Invasion, isolation and evolution shape population genetic structure in Campanula rotundifolia.

The distribution and genetic structure of most plant species in Britain and Ireland bear the imprint of the last ice age. These patterns were largely shaped by random processes during recolonization but, in angiosperms, whole-genome duplication may also have been important. We investigate the distribution of cytotypes of Campanula rotundifolia, considering DNA variation, postglacial colonization, environmental partitioning and reproductive barriers. Cytotypes and genome size variation from across the species' range were determined by flow cytometry and genetic variation was assessed using cpDNA markers. A common garden study examined growth and flowering phenology of tetraploid, pentaploid and hexaploid cytotypes and simulated a contact zone for investigation of reproductive barriers. Irish populations were entirely hexaploid. In Britain, hexaploids occurred mostly in western coastal populations which were allopatric with tetraploids, and in occasional sympatric inland populations. Chloroplast markers resolved distinct genetic groups, related to cytotype and geographically segregated; allopatric hexaploids were distinct from tetraploids, whereas sympatric hexaploids were not. Genome downsizing occurred between cytotypes. Progeny of open-pollinated clones from the contact zone showed that maternal tetraploids rarely produced progeny of other cytotypes, whereas the progeny of maternal hexaploids varied, with frequent pentaploids and aneuploids. The presence of distinctive hexaploid chloroplast types in Ireland, Scottish islands and western mainland Britain indicates that its establishment preceded separation of these land masses by sea-level rise c. 16 000 years BP. This group did not originate from British tetraploids and probably diverged before postglacial invasion from mainland Europe. The combination of cytotype, molecular, contact zone and common garden data shows an overall pattern reflecting postglacial colonization events, now maintained by geographic separation, together with more recent occasional local in situ polyploidisation. Reproductive barriers favour the persistence of the tetraploid to the detriment of the hexaploid.

A DELLA in disguise: SPATULA restrains the growth of the developing Arabidopsis seedling.

The period following seedling emergence is a particularly vulnerable stage in the plant life cycle. In Arabidopsis thaliana, the phytochrome-interacting factor (PIF) subgroup of basic-helix-loop-helix transcription factors has a pivotal role in regulating growth during this early phase, integrating environmental and hormonal signals. We previously showed that SPATULA (SPT), a PIF homolog, regulates seed dormancy. In this article, we establish that unlike PIFs, which mainly promote hypocotyl elongation, SPT is a potent regulator of cotyledon expansion. Here, SPT acts in an analogous manner to the gibberellin-dependent DELLAs, REPRESSOR OF GA1-3 and GIBBERELLIC ACID INSENSITIVE, which restrain cotyledon expansion alongside SPT. However, although DELLAs are not required for SPT action, we demonstrate that SPT is subject to negative regulation by DELLAs. Cross-regulation of SPT by DELLAs ensures that SPT protein levels are limited when DELLAs are abundant but rise following DELLA depletion. This regulation provides a means to prevent excessive growth suppression that would result from the dual activity of SPT and DELLAs, yet maintain growth restraint under DELLA-depleted conditions. We present evidence that SPT and DELLAs regulate common gene targets and illustrate that the balance of SPT and DELLA action depends on light quality signals in the natural environment.

Immobilization of pectin, xyloglucan and other soluble plant polysaccharides on blotting membranes.

Soluble polysaccharides, of natural and synthetic origins, are immobilized by nylon, nitrocellulose, and polyvinylidene difluoride blotting membranes. Retention of an acid and a neutral polysaccharide, measured using radioactive pectin and xyloglucan-rich hemicellulose, exceeded 90% on some blotting membranes eluted in aqueous media. The two polysaccharides displayed different binding characteristics. Several polyanionic uronic acid polysaccharides, bound strongly to nylon 66 (Hybond N, Amersham) and to positively charge-modified or cationic nylon blotting membranes (Hybond N +, Amersham), but much less strongly to paper, nitrocellulose or polyvinylidene difluoride. Fragments of pectin, including Rhamnogalacturonan I and Rhamnogalacturonan II, and galacturonic acid oligosaccharides as small as the dimer also bind to nylon membranes, and can be detected using cationic dyes or by means of their radioactivity. Xyloglucan-rich hemicellulose in general binds more strongly than pectin to the same substrates. Of the substrates tested charge-modified nylon gave best retention of pectin, and paper gave best retention of xyloglucan during washes in water and solutions of salts, acids and bases. The influence of pH and solutions of mono, di- and trivalent salts on the retention of some pectic polysaccharides by nylon was investigated. A large proportion, in excess of 70% of pectin applied to charge-modified nylon, remained tightly bound at all salt concentrations up to 2 M. Soluble acid polysaccharides immobilized on blotting membranes could be detected by staining with cationic dyes, such as ruthenium red, alcian blue 8GX, and coriphosphine O, providing facile detection and a simple means of characterizing the cytochemical specificity of their staining reactions. Immobilized neutral polysaccharides, which do not react with cationic dyes, could usually be detected by the periodate-Schiff reaction, or, if labelled with radionuclides, by autoradiography or scintillation counting. Soluble polysaccharides, like proteins and nucleic acids, may therefore be immobilized on blotting membranes for investigation with cytochemical, immunocytochemical, and other molecular probing and detection procedures. Quantitative binding data showed marked differences in the affinity of different polysaccharides for blotting substrates. Detailed characterization of the binding behaviour is therefore a prerequisite for optimization and rational application of polysaccharide blotting.