Measuring Osmotic Pressure of Sap within Live Cells by Means of a Visual Melting Point Apparatus

A freezing slide apparatus is described for visual observation of freezing water and melting ice within plant cells. The slide consists of an ordinary microscope slide glued into a Plexiglass jacket, through which cold 90% ethyl alcohol is pumped at varying rates for temperature control. Temperature is recorded by means of an iron-constantan thermocouple wire (25-micron diameter) connected to a recording potentiometer. Tissue strips were quick frozen (at a cooling rate of 33 C per ½ minute) and then warmed very slowly (at a rate of 2 C per minute) for observation of melting points. This apparatus has been used to determine osmotic pressures of cell sap of guard and adjoining epidermal cells of Chrysanthemum morifolium and Pelargonium hortorum. An accuracy of ± 1.2 atmospheres is possible. Wide variations among osmotic pressures of both guard and epidermal cells were found at any one stomatal aperture in both species.     

Role of Microcystins in Poisoning and Food Ingestion Inhibition of Daphnia galeata Caused by the Cyanobacterium Microcystis aeruginosa

The effects of microcystins on Daphnia galeata, a typical filter-feeding grazer in eutrophic lakes, were investigated. To do this, the microcystin-producing wild-type strain Microcystis aeruginosa PCC7806 was compared with a mcy⁻ PCC7806 mutant, which could not synthesize any variant of microcystin due to mutation of a microcystin synthetase gene. The wild-type strain was found to be poisonous to D. galeata, whereas the mcy⁻ mutant did not have any lethal effect on the animals. Both variants of PCC7806 were able to reduce the Daphnia ingestion rate. Our results suggest that microcystins are the most likely cause of the daphnid poisoning observed when wild-type strain PCC7806 is fed to the animals, but these toxins are not responsible for inhibition of the ingestion process.     

Atypical expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in subcutaneous adipose tissue of male rats.

The mRNAs encoding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mtHMG-CoA synthase), the rate limiting enzyme in ketone body production, are highly expressed in subcutaneous (SC) and, to a lesser extent, in peri-epididymal (PE) rat adipose tissues. This atypical mtHMG-CoA synthase gene expression is dependent on the age (from 9 weeks of age) and sex (higher in male than in female) of the rats. In contrast, the expression of mtHMG-CoA synthase in SC adipose deposit is independent of the nutritional state (fed versus starved) or of the thermic environment (24 degrees C versus 4 degrees C). The expression of mtHMG-CoA synthase is suppressed in SC fat pads of castrated male rats whereas treatment of castrated rats with testosterone restores a normal level of expression. Moreover, testosterone injection induces the expression mtHMG-CoA synthase in SC adipose tissue of age-matched females. The presence of the mtHMG-CoA synthase immunoreactive protein confers to mitochondria isolated from SC adipose deposits, the capacity to produce ketone bodies at a rate similar to that found in liver mitochondria (SC = 13.7 +/- 0.7, liver = 16.4 +/- 1.4 nmol/min/mg prot). mtHMG-CoA synthase is expressed in the stromal vascular fraction (SVF) whatever the adipose deposit considered. While acetyl-CoA carboxylase (ACC) is only expressed in mature adipocytes, the other lipogenic enzymes, fatty acid synthase (FAS) and citrate cleavage enzyme (CCE), are expressed both in SVF cells and mature adipocytes. The expression of lipogenic enzyme genes is markedly reduced in adipocytes but not in SVF cells isolated from 48-h starved male rats. When SVF is subfractionated, mtHMG-CoA synthase mRNAs are mainly recovered in two fractions containing poorly digested structures such as microcapillaries whereas the lowest expression is found in the pre-adipocyte fraction. Interestingly, FAS and CCE mRNAs co-segregate with mtHMG-CoA synthase mRNA. The possible physiological relevance of such atypical expression of mtHMG-CoA synthase is discussed.     

Reduced representation genome sequencing reveals patterns of genetic diversity and selection in apple

Identifying DNA sequence variations is a fundamental step towards deciphering the genetic basis of traits of interest.Here,a total of 20 cultivated and 10 wild apples were genotyped using specific-locus amplified fragment sequencing,and 39,635 single nucleotide polymorphisms with no missing genotypes and evenly distributed along the genome were selected to investigate patterns of genome-wide genetic variations between cultivated and wild apples.Overall,wild apples displayed higher levels of genetic diversity than cultivated apples.Linkage disequilibrium(LD) decays were observed quite rapidly in cultivated and wild apples,with an r~2-value below 0.2 at 440 and 280 bp,respectively.Moreover,bidirectional gene flow and different distribution patterns of LD blocks were detected between domesticated and wild apples.Most LD blocks unique to cultivated apples were located within QTL regions controlling fruit quality,thus suggesting that fruit quality had probably undergone selection during apple domestication.The genome of the earliest cultivated apple in China,Nai,was highly similar to that of Malus sieversii,and contained a small portion of genetic material from other wild apple species.This suggested that introgression could have been an important driving force during initial domestication of apple.These findings will facilitate future breeding and genetic dissection of complex traits in apple.     

Lawsonia intracellularis contains a gene encoding a functional rickettsia-like ATP/ADP translocase for host exploitation

ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1(Li)]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1(Li) were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1(Li) transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (+/- 36.5) microM for ATP and 275.2 (+/- 28.1) microM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1(Li) is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.     

Multiple models for Rosaceae genomics

The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.     

Simultaneous freeze tolerance and avoidance in individual fungus gnats, <Emphasis Type="Italic">Exechia nugatoria</Emphasis>

Freeze tolerance and freeze avoidance are typically described as mutually exclusive strategies for overwintering in animals. Here we show an insect species that combines both strategies. Individual fungus gnats, collected in Fairbanks, Alaska, display two freezing events when experimentally cooled and different rates of survival after each event (mean ± SEM: −31.5 ± 0.2°C, 70% survival and −50.7 ± 0.4°C, 0% survival). To determine which body compartments froze at each event, we dissected the abdomen from the head/thorax and cooled each part separately. There was a significant difference between temperature levels of abdominal freezing (−30.1 ± 1.1°C) and head/thorax freezing (−48.7 ± 1.3°C). We suggest that freezing is initially restricted to one body compartment by regional dehydration in the head/thorax that prevents inoculative freezing between the freeze-tolerant abdomen (71.0 ± 0.8% water) and the supercooled, freeze-sensitive head/thorax (46.6 ± 0.8% water).