Modification of gibberellin signalling metabolism

Authors: Effie MutasaGottgens Aiming Qi Ann Mathews Stephen Thomas Andrew Phillips Peter Hedden

Publish Date: 2008/08/12

Volume: 18, Issue: 2, Pages: 301-

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Abstract

Sugar beet Beta vulgaris spp vulgaris is a biennial long day plant with an obligate requirement for vernalization prolonged exposure to low temperature As a spring crop in temperate European climates it is vulnerable to vernalizationinduced premature bolting and flowering resulting in reduced crop yield and quality Gibberellins GAs play important roles in key physiological processes including stem elongation bolting and flowering and are therefore potential targets for controlling reproductive growth in sugar beet We show that the BvGA20ox gene which encodes an enzyme necessary for GA biosynthesis was transcriptionally activated in apices of sugar beet plants after vernalization and that GA metabolism can be manipulated to delay bolting in vernalized plants We demonstrate that downregulation of GA responses by transformation with the Arabidopsis thaliana gai gene which represses GA signalling under its own promoter pgaigai or deactivation of GA by overexpression of the Phaseolus coccineus bean GA2ox1 gene which inactivates GA increased the required post vernalization thermal time an accurate and stable measure of physiological time to bolt by ~300°Cd This resulted in agronomically significant bolting time delays of ~2 weeks and 3 weeks in the pgaigai and 35SPcGA2ox1 plants respectively Our data represent the first transgenic sugar beet model to 1 show that GA signalling can be used to improve crops by manipulation of the transition to reproductive growth and 2 provide evidence that GA is required for seed set in sugar beetBroom’s Barn receives financial support from the UK beet industry administered through the British Beet Research Organisation http//wwwbbrocouk/ Rothamsted Research receives grantaided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom http//wwwbbsrcacuk/ Dr Birgitta Debenham is gratefully acknowledged for contributions to the vernalization timecourse experiment Sarah Yallop and Roz Williamson are gratefully acknowledged for providing technical assistance with preparation cultivation and analysis of GM plants Kevin Sawford is acknowledged for assistance with culture and maintenance of plants in the glasshouse and controlled environment rooms We are grateful to all those who have provided us with sugar beet seeds as specified and to SES VanderHave Tienen Belgium for licensing the guard cell protoplast transformation method to Broom’s Barn Special thanks go to Guy Weyens and Marc Lefevre for assisting Dr Richard Rick Scott in getting the method originally established at Broom’s BarnPlants were grown at Broom’s Barn 52°16′ N 00°34′ E except where specified in the glasshouse at 20–22°C with a 16 h photoperiod supplemented as necessary by Son/T 400W high pressure sodium lamps supplied by Thermaforce Ltd Cockermouth Cumbria UK fixed at ~14 m above the bench The lamps were set to work automatically between 5 am and 9 pm in order to maintain light levels within the glasshouse above 228 μmol m−2 s−1 and to switch off if light levels in the glasshouse exceeded 457 μmol m−2 s−1The diploid biennial sugar beet line genotype NF supplied by SES Vanderhave formerly SES Advanta Belgium was used for guard cell protoplast transformation The bolting susceptible biennial KWS breeding line VVA/ZR10235 supplied by English Sugar Beet Seed Sleaford UK was used for the vernalization time course experimentsGuard cell donor plants were grown as previously described Hall et al 1996 except that in the growth room 23°C/16 h photoperiod light photosynthetically active radiation PAR of ~60 μmol m−2 s−1 was supplied from a mixture of Sylvania 58 W Grolux and Biolux Daystar light bulbs GEC Technology Glasgow UK Regenerated plants were transferred to compost Levington F2S Scotts Company UK Ltd and grown in the glasshouse as above after acclimation at high humidity in propagators for 1–2 weeks Seed production from biennial lines required vernalization for 18 weeks as previously described Chia et al 2008 Undesired crosspollination was avoided by using pollen isolation bags PBS International Scarborough UK When seeds ripened inflorescences were cut off and hung in a well aerated room to dry for a further 2–3 weeks before seeds were harvested For the vernalization timecourse post vernalization thermal buffering was achieved by slowly increasing temperature to ambient levels by 2°C every two days while plants remained under continuous low lightThe time taken to bolt can be measured as days or as thermal time accumulated above a threshold air temperature from the date when the fully vernalized plants are returned to the glasshouse until the date when they bolt Because it was difficult to observe the exact date of bolting and the first bolt height measurement varied from plant to plant a standard bolt height was required in order to calculate the time taken for the plants to bolt This standard bolt height was taken to be 5 cm the previously defined minimum height Smit 1983 and a functional approach was adopted to determine the time required for the elongating bolt to reach this height To account for daily temperature fluctuation in the glasshouse the accumulated thermal time above a threshold of 3°C was related to the sequentially measured bolt height Werker and Jaggard 1997 With respect to the various growth functions the expolinear growth equation H = c/rln1 + exprθ − θ b Goudriaan and Monteith 1990 was most appropriate for describing the relationship between bolt height and thermal time where H is the bolt height θ is the accumulated thermal time after vernalization r is the initial relative growth rate c is the maximum absolute growth rate and θ b is the accumulated thermal time at which the bolt passes from exponential to linear growth Once the parameters of c r and θ b were estimated the thermal time and the days taken to a standard bolt height were determined

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