Module-specific regulation of the beta-phaseolin promoter during embryogenesis

The phas promoter displays stringent spatial regulation, being very highly expressed during embryogenesis and completely silent during all phases of vegetative development in bean, Phaseolus vulgaris. This pattern is maintained in transgenic tobacco and, as shown here, Arabidopsis. Dimethyl sulphate in vivo footprinting analyses revealed that over 20 cis-elements within the proximal 295 bp of the phas promoter are protected by factor binding in seed tissues whereas none are bound in leaves. The hypothesis that this complex profile represents a summation of several module (cotyledon, hypocotyl, and radicle)-specific factor-DNA interactions has been explored by the incorporation of site-directed substitution mutations into 10 locations within the -295phas promoter. Only 2.6% of -295phas promoter activity remained after mutation of the G-box; the CCAAAT box, the E-box and the RY elements were also found to mediate high levels of expression in embryos. Whereas the CACA element has dual positive and negative regulatory roles, the vicilin box was identified as a strong negative regulatory element. The proximal (-70 to -64) RY motif was found to bestow expression in the hypocotyl while all the RY elements contribute to expression in cotyledons but not to vascular tissue expression during embryogenesis. RY elements at positions -277 to -271, -260 to -254, and -237 to -231 were found to orchestrate radicle-specific repression. The G-box appears to be the functional abscisic acid responsive element and the E-site may be a coupling element. The results substantiate the concept that autarkical cis-element functions generate modular patterning during embryogenesis. They also reflect the existence of both redundancy and hierarchy in cis-element interactions. Importantly, the virtually identical expression patterns observed for the two distantly related plants studied argue strongly for the generality of function for the observed factor-element interactions.     

Cloning and molecular characterisation of a potato SERK gene transcriptionally induced during initiation of somatic embryogenesis

Somatic embryogenesis offers great potential in plant propagation, long-term germplasm conservation, and as a suitable model system for deciphering early events during embryogenesis. The up-regulation and ectopic expression of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene has been shown to mark and enhance embryogenic competence in somatic cells of model plant species. We have cloned and characterised a SERK gene (StSERK1) from potato (Solanum tuberosum L.), an important crop plant. Sequence analysis of StSERK1 revealed high levels of similarity to other plant SERKs, as well as a conserved intron/exon structure which is unique to members of the SERK family. Furthermore, StSERK clustered most closely with SERK gene family members such as MtSERK1, CuSERK1, AtSERK1, and DcSERK, implicated in evoking somatic embryogenesis. Monitoring of SERK expression during progression of potato somatic embryogenesis revealed increased StSERK expression during the induction phase. Subsequently, during the embryo transition phases, StSERK expression was unchanged and did not vary among embryo-forming and inhibitory conditions. However, in isolated somatic embryos StSERK expression was again up-regulated. In other plant parts (leaves, true potato seeds, microtubers and flower buds), StSERK showed different levels of expression. Expression analysis suggests that the isolated StSERK could be a functional SERK orthologue. The possible role of SERK as a marker of pluripotency, rather than embryogenesis alone, is discussed.     

Cell Ablation Reveals That Expression from the Phaseolin Promoter Is Confined to Embryogenesis and Microsporogenesis

Most previous studies of the [beta]-phaseolin (phas) gene, which encodes the major storage protein in bean (Phaseolus vulgaris L.), have shown its expression to be rigorously confined to the developing seed, both in bean and transgenic tobacco (Nicotiana tabacum L. cv Xanthi) plants. To confirm unequivocally the lack of phas expression in vegetative tissues, we placed the diphtheria toxin A-chain (DT-A) coding region under the control of [beta]-phaseolin promoter sequences. Tobacco plants transgenic for phas/DT-A were phenotypically normal until flowering, when they produced anthers that were externally normal but contained no viable pollen. Microscopic examination of immature anthers revealed a normal tapetum, but the pollen mother cells did not undergo meiosis and subsequently degenerated, resulting in male-sterile plants. This demonstration of phas expression during microsporogenesis was corroborated by the expression of [beta]-glucuronidase in pollen of plants transformed with comparable phas/uidA constructs. Although these findings suggested that similarities in phas expression may exist between seed and pollen maturation, no phas activity could be detected in bean pollen. After fertilization of the DT-A-transformed plants with pollen from wild-type tobacco, 50% of the resulting embryos aborted at the heart stage, defining this as the earliest time for phas expression during embryogenesis.