Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies.

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate-zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.

Differential Gene Expression in Response to Auxin Treatment in the Wild Type and rac, an Adventitious Rooting-Incompetent Mutant of Tobacco.

Histological analyses of auxin-treated cuttings from the wild type and the rac mutant of tobacco (Nicotiana tabacum cv Xanthii) previously revealed that some rac phloem parenchyma or inner cortical parenchyma cells form callus in response to exogenous auxin treatment but these cells never undergo the organized divisions associated with adventitious root initiation in the wild type. Here we report the effect of the rac mutation on the temporal and spatial expression patterns of three genes previously shown to be associated with adventitious root meristems, HRGPnt3, iaa4/5, and gh3. Using histochemical staining analyses of HRGPnt3-GUS transformant cuttings, we determined that the rac mutation blocks auxin activation of the HRGPnt3 promoter. Thus, activation of the HRGPnt3 promoter occurs specifically during adventitious root initiation in tobacco cuttings. Histochemical staining analyses of iaa4/5-GUS and gh3-GUS transformant cuttings revealed that the rac mutation does not repress the auxin activation of the iaa4/5 and gh3 promoters. Based on our histochemical staining analyses, we conclude that differential gene expression occurs in response to auxin treatment during adventitious root initiation in the wild type compared with callus formation in rac cuttings. We also determined that HRGPnt3 mRNA accumulation occurs in response to components of our root-induction protocol other than auxin, indicating that HRGPnt3 expression is regulated both developmentally and environmentally.

Changes in the pattern of cell arrangement at the surface of the shoot apical meristem in Hedera helix L. following gibberellin treatment.

The changes in the pattern of cell arrangement and surface topography at the shoot apical meristem of Hedera helix L., which occur during gibberellic acid (GA3)-induced transition from spiral to distichous phyllotaxis, were examined by scanning electron microscopy of rapidly frozen tissue. The technique preserves the original shape of the cells in their turgid state. It reveals distinct sets of radially oriented cell files, about four to eight cells wide, which extend from the central region of the meristem toward leaf primordia on the meristem flanks. In apices with spiral phyllotaxis, a new emerging primordium (0) appears as an acropetal bulge between the radial files adjacent to the third (3) and the second (2) older primordia. The bulging is associated with radial or oblique cell divisions while those located at the meristem flanks and in the radial files are oriented tangentially. As the displacement of existing primordia away from the central region increases following the GA3 treatment, radial and oblique divisions as well as acropetal bulging invade the radial files adjacent to the primordium 2; consequently the angular divergence of the emerging primordium from the youngest existing primordium (1) increases. In apices with distichous phyllotaxis, the earliest bulging appears on both sides of the radial files facing primordium 2, with a slight depression at the files. The radial files therefore correspond to regions of the meristem where acropetal bulging is generally delayed, although this effect apparently diminishes with increasing distance of existing primordia from the meristem center.

Dihydroflavonol Reductase Activity in Relation to Differential Anthocyanin Accumulation in Juvenile and Mature Phase Hedera helix L.

Juvenile phase English ivy (Hedera helix L.) plants accumulate anthocyanin pigment in the hypodermis of stems and petioles, whereas genetically identical plants of the mature phase do not. The objective of this work was to assess which enzyme(s) might limit anthocyanin accumulation in mature phase ivy. Leaf discs of both juvenile and mature phase ivy accumulated comparable levels of the flavonols kaempferol and quercetin, whereas only juvenile phase discs accumulated anthocyanin. The accumulation of quercetin, but lack of accumulation of leucocyanidin or anthocyanin in mature phase discs, suggested that mature discs lacked dihydroflavonol reductase activity. There was no detectable dihydroflavonol reductase activity in mature phase discs, whereas there was an induction of activity in juvenile phase discs in response to sucrose, or photosynthetically fixed carbon, and light as a photomorphogenic signal. Phenylalanine ammonia-lyase, an enzyme early in the anthocyanin biosynthetic pathway, was induced above its basal level by sucrose and light in discs of both phases of ivy, with greater activity in mature phase discs. Phenylpropanoids, a class of compounds that are precursors to flavonoids, accumulated in leaf discs of both phases, with greater levels in mature phase discs. These results indicate that the lack of dihydroflavonol reductase activity limits the accumulation of anthocyanin in mature phase tissue.

Gibberellin-induced reorganization of spatial relationships of emerging leaf primordia at the shoot apical meristem in Hedera helix L.

The transition from spiral to distichous leaf arrangement during gibberellic-acid (GA3)-induced rejuvenation in Hedera was studied in detail by scanning electron microscopy of the shoot apical meristem. The transition, which involves the initiation of about 14 new leaf primordia, is accomplished by progressive increments in the divergence angle between the leaf primordia from an initial average value of 138.9 ° until it approaches 180 °. This process is preceded, as well as accompanied, by an increased radial displacement of young leaf primordia away from the apical meristem. Although the width of the leaf primordia also increases, this is unlikely to be a causal factor since it occurs only late in the transition. The size of the primordium-free area of the apical meristem is also unlikely to be involved. Quantitative analysis shows that the divergence angle of consecutive leaf primordia commonly fluctuates between relatively large and small values. Thus the transitional stages form a spirodistichous arrangement in which the divergence angle within each pair of leaves is large relative to that between leaf pairs. The stimulation of the radial displacement of the leaf primordia and the associated phyllotactic transition may involve GA3-induced modification in the spatial organization of cortical microtubules in the apical meristem and related changes in directional cell expansion.

Flowering in bougainvillea: a function of assimilate supply and nutrient diversion.

Reproductive development, whether expressed as first node to flower or numbers of inflorescences developing, is promoted in direct relationship to leaf area and in inverse relationship to the numbers of axillary branches developing. Per cent soluble solids in the reproductive shoots vary with reproductive development. Cytokinin treatments promote inflorescence development and per cent soluble solids, further supporting a nutritional hypothesis in the control of flowering in Bougainvillea "San Diego Red." Gibberellin treatments inhibit reproductive development completely without significant lowering of per cent soluble solids, which is counter to expectations for a nutritional hypothesis. A closer examination of the reproductive axes, the tissues in which morphogenetic change occurs, must be made for the gibberellin-treated tissues.

Control of flowering in bougainvillea "san diego red.": metabolism of benzyladenine and the action of gibberellic Acid in relation to short day induction.

Benzyladenine (BA) and short day (SD) induction promote and gibberellic acid (GA) inhibits flowering in Bougainvillea "San Diego Red." GA is an overriding vegetative signal maintaining plants in a vegetative state even when BA is applied in SD conditions. SD promotes a more rapid conversion of BA to the ribotide and other "polar derivatives" (containing adenine derivatives). This effect of SD on BA metabolism is seen in root, stem, and apical bud tissues and is completely prevented by prior or simultaneous application of GA. GA treatment reduces the rate of polar derivative formation to that found in plants held in long days. The working hypothesis is that SD promotes flowering in Bougainvillea owing to reduced transport of gibberellins from leaves to roots and apical buds permitting metabolism of cytokinin, and perhaps other purine bases, to more polar forms that are more readily translocated and active in promoting reproductive development of the inflorescences axes.

Callus and shoot formation in organ and tissue cultures of Hedera helix L., English ivy.

Whole embryos and organs excised from ungerminated embryos and from seedlings of H. helix were cultured to assess their capacity to form callus and regenerate organs. There is a high potential for shoot formation in the explanted cotyledons and in the explanted hypocotyl of the ungerminated embryo. Whole embryos and explanted organs from seedlings produced callus rather than organized structures. In contrast to the excised embryo organs, embryo-derived callus had very low organogenetic potential.

Enhanced inflorescence development in bougainvillea "san diego red" by removal of young leaves and cytokinin treatments.

Removal of young leaves and application of the cytokinin, N-benzyla-alpha-(tetrahydro-2H-pyran-2yl)-adenine promote inflorescence development in Bougainvillea "San Diego Red". Defoliation greatly increased the amount of assimilate accumulated at the shoot tip 1 to 2 days after treatment. Cytokinin applications further increased the amount accumulated and this increase was apparent 4 days before morphological changes could be detected at the inflorescence axes. Short days promoted inflorescence development and also increased assimilate accumulation at the reproductive axes; thus, it is suggested that the role of short day induction in bougainvillea may be that of redirecting the flow of assimilates, perhaps by its influence on cytokinin synthesis and distribution.

Terpene Biosynthesis in Cell-free Extracts and Excised Shoots from Wedgwood Iris.

Excised shoots and cell-free extracts prepared from Wedgwood iris (Iris hollandica Hoog. "Wedgwood") shoots metabolized (14)C-labeled mevalonic acid (MVA). By using cell-free extracts, the (14)C from MVA-1-(14)C was recovered as (14)CO(2), while that from MVA-2-(14)C was recovered as neutral terpenes, acid-hydrolyzable terpenes, or (14)CO(2). Also, under optimal incubation conditions, 12.8 nanomoles R-MVA-2-(14)C was incorporated into neutral terpenes per milligram fresh weight per hour. In contrast, excised shoots incorporated only 0.58 nanomoles R-MVA-2-(14)C per mg fresh weight per hour. Labeled products identified from the cell-free system were squalene, farnesol, geranylgeraniol, and compounds that are converted to farnesol and geranylgeraniol after alkaline hydrolysis. Squalene and a 4,4-dimethylsterol were identified as products from excised shoots but not the terpene alcohols or the alkaline-hydrolyzable compounds.