No abstract

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.The ethanolic fermentation pathway branches off the main glycolytic pathway at pyruvate. In the first step, pyruvate is the substrate of pyruvate decarboxylase (PDC), yielding CO 2 and acetaldehyde. Subsequently, acetaldehyde is reduced to ethanol with the concomitant oxidation of NADH to NAD ϩ by alcohol dehydrogenase (ADH). In the present day aerobic atmosphere, ethanolic fermentation is used only by specialized organisms or under particular conditions. In plants, it has been studied because of its relevance in flooding tolerance where plant cells switch from aerobic respiration to anaerobic fermentation (for review, see Drew, 1997).ADH has been the subject of numerous genetic studies, and adh mutants have been reported for a number of species, including maize (Zea mays; for review, see Freeling and Bennett, 1985), tobacco (Nicotiana tabacum; Rousselin et al., 1990), and Arabidopsis (Jacobs et al., 1988). Maize adh null mutants are sensitive to strict anoxia but no obvious phenotype is apparent in acclimated plants (Johnson et al., 1994). Experiments with isogenic maize lines differing in ADH activity over a approximately 200-fold range indicated that ADH activity does not limit the capacity for energy production by ethanolic fermentation unless there is a reduction in activity to less than 1% of wild-type levels (Roberts et al., 1989).These results suggest that ADH is present in large excess and are inconsistent with the idea of ADH as the regulatory enzyme of this pathway.Overexpression of a bacterial PDC in transgenic tobacco resulted in constitutive high and active protein levels under both normoxic an...

This study was conducted to investigate the response of maize (Zea mays) male and female mature reproductive tissues to temperature stress. We have tested the fertilization abilities of the stressed spikelets and pollen using in vitro pollination-fertilization to determine their respective tolerance to stress. The synthesis of heat shock proteins (HSPs) was also analyzed in male and female tissues using electrophoresis of 35S-labeled proteins and fluorography, to establish a relationship between the physiological and molecular responses. Pollen, spikelets, and pollinated spikelets were exposed to selected temperatures (4, 28, 32, 36, or 400C) and tested using an in vitro fertilization system. The fertilization rate is highly reduced when pollinated spikelets are exposed to temperatures over 360C. When pollen and spikelets are exposed separately to temperature stress, the female tissues appear resistant to 4 hours of cold stress (40C) or heat stress (40°C). Under heat shock conditions, the synthesis of a typical set of HSPs is induced in the female tissues. In contrast, the mature pollen is sensitive to heat stress and is responsible for the failure of fertilization at high temperatures. At the molecular level, no heat shock response is detected in the mature pollen.

Sperm cells were isolated from corn (Zea mays L.) tricellular pollen gains. They were released using a light osmotic chock, and separated from pollen contaminants (especially starch grains) by a Percoll gradient centrifugation. Isolated sperm cells (3 x 10' per milliliter) show a high viability score (90%) as demonstrated with the fluorochromatic reaction. They appeared as spherical cells which lack cell wall and plastids, and can be considered as haploid protoplasts.In angiosperms, sperm cells are formed either in the pollen grain (tricellular pollen) or in the pollen tube during germination (bicellular pollen). In (FCR+) after exposure to the fluorescein diacetate mixture.Light Microscopy Controls. Throughout the isolation procedure, sperm cells were examined with phase contrast or fluorescent microscopy. Sperm cells were counted using 0.05% ethidium bromide in the isolation medium. Sperm cells could also be observed using phase contrast combined with fluorescence microscopy (6).Yield of Sperm Cells. The number of pollen grains was estimated at 2500 grains/mg. The initial number of sperm cells was represented by the total weight of pollen (in mg) multiplied by 2500 and by 2 (two sperm cells per pollen grain). The

SummaryWhen plants are infected with avirulent pathogens, a selected group of plant cells rapidly die in a process commonly called the hypersensitive response (HR). Some mutations and overexpression of some unrelated genes mimic the HR lesion and associated defense responses. In all of these situations, a genetically programmed cell death pathway is activated wherein the cell actively participates in killing itself. Here we report a developmentally and environmentally regulated HR-like cell death in potato leaves constitutively expressing bacterial pyruvate decarboxylase (PDC). Lesions first appeared on the tip of fully expanded source leaves. Lesion formation was accompanied by activation of multiple defense responses and resulted in a significant resistance to Phytophthora infestans. The transgenic plants showed a five-to 12-fold increase in leaf tissue acetaldehyde and exported two-to 10-fold higher amounts of sucrose compared to the wildtype. When plants were grown at a higher temperature, both the lesion phenotype and sucrose export were restored to wild-type situations. The reduced levels of acetaldehyde at the elevated temperature suggested that the interplay of acetaldehyde with environmental and physiological factors is the inducer of lesion development. We propose that sugar metabolism plays a crucial role in the execution of cell death programs in plants.