Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

Identification of AtNDI1, an internal non-phosphorylating NAD(P)H dehydrogenase in Arabidopsis mitochondria

Plant mitochondria contain non-phosphorylating NAD(P)H dehydrogenases (DHs) that are not found in animal mitochondria. The physiological function, substrate specificity, and location of enzymes within this family have yet to be conclusively determined. We have linked genome sequence information to protein and biochemical data to identify that At1g07180 (SwissProt Q8GWA1) from the Arabidopsis Genome Initiative database encodes AtNDI1, an internal NAD(P)H DH in Arabidopsis mitochondria. Three lines of evidence are presented: (a). The predicted protein sequence of AtNDI1 has high homology with other designated NAD(P)H DHs from microorganisms, (b). the capacity for matrix NAD(P)H oxidation via the rotenone-insensitive pathway is significantly reduced in the Atndi1 mutant plant line, and (c). the in vitro translation product of AtNDI1 is imported into isolated mitochondria and located on the inside of the inner membrane.     

Hydrocarbon phytoremediation in the family Fabaceae--a review

Currently, studies often focus on the use of Poaceae species (grasses) for phytoremediation of hydrocarbon-contaminated soils. Research into the use of Fabaceae species (legumes) to remediate hydrocarbons in soils has been conducted, but these plants are commonly overlooked due to slower recorded rates of degradation compared with many grass species. Evidence in the literature suggests that in some cases Fabaceae species may increase total degradation of hydrocarbons and stimulate degradative capacity of the soil microbial community, particularly for contaminants which are normally more recalcitrant to degradation. As many recalcitrant hydrocarbons have negative impacts on human and ecosystem health, development of remediation options is crucial. Reconsideration of Fabaceae species for removal of such contaminants may lead to environmentally and economically sustainable technologies for remediation of contaminated sites.     

Rhizoremediation of residual sulfonylurea herbicides in agricultural soils using Lens culinaris and a commercial supplement

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.     

Screening of Australian Native Grasses for Rhizoremediation of Aliphatic Hydrocarbon-Contaminated Soil

Rhizoremediation involves the breakdown of contaminants in soil resulting from microbial activity that is enhanced in the plant root zone. The objective of this study was to identify Australian native grass species as suitable candidates for rhizoremediation application. Seeds of nine perennial Australian native grasses were sown in soil from a mine site and artificially contaminated with a 60:40 diesel/oil mixture at concentrations of 1% (w/w), 0.5% (w/w), and 0% (control). Seedling emergence was not adversely affected by the presence of hydrocarbon contamination for all but one grass species. Three promising species (Brachiaria decumbens, Cymbopogon ambiguus, and Microlaena stipoides var. Griffin) were assessed for growth characterization in contaminated and uncontaminated soils. The evaluated species survived for 120 days in the contaminated soil and, in some instances, produced considerably more root biomass in the presence of contamination. C. ambiguus showed growth stimulation in the presence of contamination (1% and 0.5% w/w) with significantly increased root biomass production compared with the control (p = 0.0001). B. decumbens and M. stipoides showed tolerance, without adverse growth effects in the presence of diesel/oil at the exposed concentrations. Stimulation of the rhizosphere microbial population that is capable of degrading diesel/oil was found for all of the species tested, using a most probable number method for enumeration. This investigation has identified suitable candidates for further investigation of their rhizoremediation potential.     

Selenium increases seed production in Brassica

Selenium (Se) is essential for humans and animals but is not considered to be essential for higher plants. Although researchers have found increases in vegetative growth due to fertiliser Se, there has been no definitive evidence to date of increased reproductive capacity, in terms of seed production and seed viability. The aim of this study was to evaluate seed production and growth responses to a low dose of Se (as sodium selenite, added to solution culture) compared to very low-Se controls in fast-cycling Brassica rapa L. Although there was no change in total biomass, Se treatment was associated with a 43% increase in seed production. The Se-treated Brassica plants had higher total respiratory activity in leaves and flowers, which may have contributed to higher seed production. This study provides additional evidence for a beneficial role for Se in higher plants.