Scholarly article on topic 'Decreased shoot stature and grain  -amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat'

Decreased shoot stature and grain  -amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat Academic research paper on "Biological sciences"

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Academic research paper on topic "Decreased shoot stature and grain  -amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat"

Journal of Experimental Botany, Vol. 58, No. 12, pp. 3213-3226, 2007 doi:10.1093/jxb/erm166 Advance Access publication 4 October, 2007

This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Decreased shoot stature and grain a-amylase activity following ectopic expression of a gibberellin 2-oxidase gene in transgenic wheat

Nigel E. J. Appleford*'t, Mark D. Wilkinson*, Qian Ma*, Daniel J. Evans, Marlon C. Stone, Stephen P. Pearce, Stephen J. Powers, Stephen G. Thomas, Huw D. Jones, Andrew L. Phillips, Peter Hedden§ and John R. Lenton

Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom Received 10 May 2007; Revised 22 June 2007; Accepted 26 June 2007

Experimental Botany

wwwjxb.oxfoi'djournals.org

Abstract

Ectopic expression of a gibberellin 2-oxidase gene (PcGA2ox1) decreased the content of bioactive gibber-ellins (GAs) in transgenic wheat, producing a range of dwarf plants with different degrees of severity. In at least one case, a single transformation event gave rise to T1 plants with different degrees of dwarfism, the pheno-types being stably inherited over at least four generations. The dwarf phenotype, which included dark-green leaves, increased tillering and, in severe cases, a prostrate growth habit, was replicated by the application of a GA biosynthesis inhibitor to the wild type. Ear rachis length, grain set, and grain size were also decreased in the wheat transformants, compared with an azygous (null) line. The extent of post-germination a-amylase production in grains reflected the severity of the shoot phenotype of the transformants and both developmental processes were restored to normal by the application of gibberellic acid (GA3). Expression of two GA biosynthesis genes (TaGA20ox1 and TaGA3ox2) was up-regulated, and that of two a-amylase gene families (a-Amy1 and a-Amy2) down regulated, in scutella of semi-dwarf lines, compared with controls. The marked decline in transcript abundance of both a-amylase gene families in aleurone was associated with a decreased content of bioactive GAs in grains of the semi-dwarf lines.

Key words: a-Amylase, ear development, GA 2-oxidase, gene expression, gibberellin, grain size, paclobutrazol, shoot height, tillering, transgenic wheat.

Introduction

Genetic and biochemical analysis of certain dwarf and overgrowth mutants of several mono- and dicotyledonous species has provided compelling evidence that gibberel-lins (GAs) are important regulators of stem internode growth determining final plant height. In cereals, the breeding of semi-dwarf varieties of wheat (Triticum aestivum L.) and rice (Oryza sativa L.), coupled with the increased use of nitrogen fertilizers and herbicides, resulted in increased yields worldwide during the so-called 'green revolution' of the 1960-1970s. The importance of decreasing shoot stature also led the agrochemical industry to develop various classes of plant growth retardants that act by inhibiting different steps on the GA biosynthesis pathway. For many years, it has been routine agronomic practice to treat intensively managed wheat crops with growth retardants in order to prevent the damaging effects of wind and rain that cause stem collapse (lodging) in untreated crops.

The reduced height (Rht) in hexaploid bread wheat was derived mainly from the Japanese semi-dwarf variety, Norin 10, containing the Rht-B1b and Rht-D1b mutant alleles (formally Rht1 and Rht2) (Gale and Youssefian, 1985; Borner et al., 1997). These semi-dominant Rht mutations are associated with an inability of shoot tissues to respond fully to applied GA, suggesting an interference with the normal GA signal transduction pathway. DNA sequence analysis of Rht-B1b and RhtD1b genes showed that they were orthologues of maize (Zea mays L.) dwarf-8 and Arabidopsis thaliana L. GAI (Peng et al., 1999), mutant

* These authors contributed equally to the work.

y Present address: National Blood Service, Southmead Rd., Bristol BS10 5ND, UK.

z Present address: National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.

§ To whom correspondence should be addressed. E-mail: peter.hedden@bbsrc.ac.uk

© 2007 The Author(s).

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

genes that also confer GA-insensitive dwarf phenotypes. The wild-type genes encode N-terminal DELLA-domain proteins that repress plant growth and GA relieves growth repression by targeting their degradation via the ubiquitin-proteosome pathway (reviewed in Thomas and Sun, 2004). The mutant wheat semi-dwarfing genes contain nucleotide substitutions that create stop codons in the N-terminal region 1 that, by reinitiation, are assumed to result in the formation of truncated proteins that retain a capacity to inhibit growth, but are resistant to GA-induced degradation (Peng et al, 1999).

Near-isogenic wheat lines containing different GA non-responsive Rht dwarfing alleles have decreased rates of elongation and final lengths of leaves (Keyes et al., 1989; Pinthus et al., 1989) and associated stem inter-nodes (Hoogendoorn et al., 1990), compared with GA-responsive tall lines. The primary effect of these Rht genes is to decrease cell expansion in the elongation zone of vegetative stem tissues without affecting final ear size. As a consequence, more assimilates are partitioned to developing ears thus supporting greater floret survival at anthesis and increased grain set, compared with GA-responsive, tall wheats (Youssefian et al., 1992b). Interestingly, the Rht semi-dwarfing genes do not appear to affect the timing of the transition of shoot apical meristem from a vegetative to a reproductive state (Youssefian et al., 1992a), a response that is hastened by applied GA and delayed by treatment with plant growth retardants that block GA production (Hutley-Bull and Schwabe, 1982; Evans et al., 1995).

In contrast to wheat, the commercially successful varieties of rice are GA-responsive semi-dwarfs, suggesting the presence of mutations in the biosynthetic pathway leading to the formation of bioactive GA. Recently, the basis of semi-dwarfism in four rice varieties that contributed to the 'green revolution' has been attributed to different alleles at the recessive sd-1 locus that are independent mutations in the OsGA20ox2 gene. This gene, a member of a small gene family, is expressed mainly in stems and is an important regulatory step on the pathway to active GA (reviewed in Hedden, 2003). Specific suppression of another member of the gene family, OsGA20ox1, caused semi-dwarfism, suggesting that it is also involved in the regulation of shoot stature in rice (Oikawa et al., 2004). In addition, different alleles of the GA-responsive dwarf rice, d18, are associated with mutations in the OsGA3ox2 gene that is also expressed highly in stems (Itoh et al., 2001).

In wheat, one member each of the GA 20-oxidase and GA 3-oxidase gene families (TaGA20ox1 and TaGA3ox2) has been cloned and shown to be highly expressed in elongating stems and developing and germinated embryos supporting their involvement in GA-regulated processes in these tissues (Appleford et al., 2006). In stem tissues, expression was highest in the nodal region subtending a rapidly expanding internode. At present, expression of

the orthologue of the rice sdl gene (TaGA20ox2) has not been studied in wheat.

Induction of a-amylase and other hydrolytic enzymes in the aleurone layer of germinated cereal grains is dependent on embryo-produced GAs (reviewed in Fincher, 1989). GA-deficient mutants of barley (Hordeum vulgare L.) and rice produce less post-germination a-amylase than corresponding wild types and a normal phenotype is restored by GA application (reviewed in Appleford and Lenton, 1997). In germinating wheat, high levels of expression of both TaGA20ox1 and TaGA3ox2 genes in the scutellum (Appleford et al, 2006) supported evidence that this organ is the main site of de novo GA biosynthesis (Appleford and Lenton, 1997). In rice, expression of two members each of the GA 20-oxidase and GA 3-oxidase gene families was confined to the scutellar epithelium showing that this specialized tissue is an important site of GA production (Kaneko et al., 2003) and mutant analysis confirmed that normal expression of OsGA3ox2 was essential for initiation of expression of RAmy1A in aleurone (Kaneko et al., 2002).

In the absence of single gene recessive mutants in hexaploid bread wheat, and as an alternative approach to the use of plant growth retardants, transgenic wheat was generated to determine the consequences of GA depletion on specific aspects of wheat development. We were interested in the range of phenotypes that might be produced compared with those of GA-'non-responsive', Rht semi-dwarfs and a GA-responsive tall line treated with an inhibitor of GA biosynthesis. The initial strategy involved ectopic expression of a full-length cDNA of a runner bean (Phaseolus coccineus L.) GA 2-oxidase gene (PcGA2ox1) (Thomas et al., 1999), fused behind a maize ubiquitin promoter. Based on the activity of the recombinant protein, it was reasoned that this gene product would cause a more rapid turnover of GAs by inactivation of the main biologically active GA of wheat tissues, GA1 (to GA8) and its precursor, GA20 (to GA29), by 2-oxidation. While this work was in progress, it was reported that rice plants transformed with a GA 2-oxidase, OsGA2ox1, behind a 'constitutive' actin promoter produced severely dwarfed plants that failed to set grain (Sakamoto et al., 2001). However, a more targeted approach using the promoter of the shoot-specific OsGA3ox2 gene to drive the OsGA2ox1 open reading frame produced transgenic rice plants with more moderate decreases in shoot stature and normal reproductive development (Sakamoto et al., 2003).

Materials and methods

Generation of PcGA2ox1 trangenic wheat lines

Plasmid pLAP2ox2 contained a full-length cDNA of a GA 2-oxidase from runner bean (PcGA2ox1) under transcriptional control of the maize ubiquitin-1 (Ubi-1) promoter, including its 5' untranslated exon and first intron, and terminated with the

3' untranslated sequence and polyadenylation signals of the nopa-line synthase gene (nos) from the Ti plasmid from Agrobacterium tumefaciens. Plasmid CalNEO contained the neomycin phosphotransferase (neo) marker gene (conferring resistance to geneticin) under transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the nos terminator. Scutella of immature embryos (14-16 d post anthesis) of spring wheat (cv. Canon) were co-transformed with plasmids pLAP2ox2 and pCalNEO using a particle delivery system (Sparks and Jones, 2004). Following induction of embryogenic calli, two rounds of selection in the presence of geneticin sulphate and rooting, putative primary transformants (T0 generation) were transplanted into pots containing soil and grown to maturity in a greenhouse.

Grains from selfed ears (T1 generation) from each of the six independent primary transformants were imbibed in the cold (56 0C) for 6 d before planting out in seed compost in modules. Seedlings at the 2-leaf stage were transferred to pots (100 mm diam, 1.0 l volume) containing loam-based compost with slow-release fertilizer and spaced widely in a greenhouse. All plants of Line 1, and some of Line 4, were prostrate dwarfs with dark-green leaves and produced many tillers. Line 1 plants 1-10 were treated with 2x10 pl (20 ig) GA3 solution to the base of the rosette of shoots on six occasions to stimulate stem elongation, ear emergence, and grain set. Line 4 plants 2, 3, 6, 7, and 16 were also treated similarly but only on four occasions. Young leaf material was harvested directly into liquid N2 and stored at -80 oc prior to DNA extraction. T2 grains from selfed mainstem ears of T1 plants that tested PCR positive for the PcGA2ox1 transgene were sown out, as above, for phenotypic analysis and collection of leaf material for segregation analysis and determination of copy number of inserts. The segregation analysis was repeated on selected T3 seedlings.

DNA extraction, PCR conditions, and Southern blot analysis

For screening of the transgenic lines, DNA was extracted from leaf tissue using the Nucleon Phytopure kit (GE healthcare) according to the manufacturer's instructions. The presence of PcGA2ox1 was determined by PCR: sense primer 5' -TCATAGTGAACGCCTG-TAGG-3'; antisense primer 5' -TGTTCTTCACTGCTGTAATG-3'. Reactions, using the Reddymix™ PCR system (Abgene) with ~200 ng DNA in a total of 25 pl, were heated at 95 oc for 5 min and then subjected to 30 cycles of 95 oc for 30 s, 52 oc for 30 s, and 72 oc for 30 s.

For Southern blot analysis, DNA was extracted from young leaf tissue using the CTAB method (Isaac et al., 1995) and digested with Ssp 1, BamHI or EcoRI. The digested DNA (20 pg) was separated by electrophoresis on a 1.0% (w/v) agarose gel at 35 V for ~24 h and transferred by capillary blotting on to positively charged nylon membrane (Biobond™ Nylon membrane, Sigma) (Sambrook et al., 1989). A digoxigenin-labelled probe was generated using a PCR DIG probe synthesis kit (Roche Diagnostics GmbH) and the above PcGA2ox1 primers with plasmid pLAP2ox2 as template. The cycle conditions were 95 oC for 5min followed by 35 cycles of 95 oC for 30 s, 52 oC for 30 s, 72 oC for 1 min 30 s, and the extension of 72 oc for 10 min. Hybridization and detection of probe was carried out using a non-radioactive, DIG luminescent detection kit for nucleic acids (Roche Diagnostics GmbH) according to the manufacturer's instructions.

RNA isolation, northern hybridization, and qRT-PCR

Grains of cv. Canon (wild type) and T3 Line 4 plant 10 (null) and plants 8 and 16 (semi-dwarfs) were germinated at 20 oC for 72 h and scutella dissected out and plunged into liquid N2 prior to RNA isolation and northern hybridization using methods described in Appleford et al. (2006). Briefly, poly(A)+ RNA was extracted and

purified on oligi-dT cellulose, size-fractionated by electrophoresis on formaldehyde-agarose gels, and transferred to nitrocellulose. Probes for hybridization to TaGA20ox1 and TaGA3ox2 were labelled with 32P-dCTP using random primers and the full-length cDNAs as template whereas probes for a-Amy 1 (high pI), a-Amy 2 (low pI) used specific cDNA clones for the two a-amylase gene families (Lazarus et al., 1985). In a further experiment, T4 grains of Line 4 plants 10 (null) and 8 (semi-dwarf) were imbibed at 5-6 0C for 30 h before transfer to 20 oc. Scutella and aleurone layers were dissected from grains after 42 h and 66 h and plunged into liquid N2 prior to isolation of total RNA and northern hybridization (Lenton et al., 1994) using labelled probes, as above. Total RNA for qRT-PCR was extracted from young leaves, ears, peduncles, and nodes of greenhouse-grown T4 plants according to Verwoerd et al. (1989). Real-time qPCR was carried out on an ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green as described in Griffiths et al. (2006) and employing actin as the sole reference gene. The primers for PcGA2ox1 and actin were as follows: PcGA2ox1: sense 5' -TAGCAAGAGGATTGGCC-CAAAC, antisense, 5' -GGTGTTGAGGA-GGAGGTATTCG; Actin: sense 5' -AGGCATCCTGACGCTCAAGTA, antisense 5' -GCTCGTTGTA-GAAGGTGTGGTG.

Growth conditions and phenotypic analysis of PcGA2ox1 transgenic wheat

T3 grains of Line 4 plants 10 (null), 8 and 16 (semi-dwarfs),

2 (dwarf), and Line 6 plant 21 (severe dwarf) were germinated directly in modules and potted on, as above. Seedlings (15 per genotype) were transferred to a growth room under standard summer growing conditions (19/15 0C day/night, 16 h photoperiod, 488 imol m~2 s_1 at 400 mm from pot base). Mainstem leaves

3 and 6 were tagged after emergence of the lamina and the time of emergence of the first anthers of the ear (anthesis) was recorded. Final lengths of leaf 3 (measured from the soil surface to the tip of the lamina) were measured after eight days in the growth room. Plants were grown to harvest ripeness in the growth room and dried off in a greenhouse. Final lengths of mainstem internodes plus ears were measured and grain numbers and weights recorded.

T4 grains of Line 4 plants 10, 8, and 2 were germinated and potted up, as above, and grown under summer greenhouse conditions. Mainstem leaves were tagged after emergence and total and lamina lengths measured twice weekly on a subset of randomly selected plants (12 plants per genotype) from the central area of plots of blocks of relatively densely spaced pots containing the three genotypes. Numbers of primary and secondary tillers in the axils of leaves 1-3 were recorded 6 weeks after sowing as was the timing of anthesis on mainstem ears. On another subset of randomly selected plants apical development stage was determined following careful dissection of shoot apices and examination under a binocular microscope. Four plants per genotype were harvested at weekly intervals 4-7 weeks after sowing for this purpose.

Treatment of PcGA2ox1 transgenic wheat plants with GAs

T4 grains of Line 4 plants 10, 8, and 2 were pre-imbibed in Petri dishes at 5-6 0C for 48 h and planted out into pots (70x70 mm, 12 grains per pot, six pots per genotype) containing moist vermiculite. Pots were randomized into two blocks in a controlled environment cabinet set at 20 0C with a 16 h daylength (165 imol m~2 s_1 at 50 mm above pot height). Two pots of each genotype were watered or treated with 10 iM GA1 or 10 iM GA3 solutions on days 2 and 5 (25 ml per pot, six pots per treatment). Nutrient solution (25 ml) was added to all pots on days 6 and 8 and pots were watered, as required, on days 7 and 9. Final lengths of the sheaths of leaf 1 were measured on day 12.

Treatment of wild-type wheat with paclobutrazol Individual grains of wild type, cv. Canon, were planted in modules and at the 2-leaf stage transferred to pots (150 mm diameter) containing loam-based compost plus slow-release fertilizer. Pots were spaced relatively widely on benches under summer greenhouse conditions. Seedlings were treated at 3-4 d intervals with either water (control) or 10 iM 2S,3S-paclobutrazol (treated), applied to the soil at the base of the shoot, starting two days after transfer to the greenhouse. At the seedling stage, doses were less (5x 29.4 ig per pot) but increased as the plants grew larger (18x 58.7 ig per pot). Final lengths of leaf 8 (flag leaf), main stem and ears were measured for 15 plants for each treatment.

Measurement of a-amylase activity in germinated grains

Grains of cv. Canon (control) and T4 Line 4 plants 10, 8, 16, and 2 and Line 6 plant 21 were plated out (5x5 pattern) in Petri dishes (90 mm diameter) containing two layers of Whatman No. 1 filter paper moistened with 4.5 ml distilled water. Dishes were incubated in a cold room (5-6 0C) for 48 h prior to transfer to 20 0C for 72 h in darkness. Four replicate samples of three grains with shoots and roots removed were extracted for each genotype and assayed for a-amylase activity using Phadebas dye-bound starch substrate (Barnes and Blakeney, 1974). Units of activity were determined from a standard curve using purified barley malt a-amylase. Soluble protein was also assayed using Coomassie Brilliant Blue G-250 dye substrate and quantified using catalase to generate a standard curve. To determine the change in a-amylase activity over time, grains were set up, as above, and incubated for 30 h in the cold before transfer to 20 0C. a-Amylase activity was determined on grains harvested at 42, 66, and 90 h after transfer to the warm. To determine the effect of GA treatment, intact grains were incubated in water, 1.0 iM GA1 or 1.0 iM GA3, as above, and a-amylase activity determined after 66 h at 20 oc.

Quantification of GAs in germinated grains

Grains of cv. Canon and T3 Line 4 plants 10, 8, 16, and 2 were germinated in Petri dishes, as above, at 20 0C for 96 h. Uniformly sized shoot axes plus roots were dissected from grains (scutellum plus endosperm) and the separated tissues were plunged directly into liquid N2 prior to freeze-drying. The dry masses of the shoot plus root axes ranged from 1.73-2.27 g (308-368 items per genotype) and the grains from 7.33-9.14 g (296-360 items). Subsamples of shoot plus root axes (1.0 g) and grains (2.0 g) were removed for initial quantitative GA analyses using methods described in Webb et al. (1998). Briefly, samples were extracted with 80% methanol-water and [3H]- and [2H]-labelled GA standards added. Ethyl acetate-soluble acids, obtained from an aqueous phase, were purified by QAE Sephadex anion-exchange and C18 Sep-Pak cartridge chromatography prior to resolution by C18 reverse-phase HPLC. Grouped fractions, based on the retention times of the [3H]GA standards, were methylated and the ethyl acetate-soluble neutrals passed through a NH2 Bond Elut column, reduced to dryness, trimethylsilylated and analysed by GC-SIM using a BPX-5 capillary column. Amounts of endogenous compounds were determined from calibration curves. In a repeat analysis, consisting of the remainder of the axes and 2.0 g of grains, the amounts of [2H] GA standards added were similar to those determined for the endogenous GA content in the initial experiment. Results from the second analysis are presented.

Statistical analysis

The GenStat® statistical system, version 8.2, (Lawes Agricultural Trust, Rothamsted, UK) was used to analyse growth data and measurements of a-amylase activity. A natural log transformation of

some data sets prior to analysis was required to account for heterogeneity of variance across treatments. For example, high variability was found within the paclobutrazol treatment of wildtype wheat, where a simple t test was applied to the log transformed data to make comparison. One-way Analysis of Variance (ANOVA) was applied for other measures of growth for the transformants, whilst a non-parametric Kruskal-Wallis one-way ANOVA was applied to the analysis of tiller production. The lack of variability in numbers precluded the use of standard one-way ANOVA or effective generalized linear modelling here. Hence, the ranks of the observations across the genotypes are used to provide a chi-squared test of significance between them. Two-way ANOVA was applied to growth data and natural log transformed a-amylase values from the experiments using control and transformant genotypes treated with GA. Following one-way or two-way (parametric) ANOVA, the relevant least significant difference (LSD) at the 5% level of significance was used to compare means of important biological interest.

Results

Molecular and phenotypic characterization of PcGA2ox1 transgenic wheat

Six independent primary transformants (Lines 1-6) expressing PcGA2ox1 cDNA were generated during 19992000 and subsequent generations from selfed ears were characterized in terms of presence of the transgene and shoot phenotype. Of the 20 T1 grains of Line 1 that were germinated, all tested positive for the transgene by PCR and produced extreme dwarf seedlings that were treated repeatedly with GA3 to encourage ear emergence and allow grain to set. As segregation ratios indicated that this line may have had transgene inserts at multiple loci it was not investigated further. From 20 grains each of Lines 2 and 3, several T1 plants had a relatively mild semi-dwarf shoot phenotype at anthesis. The tallest PCR-positives ranged from a decrease in shoot height of only 5-12%, compared with azygous talls, whereas the more dwarfed PCR-positives were up to 25% shorter. However, no putative homozygous sublines were recovered from the T1 PCR-positive plants of Lines 2 and 3 at the T2 seedling stage. Tx plants of Line 4 produced a wide range of shoot phenotype. Measurement of the lengths of leaf 2, as well as shoot height at anthesis, confirmed that the presence of the PcGA2ox1 transgene reflected a shorter shoot phenotype. Ten talls were PCR-negative and, of the 23 PCR-positives, 14 were classified as semi-dwarfs (33-49% decrease in shoot height, compared with nulls) and nine as more severe dwarfs, five of which were treated with GA3 to ensure ear emergence from the flag leaf and adequate grain set. Six putative homozygous sublines were recovered from Line 4 at the T2 seedling stage. From the Line 5 primary transformant, 27 Tj seedlings from 38 germinated grains tested PCR positive and produced both semi-dwarf and dwarf shoot phenotypes. However, no putative homozygous sublines were rescued at the T2 seedling stage. From the Line 6 primary transformant,

24 Tx seedlings from 40 germinated grains were PCR positives, again producing both semi-dwarf and dwarf phenotypes at maturity. Only one relatively severe dwarf putative homozygous subline was rescued at the T2 seedling stage.

More detailed molecular and phenotypic characterization focused on the putative homozygous semi-dwarf and dwarf sublines from Line 4 and the more severe dwarf from Line 6. Southern blot analysis of Ssp 1-digested DNA extracted from homozygous T3 seedlings of Line 4 showing extreme dwarf and semi-dwarf growth habits and probed with full-length PcGA2ox1 cDNA produced identical banding patterns (Fig. 1A). A tall azygous plant was analysed as a negative control and shown to produce no bands that hybridized to the probe. An identical banding pattern was obtained from several T1 plants from Line 4 showing different degrees of dwarfism, whereas an extreme dwarf plant from Line 6 gave a different pattern (data not shown). Furthermore, digestion of the DNA with other restriction enzymes also produced the same banding patterns for dwarf and semi-dwarf plants from Line 4

(data not shown). Thus, the Line 4 plants contained the same transgenic loci despite displaying differences in phenotype that were maintained for at least four generations. The abundance of PcGA2ox1 transcript was compared in the first leaf of Line 4 and 6 plants by realtime RT-PCR (Fig. 1B). Transcript abundance correlated with dwarfing severity (Figs 2A, 3), except for Line 4 plant 2, which contained less transcript than the semi-dwarf lines. However, when PcGA2ox1 mRNA abundance was measured in ears, peduncles, and peduncle nodes of the Line 4 plants 2 (severe dwarf), 8 (semi-dwarf), and 10 (azygous tall) there was a clear correlation between transgene expression and dwarfing severity (Fig. 1C).

In terms of shoot phenotype, selected T2 transgenic sublines from the original Line 4 showed decreased shoot height at anthesis and considerable variation in extent of prostrate growth habit, increased tillering, and delayed ear emergence, compared with a corresponding azygous plant (null) (Fig. 2A). When plant 10 (null) was at anthesis, plant 3 remained an extreme 'cartwheel-type' prostrate

Fig. 1. Molecular analysis of PcGA2ox1 transformants. Southern blot of SspI-digested DNA from selected T3 PcGA2ox1 transformants, Line 4 plants 2 (dwarf), 8 (semi-dwarf), and 10 (null) (A). Quantitative realtime RT-PCR analysis of relative PcGA2ox1 transcript levels in: seedlings of Line 4 plants 2, 8, 10, and 16, and Line 6, plant 21 (B) and in ears, peduncles (Ped) and peduncle nodes of Line 4 plants 2, 8, and 10 (C). The lowest value is set to 1 in each case. Error bars represent standard error of the means of three technical replicates.

Fig. 2. Phenotypic variation in shoot architecture of widely-spaced T2 plants of the original Line 4 PcGA2ox1 transformant grown under spring greenhouse conditions with supplementary illumination. Plants from the left are 10 (null), 8 (semi-dwarf), 2 (dwarf), and 3 (severe dwarf) (A). Shoot phenotypes of wild-type cv. Canon (left) and paclobutrazol-treated (right) plants 8 weeks after sowing. Treated pots had received a total of 730 ig paclobutrazol over a period of 40 d at this time (B).

dwarf with large numbers of tillers and very few emerging ears. Plant 2 was another dark-green prostrate dwarf with many tillers and delayed ear emergence whereas plant 8 was semi-dwarf with fully emerged ears from the main-stem and several tillers. In this subline, there was also recovery of the natural orthogravitropic growth response of shoots during expansion of the peduncle, the last formed stem internode (compare plants 8 and 10 in Fig. 2A). Developmental plasticity was also observed within individual Line 4 sublines depending on plant density and light quality. In general, plants with a more prostrate growth habit and many more tillers were produced when plants were widely spaced and grown under artificial light.

Repeated applications of low doses of the GA biosynthesis inhibitor, 2S,3S-paclobutrazol, to relatively widely spaced wild-type plants grown under summer greenhouse conditions produced dark-green, prostrate dwarf plants with many tillers, phenotypically similar to the PcGA2ox1 transformants (Fig. 2B). In control plants, anthesis occurred between 59-63 d after sowing but was delayed to between 69-78 d in paclobutrazol-treated plants. The cumulative effect of increasing paclobutrazol dosage caused highly significant (P <0.001, t tests) decreases of 35-38% in the lengths of leaf 8 (flag leaf) and mainstem and of 23% in the size of the ear, compared with controls (Table 1).

Growth of the transformants was measured on T3 plants grown in a controlled environment simulating average August growing conditions. The decrease in length of leaf 3 (up to 38%) in sublines 2, 16, and 8 from Line 4, compared with a non-transgenic (null) segregant (Line 4, subline 10) was highly significant (P <0.05, LSD following ANOVA), but less severe than Line 6 plant 21 (66% decrease) (Fig. 3). In Line 4, plant 16 exhibited a slightly more prostrate growth habit than plant 8 whereas plant 2 and Line 6 plant 21 were even more extreme prostrate dwarfs. Main stem height of Line 4 plants 8 and 16 was decreased by 40-44%, compared with a null (plant 10), and corresponding highly significant (P <0.05, LSD) decreases of 54% and 61% were observed in Line 4 plant 2 and Line 6 plant 21, respectively. The lengths of the peduncle, the last-formed stem internode, reflected those of the main stem as a whole. The transformants also produced one extra main stem leaf, compared with the null, under these growing conditions.

Table 1. Mean lengths (mm ±SE) of wheat cv. Canon tissues after treatment with water (control) or 10 iM 2S,3S-paclobu-trazol

T-tests on log transformed values showed highly significant differences (P <0.001) between control and paclobutrazol-treated tissues.

Treatment Leaf 8 Main stem Ear

Water 888.564.7 794.5611.1 102.060.63

Paclobutrazol 576.567.8 491.767.4 78.561.41

Initially, the rate of floral development was much slower in the PcGA2ox1 transformants, but increased later so that timing to anthesis was delayed by only 1-2 d in Line 4, plant 8 and 5-7 d in Line 6, plant 21, compared with the null (plant 10). In Line 4 plants, there was a significant (P <0.05) 4-8% decrease in final ear length (Fig. 3) and ears also contained one additional spikelet (Table 2). Line 6, plant 21 produced a 'club-like' ear (30% decrease in length) which contained one less spikelet (Table 2). There was a significant (P <0.05) decrease in grain number per spikelet in Line 4 plant 2 and Line 6 plant 21 and the decrease in mean grain mass for these two lines was associated with the degree of severity of dwarfing of the main stem of the transformants, compared with the null (Table 2; Fig. 3).

A more detailed developmental analysis was conducted on selected Line 4 T4 plants grown under summer greenhouse conditions. As expected, lengths of main stem leaves 3 and 5 were shorter than the null (plant 10) with the effects of the transgene being greater on the sheath than on the lamina (Table 3). Although the lengths of the

Fig. 3 Final lengths (mm) of leaf 3, main stem and ear of selected T3 PcGA2ox1 transformants from Lines 4 and 6 grown in a controlled environment simulating average UK August growing conditions. (LSDs for comparison of lines within tissue, at the 5% significance level, are 7.6 mm for leaf 3, 12.9 mm for peduncle, 28.8 mm for main stem, and 3.6 mm for ear.)

Table 2. Yield components of main stem ears of selected T3 PcGA2ox1 transformants from Lines 4 and 6 grown in a controlled environment simulating August growing conditions in the UK

Spikelet Grain Grains/ Grain Mass/grain

no. no. spikelet mass (mg) (mg)

Line 4 plant 10 (null) 21.0 69.6 3.32 2539.0 36.5

Line 4 plant 8 21.9 70.7 3.22 2168.0 30.7

(semi-dwarf)

Line 4 plant 16 21.9 68.8 3.15 2029.0 29.6

(semi-dwarf)

Line 4 plant 2 (dwarf) 21.9 57.6 2.63 1470.0 25.5

Line 6 plant 21 (dwarf) 20.1 48.5 2.41 1251.0 25.8

LSD (95%)(60 df) 0.77 5.6 0.24 194.4 1.7

sheath of leaf 5 of the two tranformants were significantly different (P <0.05), this was not the case for the earlier produced leaf 3 (P >0.05). Even in these relatively densely spaced plants, there was a highly significant increase in tiller numbers in the axil of leaf 2 and to a lesser extent of leaf 1 of the transgenic plants, compared with the null (Table 4), differences between lines being significant (P <0.05, Kruskal-Wallis ANOVA).

Shoot apices were dissected from plants at weekly intervals to determine the timing of transition from the vegetative state to flowering. No genotypic differences in the timing to 'double ridges', the transition to flowering, were detected, although the subsequent rate of floral development was delayed in plant 8 and, to a greater extent, in plant 2, compared with the null. For example, at 35 d after sowing, apices of the null were at glume primordium stage, plant 8 was at terminal spikelet stage whereas plant 2 was only at the floret primordium stage. However, under these growing conditions, timing to anthesis was only delayed by 1-3 d in the transgenics, compared with the null.

Reversal of the dwarf shoot phenotype with applied GA As expected, sheath length of the two Line 4 transformants was much shorter than the null and root application of GA1 or GA3 stimulated growth of all three sublines (Table 5). Genotype, GA treatment and the interaction of these two factors were highly significant (P <0.001, ANOVA). The prediction was that GA1 would be less effective than GA3 at restoring leaf growth of the transformants because of increased metabolism to an inactive GA, GA8, whereas GA3 is not a substrate for PcGA2ox1 (SG

Table 3. Lengths (mm) of leaf components of selected T4 Line 4 PcGA2ox1 transformants grown under summer greenhouse conditions

Transformant Leaf 3 Leaf 5

Lamina Sheath Lamina Sheath

4.10 (null) 233.6 71.8 335.2 152.7

4.8 (semi-dwarf) 177.9 41.4 277.9 103.6

4.2 (dwarf) 158.2 36.2 242.8 88.5

LSD (95%)(33 df) 12.4 7.0 18.6 9.6

Table 4. Mean numbers ±SE of tillers in selected T4 Line 4 PcGA2ox1 transformants 6 weeks after sowing under summer greenhouse conditions

Transformant Leaf 1 Leaf 2 Leaf 3

4.10 (null) 2.6760.188 1.9260.193 1.1760.112

4.8 (semi-dwarf) 3.1760.122 2.7560.131 1.4260.149

4.2 (dwarf) 3.5060.261 2.9260.149 1.6760.142

P-valuea 0.024 <0.001 0.05

a P-value from %2 (2 df) test (Kruskal-Wallis one-way ANOVA).

Thomas, AL Phillips, P Hedden, unpublished data). In the null line (plant 10), GA3 was slightly more effective than GA1 in stimulating sheath length, but was much more effective in the transformants, as expected. In addition, GAx was less effective at restoring growth of the dwarf (plant 2), compared with the semi-dwarf (plant 8). Application of GA3 to the transgenic plants restored sheath length almost completely to that of the treated null (Table 5).

Germination capacity of grains of PcGA2ox1 transgenic plants

Germination tests on Line 4 T4 grains hand-threshed two weeks after harvest ripeness showed that the PcGA2ox1 transgene affected both embryo dormancy and seedling vigour. Whereas the null (plant 10) gave 76% germination after three days at 20 °C, the corresponding germination percentages were 40%, 32%, and 8% for plants 8, 16, and 2, respectively. Radicle lengths ranged from c. 20 mm in the null to 1-2 mm just rupturing the pericarp in plant 2. These results were not a consequence of poor embryo viability since all sublines gave 100% germination after five days at 15 °C. After a further three weeks of after-ripening in the warm, grains from all sublines showed 100% germination after three days at 20 °C, although differences in seedling vigour remained evident in material after-ripened up to seven weeks.

a-Amylase production in germinated grains of PcGA2ox1 transgenic plants

After-ripened T4 grains were imbibed in the cold for 48 h to remove any residual dormancy and germinated for 72 h at 20 °C. a-Amylase activity was determined in extracts of grains following excision of root and shoot axes. In addition, activity was also determined on a unit protein basis in order to account for genotypic differences in grain size. Using a natural log transformation to correct for variance heterogeneity across lines, overall highly significant differences (P <0.001) in a-amylase activity on both a per grain and unit protein basis were found (Fig. 4). All lines were significantly different from the wild type and the azygous (null) line, grains of which produced similar amounts of a-amylase per grain, whereas activity in the two semi-dwarfs (plants 8 and 16) was decreased by 35% and in the dwarf (plant 2) by 50% (Fig. 4). The more

Table 5. Length (mm) of the sheath of leaf 1 of selected T4 Line 4 PcGA2ox1 transformants after treatment with water, GA1 or GA3 (each 10 iM)

Transformant Water GAj GA3

4.10 (null) 57.4 85.9 90.0

4.8 (semi-dwarf) 38.7 72.8 83.6

4.2 (dwarf) 32.5 60.6 82.5

LSD (95%) (8 df) 3.71

extreme dwarf, Line 6 plant 21, showed a 65% decrease in activity, compared with the wild-type and null controls. Even in dwarf sublines containing smaller grains (Table 2), genotypic differences in enzyme activity per unit protein remained substantial (Fig. 4).

In the time-course experiment, grains of Line 4 plant 10 (null), Line 4 plant 8 (semi-dwarf), and Line 4 plant 2 (dwarf) were imbibed in the cold for 30 h before transfer to 20 °C. a-Amylase activity was measured after 42, 66, and 90 h at 20 °C. There were highly significant main effects of both genotype and time (P <0.001) and a significant interaction (P =0.008) between these two factors. By 42 h, significant (P <0.05, LSD) decreases of 31% and 48% in a-amylase activity per grain were observed in plants 8 and 2, respectively, compared with the null. The relative differences between genotypes became greater over time up to 90 h (Table 6).

In order to determine if the effect of the transgene could be rescued by GA treatment, grains of the three lines used above were imbibed in water, 1.0 iM GAx or 1.0 iM

Fig. 4. a-Amylase activity in grains of wild-type cv. Canon and selected T4 PcGA2ox1 transformants after incubation at 5-6 °C for 48 h before transfer to 20 °C for 72 h. (Numbers above each column are the mean log-transformed values. LSDs for comparisons at the 5% significance level are 0.115 for Units per grain and 0.140 for mU per 1g protein.)

GA3 solutions in the cold for 30 h before transfer to the warm and measurement of a-amylase activity after a further 66 h. When analysing the log transformed a-amylase activities, the main effects of genotype and GA treatment of were significant (P <0.001, ANOVA) and there was a weak genotype by treatment interaction (P=0.036 ANOVA). As expected, when using the LSD (5%) values for comparisons, there were highly significant decreases in activity in the two transgenic lines incubated in water, compared with the null (Table 7). Neither GA caused a significant (P <0.05) increase in enzyme activity in the null and GA1 was also ineffective in the transformants. Application of GA3 restored amylase activity in the semi-dwarf (plant 8) to that in the water-treated null line (plant 10) and caused a large increase in the dwarf (plant 2), compared with water or GA1-treated plants (P <0.05).

Gibberellin content of PcGA2ox1 transgenic plants

Several GAs of the early 13-hydroxylation pathway were quantified in shoot and root axes of T3 grains germinated for 4 d at 20 °C in darkness. Comparisons were made between two independent 'controls' (Canon and null, Line 4 plant 10), two independent semi-dwarfs (Line 4 plants 8 and 16), and a single more severe dwarf (Line 4 plant 2). Similar results were obtained from two separate analyses, but data from only one experiment are presented in Fig. 5

Table 6. a-Amylase activity in grains of selected T4 Line 4 PcGA2ox1 transformants at different times after imbibition

Grains were pre-imbibed at 5-6 °C for 30 h prior to transfer to 20 °C. Statistical analysis was on the log-transformed data. The back-transformed means are shown in parenthesis.

Transformants Loge a-amylase activity (back-transformed

means, U grain

42 h 66 h 90 h

4.10 (null) 2.470 (11.8) 3.531 (34.2) 4.168 (64.6)

4.8 (semi-dwarf) 2.093 (8.1) 2.909 (18.3) 3.449 (31.5)

4.2 (dwarf) 1.791 (6.0) 2.678 (14.6) 2.867 (17.6)

LSD (95%) (27 df) 0.214

Table 7. a-Amylase activity in grains of selected T4 Line 4 PcGA2ox1 transformants after treatment with water, GA1 or GA3 (each at 1.0 iM)

Grains were pre-imbibed at 5-6 °C for 30 h prior to transfer to 20 °C for 66 h. Statistical analysis was on the log-transformed data. The back-transformed means are shown in parenthesis.

Transformant Loge a-amylase activity (back-transformed means, U grain-1)

Water GAj GA3

4.10 (null) 4.8 (semi-dwarf) 4.2 (dwarf) LSD (95%) (27 df) 3.661 (38.9) 3.109 (22.4) 2.893 (18.1) 0.182 3.663 (39.0) 3.285 (26.7) 2.982 (19.7) 3.833 (46.2) 3.680 (39.7) 3.392 (29.7)

Quantification of several GAs in the scutellum plus endosperm of germinated grains of the transgenic plants was problematic due to the large amount of impurities present. Of those GAs that could be quantified reliably, there was an overall 4-fold decrease in the combined GAx and GA3 concentration in the semi-dwarf (Line 4 plants 8 and 16) and a >6-fold decrease in the dwarf (Line 4 plant 2), compared with the controls (Fig. 5B). These differences reflected 4.4-fold and 9.2-fold decreases in biologically active GA on a per grain basis, for the semi-and dwarf plants, respectively.

Expression of GA dioxygenase and a-amylase genes in germinated grains

Expression of two GA biosynthesis dioxygenase genes was examined in T3 grains of two independent semi-dwarf lines and controls (Canon and Line 4 plant 10) that were germinated for 3 d at 20 °C in darkness. In scutella, expression of TaGA3ox2 was higher than TaGA20ox1 and both were up-regulated in the semi-dwarf lines, compared with controls (Fig. 6A). Interestingly, there was an apparent decrease in abundance of transcripts for both a-amylase gene families in scutella of the semi-dwarfs, compared with controls.

In a further experiment, T4 grains of the null (plant 10) and semi-dwarf (plant 8) were preincubated in the cold for 30 h before transfer to 20 °C. Decreases in abundance of 35-40% for a-Amy 1 and 60-70% for a-Amy 2 transcripts were observed in aleurone of the semi-dwarf, compared with the null, after 42 h and 66 h in the warm (Fig. 6B). There was also a slight decrease in a-Amy 1 transcripts in scutella (data not shown). No transcripts for either of the wheat GA biosynthesis genes (TaGA20ox1 and TaGA3ox2) were detected in aleurone under these conditions. The PcGA2ox1 transgene was expressed in both scutellum and aleurone of the semi-dwarf (plant 8) but, as expected, was

Fig. 5. Gibberellin concentration (ng g-1 dry mass) in shoot plus root undetected in the nuU (plant 10) (data not shown).

axes (A) and grains (scutellum plus endosperm) (B) of wild-type cv.

Canon and selected T3 Line 4 PcGA2ox1 transformants grown at 20 °C

for 4 d in darkness. (Asterisk) Not determined due to interference. DiSCUSSiOn

because values for GA3 and GA8 were not obtained from Genetic dissection of the GA biosynthesis (reviewed in

the first analysis. Overall, the concentrations of GA44, Sakamoto et al., 2004) and signal transduction pathways

GA19, GAi, GA3, and GA8 were substantially reduced in (reviewed in Hartweck and Olszewski, 2006) has provided

axes of the transgenic plants compared with the two compelling evidence for the involvement of GAs in the

control lines (Fig. 5A). The largest differences were for regulation of plant stature in both Arabidopsis and diploid

GA19 and GAj concentrations, for which there were >5- cereals such as rice and barley. As might be predicted,

fold decreases in Line 4 plants 8 and 16 (semi-dwarfs) and ectopic expression of PcGA2ox1 in wheat produced a

approximately 15-fold decreases in the same GAs in Line range of dark-green, dwarf shoot phenotypes (Fig. 2A)

4 plant 2 (dwarf). As a by-product of GA 3-oxidase similar to those in which OsGA2ox1 was over-expressed

activity, GA3 contents reflected those of GAx but geno- in transgenic rice (Sakamoto et al., 2001). In the present

typic differences were much less as this GA is not a wheat transformants it is difficult to reconcile why a range

substrate for PcGA2ox1 activity. In addition, there was a of shoot phenotypes arose from a single transformation

1.7-fold increase in the concentration of GA29 in the semi- event and has remained stable over four generations.

dwarfs, compared with controls, but only a modest de- Southern blot analysis showed that the Line 4 trans-

crease in its immediate precursor, GA20. formants contained the same number of copies of the

Fig. 6. Expression of GA biosynthesis and a-amylase genes in scutella of wild-type cv. Canon and selected Line 4 PcGA2ox1 transformants germinated at 20 °C for 3 d. Relative transcript abundance was determined from northern blots of equal loadings of poly(A)+ RNA and the signal detected and quantified using a phosphoimager (A). Expression of a-Amy1 (high pi) and a-Amy2 (low pi) a-amylase gene families in aleurone layers from T4 grains of Line 4 PcGA2ox1 transformants plants 10 (null) and 8 (semi-dwarf) pre-incubated at 5-6 °C for 30 h prior to transfer to 20 °C for 42 h and 66 h. Relative transcript abundance was determined from northern blots of total RNA (3 ig per lane). Blots were stripped and reprobed with a 25S rRNA probe and signals quantified using a phosphoimager.

insert (Fig. 1A) and qRT-PCR clearly indicated different levels of expression of the transgene (Fig. 1B, C). We are not aware of previous reports of different degrees of transgene expression being stably inherited from a single transformation event, but assume it is due to differential transcriptional silencing in the T1 generation, as has been described during generation of a newly synthesized wheat allotetraploid (Kashkush et al., 2002). Furthermore, the degree of silencing appears to be tissue-specific since, although there was a clear correlation between transgene expression in ears, peduncles, and peduncle nodes with the severity of the mature plant phenotype in Line 4 sublines 2 and 8 (Figs 1C, 2A), expression in young leaves of these plants did not reflect the final phenotype (Fig. 1B). Tissue-specific transgene silencing has been noted previously (Kloti et al., 2002).

At the whole plant level, a similar severe dwarf shoot phenotype was also produced when wild-type wheat was treated with the GA biosynthesis inhibitor, paclobutrazol (Fig. 2B; Table 1). The decreased shoot stature of the present wheat transformants was relatively severe, ranging from 40-60% that of the null (Fig. 3), similar to that of the more extreme 'GA-insensitive' Rht dwarfs, Rht-B1c and Rht-D1b+Rht-B1c (Youssefian et al, 1992b; Flintham et al, 1997). By comparison, shoot height is decreased by

only 15-20% when the commercially successful semi-dwarfing Rht-B1b or Rht-D1b alleles are introduced into tall wheat.

During the vegetative stage of development, leaf growth of the wheat transformants was decreased with a greater effect being on the sheath than on the lamina (Fig. 3; Table 3). Growth of the first leaf sheath of the transformants was restored by application of GA3, which is not a substrate for PcGA2ox1. However, growth was restored only partially by application of GA1 and to a lesser extent in the more dwarf line (plant 2), whereas both GAs stimulated growth of the null (Table 5). Although not measured directly, the extent of deactivation of applied (and endogenous) GAx appeared to reflect the degree of shoot dwarfism (Fig. 3; Table 5). Similar results were observed when seedlings of transgenic rice expressing OsGA2ox1 cDNA were treated with the same GAs (Sakamoto et al., 2001).

Another feature of the present wheat transformants, when grown widely-spaced, was a prostrate growth habit with loss of apical dominance and increased outgrowth of lateral shoots (tillers) (Fig. 2A). This shoot branching pattern was replicated by application of paclobutrazol to wildtype plants (Fig. 2B) and is also characteristic of several 'GA-insensitive' Rht wheat varieties (Gale and Marshall,

1973). Recent genetic evidence from rice showed that shoot dwarfism may arise from excessive tillering and that transport of apically-produced auxin is required to up-regulate a gene involved in the production of a carotenoid cleavage signal that normally suppresses outgrowth of lateral shoots (Zou et al., 2006). Outgrowth of tiller buds in wheat and barley is also influenced strongly by environmental factors such as light quality and nutrient status. Decreasing the far-red component of light in widely spaced plants resulted in increased tiller production (less apical dominance) in wheat (Barnes and Bugbee, 1991). Conversely, increased far-red light signalling at the base of a canopy decreased tiller numbers in densely spaced barley plants (Skinner and Simmons, 1993). Similarly, in the present experiments, a more upright growth habit with less tillering (more apical dominance) was observed when the wheat transformants were densely spaced, which relates to a normal shade avoidance response to increased far-red light reflected down the canopy (Table 4). Taken together, these results provide supporting evidence that besides a primary effect on shoot elongation, GAs are also involved, either directly or indirectly, in apical dominance of wheat shoots. Since auxin, cytokinin, and other hormonal signals are also known to affect axillary bud outgrowth, tillering in wheat is likely to be controlled, in part, by a complex interaction between hormone and phyto-chrome signalling pathways.

Auxin transport and signalling at the base of the leaf sheath (pulvinus) are known to be important components of the natural orthogravitropic growth response of cereal shoots when displaced from the vertical (Dayanandan et al., 1976). Wolbang et al. (2007) found a significant increase in iAA concentration in the lower half of barley p-node pulvini within 2.5 h of gravistimulation, whereas an increase in GA1 content occurred later when bending was well underway. in addition, the GA1-deficient, grd2c barley mutant was a prostrate dwarf, phenotypically similar to the present PcGA2ox1 transgenic wheat (Fig. 2A) and wild type treated with a growth retardant (Fig. 2B), and showed much reduced pulvinar bending upon gravi-stimulation. in contrast, the constitutive GA-signalling, slender barley mutant, sln1c, lacking a functional DELLA protein showed greater pulvinar bending following gravi-tropic stimulation. Taken together, these results suggest that bending of the pulvinus following gravistimulation is driven primarily by an increase in auxin concentration on the lower side of the pulvinus, but that GA is also required for this response. it was assumed that differences in the setpoint angle of the tillers in the present PcGA2ox1 trans-genic wheat (Fig. 2A) reflects the decreased GA1 content of the different lines (Fig. 5A), as was the case for the grd2b and grd2c barley mutants (Wolbang et al., 2007).

Whilst a delay in ear emergence was evident in the present wheat transformants (Fig. 2A), and wild type treated with paclobutrazol (Fig. 2B), the precise cause(s)

remains to be established. The production of one extra leaf at the shoot apex following GA depletion was consistent with previous observations on early applications of growth retardants to wheat during the vegetative growth stage of growth (Hutley-Bull and Schwabe, 1982). In the present experiments with plants growing under long-day conditions, no difference in the timing of floral transition was detected between the wheat transformants and the null line. increased allocation of resources to support growth of the additional leaf and tillers, rather than the young developing ear, might explain the observed initial delay in floral development, which recovered later such that timing to anthesis was only delayed by a few days. More frequent sampling of material grown under different daylengths is required to clarify the precise role of GA in determining the rate of both leaf and spikelet primordium initiation at the shoot apex, as well as the timing of the transition from a vegetative to a reproductive state. it is, however, interesting to note that GAs have been implicated previously in the timing of vegetative phase change and reproductive maturity in maize (Evans and Poethig, 1995).

One unexpected aspect of the shoot phenotype of both the present wheat transformants and a wild type treated with paclobutrazol was a decrease in final ear size (Fig. 3; Table 1), a response not observed even in wheat lines containing the more severe 'GA-insensitive' Rht-B1c allele (Youssefian et al., 1992b; Webb et al., 1998). In the present experiments, induction of GA deficiency, either genetically or chemically, clearly indicated some degree of GA-dependency for normal ear growth. It is not immediately clear, therefore, why introduction of so-called 'GA-insensitivity' Rht dwarfing alleles into wheat does not also decrease final ear size. If the growth inhibiting N-terminal deleted DELLA proteins are expressed in developing ears of Rht lines then factor(s) other than GA (possibly auxin) must be able to promote their degradation in order to produce an ear phenotype similar to that of a wild type. Alternatively, changes in the pattern of cell division during rachis development in the ear may involve a GA signalling pathway independent of DELLA proteins whose primary function may be to restrict cell expansion in the extension zone of vegetative tissues. Whatever the case, further work is required to elucidate the hormonal signalling pathways regulating ear development in commercially important Rht wheat lines.

Another subtle difference between the present wheat transformants and semi-dwarf wheats was the degree of grain set within an ear. Although grain numbers per ear were similar in the null and the two semi-dwarf wheat transformants (plants 8 and 16) they were significantly decreased in the more extreme dwarfs (Table 2). Decreased grain set of the more extreme dwarf wheat transformants may have been a consequence of more severe GA deficiency restricting pollen tube growth and resulting in male sterility, as occurs in Arabidopsis following ectopic

expression of a pea (Pisum sativum L.) GA 2-oxidase (PsGA2ox2) (Singh et al., 2002). By contrast, increases in grain number per ear accounted for higher yields of field-grown wheat containing different Rht dwarfing alleles (Flintham et al., 1997). In this instance, increased resource allocation to developing ears supported greater floret survival at anthesis and hence higher grain numbers per ear in 'GA-insensitive' Rht wheat (Youssefian et al., 1992b).

Decreased grain size of main stem ears (Table 2) was associated with the reduction in shoot height of the wheat transformants (Fig. 3) and may have been a consequence of an overall decrease in total dry matter production. However, it is also possible that GA depletion has a more direct effect on grain development. For example, an analysis of the lh-2 mutant of pea showed that GA produced during early seed development was required for normal seed growth and survival (Swain et al., 1997). Moreover, ectopic expression of PsGA2ox2 in Arabidopsis also caused abortion of developing seeds, similar to that observed in the GA-deficient pea mutant (Singh et al., 2002). Besides affecting grain size, preliminary results with the present wheat transformants showed that GA-depletion also resulted in increased post-harvest embryo dormancy. In addition, post-germination seedling vigour was associated with decreased mobilization of endosperm reserves (Fig. 4; Table 6). Induction of GA deficiency, either genetically or chemically, in developing kernels of an ABA-deficient maize mutant prevented premature embryo germination on the cob (vivipary) (White et al., 2000). This suggests that the balance between ABA and GA production and signalling is important for the maintenance and loss of embryo dormancy in cereals. Further evidence for a direct involvement of GAs in grain development also comes from the observation that pre-maturity a-amylase production and grain shrivelling were associated with increased GA content in a barley mutant (Green et al., 1997).

Direct evidence for decreased content of active GA in the present wheat transformants came from quantitative GC-MS analysis of GAs in etiolated embryonic axes and partially digested endosperm of germinated grains that were shown previously to be a rich source of GAs (Lenton et al., 1994). In the present work, there was a >5-fold decrease in GA1 concentration in both these tissues for two independent semi-dwarf lines, compared with a null and wild type (controls), and a much greater decrease in a more severe dwarf line, confirming that the PcGA2ox1 transgene was functional in planta (Fig. 5A, B). The decrease in GA1 content in the transgenic lines was associated with a decrease in GA19 (Fig. 5), particularly in axes, consistent with up-regulation of TaGA20ox1 expression due to relief of feedback repression, as was observed in scutella (Fig. 6A). The abundance of GA3 and GA8 were, like GAi, reduced in the transgenic plants (Fig. 6), suggesting that a major consequence of the enhanced 2-oxidase activity may be increased turnover of

their common precursor GA2o, thereby reducing its availability for 3-oxidation to GAx and GA3. However, GA29, the product formed from GA20 by 2ß-hydroxylation, did not accumulate in the transgenic plants as much as might be expected, perhaps due to its further oxidation to GA29-catabolite (Thomas et al., 1999), for which no internal standard was available. Clearly, metabolic studies with labelled precursors are required to explore these possibilities. Ectopic expression of OsGA2ox1 cDNA in transgenic rice, as well as causing a 4-fold decrease in GA1 concentration in light-grown leaves, resulted in 2.5-fold and 6.7-fold increases in the concentration of GA8 and GA29, respectively, compared with wild type (Sakamoto et al., 2001).

In grains of the controls, GA19 and GA20 were much less abundant than GA1 (Fig. 5B), probably reflecting the high level of expression of TaGA3ox2 and, to a lesser extent, TaGA20ox1 in scutella (Fig. 6A; Appleford et al., 2006). Previous evidence suggested that the scutellum of wheat was a major site of de novo GA biosynthesis, based on the accumulation of ent-kaurene in grains germinated in the presence of an ent-kaurene oxidase inhibitor, paclo-butrazol (Lenton et al., 1994; Appleford and Lenton, 1997). More recently, using GA promoter-reporter constructs, expression of two GA 20-oxidases and two GA 3-oxidases was shown to be confined to the epithelial layer of the scutellum of germinated rice (Kaneko et al., 2003). In future, it will be interesting to determine if expression of all genes of the GA-biosynthetic pathway is confined to the scutellar epithelium and if this is also the case in temperate cereals, such as wheat and barley.

In the two semi-dwarf wheat lines, expression of TaGA20ox1 and TaGA3ox2 increased in scutella on day 3 (Fig. 6A) and was associated with a 4-fold decrease in active GAs in grains on day 4 (Fig. 5B). A similar up-regulation of expression of these GA biosynthesis genes was also observed in scutella of wild type incubated in the presence of paclobutrazol, compared with untreated plants (NEJ Appleford and JR Lenton, unpublished results). One interpretation of these results is that the decreased concentration of active GAs relieved, in part, a homeostatic mechanism whereby repression of these two GA biosynthesis genes normally occurs as a consequence of GA action (reviewed in Hedden and Phillips, 2000). An alternative, but not exclusive, possibility is that decreased carbohydrate availability in scutella may partially relieve a postulated negative feedback loop imposed by sugars on GA biosynthesis (Yu et al, 1996; Perata et al, 1997).

The decreased content of active GAs in grains of the transformants, compared with controls, (Fig. 5B) was associated with a decline in abundance of transcripts for both a-amylase gene families in aleurone (Fig. 6B) and in grain a-amylase activity (Fig. 4; Table 6). Previously, a decrease in GA content of endosperm was associated with a decline in abundance of transcripts for both the high- and low-pI

a-amylase gene families in aleurone of wheat grains imbibed in the presence of paclobutrazol (Lenton et al., 1994). Application of GA3 largely reversed the decline in a-amylase production (Table 7). The reduced effectiveness of GA1 was assumed to reflect increased catabolism as a consequence of the activity of the transgene. Taken together, these results provide strong evidence for the GA-dependency of a-amylase production in aleurone, the main source of the enzyme, in intact germinating wheat grains. Previous work with a GA-deficient barley mutant established the dependency of a-amylase gene expression in aleurone on embryo-produced GA (Chandler and Mosleth, 1990; Zwar and Chandler, 1995).

The decreased GA content of grains of the semi-dwarf wheat lines on day 4 (Fig. 5B) was also associated with a decline in abundance of transcripts for both a-amylase gene families in scutella on day 3 (Fig. 6A). This observation, like that for the high pI gene family in barley (Chandler and Mosleth, 1990), would also suggest that scutellum-produced a-amylase was GA-dependent. However, an alternative interpretation of these observations is also possible. For example, there is increasing evidence that sugar signalling can also cause repression of a-amylase gene expression in rice and barley embryos (Yu et al., 1996; Perata et al., 1997; Loreti et al., 2000). It is conceivable, therefore, that decreased mobilization of endosperm reserves may limit availability of sugars thus relieving repression of a-amylase gene expression in scutella of the wheat transformants (Fig. 6A). In fact, the present range of PcGA2ox1 transgenic wheat provides useful material in which to study regulation of expression of both GA biosynthesis and a-amylase genes in relation to changes in GA and carbohydrate status in wheat scutellar tissues post germination.

In future, a more targeted approach is required to perturb GA production in specific tissues of stem internodes and/or developing grains, in order to manipulate processes that result in increased grain yield and quality. Support for such an approach comes from the generation of dwarf rice with normal grain production by restricting increased GA turnover to vegetative tissues (Sakamoto et al., 2003).

Acknowledgements

Rothamsted Research receives support from the Biotechnology and Biological Sciences Research Council of the UK.

References

Appleford NEJ, Evans DJ, Lenton JR, Gaskin P, Croker SJ, Devos K, Phillips AL, Hedden P. 2006. Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat. Planta 223, 568-582.

Appleford NEJ, Lenton JR. 1997. Hormonal regulation of alpha-amylase gene expression in germinating wheat (Triticum aesti-vum) grains. Physiologia Plantarum 100, 534-542.

Barnes C, Bugbee B. 1991. Morphological responses of wheat to changes in phytochrome photoequilibrium. Plant Physiology 97, 359-365.

Barnes WC, Blakeney AB. 1974. Determination of cereal a-amylase using a commercially available dye-labeled substrate. Starke 26, 193-197.

Börner A, Roder M, Korzun V. 1997. Comparative molecular mapping of GA insensitive Rht loci on chromosomes 4B and 4D of common wheat (Triticum aestivum L.). Theoretical and Applied Genetics 95, 1133-1137.

Chandler PM, Mosleth E. 1990. Do gibberellins play an in vivo role in controlling alpha-amylase gene expression? In: Ringlund K, Mosleth E, Mares DJ, eds. Fifth international symposium on pre-harvest sprouting in cereals. Boulder CO, USA: Westview Press, 100-109.

Dayanandan P, Hebard FV, Kaufman PB. 1976. Cell elongation in grass pulvinus in response to geotropic stimulation and auxin application. Planta 131, 245-252.

Evans LT, Blundell C, King RW. 1995. Developmental responses by tall and dwarf isogenic lines of spring wheat to applied gibberellins. Australian Journal of Plant Physiology 22, 365-371.

Evans MMS, Poethig RS. 1995. Gibberellins promote vegetative phase-change and reproductive maturity in maize. Plant Physiology 108, 475-487.

Fincher GB. 1989. Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annual Review of Plant Physiology and Plant Molecular Biology 40, 305-346.

Flintham JE, Borner A, Worland AJ, Gale MD. 1997. Optimizing wheat grain yield: effects of Rht (gibberellin-insensitive) dwarfing genes. Journal of Agricultural Science 128, 11-25.

Gale MD, Marshall GA. 1973. Insensitivity to gibberellin in dwarf wheats. Annals of Botany 37, 729-735.

Gale MD, Youssefian S. 1985. Dwarfing genes in wheat. In: Russell GE, ed. Progress in plant breeding. London: Butterworths, 1-35.

Green LS, Faergestad EM, Poole A, Chandler PM. 1997. Grain development mutants of barley: alpha-amylase production during grain maturation and its relation to endogenous gibberellic acid content. Plant Physiology 114, 203-212.

Griffiths J, Murase K, Rieu I, et al. 2006. Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis. The Plant Cell 18, 3399-3414.

Hartweck LM, Olszewski NE. 2006. Rice GIBBERELLIN INSENSITIVE DWARF1 is a gibberellin receptor that illuminates and raises questions about GA signaling. The Plant Cell 18, 278-282.

Hedden P. 2003. The genes of the Green Revolution. Trends in Genetics 19, 5-9.

Hedden P, Phillips AL. 2000. Gibberellin metabolism: new insights revealed by the genes. Trends in Plant Science 5, 523-530.

Hoogendoorn J, Rickson JM, Gale MD. 1990. Differences in leaf and stem anatomy related to plant height of tall and dwarf wheat (Triticum aestivum L.). Journal of Plant Physiology 136, 72-77.

Hutley-Bull PD, Schwabe WW. 1982. Some effects of low-concentration gibberellic acid and retardant application during early growth on morphogenesis in wheat. In: McLaren JS, ed. Chemical manipulation of crop growth and development. London: Butterworths, 329-342.

Isaac PG, Stacey J, Clee CM. 1995. Nonradioactive probes. Molecular Biotechnology 3, 259-265.

Itoh H, Ueguchi-Tanaka M, Sentoku N, Kitano H, Matsuoka M, Kobayashi M. 2001. Cloning and functional analysis of two gibberellin 3ß-hydroxylase genes that are differently expressed during the growth of rice. Proceedings of the National Academy of Sciences, USA 98, 8909-8914.

Kaneko M, Itoh H, Inukai Y, Sakamoto T, Ueguchi-Tanaka M, Ashikari M, Matsuoka M. 2003. Where do gibberellin biosynthesis and gibberellin signaling occur in rice plants? The Plant Journal 35, 104-115.

Kaneko M, Itoh H, Ueguchi-Tanaka M, Ashikari M, Matsuoka M. 2002. The alpha-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium. Plant Physiology 128, 1264-1270.

Kashkush K, Feldman M, Levy AA. 2002. Gene loss, silencing and activation in a newly synthesized wheat allotetraploid. Genetics 160, 1651-1659.

Keyes GJ, Paolillo DJ, Sorrells ME. 1989. The effects of dwarfing genes Rht1 and Rht2 on cellular dimensions and rate of leaf elongation in wheat. Annals of Botany 64, 683-690.

Kloti A, He X, Potrykus I, Hohn T, Fiitterer J. 2002. Tissue-specific silencing of a transgene in rice. Proceedings of the National Academy of Sciences, USA 99, 10881-10886.

Lazarus CM, Baulcombe DC, Martienssen RA. 1985. Alpha-amylase genes of wheat are two multigene families which are differentially expressed. Plant Molecular Biology 5, 13-24.

Lenton JR, Appleford NEJ, Croker SJ. 1994. Gibberellins and a-amylase gene expression in germinating wheat grains. Plant Growth Regulation 15, 261-270.

Loreti E, Alpi A, Perata P. 2000. Glucose and disaccharide-sensing mechanisms modulate the expression of alpha-amylase in barley embryos. Plant Physiology 123, 939-948.

Oikawa T, Koshioka M, Kojima K, Yoshida H, Kawata M. 2004. A role of 0sGA20ox1, encoding an isoform of gibberellin 20-oxidase, for regulation of plant stature in rice. Plant Molecular Biology 55, 687-700.

Peng JR, Richards DE, Hartley NM, et al. 1999. 'Green revolution' genes encode mutant gibberellin response modulators. Nature 400, 256-261.

Perata P, Matsukura C, Vernieri P, Yamaguchi J. 1997. Sugar repression of a gibberellin-dependent signaling pathway in barley embryos. The Plant Cell 9, 2197-2208.

Pinthus MJ, Gale MD, Appleford NEJ, Lenton JR. 1989. Effect of temperature on gibberellin (GA) responsiveness and on endogenous GA1 content of tall and dwarf wheat genotypes. Plant Physiology 90, 854-859.

Sakamoto T, Kobayashi M, Itoh H, Tagiri A, Kayano T, Tanaka H, Iwahori S, Matsuoka M. 2001. Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice. Plant Physiology 125, 1508-1516.

Sakamoto T, Miura K, Itoh H, et al. 2004. An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiology 134, 1642-1653.

Sakamoto T, Morinaka Y, Ishiyama K, Kobayashi M, Itoh H, Kayano T, Iwahori S, Matsuoka M, Tanaka H. 2003. Genetic manipulation of gibberellin metabolism in transgenic rice. Nature Biotechnology 21, 909-913.

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, USA: Cold Spring Harbor Laboratory Press.

Singh DP, Jermakow AM, Swain SM. 2002. Gibberellins are required for seed development and pollen tube growth in Arabidopsis. The Plant Cell 14, 3133-3147.

Skinner RH, Simmons SR. 1993. Modulation of leaf elongation, tiller appearance and tiller senescence in spring barley by far-red light. Plant, Cell and Environment 16, 555-562.

Sparks CA, Jones HD. 2004. Transformation of wheat by biolistics. In: Curtis IP, ed. Transgenic crops of the world: essential protocols. Dordrecht: Kluwer Academic Publishers, 19-35.

Swain SM, Reid JB, Kamiya Y. 1997. Gibberellins are required for embryo growth and seed development in pea. The Plant Journal 12, 1329-1338.

Thomas SG, Phillips AL, Hedden P. 1999. Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation. Proceedings of the National Academy of Sciences, USA 96, 4698-4703.

Thomas SG, Sun TP. 2004. Update on gibberellin signaling. A tale of the tall and the short. Plant Physiology 135, 668-676.

Verwoerd TC, Dekker BMM, Hoekema A. 1989. A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research 17, 2362.

Webb SE, Appleford NEJ, Gaskin P, Lenton JR. 1998. Gibberellins in internodes and ears of wheat containing different dwarfing alleles. Phytochemistry 47, 671-677.

White CN, Proebsting WM, Hedden P, Rivin CJ. 2000. Gibberellins and seed development in maize. I. Evidence that gibberellin/abscisic acid balance governs germination versus maturation pathways. Plant Physiology 122, 1081-1088.

Wolbang CM, Davies NW, Taylor SA, Ross JJ. 2007. Gravis-timulation leads to asymmetry of both auxin and gibberellin levels in barley pulvini. Physiologia Plantarum doi: 10.1111/ j.1399—3054.2007.00931.x.

Youssefian S, Kirby EJM, Gale MD. 1992a. Pleiotropic effects of the GA-insensitive Rht dwarfing genes in wheat. 1. Effects on development of the ear, stem and leaves. Field Crops Research 28, 179-190.

Youssefian S, Kirby EJM, Gale MD. 1992b. Pleiotropic effects of the GA-insensitive Rht dwarfing genes in wheat. 2. Effects on leaf, stem, ear, and floret growth. Field Crops Research 28, 191-210.

Yu SM, Lee YC, Fang SC, Chan MT, Hwa SF, Liu LF. 1996. Sugars act as signal molecules and osmotica to regulate the expression of alpha-amylase genes and metabolic activities in germinating cereal grains. Plant Molecular Biology 30, 1277-1289.

Zou JH, Zhang SY, Zhang WP, Li G, Chen ZX, Zhai WX, Zhao XF, Pan XB, Xie Q, Zhu LH. 2006. The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds. The Plant Journal 48, 687-696.

Zwar JA, Chandler PM. 1995. Alpha-amylase production and leaf protein synthesis in a gibberellin-responsive dwarf mutant of Himalaya barley (Hordeum vulgare L.). Planta 197, 39-48.

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