Transcriptional landscapes of floral meristems in barley.

Organ development in plants predominantly occurs postembryonically through combinatorial activity of meristems; therefore, meristem and organ fate are intimately connected. Inflorescence morphogenesis in grasses (Poaceae) is complex and relies on a specialized floral meristem, called spikelet meristem, that gives rise to all other floral organs and ultimately the grain. The fate of the spikelet determines reproductive success and contributes toward yield-related traits in cereal crops. Here, we examined the transcriptional landscapes of floral meristems in the temperate crop barley (Hordeum vulgare L.) using RNA-seq of laser capture microdissected tissues from immature, developing floral structures. Our unbiased, high-resolution approach revealed fundamental regulatory networks, previously unknown pathways, and key regulators of barley floral fate and will equally be indispensable for comparative transcriptional studies of grass meristems.

A novel DNA binding protein with homology to Myb oncoproteins containing only one repeat can function as a transcriptional activator.

A cDNA clone encoding a novel Myb-related protein, designated MybSt1, was isolated from a potato cDNA expression library by South Western screening using the CaMV 35S promoter domain A as a probe. Sequence comparison shows a small region with some homology to the highly conserved DNA binding domain of the c-myb proto-oncogene consisting of three imperfect repeats. The Myb motif of the MybSt1 protein is distinct from the plant Myb DNA binding domain described so far. In contrast to the known plant Myb proteins, with two repeats required for the DNA binding activity, the clone mybSt1 contains only one such repeat. Nevertheless, the Myb-related protein MybSt1 is able to bind to DNA in a sequence-specific manner. In addition to the Myb-like region, the protein MybSt1 contains an acidic segment in its central region as well as a proline-rich region near the C-terminus. Applying the random binding site selection technique, high-affinity DNA binding sites for MybSt1 were identified, sharing the core motif GGATA. In transient expression assays using plant protoplasts, clear evidence was obtained for this myb clone functioning as a transcriptional activator.

Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants.

A cauliflower mosaic virus (CaMV) 35S promoter derivative, which is tightly repressed by the Tn 10 encoded Tet repressor in a transient expression system as well as in transgenic plants has been constructed. After treatment of transgenic plants with tetracycline (Tc) the activity of the reporter enzyme beta-glucuronidase (GUS) increased up to 500-fold in tissue culture as well as under greenhouse conditions. Efficient de-repression was achieved by Tc uptake through the roots as well as by Tc treatment of leaves of intact plants. As Tc is not very stable in the plants, this system can also be used for a transient expression of a transgene. This system provides a unique tool for regenerating transgenic plants carrying a repressed transgene and for efficiently de-repressing its activity by a specific inducer at any time point of further development.

The Tn10-encoded Tet repressor blocks early but not late steps of assembly of the RNA polymerase II initiation complex in vivo.

We have studied the effect of the Tn10-encoded Tet repressor on expression from 13 cauliflower mosaic virus (CaMV) 35S promoter derivatives that contain a tet operator sequence in various positions downstream of the TATAbox. When the operator sequence was inserted less than 33 bp away from the TATAbox (position +9 with respect to the transcription start site), the repressor interfered with transcription, whereas increasing the distance to 35 bp (position +11) abolished repression. This result indicates that initiation of transcription from the CaMV 35S promoter occurs in at least two different steps: (1) binding of transcription factors, involving sequences extending to position +9; this step can be inhibited by binding of the Tet repressor protein; and (2) initiation of transcription from this complex, which is not affected by the repressor protein. We suggest that the Tet repressor can be used to investigate whether transcription conditions in vitro truly reflect the in vivo situation.

Characterization of the interaction of plant transcription factors using a bacterial repressor protein.

Transcription initiation from a eukaryotic polymerase II promoter requires a functional interaction of regulatory transcriptional activators with at least one of the basal transcription factors binding in the vicinity of the TATA box. To characterize this type of interaction in vivo, we have inserted the bacterial Tet repressor-operator complex in nine different positions between an enhancer element (as-1) and the TATA box of the cauliflower mosaic virus (CaMV) 35S RNA promoter. A direct contact between the transcriptional activator ASF-1, which binds to as-1, and the transcriptional machinery should be affected by a repressor protein bound between them, as the spacing of only 34 base pairs (bp) between as-1 and the TATA box is too short to allow looping of the DNA around the repressor. In each construct, the distance of 34 bp was kept constant, while the position of the 19-bp tet operator relative to the TATA box differed by 2 bp. Thus, the position of the Tet repressor relative to the plant transcription factors was consecutively changed by 72 degrees, which allowed us to investigate whether repression depended on the stereospecific alignment of the repressor with the transcription factors. Binding of the Tet repressor to the operator blocked transcription only when the operator was inserted less tha 5 bp from the TATA box. In all other promoter derivatives, no inhibitory effect of the repressor was observed, which suggests that ASF-1 does not directly interact with the general transcription machinery.