Developmental regulation of expression of the malate synthase gene in transgenic plants

The cucumber malate synthase (MS) gene, including 1856 bp of 5 non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5 sequence was linked to the -glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of -glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.     

Direct evidence for the cell surface location of a protease-inhibitor complex on intact leukaemia cells

The interaction of a protease with two fluorescent inhibitors has been studied using intact fixed leukaemia cells as the source of the membrane bound enzyme. Fresh rat leukaemia cells were disrupted and the cytosol collected; this extract was known to contain a protein inhibitor of guanidinobenzoatase (GB) associated with leukaemia cells. All the cytosolic proteins were derivatised with Texas red acid chloride. Leukaemia cells with latent GB failed to bind the Texas red inhibitor protein but did so after activation of GB. Competition experiments with 9-amino acridine (a fluorescent marker for the active site of GB) demonstrated that the Texas red-inhibitor protein could only bind to intact leukaemia cells when the active centre of GB was not already occupied by 9-amino acridine. This competition between these two fluorescent inhibitors demonstrated their specificity for GB. The use of intact leukaemia cells and the high molecular weight of the inhibitor protein precludes the possibility of any interaction between GB and inhibitor within the cells. It is concluded that GB and the GB-inhibitor complex of latent GB are located on the external surface of intact leukaemia cells.     

Misting and nitrogen fertilization of shoots of a saltmarsh grass: effects upon fungal decay of leaf blades

We conducted a 12-week field manipulation experiment in which we raised the nitrogen availability (ammonium sulfate fertilization to roots) and/or water potential (freshwater misting) of decaying leaf blades of a saltmarsh grass (smooth cordgrass, Spartina alterniflora) in triplicate 11-m² plots, and compared the manipulated plots to unmanipulated, control plots. The ascomycetous fungi that dominate cordgrass leaf decomposition processes under natural conditions exhibited a boosting (>2-fold) of living standing crop (ergosterol content) by misting at the 1 st week after tagging of senescent leaves, but afterwards, living-fungal standing crop on misted blades was equivalent to that on control blades, confirming prior evidence that Spartina fungi are well adapted to natural, irregular wetting. Misting also caused 2-fold sharper temporal declines than control in instantaneous rates of fungal production (ergosterol synthesis), 5-fold declines in density of sexual reproductive structures that were not shown by controls, and 2-fold higher rates of loss of plant organic mass. Extra nitrogen gave a long-term boost to living-fungal standing crop (about 2-fold at 12 weeks), which was also reflected in rates of fungal production at 4 weeks, suggesting that saltmarsh fungal production is nitrogen-limited. Although bacterial and green-microalgal crops were boosted by manipulations of nitrogen and/or water, their maximal crops remained 0.3 or 2% (bacteria or green microalgae, respectively) of contemporaneous living-fungal crop. The fungal carbon-productivity values obtained, in conjunction with rates of loss of plant carbon, hinted that fungal yield can be high (>50%), and that it is boosted by high availability of nitrogen. We speculate that one partial cause of high fungal yield could be subsidy of fungal growth by dissolved organic carbon from outside decomposing leaves.     

Specificity studies of 3-mercaptopyruvate sulfurtransferase

3-Mercaptopyruvate sulfurtransferase (E.C. 2.8.1.2; MST) is an enzyme believed to function in the endogenous cyanide (CN) detoxification system because it is capable of transferring sulfur from 3-mercaptopyruvate (3-MP) to CN, forming the less toxic thiocyanate (SCN). To date, 3-MP is the only known sulfur-donor substrate for MST. In an effort to increase the understanding of what chemical properties of 3-MP affect its utilization as a substrate, in vitro enzyme kinetic studies of MST were conducted using two mercaptic acids that are structurally related to 3-MP. Neither of these compounds was able to serve as a sulfur-donor substrate for MST. Inhibitor studies determined that 3-mercaptopropionic acid did not affect the K_m of MST for 3-MP but did decrease V_max and, thus, was determined to be a noncompetitive inhibitor. Alternatively, 2-mercaptopropionic acid 2-MPA decreased K_m and V_max and was determined to be an uncompetitive inhibitor of MST with respect to 3-MP. These data indicate that the α-keto group of 3-MP is necessary for its utilization as a substrate, and the inhibitor studies suggest that the position of the sulfur may also affect the binding of these compounds to the enzyme. These observations increase the understanding of what factors can affect the utilization of a compound as a sulfur-donor substrate for MST and may aid in the development of alternative sulfur-donor substrates for MST. © 1996 John Wiley & Sons, Inc.     

Diurnal rhythm of galanin-like immunoreactivity in the paraventricular and suprachiasmatic nuclei and other hypothalamic areas

The peptide galanin (GAL), when injected into the rat hypothalamus, is known to stimulate feeding behavior and affect the secretion of various hormones, including insulin and the adrenal steroid, corticosterone. To determine whether endogenous peptide levels shift in relation to natural rhythms of feeding and circulating hormone levels, rats were sacrificed at different times of the light/dark cycle, and their GAL levels were measured, via radioimmunoassay, in medial hypothalamic dissections and micropunched hypothalamic areas. The results suggest the existence of two distinct diurnal rhythms for hypothalamic GAL. One rhythm, detected exclusively in the area of the SCN, is characterized by bimodal peaks of GAL, threefold higher than basal peptide levels, around the onset of the dark and light periods. The second rhythm shows a single peak of GAL towards the middle of the nocturnal feeding cycle, specifically between the third and sixth hour. This latter rhythm is evident in the dorsal region of the medial hypothalamus, localized specifically to the lateral portion of the PVN. Moreover, it is inversely related to circulating insulin but unrelated to the adrenal steroids, suggesting a possible association between this pancreatic hormone and GAL in the PVN.     

Mating system parameters and population genetic structure in pioneer populations of Prosopis velutina (Leguminosae)

This study describes the genetic structure and provides estimates of mating system parameters in three natural populations of Prosopis velutina Woot. in southeastern Arizona. F statistics derived from isozyme data revealed the presence of both interpopulation and intrapopulation genetic differentiation. This population structure is discussed in relation to the ecological history of these populations that invaded the grasslands sites from adjacent riparian areas within the last 90 years, and possible modes of seed dispersal. The multilocus estimation program MLT of Ritland (Journal of Heredity 8: 235–237, 1990) was used to provide estimates of t_s, t_m, and the fixation index (F) of the maternal parents. Average estimates of t_s ranged from 0.591 to 0.912. Estimates of t_m ranged from 0.609 to 1.004, and averaged 0.758. The difference between t_m and t_s, which provides a measure of biparental inbreeding, averaged 0.058. This last result, together with significant heterogeneity found in pollen allele frequencies, suggests that a family structure has developed in the populations. A negative F value for one of the populations suggests selection against homozygotes between the seedling stage and maturity.     

Inoculum Density-Dependent Mortality and Colonization of the Phyllosphere by Pseudomonas syringae

Pseudomonas syringae inocula containing cell concentrations ranging from 10⁵ to 10⁹ cells per ml were applied to the primary leaves of bean plants. The plants were incubated under conditions of high temperature and illumination and low relative humidity. Bacterial mortality rates and the proportional population decline of the inoculum were lowest at the highest inoculum concentrations. Addition of a high concentration of heat-killed cells to the inoculum containing a low concentration of viable cells significantly reduced both the mortality rate and the proportional population decline of the viable cells. The mechanisms underlying this density-dependent mortality may include cooperative protective effects of extracellular factors, such as bacterial extracellular polysaccharides, and physical protection by neighboring cells. Although epiphytic populations derived from inoculum concentrations of 10⁸ or 10⁹ cells per ml tended toward 10⁶ CFU/g, the presumed carrying capacity of the leaf, populations derived from lower inoculum concentrations never achieved this carrying capacity. Assuming that epiphytic populations of P. syringae reside in discrete protected sites, our results suggest that at low inoculum concentrations, following a period of environmental stress, the number of viable cells may have dropped to zero in some sites; hence, the carrying capacity of the leaf could not be achieved.     

Ecological Similarity and Coexistence of Epiphytic Ice-Nucleating (Ice+) Pseudomonas syringae Strains and a Non-Ice-Nucleating (Ice-) Biological Control Agent

De Wit replacement series were used to study competitive interactions between epiphytic Ice⁺Pseudomonas syringae strains and the biological frost control agents Ice^-P. syringae TLP2del1 and Pseudomonas fluorescens A506. Mixtures containing two strains in different proportions but at a constant total population size were inoculated onto potato leaves. The population sizes of each strain and the total population size were determined when the community had reached equilibrium. A near-isogenic P. syringae strain pair exhibited an interaction similar to that expected for strains competing equally for limiting environmental resources. Replacement series with nonisogenic Ice⁺ and Ice^-P. syringae strain pairs suggested that these strains competed for limiting resources according to their relative competitive abilities. There was no evidence of any niche differentiation between the Ice⁺P. syringae strains and the Ice^-P. syringae strain. The growth responses of epiphytes following addition of nutrients to the phyllosphere indicated that the epiphytic P. syringae populations were nutrient limited and that, under growth chamber conditions, the populations were more limited by the availability of carbon than by the availability of nitrogen. Determination of in vitro carbon source utilization profiles provided further evidence for the lack of niche differentiation between the Ice⁺ and the Ice^-P. syringae strains. Niche overlap indices calculated for the Ice⁺P. syringae strains with respect to Ice^-P. syringae TLP2del1 were uniformly high, indicating ecological similarity, and were consistent with the observed low level of coexistence. The biological frost control agent P. fluorescens A506 replaced P. syringae. This was correlated with a high degree of niche overlap between these species.