This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain-leucine zipper (HD-Zip) class of putative transcription factors. Oshox1 maps to chromosome 10 and belongs to a family of related rice genes. Two-hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD-Zip protein. This suggests that protein-protein interactions may also occur between different HD-Zip proteins in rice, which would provide enormous versatility for generating specific gene-control mechanisms. Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants. Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator. In vitro and in vivo DNA-binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor. Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression. Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N-terminal region of Oshox1, preceding the homeodomain.

Transposition of the maize En/Spm system in rice was investigated using a two-component construct consisting of an immobilised transposase source driven by the CaMV 35S-promoter, and a modified I/dSpm transposon. Mobilization of I/dSpm in somatic sectors was demonstrated by sequencing of excision products and isolation of flanking genomic sequences in T0 and T1 progeny plants. Since the transposition efficiency appeared to be considerably lower than that observed in maize or in other heterologous systems like Arabidopsis, we examined En/Spm transcription and splicing in the transgenic rice plants. Northern analysis revealed the presence of transcripts encoding the active TnpA and TnpD transposases, with the latter predominating; this is the reverse of what is seen in maize and Arabidopsis. RT-PCR analysis confirmed the occurrence of correct splicing and the formation of the two other alternatively spliced transcripts (TnpB and TnpC), as previously described for maize. Two alternative splice donor sites at the end of exon 1 were identified in maize at positions 578 and 704. We observe that rice is similar to maize in that TnpA is preferentially spliced at position 578. We also show that in Arabidopsis splicing occurs preferentially at position 704, as in other dicots like tobacco. These observations indicate differences in the splicing of transcripts of the maize En/Spm element between dicot and monocot hosts. Nevertheless, the ratio in which the transcripts for the active transposases are produced seems to determine the efficiency of transposition, irrespective of the host considered. A limiting amount of TnpA might therefore be responsible for the lower transposition activity of En/Spm in rice. Alternatively, reduced mobility of the modified I/dSpm element used may have resulted from the absence of critical sequences necessary for transposition. The influence of endogenous, autonomous, En/Spm -related elements present in the rice genome on the transposition behaviour of the exogenous maize element is also considered.

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