Ectopic expression of Zmhox1b alters the development in transgenic tobacco
--Bärbel Überlacker, Claudia Mehlem and Wolfgang Werr

Zmhox1a (Zea mays homeobox gene 1a), our first homeobox gene, was isolated by screening a Shrunken promoter. Its homeodomain is only distantly related to other plant homeodomains, including the maize Knotted class. Therefore Zmhox1a is a member of an unrelated class of maize homeobox genes. A close relative, Zmhox1b, was isolated using the Zmhox1a homeobox as a probe (Bellmann and Werr, 1992, EMBO J. 11:3367-3374). Both genes represent a highly related gene pair, the gene products share 91% similarity on the protein level. They are not alleles because Zmhox1a maps on chromosome 8, while Zmhox1b is located on chromosome 6.

Northern experiments show that Zmhox1a and Zmhox1b are transcribed at low level in most tissues. In contrast to Zmhox1bprobes, with which similar RNA amounts were detected in all tissues except in leaves, Zmhox1a transcript levels peaked in suspension cells and during embryonic development. From young to adult leaves the Zmhox1a/b transcripts decrease, 1a drops below the detection level while the 1b RNA remains visible at a very low level (see Fig. 1).

To gain insight into biological functions we raised transgenic tobacco plants ectopically expressing the Zmhox1a/b gene products. Only the protein coding regions were expressed to exclude posttranscriptional regulation, which may involve the 5' and 3' untranslated sequences of Zmhox1a or b cDNA clones. The open reading frames were fused behind the 1 leader sequence of TMV and expressed under the control of the CaMV 35S promoter. Due to differences in the cloning strategy, so far the experiment with Zmhox1b is most advanced.

Ectopic expression of the Zmhox1b gene affects the development of the vegetative plant body and the flower. Out of 137 independent primary transformants 70% show an abnormal flower phenotype. Two whorls are affected: the stamen and the carpel. Different flowers contain between 1-5 petaloid stamens which often are also fused. The filament is unchanged but petals grow out below the anthers; the pollen of these transformed stamens is sterile. Severe phenotypes carry the homeotic transformation in all stamens and are sterile even in wild-type backcrosses. In addition these plants show alterations in the carpel. The stigma is changed in shape, the position of the ovary is displaced from the receptacle towards the stigma and the number of ovules per placenta is highly reduced. Besides changes in the flower, the ectopically expressing tobacco plants are often more branched than control plants. Most plants are shorter than the wildtype and exhibit outgrowth of additional flowering side shoots below the main flower. Severely affected plants grow adventitious shoots from every axillary bud, but unfortunately are often sterile. The abnormal flower development, the weaker bushy growth habit and plant shortness are stably inherited into the next plant generation. 

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