Co-Permeability of 3H-Labeled Water and 14C-Labeled Organic Acids across Isolated Plant Cuticles1 : Investigating Cuticular Paths of Diffusion and Predicting Cuticular Transpiration

Penetration of 3H-labeled water (3H2O) and the 14C-labeled organic acids benzoic acid ([14C]BA), salicylic acid ([14C]SA), and 2,4-dichlorophenoxyacetic acid ([14C]2,4-D) were measured simultaneously in isolated cuticular membranes of Prunus laurocerasus L., Ginkgo biloba L., and Juglans regia L. For each of the three pairs of compounds (3H2O/[14C]BA, 3H2O/[14C]SA, and 3H2O/[14C]2,4-D) rates of cuticular water penetration were highly correlated with the rates of penetration of the organic acids. Therefore, water and organic acids penetrated the cuticles by the same routes. With the combination 3H2O/[14C]BA, co-permeability was measured with isolated cuticles of nine other plant species. Permeances of 3H2O of all 12 investigated species were highly correlated with the permeances of [14C]BA (r2 = 0.95). Thus, cuticular transpiration can be predicted from BA permeance. The application of this experimental method, together with the established prediction equation, offers the opportunity to answer several important questions about cuticular transport physiology in future investigations.

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots1

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27).

Biological impact on mineral dissolution: Application of the lichen model to understanding mineral weathering in the rhizosphere

Microorganisms modify rates and mechanisms of chemical and physical weathering and clay growth, thus playing fundamental roles in soil and sediment formation. Because processes in soils are inherently complex and difficult to study, we employ a model based on the lichen–mineral system to identify the fundamental interactions. Fixed carbon released by the photosynthetic symbiont stimulates growth of fungi and other microorganisms. These microorganisms directly or indirectly induce mineral disaggregation, hydration, dissolution, and secondary mineral formation. Model polysaccharides were used to investigate direct mediation of mineral surface reactions by extracellular polymers. Polysaccharides can suppress or enhance rates of chemical weathering by up to three orders of magnitude, depending on the pH, mineral surface structure and composition, and organic functional groups. Mg, Mn, Fe, Al, and Si are redistributed into clays that strongly adsorb ions. Microbes contribute to dissolution of insoluble secondary phosphates, possibly via release of organic acids. These reactions significantly impact soil fertility. Below fungi–mineral interfaces, mineral surfaces are exposed to dissolved metabolic byproducts. Through this indirect process, microorganisms can accelerate mineral dissolution, leading to enhanced porosity and permeability and colonization by microbial communities.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

Organic protomolecule assembly in igneous minerals

C—H stretching bands, νCH, in the infrared spectrum of single crystals of nominally high purity, of laboratory-grown MgO, and of natural upper mantle olivine, provide an “organic” signature that closely resembles the symmetrical and asymmetrical C—H stretching modes of aliphatic —CH2 units. The νCH bands indicate that H2O and CO2, dissolved in the matrix of these minerals, converted to form H2 and chemically reduced C, which in turn formed C—H entities, probably through segregation into defects such as dislocations. Heating causes the C—H bonds to pyrolyze and the νCH bands to disappear, but annealing at 70°C causes them to reappear within a few days or weeks. Modeling dislocations in MgO suggests that the segregation of C can lead to Cx chains, x = 4, with the terminal C atoms anchored to the MgO matrix by bonding to two O−. Allowing H2 to react with such Cx chains leads to [O2C(CH2)2CO2] or similar precipitates. It is suggested that such Cx—Hy—Oz entities represent protomolecules from which derive the short-chain carboxylic and dicarboxylic and the medium-chain fatty acids that have been solvent-extracted from crushed MgO and olivine single crystals, respectively. Thus, it appears that the hard, dense matrix of igneous minerals represents a medium in which protomolecular units can be assembled. During weathering of rocks, the protomolecular units turn into complex organic molecules. These processes may have provided stereochemically constrained organics to the early Earth that were crucial to the emergence of life.

Seed and vascular expression of a high-affinity transporter for cationic amino acids in Arabidopsis.

In most plants amino acids represent the major transport form for organic nitrogen. A sensitive selection system in yeast mutants has allowed identification of a previously unidentified amino acid transporter in Arabidopsis. AAT1 encodes a hydrophobic membrane protein with 14 membrane-spanning regions and shares homologies with the ecotropic murine leukemia virus receptor, a bifunctional protein serving also as a cationic amino acid transporter in mammals. When expressed in yeast, AAT1 mediates high-affinity transport of basic amino acids, but to a lower extent also recognizes acidic and neutral amino acids. AAT1-mediated histidine transport is sensitive to protonophores and occurs against a concentration gradient, indicating that AAT1 may function as a proton symporter. AAT1 is specifically expressed in major veins of leaves and roots and in various floral tissues--i.e., and developing seeds.

Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1.

The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(II) sepulchrate trichloride] for the electrocatalytically driven omega-hydroxylation of lauric acid. Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 [mRat4A1/mRatOR]L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase. Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor. These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals.