Seed dispersal in Cakile edentula var. lacustris : decoupling the fitness effects of density and distance from the home site

A factorial design of three densities of siblings at three local distances from seed parents was employed to distinguish effects of density from effects of dispersal distance on lifespan and fruit production of Cakile edentula var. lacustris, a plant with heteromorphic seeds. The segmented fruits produce two seed types: proximal and distal, with distal seeds having greater mass and greater dispersibility. Effects of longer distances (0.5 km and 30 km) on lifespan and fruit production were investigated using plants at low density. The prediction was tested that the greater seed mass of distal seeds increases fitness when seeds are dispersed into sites of unknown quality away from the home site or when seeds are dispersed to low density. High density caused earlier mortality and lower probability of reproduction. Distance from the maternal plant did not influence lifespan or reproduction at distances of 15 m or less, but lifespan was longer 0.5 km from the home site. No interaction was detected between the effects of density and distance on either lifespan or total fitness. Environmental conditions that influence fitness did not vary as a function of dispersal distance in this system, and favorable conditions at the home site did not persist between generations. Therefore, selection on dispersion patterns in natural conditions is likely to be through effects of density rather than dispersal distance. Proximal seeds had greater reproduction than distal seeds at the home site, and distal seeds had greater reproduction at the more distant sites (but not the most distant site), as expected, but these performance differences could not be attributed to differences in mass between the two seed types. Reduced seed mass was favored at the most distant site, but larger seed mass was favored most strongly at low density. Seeds that are dispersed to low density are larger, suggesting that although kin selection may limit the effectiveness of individual selection to increase seed mass under conditions of sibling competition, density-dependent individual selection on seed mass, rather than distant-dependent selection, also contributes to the observed associations among seed type, seed mass and dispersal ability. Received: 21 October 1996 / Accepted: 4 December 1996     

Structure-function studies of heparin-binding (acidic fibroblast) growth factor-1 using site-directed mutagenesis.

The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF activities. In this report, we summarize evidence that indicates that the heparin-binding and mitogenic activities of HBGF-1 can be dissociated from the receptor-binding activities of the growth factor by site-directed mutagenesis of a single lysine residue. Thus, the mutant HBGF-1 has normal receptor-binding activity and is capable of stimulating tyrosine kinase activity and proto-oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto-oncogene expression from the mitogenic response is observed when the human wild-tupe HBGF-1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild-type or mutant HBGF-1. Production of wild-type induces a transformed phenotype, whereas over-production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site-directed mutagenesis of putative nuclear translocation sequences in the wild-type protein do not affect mitogenic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function.

Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.     

Interleukin-3 induces proliferation but not lymphokine activated killer activity from human and murine mononuclear cells

Recombinant IL-3 (rIL-3) is a potent colony stimulating factor capable of stimulating early hematopoietic pluripotential progenitor cells and of supporting the differentiation of multiple cells. IL-3 has also been shown to have effects on mature, differentiated circulating cells including eosinophils and T cells. We evaluated the role of exogenous rIL-3 in the generation of cells with LAK activity from murine splenocytes and human bone marrow, spleen, unseparated PBMC and purified null cell preparations. rIL-3 was unable to generate lytic activity from any of these populations by itself and appeared to decrease LAK activity in bone marrow cultures containing high dose IL-2, (bone marrow derived cells (n = 3) with LAK activity for fresh tumor, mean lytic units(LU) 94.6 +/- 63.5 vs 32.8 +/- 44.8 for IL-2 and IL-2 plus IL-3 cultures, respectively p2 less than 0.05). Unlike previous reports testing murine cells, IL-3 priming and subsequent culture in IL-2 of human unseparated bone marrow cells or human or murine splenocytes, failed to generate long-term cultures with lytic activity. IL-3 did, however, induce a dose dependent stimulation of bone marrow and null cell preparations (mean null cell stimulation (3H Thymidine incorporation) with IL-3, 436 +/- 168 cpm vs 9802 +/- 9799 cpm, for 0 vs 10(3) units of IL-3, respectively n = 4, p2 less than 0.05). Furthermore, in bone marrow, unseparated PBMC and null cell cultures, the addition of rIL-3 generated characteristic large blastic appearing cells with prominent basophilic granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of in vitro tumor-infiltrating lymphocyte cytotoxicity by heteroconjugated antibodies.

Tumor-infiltrating lymphocytes (TIL) were obtained from a mouse melanoma cell line (CL 62) transfected with the gene for the human melanoma Ag p97. TIL were cultured with anti-CD3 antibody and IL-2 for up to 38 days. Flow cytometry identified these TIL as Thy-1.2 + ve/CD4-ve/CD8 + ve cells. A heteroconjugated antibody 500A2 x 96.5, specific for both the CD3 Ag on TIL and the p97 Ag on CL 62 melanoma cells, was prepared using N-succinimidyl-3-(2-pyridyldithio)-propionate as a linking agent. TIL alone demonstrated low levels of cytotoxicity against autologous CL 62 tumor and also against the parental K1735 tumor and an allogeneic murine melanoma (B16). The addition of 500A2 x 96.5 heteroconjugated antibody enhanced TIL-mediated lysis of CL 62 tumor, but not of the K1735 or B16 tumors. This enhanced cytotoxicity was elicited at E:T ratios as low as 0.4:1, and in TIL cultured for 7 to 38 days. These results suggest that hetero-conjugated antibody may enhance the anti-tumor effect of TIL in vivo.     

Synthesis of Rhodobacter sphaeroides cytochrome c2 in Escherichia coli

The cytochrome c2 structural gene, cycA, from Rhodobacter sphaeroides was expressed in Escherichia coli. CycA-specific mRNA was detected in E. coli both under aerobic and anaerobic conditions with trimethylamine-N-oxide as electron acceptor. However mature holocytochrome c2 was only detected in anaerobically-grown cells. The mature form of cytochrome c2 (Mr = 12,500) was secreted into the periplasm of E. coli suggesting that the signal polypeptide was processed. The cytochrome c2 synthesized in E. coli exhibited absorbance maxima in the reduced form at 550 nm (alpha-band) and 521 nm (beta-band) and contained covalently attached haem c. The results indicate that a foreign c-type cytochrome can be secreted and assembled in E. coli under anaerobic conditions.     

Seed Dispersal as a Maternally Influenced Character: Mechanistic Basis of Maternal Effects and Selection on Maternal Characters in an Annual Plant

Maternal influences on progeny characters affect phenotypic correlations between characters expressed in maternal and progeny generations and consequently influence evolutionary responses to selection. Net selection on maternally influenced characters depends on selection both on the progeny character and on the maternal characters that influence it. I used seed dispersal in Cakile edentula as a system in which to identify the mechanisms of environmentally mediated maternal effects and to determine how selection on maternal characters alters the adaptive value of dispersal. In C. edentula, maternal morphology responds to conspecific density experienced by the mother. Maternal morphology in turn affects offspring (seed) dispersal and density and thereby offspring morphology and fitness. I estimated the magnitude of density-mediated maternal effects on dispersal and identified their mechanism by characterizing the plasticity of maternal morphology to density. I also measured density-dependent selection on maternal characters that influence dispersal. Maternal plasticity to density was caused by both allometric and nonallometric variation in morphology, and this plasticity resulted in a negative correlation between maternal and progeny density. Such negative maternal effects are expected to retard responses to selection. Maternal morphology influenced maternal fitness, in part through the relationship of fitness to maternal plant size and in part through size-independent fitness effects. Maternal phenotypes that promote dispersal, and thereby increase progeny fitness, were associated with decreased maternal fitness. Selection on dispersal at the level of progeny favors increased dispersal; maternal influences on dispersal, however, not only cause a greatly reduced adaptive value of dispersal but lead to the prediction of a slower response to selection.     

Diversification of phytochrome contributions to germination as a function of seed-maturation environment

Environmental conditions during seed maturation influence germination, but the genetic basis of maternal environmental effects on germination is virtually unknown. Using single and multiple mutants of phytochromes, it is shown here that different phytochromes contributed to germination differently, depending on seed-maturation conditions. Arabidopsis thaliana wild-type seeds that were matured under cool temperatures were intensely dormant compared with seeds matured at warmer temperature, and this dormancy was broken only after warm seed-stratification followed by cold seed-stratification. The warm-cold stratification broke dormancy in fresh seeds but not in dry after-ripened seeds. Functional PHYB and PHYD were necessary to break cool-induced dormancy, which indicates a previously unknown and ecologically important function for PHYD. Disruption of PHYA in combination with PHYD (but not PHYB) restored germination to near wild-type levels, indicating that PHYA contributes to the maintenance of cool-induced dormancy on a phyD background. Effects of seed-maturation temperature were much stronger than effects of seed-maturation photoperiod. PHYB contributed to germination somewhat more strongly in seeds matured under short days, whereas PHYD contributed to germination somewhat more strongly in seeds matured under long days. The variable contributions of different phytochromes to germination as a function of seed-maturation conditions reveal further functional diversification of the phytochromes during the process of germination. This study identifies among the first genes to be associated with maternal environmental effects on germination.     

Regulation of isoprene emission in Populus trichocarpa leaves subjected to changing growth temperature

The hydrocarbon isoprene is emitted in large quantities from numerous plant species, and has a substantial impact on atmospheric chemistry. Temperature affects isoprene emission at several levels: the temperature at which emission is measured, the temperature at which leaves develop, and the temperatures to which a mature leaf is exposed in the days prior to emission measurement. The molecular regulation of the response to the last of these factors was investigated in this study. When plants were grown at 20 degrees C and moved from 20 to 30 degrees C and back, or grown at 30 degrees C and moved from 30 to 20 degrees C and back, their isoprene emission peaked within 3 h of the move and stabilized over the following 3 d. Trees that developed at 20 degrees C and experienced 30 degrees C episodes had higher isoprene emission capacities than did leaves grown exclusively at 20 degrees C, even 2 weeks after the last 30 degrees C episode. The levels and extractable activities of isoprene synthase protein, which catalyses the synthesis of isoprene, and those of dimethylallyl diphosphate (DMADP), its substrate, alone could not explain observed variations in isoprene emission. Therefore, we conclude that control of isoprene emission in mature leaves is shared between isoprene synthase protein and DMADP supply.