rht (reduced height) alleles have reduced sensitivity to endogenous or exogenous gibberellin (GA). The Rht isolines are also less sensitive to the effects of chlormequat and other compounds which cause dwarfing by depressing GA synthesis. Abscisic acid (ABA) inhibits cereal seed germination and is in dynamic tension with GA, which enhances germination. Tetcyclacis is a cytochrome P450 mono-oxygenase inhibitor of GA synthesis. It also inhibits ABA hydroxylase, thus extending ABA activity. In preliminary experiments, wheat varieties differing in Rht complement responded differently to ABA and/or tetcyclacis. We therefore investigated the effect of seed applications of ABA (100 µM), with or without tetcyclacis (25 µM), on germination and initial growth of Rht wheat isolines Rht-B1b, Rht-B1a (dwarf); Rht-B1b,Rht-D1b (dwarf); Rht-B1b + Rht-D1b (double-dwarf); and Rht-B1a, Rht-D1a (tall) to determine a possible genetic basis for the observed varietal differences. Treating seeds with ABA, tetcyclacis, or both, delayed emergence by 1, 1.8, or 3.7 d, respectively, compared with controls. ABA had little or no effect on final emergence, shoot height, or fresh weight, but led to a slight reduction in dry weight of Rht-D1b and Rht-B1b+D1b. Tetcyclacis reduced growth rate (mm/d), shoot height, fresh weight, and percentage dry weight in proportion to Rht dosage, as is typical of GA synthesis inhibitors. Synergistic effects of ABA and tetcyclacis were not detected for any parameter measured, except for rate of emergence. ABA did not exert a profound effect on seedling germination or initial growth of the Rht wheat isolines examined in this study.
Abbreviations: Rht, reduced-height • ABA, abscisic acid • GA, gibberellin
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INTRODUCTION
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WHEAT VARIETIES with the dwarfing Rht (reduced height) alleles have reduced sensitivity to endogenous gibberellin (GA) and to exogenous GA applications which would otherwise enhance plant growth (Gale and Youssefian, 1985). The Rht alleles also confer decreased sensitivity to inhibitors of GA synthesis such as ancymidol (Paolillo et al., 1991) or chlormequat (Beharav et al., 1994) that reduce plant growth in non-dwarf (tall) wheat. Although growth of Rht plants is decreased, seed germination is the same regardless of Rht complement (Allan, 1986).
Abscisic acid (ABA) inhibits cereal seed germination by repressing the transcription of 
-amylase genes in the aleurone layer (Fincher, 1989). ABA is in dynamic tension with GA, which stimulates transcription of -amylase genes and thus enhances germination. Practical use of ABA as a growth regulator is limited by its rapid degradation under field conditions (or after a single application, rather than a constant supply, under controlled conditions) to phaseic acid (Zeevaart et al., 1990). The initial inhibition of germination or growth resulting from a single application of ABA quickly dissipates as a result of oxidation by ABA hydroxylase. ABA hydroxylase is a cytochrome P450 monooxygenase (Zeevaart et al., 1990), and as such can be inhibited by tetcyclacis, thus extending or possibly even enhancing the effect of exogenous ABA (Dorffling et al., 1989; Daeter and Hartung, 1990). Tetcyclacis also limits the oxidation of ent-kaurene to kaurenoic acid, thus inhibiting GA synthesis, which in turn decreases the rates of seed germination and seedling growth. In GA-insensitive Rht dwarf wheat, however, there is less likelihood of a confounding effect of GA synthesis inhibition on the effect of ABA.
The effect of ABA on germination and initial growth of most Rht wheat isolines has been not been investigated. King and co-workers (1983) found that ABA content of leaves was not affected by the Rht complement, while Blum et al. (1997) showed that growth of dwarf wheat was less sensitive to a constant supply of exogenous ABA than was that of tall isolines. On the other hand, Flintham (1981) demonstrated that ABA delayed the germination of seeds of Rht3 [now known as Rht-B1c (Flintham et al., 1997)] wheat to the same extent as seeds of the non-dwarf isoline.
Blum and co-workers (1995, 1997) have suggested that there may be a genetic basis for varietal differences in response to ABA. Germination and growth of `Dariel' wheat was less sensitive to exogenous ABA than was that of `Bet Hashita' (Klein et al., 1998). Dariel has one dose of Rht, while Bet Hashita has a double dose of the gene (M. Pinthus, 1997, pers. comm.), although there are other differences in the cultivars' genetic composition. In order to concentrate specifically on interactions between Rht genes and ABA, we applied ABA to seeds of wheat isolines with single and double dosages of Rht alleles and measured seedling emergence and growth. We also investigated whether tetcyclacis could enhance the ABA effect, as was shown previously in different systems (Dorffling et al., 1989; Daeter and Hartung, 1990).
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Materials and methods
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Plant Materials
Five isolines of wheat carrying the alleles Rht-B1b, Rht-B1a; Rht-B1b, Rht-D1b; Rht-B1b + Rht-D1b; and Rht-B1a, Rht-B1a from Norin 10 / Brevor 14 in a background of the cultivar Burt (Allan and Pritchett, 1975), plus an additional line containing each of the Rht alleles from Suwon 92 in a background of Burt were obtained from R.E. Allan of Washington State University, Pullman, WA. Rht alleles from Norin 10 or Suwon 92 sources yield isolines that are sufficiently similar that the data could be analyzed together (Allan, 1986, and 1998, pers. comm.). The dwarfing alleles in these isolines were previously known as Rht1 (semi-dwarf), Rht2 (semi-dwarf), Rht1+Rht2 (double-dwarf), and rhtl (tall) (Flintham et al., 1997).
Seed Treatments
Seeds from each isoline were weighed, placed in plastic mesh bags, and imbibed for 2 h at 20°C in solutions of 25 µM tetcyclacis, 50 µM abscisic acid (ABA), or a combination of 25 µM tetcyclacis and 50 µM ABA. These concentrations were chosen after preliminary experiments indicated that they did not affect the final percentage germination of the seeds (Klein and Hebbe, 1998). Seeds were removed from solution and reweighed after excess moisture was blotted away, after which they were air-dried at 25°C for 24 h before sowing. Unimbibed seeds served as controls.
The four treatments were replicated in each of six allelically similar isolines in a completely randomized design. Ten seeds of each isoline-treatment combination were sown 1.5 cm deep in 7-cm-diam pots containing a 3:1 (v:v) sand-peat mix. Pots were placed in a growth chamber with a 16:8 h day:night photoperiod at 20°C and 100 µmol m-2.s-1, and were watered three times weekly with 25 mL of water. Daily measurements were made of emergence and of plant height from shoot base to tip of first leaf. After 3 wk, shoots were harvested, weighed, dried for 18 h at 80°C, and reweighed.
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Results
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Rate of Emergence and Final Emergence
Rht complement did not affect the rate of emergence of the wheat seedlings, which attained 50% of final emergence by 3.4 d after sowing untreated seeds (Fig. 1A)
. Treating seeds with 25 µM tetcyclacis delayed emergence by 1.8 d, while treating with 100 µM ABA delayed emergence by approximately 1 d, compared with controls. Combining tetcyclacis and ABA resulted in a synergistic delay in emergence of 3.7 d, compared with controls.
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Fig. 1 Rate of emergence (a) and total emergence (b) of `Burt' wheat seedlings with different complements of Rht alleles. Seeds were pretreated with 50 µM abscisic acid (ABA), 25 µM tetcyclacis, or a combination of the two (Tet+ABA). Standard error is indicated
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Final emergence of all isolines was greater than 85% (Fig. 1B). Tetcyclacis, alone or in combination with ABA, significantly reduced the final emergence of tall genotypes by 13%. The emergence of dwarf (Rht-D1b and Rht-B1b) seedlings was reduced by tetcyclacis or ABA, but the difference from controls was only a few percent. The double-dwarfs were virtually unaffected by separate tetcyclacis or ABA treatments, but had a synergistic reduction in emergence in the combined presence of ABA and tetcyclacis, as did Rht-B1b.
Growth Rate and Final Height
The growth rate (Fig. 2A)
and final height (Fig. 2B) of emerged seedlings were dictated by the genetic complement and by the presence of tetcyclacis. Untreated and ABA-treated seeds produced seedlings of similar height and growth rate, with tall seedlings averaging 14 mm/d growth and 24.5-cm final height, semi-dwarf seedlings averaging 10.5 mm/d and 21-cm height, and double-dwarfs growing only 7 mm/d, and attaining 15-cm height at the end of 3 wk. In the presence of tetcyclacis, however, the growth rate of all genotypes was only 4 mm/d. The final height of all seedlings from tetcyclacis-treated seeds was approximately 6.5 cm. Compared with seedlings from untreated or ABA-treated seeds, double-dwarf seedlings were proportionately less affected by seed treatment with tetcyclacis than were tall or semi-dwarf seedlings. The synergistic effect resulting from combining ABA with tetcyclacis that was noted in seedling emergence rate (Fig. 1A) was not evident in subsequent seedling growth (Fig. 2A).
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Fig. 2 Growth rate (a) and final height (b) of `Burt' wheat seedlings with different complements of Rht alleles. Seeds were pretreated with 50 µM abscisic acid (ABA), 25 µM tetcyclacis, or a combination of the two (Tet+ABA). Standard error is indicated
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Fresh Weight and Percent Dry Weight
Shoot fresh weight followed the same general pattern as that of seedling growth (Fig. 3A)
. ABA-treated seeds produced seedlings with the same shoot fresh weight as untreated controls. Tall seedlings averaged 126 mg, semi-dwarfs averaged 114 mg, and double-dwarfs averaged 96 mg. Regardless of genotype, tetcyclacis-treated seedlings weighed 82 mg, while seeds treated with the combination of tetcyclacis and ABA produced seedlings weighing 72 mg. As was the case with seedling height, double-dwarf seedlings were least sensitive to tetcyclacis.
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Fig. 3 Shoot fresh weight (a) and percentage dry matter (b) of `Burt' wheat seedlings with different complements of Rht alleles. Seeds were pretreated with 50 µM abscisic acid (ABA), 25 µM tetcyclacis, or a combination of the two (Tet+ABA). Standard error is indicated
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Seedling dry weight expressed as a proportion of fresh weight ranged from 1.3 to 1.4 g kg-1 in control and ABA-treated seeds (Fig. 3B), with tetcyclacis-treated seedlings (regardless of ABA) averaging about 0.1 g kg-1 less dry weight. Rht-B1b and null seedlings accumulated slightly more dry weight on a percentage basis than did Rht-D1b or the double-dwarf seedlings, but these differences were essentially eliminated in the combined tetcyclacis and ABA treatment.
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Discussion
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The Rht alleles are considered to confer relative insensitivity to GA synthesis inhibitors such as tetcyclacis (Beharav et al., 1994; Paolillo et al., 1991) but evidently such insensitivity becomes more prominent after germination. Seedling emergence of both dwarf and tall genotypes was equally limited by tetcyclacis, which probably delayed emergence by inhibiting the synthesis and accumulation of GA that triggers germination. Given the ineffectiveness of ABA alone compared with tetcyclacis, the synergistic effect of tetcyclacis and ABA in delaying emergence could have been the result of a decrease in ABA breakdown by the inhibition of ABA hydroxylase in the presence of tetcyclacis (Zeevaart et al., 1990) and a resulting enhancement of seed dormancy by ABA. Tetcyclacis itself can inhibit the synthesis of GA enough to bring about a lag in germination (Halmann, 1990), but this effect was apparently enhanced by the increased amounts of ABA present in the seed as a result of imbibition.
Although the rates of emergence were the same across genotypes within treatments (Fig. 1A), final emergence was related to Rht complement, with the double dwarf being least affected by ABA and/or tetcyclacis (Fig. 1B). Seeds with Rht-D1b seemed more affected by ABA than by tetcyclacis, while null genotype seedlings were clearly most affected by tetcyclacis. Lu et al. (1989) reported isolating genetic variants of wheat that exceeded parental lines in growth rate and yield when exposed to ABA. However, these results were not substantiated in a survey of the differential sensitivity of growth rate to ABA in a range of wheat varieties (Blum and Sinmena, 1995). Unlike the present report, the broad range of sensitivity to ABA described in the cultivar survey (Blum and Sinmena, 1995) was not ascribed to any aspect of the genetic background of the varieties.
ABA alone or in combination with tetcyclacis had no effect on growth rate and final height in our study. Other reports showing that ABA decreases plant growth were based on a constant supply of the hormone to the plants (Blum and Sinmena, 1995; Blum et al., 1997) or on a spray directly on the growing plant (Todorov et al., 1998). The amounts of ABA absorbed by the seeds during the pulse treatment may have broken down to inactive metabolites (Zeevaart et al., 1990) or just may have been insufficient to maintain an ABA effect after emergence. Tetcyclacis should have prevented ABA breakdown and/or inactivity even after a one-time pulse-loading of the seed (Dorffling et al., 1989), and indeed, a synergistic effect was observed in seedling emergence (Fig. 1A and B). In measurements of growth rate and final height, however, (Fig. 2A and B), 25 µM tetcyclacis completely masked any contribution by ABA. Perhaps this amount of tetcyclacis was insufficient to prevent ABA breakdown after seedling emergence and during subsequent growth. Wheat seed treatments that combined higher concentrations of tetcyclacis (50 to 100 µM) and ABA (200 µM) significantly slowed the growth rate and diminished final plant height in seedlings grown for 30 d (Klein et al., 1998).
The decrease in growth as affected by tetcyclacis was proportional to Rht dosage, with reductions of 75, 66, and 50% in tall, dwarf, and double-dwarf genotypes, respectively. Seedlings with Rht-B1b alleles were slightly less sensitive to tetcyclacis than were seedlings with Rht-D1b, since they had less of a proportional decrease in height or growth, compared with plants from untreated or ABA-treated seeds. There are no reports in the literature of significant consistent physical or physiological differences between plants with one or the other allele. Wheat plants from tetcyclacis-treated seeds sown in the field eventually attained 80% of the height of plants from untreated seeds (Klein and Hebbe, 1998, unpublished data).
The decrease in shoot fresh weight resulting from tetcyclacis (Fig. 3A) may be the result of the overall decrease in growth rate noted with plant height (Fig. 2B). However, seedlings from seeds treated with GA-synthesis inhibitors such as triadimefon (Gao et al., 1988) or tetcyclacis (Klein et al., 1998) had thicker and wider leaves , indicating that the decrease in leaf length is compensated by an increase in other dimensions. The decrease in shoot fresh weight compared with controls has been noted with other GA-synthesis inhibitors such as triazoles (Fletcher et al., 1986) and trinexapac-ethyl (Zhang and Schmidt, 2000). The ratio of weight to height in tall and semi-dwarf genotypes more than doubled in response to tetcyclacis (5.5 g cm-1 without tetcyclacis vs. 12.1 g cm-1 with tetcyclacis), indicating a proportional thickening of the leaves. In double-dwarf plants, which are less sensitive to GA synthesis inhibition, the weight:height ratio increased by only 50%, from 6.4 g cm-1 without tetcyclacis to 9.6 g cm-1 in the presence of the inhibitor. The fresh weight of plants from tetcyclacis-treated seeds sown in the field did not differ from that of control plants at maturity (Klein and Hebbe, 1998, unpublished).
Although ABA did not affect seedling fresh weight, it did cause a decrease in dry matter accumulation in the semi-dwarf Rht-D1b and in the double-dwarf Rht-B1b+D1b. ABA accumulation can decrease photosynthetic rate, and thus decrease assimilate accumulation and partitioning. The genetic specificity of this effect in wheat for Rht genotypes has not previously been reported, although there are varietal differences in transpiration in response to exogenous ABA (Blum and Sinmena, 1995).
In conclusion, the Rht genes investigated in this study affected wheat seedling response to ABA and to tetcyclacis applied separately or together. The effects are mostly proportional to gene dosage, and may therefore have been associated more with potential plant size rather than specific genetic complement. The Rht-B1c allele, which results in smaller plants than the double-dwarf Rht-B1b+D1b, conferred decreased sensitivity to ABA in three-week old seedlings (Blum et al., 1997), although Flintham (1981) found that Rht-B1c did not significantly affect coleoptile elongation in response ABA. Other Rht genes such as Rht-D1c (formerly known as Rht10) or other genes that affect plant size should also be examined for their effect on the response of wheat seedlings to ABA.King Gale Quarrie 1983
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NOTES
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Contribution no. 118/99 from the Institute of Field Crops, ARO- The Volcani Center, Bet Dagan, 50250 ISRAEL.
Received for publication July 27, 1999.
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ABSTRACT
INTRODUCTION
