Auxin and physical constraint exerted by the perianth promote androgynophore bending in Passiflora mucronata L. (Passifloraceae).

The androgynophore column, a distinctive floral feature in passion flowers, is strongly crooked or bent in many Passiflora species pollinated by bats. This is a floral feature that facilitates the adaptation to bat pollination. Crooking or bending of plant organs are generally caused by environmental stimulus (e.g. mechanical barriers) and might involve the differential distribution of auxin. Our aim was to study the role of the perianth organs and the effect of auxin in bending of the androgynophore of the bat-pollinated species Passiflora mucronata. Morpho-anatomical characterisation of the androgynophore, including measurements of curvature angles and cell sizes both at the dorsal (convex) and ventral (concave) sides of the androgynophore, was performed on control flowers, flowers from which perianth organs were partially removed and flowers treated either with auxin (2,4-dichlorophenoxyacetic acid; 2,4-D) or with an inhibitor of auxin polar transport (naphthylphthalamic acid; NPA). Asymmetric growth of the androgynophore column, leading to bending, occurs at a late stage of flower development. Removing the physical constraint exerted by perianth organs or treatment with NPA significantly reduced androgynophore bending. Additionally, the androgynophores of plants treated with 2,4-D were more curved when compared to controls. There was a larger cellular expansion at the dorsal side of the androgynophores of plants treated with 2,4-D and in both sides of the androgynophores of plants treated with NPA. This study suggests that the physical constraint exerted by perianth and auxin redistribution promotes androgynophore bending in P. mucronata and might be related to the evolution of chiropterophily in the genus Passiflora.

The Arabidopsis SHAGGY-related protein kinase (ASK) gene family: structure, organization and evolution.

Higher plants contain a multigene family encoding proteins that share a highly conserved catalytic protein kinase domain about 70% identical to SHAGGY protein kinase (SGG) and glycogen synthase kinase-3 (GSK-3), respectively, from Drosophila and mammals. In this study we have characterized the structure and evolution of the Arabidopsis SHAGGY-related protein kinase (ASK) gene family. At least ten ASK genes are present per haploid genome of Arabidopsis. The genomic sequences of five ASK genes show a strikingly high conservation of intron positions and exon lengths. Phylogenetic analyses suggested that the Arabidopsis gene family contains at least three ancient classes of genes that diverged early in land plant evolution. The different classes may reflect specificity of substrates and/or biological functions. Eight out of the ten predicted ASK genes were mapped and shown to be dispersed over the five Arabidopsis chromosomes. A tentative model for the organization and evolution of the Arabidopsis ASK genes is presented.

Plant regeneration from protoplast cultures of Passiflora edulis var. flavicarpa Deg., P. amethystina Mikan. and P. cincinnata Mast.

Protoplasts isolated from seedling cotyledons of yellow passionfruit (Passiflora edulis var. flavicarpa Deg.) and two related wild species, P. amethystina Mikan. and P. cincinnata Mast., divided in culture and produced calli. Shoot regeneration was obtained in MS medium (Murashige and Skoog 1962) containing 2.0 mg/l 6-benzylaminopurine (BAP). Regenerated plants produced roots in half-strength hormone-free MS medium and could be transferred to soil after being acclimatized.

Arabidopsis thaliana SHAGGY-related protein kinases (AtSK11 and 12) function in perianth and gynoecium development.

In higher plants, the correct patterning of the floral meristem in terms of organ type, number and form is the result of a concerted expression of a network of genes. We describe phenotypes of flower patterning, resulting from a reduction of transcript levels of the Arabidopsis SHAGGY-related protein kinase genes AtSK11(ASKalpha) and AtSK12(ASKgamma). The AtSK genes are plant homologues of the Drosophila shaggy (SGG) gene and the mammalian Glycogen-Synthase Kinase-3 (GSK-3). The SGG protein kinase is a key component of the wingless signalling pathway and is required for the establishment of tissue patterning and cell fate determination. The expression patterns of the AtSK11(ASKalpha) and AtSK12(ASKgamma) genes during wild-type Arabidopsis inflorescence development, detected by in situ hybridisation, have been shown to be consistent with a possible role in floral meristem patterning. AtSK11(ASKalpha) and AtSK12(ASKgamma) transcripts were detected at the periphery of the inflorescence meristem and in the floral meristem. At later stages the expression of the AtSK genes became localised in specific regions of developing flower organ primordia. Furthermore, we have obtained and analysed transgenic plants containing AtSK11(ASKalpha) and AtSK12(ASKgamma) gene specific antisense constructs. These plants developed flowers showing a higher number of perianth organs and an alteration of the apical-basal patterning of the gynoecium.

Plant regeneration from protoplast fusion inPassiflora spp.

Protoplasts were isolated from leaf explants ofPassiflora edulis var.flavicarpa (the yellow passion fruit) and from cell suspensions of fivePassiflora species. Chemical fusion was performed using polyethylene glycol and the microcolonies obtained were transferred to growth medium to produce calli. Electrophoresis of soluble proteins and analysis of isoenzymes from calli produced from the fusion experiments were performed to select somatic hybrids. Specific polypeptide bands allowed the identification of somatic hybrids betweenP. edulis var.flavicarpa (+)P. alata, P. edulis var.flavicarpa (+)P. amethystina, P. edulis var.flavicarpa (+)P. cincinnata, P. edulis var.flavicarpa (+)P. giberti andP. edulis var.flavicarpa (+)P. coccinea. An average of 3 to 5% hybrid calli were obtained. With the exception of theP. edulis var.flavicarpa (+)P. coccinea, whole plants were recovered from all hybrids. These somatic hybrids showed 4n=36 chromosomes, which represents a further evidence of their hybridity.

Characterization of three novel members of the Arabidopsis SHAGGY-related protein kinase (ASK) multigene family.

In this paper we report the characterization of three novel members of the Arabidopsis shaggy-related protein kinase (ASK) multigene family, named ASKdzeta (ASKzeta), ASKetha (ASKeta) and ASKiota (ASKiota). The proteins encoded by the ASK genes share a highly conserved catalytic protein kinase domain and show about 70% identity to SHAGGY (SGG) and glycogen synthase kinase-3 (GSK-3) from Drosophila and rat respectively. SGG is an ubiquitous intracellular component of the wingless signalling pathway that establishes cell fate and/or pattern formation in Drosophila. At least ten different ASK genes are expected to be present per haploid genome of A. thaliana. Different amino- and carboxy-terminal extensions distinguish different ASK family members. Five ASK gene sequences were analysed and shown to be present as single-copy genes in the Arabidopsis genome. A comparison based on the highly conserved catalytic domain sequences of all known sequences of the GSK-3 subfamily of protein kinases demonstrated a clear distinction between the plant and the animal kinases. Furthermore, we established the presence of at least three distinct groups of plant homologues of SGG/GSK-3. These different groups probably reflect biochemical and/or biological properties of these kinases. The differential expression patterns of five ASK genes were accessed by northern and in situ hybridization experiments using gene-specific probes. While ASKzeta is expressed in the whole embryo during its development, ASKeta expression is limited to the suspensor cells. No signal was detected for ASKalpha, ASKgamma and ASKiota in developing embryos.