Modification of gibberellin signalling (metabolism & signal transduction) in sugar beet: analysis of potential targets for crop improvement

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 vernalization-induced 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 down-regulation of GA responses by transformation with the Arabidopsis thaliana gai gene (which represses GA signalling), under its own promoter (pgai::gai) or deactivation of GA by over-expression 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 pgai::gai and 35S::PcGA2ox1 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 beet.

Appendix

Photoperiod and light intensity

Plants 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 ~1.4 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⁻¹.

Plant material

The 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 VV-A/ZR10235 supplied by English Sugar Beet Seed, Sleaford, UK was used for the vernalization time course experiments.

Plant culture and propagation

Guard 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 cross-pollination 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 time-course, 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 light.

Determining time taken to bolt

The 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/r)ln(1 + exp[r(θ − 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.