Metabolically engineered male sterility in rapeseed (Brassica napus L.).

Male sterility is of special interest as a mechanism allowing hybrid breeding, especially in important crops such as rapeseed (Brassica napus). Male sterile plants are also suggested to be used as a biological safety method to prevent the spread of transgenes, a risk that is high in the case of rapeseed due to the mode of pollination, out-crossing by wind or insects, and the presence of related, cross-pollinating species in the surrounding ecosystem in Europe. Different natural occurring male sterilities and alloplasmic forms have been tried to be used in rapeseed with more or less success. Due to the difficulties and limitations with these systems, we present a biotechnological alternative: a metabolically engineered male sterility caused by interference with anther-specific cell wall-bound invertase. This is an essential enzyme for carbohydrate supply of the symplastically isolated pollen. The activity of this enzyme is reduced either by antisense interference or by expressing an invertase inhibitor under control of the anther-specific promoter of the invertase with the consequence of a strong decrease of pollen germination ability.

Anther-specific carbohydrate supply and restoration of metabolically engineered male sterility.

Male-sterile plants are used in hybrid breeding as well as for gene confinement for genetically modified plants in field trials and agricultural production. Apart from naturally occurring mutations leading to male sterility, biotechnology has added new possibilities for obtaining male-sterile plants, although so far only one system is used in practical breeding due to limitations in propagating male-sterile plants without segregations in the next generation or insufficient restoration of fertility when fruits or seeds are to be harvested from the hybrid varieties. Here a novel mechanism of restoration for male sterility is presented that has been achieved by interference with extracellular invertase activity, which is normally specifically expressed in the anthers to supply the developing microspores with carbohydrates. Microspores are symplastically isolated in the locular space of the anthers, and thus an unloading pathway of assimilates via the apoplasmic space is mandatory for proper development of pollen. Antisense repression of the anther-specific cell wall invertase or interference with invertase activity by expressing a proteinacious inhibitor under the control of the anther-specific invertase promoter results in a block during early stages of pollen development, thus causing male sterility without having any pleiotropic effects. Restoration of fertility was successfully achieved by substituting the down-regulated endogenous plant invertase activity by a yeast invertase fused to the N-terminal portion of potato-derived vacuolar protein proteinase II (PiII-ScSuc2), under control of the orthologous anther-specific invertase promoter Nin88 from tobacco. The chimeric fusion PiII-ScSuc2 is known to be N-glycosylated and efficiently secreted from plant cells, leading to its apoplastic location. Furthermore, the Nin88::PiII-ScSuc2 fusion does not show effects on pollen development in the wild-type background. Thus, such plants can be used as paternal parents of a hybrid variety, thereby the introgression of Nin88::PiII-ScSuc2 to the hybrid is obtained and fertility is restored. In order to broaden the applicability of this male sterility/restoration system to other plant species, a phylogenic analysis of plant invertases(beta-fructofuranosidases) and related genes of different species was carried out. This reveals a specific clustering of the cell wall invertases with anther-specific expression for dicotyl species and another cluster for monocotyl plants. Thus, in both groups of plants, there seems to be a kind of co-evolution, but no recent common ancestor of these members of the gene family. These findings provide a helpful orientation to classify corresponding candidate genes in further plant species, in addition to the species analysed so far (Arabidopsis, tobacco, tomato, potato, carrots, rice, and wheat).

Interspecies compatibility of the anther specific cell wall invertase promoters from Arabidopsis and tobacco for generating male sterile plants.

Histochemical GUS-staining and fluorometric analyses revealed strong tissue specific activities of the cell wall invertase promoters Nin88 from Nicotiana tabacum and AtcwINV2 from Arabidopsis thaliana that are restricted tightly to anthers and pollen, respectively. Both in A. thaliana and N. tabacum repression of invertase activity by anther specific RNA-interference turned out to be an efficient method to circumvent carbohydrate supply of the symplastically isolated pollen with subsequent strong decrease of pollen germination ability and seed setting. In the case of tobacco, comparable results were also obtained by expressing a proteinaceous invertase inhibitor, whereas this approach was less efficient in Arabidopis. The present study revealed that anther specific interference with invertase-activity in order to generate male sterile plants can be applied to members of the two different plant families Solanaceae (N. tabacum) and Brassicaceae (A. thalaina) and the strategy seems to be a general tool for practical application in hybrid breeding or as biological safety precautions. To elucidate the compatibility of the isolated promoters beyond plant families, we transferred the regulatory sequences into the respectively heterologous systems, i.e. the Nin88 promoter into Arabidopsis and the AtcwINV2 promoter into tobacco. The specificities of both promoters are maintained in the heterologous backgrounds, but their activities are strongly reduced as GUS-stainings of flowers and pollen revealed and fluorometrical quantification confirmed.