John R. Lenton

Transcripts of Vp-1 homeologues are misspliced in modern wheat and ancestral species

The maize (Zea mays) Viviparous 1 (Vp1) transcription factor has been shown previously to be a major regulator of seed development, simultaneously activating embryo maturation and repressing germination. Hexaploid bread wheat (Triticum aestivum) caryopses are characterized by relatively weak embryo dormancy and are susceptible to preharvest sprouting (PHS), a phenomenon that is phenotypically similar to the maize vp1 mutation. Analysis of Vp-1 transcript structure in wheat embryos during grain development showed that each homeologue produces cytoplasmic mRNAs of different sizes. The majority of transcripts are spliced incorrectly, contain insertions of intron sequences or deletions of coding region, and do not have the capacity to encode full-length proteins. Several VP-1-related lower molecular weight protein species were present in wheat embryo nuclei. Embryos of a closely related tetraploid species (Triticum turgidum) and ancestral diploids also contained misspliced Vp-1 transcripts that were structurally similar or identical to those found in modern hexaploid wheat, which suggests that compromised structure and expression of Vp-1 transcripts in modern wheat are inherited from ancestral species. Developing embryos from transgenic wheat grains expressing the Avena fatua Vp1 gene showed enhanced responsiveness to applied abscisic acid compared with the control. In addition, ripening ears of transgenic plants were less susceptible to PHS. Our results suggest that missplicing of wheat Vp-1 genes contributes to susceptibility to PHS in modern hexaploid wheat varieties and identifies a possible route to increase resistance to this environmentally triggered disorder.

Genetic map locations for orthologous Vp1 genes in wheat and rice

Abstract Chromosome locations for gene orthologues of the dormancy-related maize transcription factor VIVIPAROUS-1, encoded by the Vp1 locus on maize chromosome 3, were determined in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) via linkage to markers on existing molecular maps using a cDNA of a wheat Vp1 orthologue as a probe in genomic Southern analyses. Vp1-orthologous loci were detected on the long arms of wheat chromosomes 3A, 3B and 3D [Xlars10 (taVp1) loci] and rice chromosome 1 (osVp1), in line with previous evidence of synteny between these regions of the rice and wheat genomes and chromosome 3 of maize. The wheat loci mapped some 30 cM from the centromeres and some 30 cM proximal to the red grain (R) loci that control seed colour and coat-imposed dormancy. This unequivocal, genetic separation of the Vp1 and R loci may offer an opportunity for improving resistance to pre-harvest sprouting in wheat by combining the coat-imposed dormancy associated with red seed colour and true embryo dormancy regulated by Vp1.

Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat.

The enzymes gibberellin (GA) 20-oxidase and 3-oxidase are major sites of regulation in GA biosynthesis. We have characterised one member of each of the gene families encoding these enzymes that are highly expressed in elongating stems and in developing and germinating grains of wheat and are therefore likely to have prominent developmental roles in these tissues. We mapped the three homoeologues of the GA 20-oxidase gene TaGA20ox1 to chromosomes 5BL, 5DL and 4AL. TaGA20ox1 is expressed mainly in the nodes and ears of the elongating stem, and also in developing and germinating embryos. Expression in the nodes, ears and germinating embryos is predominantly from the A and D genomes. Each homoeologous cDNA encodes a functional enzyme that catalyses the multi-step conversions of GA12–GA9, and GA53–GA20. Time course and enzyme kinetic studies indicate that the initial oxidation steps from GA12 and GA53 to the free alcohol forms of GA15 and GA44, respectively, occur rapidly but that subsequent steps occur more slowly. The intermediate GA19 has an especially low affinity for the enzyme, consistent with its accumulation in wheat tissues. The three homoeologous cDNAs for the 3-oxidase gene TaGA3ox2 encode functional enzymes, one of which was shown to possess low levels of 2β-hydroxylase, 2,3-desaturase, 2,3-epoxidase and even 13-hydroxylase activities in addition to 3β-hydroxylase activity. In contrast to TaGA20ox1, TaGA3ox2 is expressed in internodes, as well as nodes and the ear of the elongating stem. It is also highly expressed in developing and germinated embryos.

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 2β-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.

Isolation of gibberellin metabolic pathway genes from barley and comparative mapping in barley, wheat and rice

Gene sequences encoding gibberellin (GA) biosynthetic and catabolic enzymes were isolated from ‘Himalaya’ barley. These genes account for most of the enzymes required for the core pathway of GA biosynthesis as well as for the first major catabolic enzyme. By means of DNA gel blot analysis, we mapped coding sequences to chromosome arms in barley and wheat using barley-wheat chromosome addition lines, nulli-tetrasomic substitution and ditelosomic lines of wheat. These same sequences were used to identify closely related sequences from rice, which were mapped in silico, thereby allowing their syntenic relationship with map locations in barley and wheat to be investigated. Determination of the chromosome arm locations for GA metabolic genes provides a framework for future studies investigating possible identity between GA metabolic genes and dwarfing genes in barley and wheat.

GIBBERELLIN INSENSITIVITY AND DEPLETION IN WHEAT - CONSEQUENCES FOR DEVELOPMENT

Near-isogenic lines of wheat containing the semi-dwarfing (Rht1) and dwarfing (Rht3) genes are reduced in height by 18% and 56% respectively, compared with the tall (rht) parent, Maris Huntsman. Variation in stem height in these lines is due to differences in rates of elongation of individual stem internodes. By contrast, the Rht genes do not affect rachis internode elongation in the developing ear. The differential effect of Rht genes on growth of stem and ear internodes results in a greater survival of florets in ears of Rht genotypes at anthesis. Associated with the reduction in stem height in Rht genotypes is an insensitivity of vegetative tissues to applied GA and an increase in the concentration of endogenous GA1. The relative effectiveness of chlormequat chloride and paclobutrazol as growth retardants and inhibitors of GA biosynthesis is examined in wheat seedlings. Low concentrations of 2S,3S paclobutrazol are shown to inhibit the accumulation of GA1 in the GA-insensitive, Rht3 genotype without affecting shoot growth. This inhibitor is used to explore the relationship between GA1 concentration in the expansion zone and final length of leaves in the GA-responsive, rht genotype. The merit of a combined genetical and chemical approach to examining the relative importance of GA concentration and tissue sensitivity in the development of wheat is discussed critically.

An analysis of dormancy, ABA responsiveness, after-ripening and pre-harvest sprouting in hexaploid wheat (Triticum aestivum L.) caryopses

Embryo and caryopsis dormancy, abscisic acid (ABA) responsiveness, after-ripening (AR), and the disorder pre-harvest sprouting (PHS) were investigated in six genetically related wheat varieties previously characterized as resistant, intermediate, or susceptible to PHS. Timing of caryopsis AR differed between varieties; AR occurred before harvest ripeness in the most PHS-susceptible, whereas AR was slowest in the most PHS-resistant. Whole caryopses of all varieties showed little ABA-responsiveness during AR; PHS-susceptible varieties were responsive at the beginning of the AR period whereas PHS-resistant showed some responsiveness throughout. Isolated embryos showed relatively little dormancy during grain-filling and most varieties exhibited a window of decreased ABA-responsiveness around the period of maximum dry matter accumulation (physiological maturity). Susceptibility to PHS was assessed by overhead misting of either isolated ears or whole plants during AR; varieties were clearly distinguished using both methods. These analyses allowed an investigation of the interactions between the different components of seed development, compartments, and environment for the six varieties. There was no direct relationship between speed of caryopsis AR and embryo dormancy or ABA-responsiveness during seed maturation. However, the velocity of AR of a variety was closely associated with the degree of susceptibility to PHS during AR suggesting that these characters are developmentally linked. Investigation of genetic components of AR may therefore aid breeding approaches to reduce susceptibility to PHS.

Endogenous gibberellins and kauranoids identified from developing and germinating barley grain

Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA1 (trace), GA4 (trace), GA8 (trace), GA12, GA17, GA20 (tentative) (trace), GA25, GA34, GA48, 18-hydroxy-GA4, 12β-hydroxy-GA9, and 18-hydroxy-GA34 (tentative). A hydrolyzed butanol extract contained GA17, GA20, GA48, and 18-hydroxy-GA34 (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA1, GA3 (possibly a contaminant), GA17, GA19, GA20, GA34, GA48, and 18-hydroxy-GA34 (tentative). A hydrolyzed butanol extract contained GA34, GA48, and 18-hydroxy-GA34 (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1β-Hydroxylated GAs previously found in wheat were not found in barley in this study.

Gibberellins and α-amylase gene expression in germinating wheat grains

Gibberellins (GAs), GA8, GA17, GA19, GA20, GA29, and GA79 were identified by full-scan gas chromatography-mass spectrometry in a purified acidic fraction and GA8, GA20, GA79, and GA90 in a hydrolysed conjugate fraction from mature wheat grains. Gibberellin A20-13-O-glucoside was also quantified directly as the permethyl derivative in dry seed. The scutellum was identified as the major site of de novo GA biosynthesis by measuring ent-kaurene accumulation in vivo in grains treated with 2S,3S-paclobutrazol. Several GAs of the early 13-hydroxylation GA pathway began to accumulate in the axis and scutellum between 24 and 48 hours in untreated grains germinated at 15°C. Bioactive GA1 and GA3 also increased in the endosperm during this period, whereas abscisic acid contents of embryo and endosperm declined rapidly over 48 hours following imbibition. Treating grains with 2S,3S-paclobutrazol reduced GA1 plus GA3 content of scutella by 70–80% over 4 days without affecting significantly the steady-state pool of α-amylase mRNA transcripts. In contrast, a 50–80% reduction in the content of bioactive GAs in the endosperm was associated with a 70–78% decrease in transcripts for both α-amylase gene families in aleurones of paclobutrazol-treated grains. It was concluded that the initiation of α-amylase gene expression in wheat scutella was independent of de novo GA biosynthesis, whereas that in the aleurone was largely dependent on embryo-produced GAs.

Inhibition of celery cell growth and sterol biosynthesis by the enantiomers of paclobutrazol

Abstract The four enantiomeric forms of the triazole plant growth retardant, paclobutrazol, [(2 RS , 3 RS )-1-(4chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)-pentan-3-ol] were tested as inhibitors of cell growth and sterol composition using a celery suspension culture. The (2 R ,3 R )- and (2 R ,3 S )-enantiomers were potent inhibitors of cell growth and caused a large accumulation of the 14α-methylsterols obtusifoliol, cycloeucalenol, 14α,24-dimethylcholest-8-en-3β-ol and 14α,24-dimethylcholesta-8,24(28)-dien-3β-ol. The (2 S ,3 S )- and (2 S ,3 R )-enantiomers on the other hand were only inhibitory to growth and sterol 14α-demethylation at higher concentrations. It is concluded that the (2 R )-configuration confers the highest potency both for retarding cell proliferation and for inhibition of the cytochrome P-450 dependent sterol 14α-demethylation reaction in celery cells. A lowering of the stigmasterol: sitosterol ratio was noted, particularly with the (2 R ,3 R )- and (2 S ,3 S )-enantiomers, which suggested that the sterol Δ 22 -desaturase may also be a target for inhibition by triazoles. The relevance of these results to the mode of action of (2 RS ,3 RS )-paclobutrazol as a plant growth retardant is discussed.