ABSTRACT

The early stage of rapeseed embryo development is characterized by starch accumulation. With the onset of oil and storage protein deposition, this starch is degraded (da Silva et al.,1997). The physiological function of this “transient” starch accumulation is still obscure. We hypothesize that repression of starch synthesis might provide energy (ATP), as well as carbon skeletons for fatty acid-synthesis and therefore probably increase the oil deposition. To test this assumption we transformed rapeseed hypocotyl segments with a cDNA encoding the small subunit of ADP-glucose pyrophosphorylase (AGPase) from Brassica napus inserted into an embryo-specific thioesterase-promoter-terminator cassette. The cDNA was either inserted in antisense, or in sense orientation to exploit the effect of cosuppression. Analysis of transformants revealed several lines with decreased AGPase-activity as well as decreased starch contents. Furthermore, we have indications for an increase in oil content in these lines.

A different approach to alter the energy (ATP) supply of developing rapeseed embryos was performed by modulating the activity of the plastidic ATP/ADP translocator. Recently, we demonstrated that altering activities of this transporter protein influence the quantity and quality of starch in transgenic potato tubers (Tjaden et al., 1998). To analyse whether a similar effect can be obtained with respect to the storage products of rapeseed we used the plastidic ATP/ADP transporters from Solanum tuberosum (sense) and B. napus (antisense) to transform plants as described above. Results from primary transformants are presented.

Introduction

Seeds contain different amounts of storage compounds: oil, protein and starch. Often one of these compounds dominates the others, like starch in cereal seeds e.g. maize, wheat, or oil in seeds of rapeseed or sunflower, but the other storage compounds are also present in larger or smaller amounts. For example, the starch storing maize endosperm contains 4% oil, (Doehlert, 1990; Earle et al., 1946) whereas the oil storing maize germ also contains about 8% starch (Earle et al., 1946). Understanding the mechanisms resulting in different ratios of storage compounds in seeds and the cross-talk between the pathways leading to these compounds is a prerequisite to alter their composition by biotechnological means in order to meet the demands of nutrition and non-food applications apart from being very interesting as a fundamental question.

In rapeseed, the embryo is the site of synthesis and deposition of storage compounds. Oil and protein make up up to 80-85% of the dry weight of the mature embryo (Murphy and Cummins, 1989). In the course of embryo development a transient accumulation of starch occurs, which at maximum content makes up 10% of final oil content on a dry weight basis (da Silva et al., 1997). This profile of starch accumulation is similar to that reported for Sinapis alba, a related species (Fischer et al.,1988) but differs from that of soybean where the maximum amount of starch accumulation is much higher (up to 50% of the final lipid content on a dry weight basis, Adams et al., 1980). Further indications that this transient starch accumulation is not the result of breeding efforts are provided by the closely related species Arabidopsis, which is a weedy plant. In Arabidopsis a similar profile of starch accumulation was observed during seed developement (Focks and Benning, 1998). Since starch accumulation and synthesis of fatty acids (major constituents of lipids) are exclusively located in plastids (Preiss, 1982; Stumpf, 1980), an interaction between both pathways is likely.

INTERACTION OF THE PATHWAYS LEADING TO STARCH AND LIPID SYNTHESIS

Early evidence for an interaction of starch and lipid synthesis came from analyses of pea embryos which were mutant at the r-locus. Betty and Smith (1990) found that a significantly lowered starch content correleated with doubled lipid contents. Recently, in Arabidopsis a low seed oil mutant was discovered (wri1, Focks and Benning, 1998), which transiently accumulated larger amounts of starch when compared to the wildtype.

Using isolated plastids from cauliflower bud inflorescences we analysed the capacity for simultaneous synthesis of starch and fatty acids (Möhlmann et al., 1994). Increasing the rate of Glucose-6-phosphate (Glc6P) dependent starch synthesis 2,5 fold by adding 3-Phosphoglycerate (which acts as an allosteric activator of plastidic ADP-glucose-pyrophosphorylase (AGPase)) the concurrent rate of fatty acid synthesis was reduced by 40%.

Developing embryos of rapeseed are able to perform high rates of starch and fatty acid synthesis from exogenously supplied Glc6P, although higher rates of fatty acid synthesis are obtained with pyruvate (Kang and Rawthorne, 1994). Both pathways are dependent on the supply of exogenous ATP (Kang and Rawthorne, 1996). We propose that interaction between starch and fatty acid synthesis is likely to occur in vivo due to competition for carbohydrate precursors (Glc6P) and energy (ATP, Betty and Smith (1990); Focks and Benning, (1998); Möhlmann et al., (1994)), although Kang and Rawthorne (1996) observed only little competition for ATP in vitro.

To increase the oil content of rape seeds we followed two strategies:

1. downregulation of AGPase activity to reduce starch synthesis and channel carbon skeletons and ATP to fatty acid synthesis.

2. Increasing the activity of the plastidic ATP/ADP transporter to supply the plastidic stroma with additional ATP for anabolic reactions.

REPRESSING STARCH SYNTHESIS BY REDUCING THE ACTIVITY OF THE PLASTIDIC AGPase

AGPase is thought to be a key enzyme in starch synthesis. A reduction of the activity of this enzyme leads to a drastic decrease of starch content in several tissues like pea embryos, potato tubers and Arabidopsis leaves (Smith et al., 1989; Müller-Röber et al., 1992; Neuhaus and Stitt, 1990).

Recently, it was discovered that a high percentage of AGPase is located in the cytosol of cereal endosperms of barley and maize (Thorbjornsen et al., 1996; Denyer et al., 1996). That AGPase in rapeseed would be located in the plastid is likely for two reasons: Firstly, measurements of AGPase activity in isolated rapeseed plastids suggest a plastidic localisation (Kang and Rawthorne, 1994), secondly, identification of a starchless mutant of the closely related species Arabidopsis is lacking plastidic phosphoglucose-mutase, necessary for conversion of Glc6P to Glc1P (Casper et al., 1985).

The plant AGPase is a heterotetrameric enzyme consisting of two large and two small subunits (Preiss, 1993). The small subunit is the catalytic one and necessary for large subunit stability (Wang et al., 1998). Therefore we decided to reduce the amount of small subunit protein via an antisense approach as well as by cosuppression (Matzke et al., 1995). We isolated a cDNA encoding the small subunit of AGPase from an embryospecific cDNA library (Elborough et al., 1994). This cDNA was put under the control of a thioesterase promoter from Cuphea lanceolata, ClFatB4, already known to be embryo-specific in transgenic rapeseed (G. Hemmann, J. Bautor and N. Martini, unpublished results). Hypocotyl segments from canola plants (var. DRAKKAR) were transformed essentially according to De Block et al. (1989). After regeneration of plants and verification of the transformed condition via PCR and resistance against glufosinate-ammonium, starch content and AGPase activity were measured in developing embryos according to the method of Da Silva et al., (1997). The oil content of mature embryos was quantified using near infra-red spectroscopy. Three lines constructed for cosuppression of AGPase showed low AGPase activities (30-60% of wildtype activity) as well as lowered (10-40% of wildtype) starch contents. At least one line is characterised by a relative increase in oil content of about 5% above vector controls. It remains to be demonstrated that these effects in plants are stable in following generations and in field trials.

INCREASING THE ACTIVITY OF THE PLASTIDIC ATP/ADP TRANSPORTER IN RAPESEED EMBRYOS

The plastidic ATP/ADP transporter imports ATP from the cytosol in exchange to stromal ADP, thus supplying the plastid stroma with energy equivalents for different anabolic reactions, especially in storage plastids (for review see: Emes and Neuhaus, 1998; Winkler and Neuhaus, 1999). Recently we could demonstrate that this transporter has a superior impact on starch synthesis in potato tuber amyloplasts, exhibiting a control coefficient for starch synthesis of 0.78 (Tjaden et al., 1998). During this work we identified a cDNA from potato (Solanum tuberosum) encoding a plastidic ATP/ADP transporter homolog. In order to provide more ATP for fatty acid synthesis, this cDNA was used for the generation of a sense construct under the control of the ClFatB4 promotor (see above) and the subsequent transformation of rapeseed plants. Several lines of primary transformants accumulated increased levels of corresponding mRNA. ATP/ADP transporter antisense plants were also generated to study the effect of this transporter on the metabolism of an oil storing tissue in more detail.

Conclusions

Although we have to be careful interpreting these preliminary results, there are indications for an interaction of fatty acid and starch synthesis in developing rapeseed embryos. Three lines of transgenic plants transformed with constructs allowing a cosuppression of AGPase exhibited significantly lowered activities of this enzyme as well as lowered starch contents. In one line, showing the lowest activity and strongest reduction of starch, the amount of oil increased by 5% relative to vector controls. It is still too soon to discuss the promising preliminary data from transgenic rapeseed expressing a plastidic ATP/ADP transporter.

Acknowledgements:

We gratefully acknowledge Jaqueline Bautor (MPI Cologne) for her skillful assistance in rapeseed transformation and Dr. Werner Paulmann (Saatzucht Hans Lembke, Malchow) for near infra-red spectroscopic analyses of transgenic seeds. T.M. was financially supported by the Bundesministerium für Ernährung, Landwirtschaft und Forsten through the Fachagentur Nachwachsende Rohstoffe.

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