Synthesis and confirmation of structure for a new gibberellin, 2 beta-hydroxy-GA12 (GA110), from spinach and oil palm.

The identity of a new gibberellin (GA) in spinach and oil palm sap has been confirmed as 2 beta-hydroxy-GA12 (GA110) by comparisons of GC-mass spectral data obtained for the trimethylsilyl ether methyl ester derivatives with those of a synthetic sample prepared by means of a 24 step sequence from gibberellic acid; 2 beta-hydroxy-GA24 was also prepared. Experimental details for the latter part of the syntheses are described.

Gibberellins and leaf expansion in near-isogenic wheat lines containing Rht1 and Rht3 dwarfing alleles.

In near-isogenic lines of winter wheat (Triticum aestivum L. cv. Maris Huntsman) grown at 20° C under long days the reduced-height genes, Rht1 (semi-dwarf) and Rht3 (dwarf) reduced the rate of extension of leaf 2 by 12% and 52%, respectively, compared with corresponding rht (tall) lines. Lowering the growing temperature from 20° to 10° C reduced the rate of linear extension of leaf 2 by 2.5-fold (60% reduction) in the rht3 line but by only 1.6-fold (36% reduction) in the Rht3 line. For both genotypes, the duration of leaf expansion was greater at the lower temperature so that final leaf length was reduced by only 35% in the rht3 line and was similar in the Rht3 line at both temperatures. Seedlings of the rht3 (tall) line growing at 20° C responded positively to root-applied gibberellin A1 (GA1) in the range 1-10 µM GA1; there was a linear increase in sheath length of leaf 1 whereas the Rht3 (dwarf) line remained unresponsive. Gibberellins A1, 3, 4, 8, 19, 20, 29, 34, 44 and 53 were identified by full-scan gas chromatography-mass spectrometry in aseptically grown 4-d-old shoots of the Rht3 line. In 12-d-old seedlings grown at 20° C, there were fourfold and 24-fold increases in the concentration of GA1 in the leaf expansion zone of Rht1 and Rht3 lines, respectively, compared with corresponding rht lines. Although GA3 was present at a similar level to GA1 in the rht3 (tall) line it accumulated only fivefold in the Rht3 (dwarf) line. The steady-state pool sizes of endogenous GAs were GA19 ≫ GA20 = GA1 in the GA-responsive rht3 line whereas in the GA non-responsive Rht3 line the content of GA19≈ GA20 ⋘ GA1. It is proposed that one of the consequences of GA1 action is suppression of GA19-oxidase activity such that the conversion of GA19 to GA20 becomes a rate-limiting step on the pathway to GA1 in GA-responsive lines. In the GA-non-responsive Rht lines it is suggested that GA19 oxidase is not downregulated to the same extent and GA1 accumulates before the next rate-limiting step on the pathway, its 2ß-hydroxylation to GA8. The steady-state pool sizes of GA19, 20, 1, 3 and 8 were similar in developmentally equivalent tissues of the rht3 (tall) line growing at 10° C and 20° C despite a 2.5-fold difference in the rate of leaf expansion. In contrast, in the Rht3 (dwarf) line, the extent of accumulation of GA1 reflected the severity of the phenotype at the two temperatures with slower growing tissues accumulating less, not more, GA1. These results are interpreted as supporting the proposed model of regulation of the GA-biosynthetic pathway rather than previous suggestions that GA1 accumulates in GA-insensitive dwarfs as a consequence of reduced growth rates.

Comparison of gibberellins in normal and slender barley seedlings.

Gibberellins A(1), A(3), A(8), A(19), A(20), and A(29) were identified by full scan gas chromatography-mass spectrometry in leaf sheath segments of 7-day-old barley (Hordeum vulgare L. cv Golden Promise) seedlings grown at 20 degrees C under long days. In a segregating population of barley, cv Herta (Cb 3014), containing the recessive slender allele, (sln 1) the concentration of GA(1) and GA(3) was reduced by 10-fold and 6-fold, respectively, in rapidly growing homozygous slender, compared with normal, leaf sheath segments. However, the concentration of the C(20) precursor, GA(19), was nearly 2-fold greater in slender than in normal seedlings. There was little difference in the ABA content of sheath segments between the two genotypes. The gibberellin biosynthesis inhibitor, paclobutrazol, reduced the final sheath length of normal segregants (50% inhibition at 15 micromolar) but had no effect on the growth of slender seedlings at concentrations below 100 micromolar. There was a 15-fold and 4-fold reduction in GA(1) and GA(3), respectively, in sheath segments of 8-day-old normal seedlings following application of 10 micromolar paclobutrazol. The same treatment also reduced the already low concentrations of these gibberellins in slender segregants. The results show that the pool sizes of gibberellins A(1) and A(3) are small in slender barley and that leaf sheath extension in this genotype appears to be gibberellin-independent. The relationship between gibberellin status and tissue growth-rate in slender barley is contrasted with other gibberellin nonresponsive, but dwarf, mutants of wheat (Triticum aestivum) and maize (Zea mays).

Effect of Temperature on Gibberellin (GA) Responsiveness and on Endogenous GA(1) Content of Tall and Dwarf Wheat Genotypes.

Near-isogenic wheat (Triticum aestivum L.) lines differing in height-reducing (Rht) alleles were used to investigate the effects of temperature on endogenous gibberellin (GA) levels and seedling growth response to applied GA(3). Sheath and lamina lengths of the first leaf were measured in GA treated and control seedlings, grown at 11, 18, and 25 degrees C, of six Rht genotypes in each of two varietal backgrounds, cv Maris Huntsman and cv April Bearded. Endogenous GA(1) levels in the leaf extension zone of untreated seedlings were determined by gas chromatography-mass spectrometry with a deuterated internal standard in the six Maris Huntsman Rht lines grown at 10 and 25 degrees C. Higher temperature increased leaf length considerably in the tall genotype, less so in the Rht1 and Rht2 genotypes, and had no consistent effect on the Rht1+2, Rht3 and Rht2+3 genotypes. In all genotypes, endogenous GA(1) was higher at 25 degrees C than at 10 degrees C. At 10 degrees C the endogenous GA(1) was at a similar level in all the genotypes (except Rht2+3). At 25 degrees C it increased 1.6-fold in the tall genotype, 3-fold in Rht1 and Rht2, 6-fold in Rht3, and 9-fold in Rht1+2. Likewise, the genotypic differences in leaf length were very conspicuous at 25 degrees C, but were only slight and often unsignificant at 11 degrees C. The response of leaf length to applied GA(3) in the Rht1, Rht2, and Rht1+2 genotypes increased significantly with lowering of temperature. These results suggest the possibility that the temperature effect on leaf elongation is mediated through its effect on the level of endogenous GA(1) and that leaf elongation response to endogenous or applied GAs is restricted by the upper limits set by the different Rht alleles.

Determination of endogenous abscisic acid levels in immature cereal embryos during in vitro culture.

Levels of endogenous abscisic acid (ABA) in immature wheat (Triticum aestivum cv. Timmo) and barley (Hordeum vulgare cv. Golden Promise) embryos have been determined by enzyme-linked immunosorbent assay. Embryos of both cereal species showed an increase in ABA content during development on the parent plant. Immature embryos were excised and cultured in vitro on nutrient media that led to precocious germination or on media containing 9% (w/v) mannitol that maintained their developmental arrest. Barley and wheat embryos responded to these culture conditions in an identical manner with respect to changes in morphology, fresh weight, protein and lectin content. However, in complete contrast, the ABA content of barley embryos increased by an order of magnitude during culture on mannitol, whereas that of wheat embryos showed no significant change. The results are discussed within the context of the role of ABA in the regulation of embryo development.

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato.

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO 3 (-) absorption increased two-to five fold within 2 d of being deprived of NO 3 (-) supply. Nitrogen-starved barley roots continued to maintain a high potential for NO 3 (-) absorption, whereas NO 3 (-) absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO 3 (-) solution, roots of both species transported more NO 3 (-) and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO 3 (-) stores took precedence over NO 3 (-) transport to the xylem. Consequently, as N stress became more severe, transport of NO 3 (-) and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L p) and exudate volume (J v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L p and J v were increased, but NO 3 (-) absorption and NO 3 (-) flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO 3 (-) absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO 3 (-) absorption and transport and L p had been observed (wild-type tomato); (2) changes in NO 3 (-) absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO 3 (-) transport to those observed in the wild type.

Paclobutrazol inhibition of sterol biosynthesis in a cell suspension culture and evidence of an essential role for 24-ethylsterol in plant cell division.

Growth of a celery (Apium gravidens) cell suspension culture was inhibited by the synthetic plant growth regulator paclobutrazol. Paclobutrazol caused a reduction in the incorporation of [2-14C]acetate into the 4-demethyl sterols (campesterol, sitosterol, stigmasterol) but radioactivity accumulated in the 4 alpha-methylsterols. The accumulating 4 alpha-methylsterols were identified as obtusifoliol and cycloeucalenol indicating that paclobutrazol was inhibiting sterol biosynthesis by blocking 14 alpha-demethylation. The inhibition of celery cell growth by paclobutrazol could be partially overcome by addition of cholesterol to the culture medium. However, addition of stigmasterol restored growth to the control value suggesting an essential role for a 24-ethylsterol to support plant cell division.

T-DNA genes to study plant development: precocious tuberisation and enhanced cytokinins in A. tumefaciens transformed potato.

Potato Line Mb1501B is a derivative of the cultivar Maris Bard (Solanum tuberosum), transformed with T-DNA from A. tumefaciens strain LBA1501. In culture it grew as frequently branching stunted shoots with a basal callus, lacking roots. These shoots did not form tubers. When grafted, Mb1501B shoots gradually became morphologically more normal and aerial tubers formed readily. Cultured Mb1501B shoots contained 100-200-fold higher concentrations of the biologically-active cytokinins zeatin, zeatin riboside and their corresponding side-chain o-glucosides than untransformed Maris Bard shoots. Cultured Mb1501B shoots contained approximately a 3-fold lower concentration of indole acetic acid (IAA). In grafted Mb1501B plants a 3-10-fold higher concentration of the active cytokinins was found compared with untransformed plants and no difference in IAA concentration.

Endogenous abscisic acid in relation to photoperiodically induced bud dormancy.

The abscisic acid contents of birch, maple and sycamore plants growing under long and short photoperiods were measured by gas-liquid chromatography. No increase was observed in the abscisic acid content of extracts when plants were transferred to dormancy-inducing conditions.

The identification and quantitative analysis of abscisic acid in plant extracts by gas-liquid chromatography.

New techniques are described which permit the quantitative analysis of microgram quantities of abscisic acid in plant extracts by gas chromatography. Presumptive methyl abscisate peaks on gas chromatograms are positively identified by photosensitised isomerisation to methyl 2-trans-abscisate. Losses of abscisic acid during pre-purification are corrected by using 2-trans-abscisic acid as an internal standard.