Non-linearity in rate-temperature relations of germination in oilseed rape

Predominantly linear relations between germination rate andtemperature (over specific ranges) have hitherto been foundamong the main crop plants. Given linearity, accumulated temperatureabove a base (T_b) is a reliable predictor of germination time.In contrast, this paper shows marked non-linearities in theresponse of germination to temperature for four oilseed rapecultivars, two ‘double high’ (Martina and Askari)and ‘two double low’ (Rocket and Express). Linearregressions of 1/time to germination of given percentiles indicateT_b is about 3 C for all cultivars. However, consistent deviationsfrom linearity occur especially at low temperature, such thatgermination time at 5C is up to twice as fast as predictedfrom a linear model. Moreover, two of the cultivars show anincreasing proportion of seeds not germinating as temperatureis lowered. These seeds were still viable as demonstrated bytheir ability to germinate when transferred to warmer temperature.The consequence of non-linearity was to increase the spreadof time over which seeds germinated at low temperature. Thispaper defines the effects and indicates the margin of errorlikely to follow from applying linear rate/temperature modelsto whole populations. The findings have implications for thepropensity of different oilseed rape cultivars to become volunteerweeds and feral populations. Key words: Oilseed rape, germination, temperature, model, non-linear     

Homeostatic gas-exchange parameters inferred from 13C/12C in tree rings of conifers

The CO₂ concentration of the atmosphere has increased by almost 30% in the past two centuries, with most of the increase (>5 Pa) during the past 60 years. Controlled environment studies of crop plants dependent on the C₃ photosynthetic pathway indicate that an increase of this magnitude would enhance net photosynthesis, reduce stomatal conductance, and increase the difference in CO₂ concentration across the stomata, i.e., CO₂ concentration outside the leaf to that within (c _a-c _i). Here we report evidence, based on stable isotope composition of tree rings from three species of field-grown, native conifer trees, that the trees have indeed responded. However, rather than increasing c _a-c _i, intercellular CO₂ concentrations have shifted upward to match the rise in atmospheric concentrations, holding c _a-c _i constant. No differences were detected among Douglas-fir (Pseudotsuga menziesii), ponderosa pine (Pinus ponderosa), or western white pine (Pinus monticola). The values of c _a-c _i were inferred from stable carbon isotope ratio (¹³C) of tree ring holocellulose adjusted for the 0.6–2.6 difference between holocellulose and whole sapwood. The cellulose extraction removed contaminants deposited in the tree ring after it formed and the adjustment corrected for the enrichment of cellulose relative to whole tissue. The whole sapwood values were then adjusted for bublished estimates of past atmospheric ¹³CO₂ and CO₂ concentrations. To avoid confounding tree age with CO₂, cellulose deposited by saplings in the 1980s was compared to cellulose deposited in the inner rings of nature trees when the mature trees were saplings, between 1910–1929 and 1941–1970; thus saplings were compared to saplings. In a separate analysis, the juvenile effect, which describes the tendency for ¹³C to increase in the first decades of a tree's life, was quantified independent of source CO₂ effects. This study provides evidence that conifers have undergone adjustments in the intercellular CO₂ concentration that have maintained c _a-c _i constant. Based on these results and others, we suggest that c _a-c _i, which has also been referred to as the intrinsic water-use efficiency, should be considered a homeostatic gas-exchange set point for these conifer species.     

The Candida boidinii peroxisomal membrane protein Pmp30 has a role in peroxisomal proliferation and is functionally homologous to Pmp27 from Saccharomyces cerevisiae.

The mechanism of peroxisome proliferation is poorly understood. Candida boidinii is a methylotrophic yeast that undergoes rapid and massive peroxisome proliferation and serves as a good model system for this process. Pmp30A and Pmp30B (formerly designated Pmp31 and Pmp32, respectively) are two closely related proteins in a polyploid strain of this yeast that are strongly induced by diverse peroxisome proliferators such as methanol, oleate, and D-alanine. The function of these proteins is not understood. To study this issue, we used a recently described haploid strain (S2) of C. boidinii that can be manipulated genetically. We now report that strain S2 contains a single PMP30 gene very similar in sequence (greater than 93% identity at the DNA level) to PMP30A and PMP30B. When PMP30 was disrupted, cell growth on methanol was greatly inhibited, and cells grown in both methanol and oleate had fewer, larger, and more spherical peroxisomes than wild-type cells. A similar phenotype was recently described for Saccharomyces cerevisiae cultured on oleate in which PMP27, which encodes a protein of related sequence that is important for peroxisome proliferation, was disrupted. To determine whether Pmp27 is a functional homolog of Pmp30, gentle complementation was performed. PMP30A was expressed in the PMP27 disruptant of S. cerevisiae, and PMP27 was expressed in the PMP30 disruptant of C. boidinii S2. Complementation, in terms of both cell growth and organelle size, shape, and number, was successful in both directions, although reversion to a wild-type phenotype was only partial for the PMP30 disruptant. We conclude that these proteins are functional homologs and that both Pmp30 and Pmp27 have a direct role in proliferation and organelle size rather than a role in a specific peroxisomal metabolic pathway of substrate utilization.     

Evolutionary analysis of aspartate aminotransferases

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family. Correspondence to: C.J. Marshall     

Habitat temperature and potential locomotor activity of the continental Antarctic mite,Maudheimia petronia Wallwork (Acari: Oribatei)

Microhabitat recordings suggest that the continental Antarctic mite Maudheimia petronia Wall-work experiences temperatures above 0°C for 60% of the time during summer (about 2 months). Summer daily maximum temperatures are, however, often relatively high (the highest recorded temperature was 27.7°C). Because the locomotor activity of this mite is suppressed at freezing temperatures, the time available for activity, and probably also feeding, is restricted. Temperature relations of potential locomotor activity rate suggest alleviation of this time constraint through the maximization of the rate. The locomotor activity rate of M. petronia is positively sensitive to the entire range of above-zero temperatures that it naturally experiences, being particularly accelerated at lower temperatures (Q_100°–5°C values were above 13, whereas Q_{1025°–30°C} values were below 2). Also, comparisons between mites acclimated at -15°C and 10°C suggest an inverse temperature acclimation of this rate. We hypothesize that potential feeding rate is similarly related to temperature. A relative enhancement of food intake would seem important, not only for the maintenance of a daily positive energy balance in summer, but also for the building up of energy reserves for the relatively long winter, when feeding is impossible.     

Tracheal cytotoxin structural requirements for respiratory epithelial damage in pertussis

The respiratory epithelial pathology of pertussis (whooping cough) can be reproduced by tracheal cyto-toxin (TCT), a disaccharide-tetrapeptide released by Bordetella pertussis. TCT is a muramyl peptide, a class of peptidoglycan-derived compounds which have many biological activities including adjuvanticity, somnogenicity, pyrogenicity, and cytotoxicity. The structural requirements for muramyl peptides to produce some of these biological effects have been partially characterized. Using in vitro assays with respiratory epithelial cells and tissue, we have previously determined that the disaccharide moiety of TCT is not involved in toxicity and that the side-chain functional groups of diaminopimelic acid (A₂pm) are crucial for toxicity. In this study, we determine the importance of every amino acid, functional group and chiral centre in the peptide portion of TCT. Although lactyl tetrapeptides are the most toxic of the TCT fragments, producing dose-response curves identical to TCT, the smallest analogues of TCT which are active in our assay are of the form X-γ-(d )-Glu-meso-A₂pm, where X may be an amino acid or a blocking group. Within this active substructure, main-chain chirality and all functional groups are essential for toxicity. This definition of the core region of TCT indicates that the TCT interaction site is unlike almost all other muramyl peptide interaction sites for which structure-activity data are available.     

Primary partitioning and storage of photosynthate in sucrose and starch in leaves of C4 plants

A procedure involving pulse labelling of leaves with ¹⁴CO₂ was developed to measure the primary (initial) partitioning of photosynthate between sucrose and starch. Partitioning of photosynthate into sucrose and starch was determined in leaves of C₄ plants and compared with the patterns of storage of carbon in these products during the light period. The ratio of primary partitioning into sucrose and starch varied from about 0.5 in those species that accumulated mostly starch in the leaves (Amaranthus edulis L., Atriplex spongiosa F. Muell. and Flaveria trinervia (Spreng.) C. Mohr) to about 8 in Eleusine indica (L.) Gaertn., which accumulated mostly sucrose. No label was detected in free glucose or fructose. Generally there was a reasonable link between the primary partitioning of photosynthate and the type of carbohydrate stored in the leaf during the day. However, the ratio of carbon initially partitioned into sucrose versus starch was about 3 to 4 times higher in leaves of NADP-malic enzyme-type monocotyledonous species compared with phosphoenolpyruvate carboxykinase-type species, although the ratio of sucrose to starch accumulated in leaves during the day was very similar in the two groups. Sucrose and starch were the principal carbohydrates accumulated in leaves during the day. None of the species examined contained significant amounts of fructan and only one species, Atriplex spongiosa, contained substantial amounts of hexose sugars. In most of the species studied, the proportion of photosynthate partitioned into starch was greater at the end of the day than at the beginning. With the exception of Flaveria trinervia, the rate of CO₂ assimilation did not decline during the day, showing that, under our conditions, accumulation of carbohydrate in the leaves did not lead to feedback inhibition of photosynthesis in these C₄ species.Abbreviations Chl chlorophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK phosphoenolpyruvate carboxykinase We thank Prof. H.W. Heldt (Pflanzenphysiologisches Institut, Universität Göttingen) for discussions and advice during the course of this work.     

Telomeres Cluster De Novo before the Initiation of Synapsis: A Three-dimensional Spatial Analysis of Telomere Positions before and during Meiotic Prophase

We have analyzed the progressive changes in the spatial distribution of telomeres during meiosis using three-dimensional, high resolution fluorescence microscopy. Fixed meiotic cells of maize (Zea mays L.) were subjected to in situ hybridization under conditions that preserved chromosome structure, allowing identification of stage-dependent changes in telomere arrangements. We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres. Quantitative measurements on the spatial arrangements of telomeres revealed that, as cells passed through premeiotic interphase and into leptotene, there was an increase in the frequency of large telomere-to-telomere distances and a decrease in the bias toward peripheral localization of telomeres. By leptotene, there was no obvious evidence of telomere grouping, and the large, singular nucleolus was internally located, nearly concentric with the nucleus. At the end of leptotene, telomeres clustered de novo at the nuclear periphery, coincident with a displacement of the nucleolus to one side. The telomere cluster persisted throughout zygotene and into early pachytene. The nucleolus was adjacent to the cluster at zygotene. At the pachytene stage, telomeres rearranged again by dispersing throughout the nuclear periphery. The stagedependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions. Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.     

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme Hind III, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori , and their relative discriminatory abilities were assessed. Four probes, (GACA)₄, (GT)₈, (GTG)₅ and (GGAT)₄, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)₄-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori .     

Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling.

Activation of the Raf serine/threonine protein kinases is tightly regulated by multiple phosphorylation events. Phosphorylation of either tyrosine 340 or 341 in the catalytic domain of Raf-1 has been previously shown to induce the ability of the protein kinase to phosphorylate MEK. By using a combination of mitogenic and enzymatic assays, we found that phosphorylation of the adjacent residue, serine 338, and, to a lesser extent, serine 339 is essential for the biological and enzymatic activities of Raf-1. Replacement of S338 with alanine blocked the ability of prenylated Raf-CX to transform Rat-1 fibroblasts. Similarly, the loss of S338-S339 in Raf-1 prevented protein kinase activation in COS-7 cells by either oncogenic Ras[V12] or v-Src. Consistent with phosphorylation of S338-S339, acidic amino acid substitutions of these residues partially restored transforming activity to Raf-CX, as well as kinase activation of Raf-1 by Ras[V12] or v-Src. Two-dimensional phosphopeptide mapping of wild-type Raf-CX and Raf-CX[A338A339] confirmed the presence of a phosphoserine-containing peptide with the predicted mobility in the wild-type protein which was absent from the mutant. This peptide could be quantitatively precipitated by an antipeptide antibody specific for the 18-residue tryptic peptide containing S338-S339 and was demonstrated to contain only phosphoserine. Phosphorylation of this peptide in Raf-1 was significantly increased by coexpression with Ras[V12]. These data demonstrate that Raf-1 residues 338 to 341 constitute a unique phosphoregulatory site in which the phosphorylation of serine and tyrosine residues contributes to the regulation of Raf by Ras, Src, and Ras-independent membrane localization.