In addition to the classical electron transport pathway coupled to ATP synthesis, plant mitochondria have an alternative pathway that involves type II NAD(P)H dehydrogenases (NDs) and alternative oxidase (AOX). This alternative pathway participates in thermogenesis in select organs of some species and is thought to help prevent cellular damage during exposure to environmental stress. Here, we investigated the function and role of one alternative path component, AtNDB2, using a transgenic approach in Arabidopsis (Arabidopsis thaliana). Disruption of AtNDB2 expression via T-DNA insertion led to a 90% decrease of external NADH oxidation in isolated mitochondria. Overexpression of AtNDB2 led to increased AtNDB2 protein abundance in mitochondria but did not enhance external NADH oxidation significantly unless AtAOX1A was concomitantly overexpressed and activated, demonstrating a functional link between these enzymes. Plants lacking either AtAOX1A or AtNDB2 were more sensitive to combined drought and elevated light treatments, whereas plants overexpressing these components showed increased tolerance and capacity for poststress recovery. We conclude that AtNDB2 is the predominant external NADH dehydrogenase in mitochondria and together with AtAOX1A forms a complete, functional, nonphosphorylating pathway of electron transport, whose operation enhances tolerance to environmental stress. This study demonstrates that at least one of the alternative NDs, as well as AOX, are important for the stress response.
Here, we describe a suppression PCR-based method for directional cloning of randomly primed cDNAs from small quantities of tissue. Synthesis of the first cDNA strand is conducted on oligonucleotide-coated magnetic beads. Synthesis of the second strand is accomplished using nonspecifically primed suppression PCR. This method is used to synthesize a cDNA library from zebrafish embryos at 6-9 h after fertilization. The sequencing of the clones and their use in an in situ hybridization screen to detect restricted patterns of gene transcription in zebrafish embryos showed that this method allows the rapid identification of genes that are important for development and genes that are expressed at levels undetectable by whole-mount in situ transcript hybridization. The random priming of cDNA alleviates the problems encountered in the identification of zebrafish genes from poly(dT)-primed cDNA clones caused by the long 3' UTRs frequently found in transcripts from this organism.
Sulfonylureas (SU) are a popular herbicide used today for controlling weeds. While beneficial for this purpose they present a persistent problem in agricultural treated areas, with this treatment proving detrimental for successive crops. This study assessed the phytoremediative properties of lentils (Lens culinaris) grown in uncontaminated and chlorsulfuron-contaminated soil, with and without the addition of a growth supplement, PulseAider™. The results show that in the presence of lentils the degradation of chlorsulfuron is enhanced and this degradation rate is significantly increased when the PulseAider™ supplement was included during seed sowing. The supplement PulseAider™ also significantly increased shoot and root biomass, root branching, and nodule number under control conditions. While this was not so for plants grown in contaminated soils, the PulseAider™ supplement seemed to alter root branching and morphology. Most Probable Number (MPN) assays showed increased numbers of potential chlorsulfuron-degrading bacteria in soil treated with PulseAider™, although this was found to be significant only in the control soil. Sequencing of the 16S ribosomal gene showed the presence of Pseudomonas fluorescens bacterial species which is a known chlorsulfuron-degrading bacterium. This study is one of the first to address the remediation of residual SU herbicides and offers an economically feasible solution that may have an impact on global food security.
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