Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1- PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.      

An optimized protocol for the identification of diatoms,flagellated algae and pathogenic protozoa with phylochips

In the past decade, molecular probe‐based methods have proved successful in improving both the efficiency and accuracy of the identification of microorganisms, especially those that are devoid of distinct morphological features. However until recently, these methods had the major drawback of being limited to the identification of only one or just a few species at a time. With the use of DNA microarrays, it is possible to identify large numbers of taxa on a single‐glass slide, the so‐called phylochip. There are numerous microarray protocols in the literature. These protocols share the same principles, but vary in details, e.g. labelling approach or detergent concentration in the washing buffer. In this study, we show that even small variations in hybridization protocols can have a strong impact on the outcome of the microarray hybridization. An optimized protocol for species identification on phylochips is presented. The optimized protocol is the result of a joined effort of three laboratories to develop phylochips for microbial species identification.     

Natural variation in ozone sensitivity among Arabidopsis thaliana accessions and its relation to stomatal conductance

Genetic variation between naturally occurring populations provides a unique source to unravel the complex mechanisms of stress tolerance. Here, we have analysed O₃ sensitivity of 93 natural Arabidopsis thaliana accessions together with five O₃‐sensitive mutants to acute O₃ exposure. The variation in O₃ sensitivity among the natural accessions was much higher than among the O₃‐sensitive mutants and corresponding wild types. A subset of nine accessions with major variation in their O₃ responses was studied in more detail. Among the traits assayed, stomatal conductance (g_st) was an important factor determining O₃ sensitivity of the selected accessions. The most O₃‐sensitive accession, Cvi‐0, had constitutively high g_st, leading to high initial O₃ uptake rate and dose received during the first 30 min of exposure. Analyzing O₃‐induced changes in stress hormone concentrations indicated that jasmonate (JA) concentration was also positively correlated with leaf damage. Quantitative trait loci (QTL) mapping in a Col‐0 × Cvi‐0 recombinant inbred line (RIL) population identified three QTLs for O₃ sensitivity, and one for high water loss of Cvi‐0. The major O₃ QTL mapped to the same position as the water loss QTL further supporting the role of stomata in regulating O₃ entry and damage.     

Evidence for the Activation of NADH-cytochrome c Reductase during Germination of Lettuce

A very rapid increase in particulate cytochrome c reductase activity during the very early stages of germination of lettuce is demonstrated. The increase in activity does not parallel water uptake or the increase in cytochrome oxidase activity. The increase is reversible on drying of imbibed seeds and is not inhibited by cyanide. It is concluded that the increase in activity is due to some kind of activation process and not to de novo synthesis of protein. Possible mechanisms of activation were investigated. It was not possible to simulate the activation process in vitro, in the isolated particulate fraction.