Functions of OsDof25 in regulation of OsC4PPDK.

Relative little is known about the functions of the so-called Dof zinc factors in plants. Here we report on the analysis of OsDof25 and show a function in regulation of the important C4 photosynthesis gene, OsC4PPDK in rice. Over-expression of OsDof25 enhanced the expression of OsC4PPDK in transient expression experiments by binding in a specific way to a conserved Dof binding site which was confirmed by yeast and in vitro binding studies. Expression studies using promoter GUS plants as well as qPCR experiments showed that OsDof25 expressed in different tissues including both photosynthetic and non-photosynthetic organs and that expression of OsDof25 was partially overlapping with the OsC4PPDK gene. Conclusive evidence for a role of OsDof25 in regulation of C4PPDK came from loss-of-function and gain-of-function experiments with transgenic rice, which showed that down-regulation or over-expression of OsDof25 correlated with OsC4PPDK expression and that OsDof25 has functions as transcriptional activator.

Transcription and somatic transposition of the maize En/Spm transposon system in rice.

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.

Transpositional behaviour of an Ac/Ds system for reverse genetics in rice.

A collection of transposon Ac/ Ds enhancer trap lines is being developed in rice that will contribute to the development of a rice mutation machine for the functional analysis of rice genes. Molecular analyses revealed high transpositional activity in early generations, with 62% of the T0 primary transformants and more than 90% of their T1 progeny lines showing ongoing active transposition. About 10% of the lines displayed amplification of the Ds copy number. However, inactivation of Ds seemed to occur in about 70% of the T2 families and in the T3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T1 plants indicated the independence of insertions in different T1 families originating from the same T0 line. Almost 80% of the insertion sites isolated showing homology to the sequenced genome, resided in genes or within a range at which neighbouring genes could be revealed by enhancer trapping. A strategy involving the propagation of a large number of T0 and T1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T2 and T3 lines produced will then provide a collection of stable insertions to be used in reverse genetics experiments. The preferential insertion of Ds in gene-rich regions and the use of lines containing multiple Ds transposons will enable the production of a large population of inserts in a smaller number of plants. Additional features provided by the presence of lox sites for site-specific recombination, or the use of different transposase sources and selectable markers, are discussed.

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics.

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.