A rice phenolic efflux transporter is essential for solubilizing precipitated apoplasmic iron in the plant stele

Iron deficiency is one of the major agricultural problems, as 30% of the arable land of the world is too alkaline for optimal crop production, rendering plants short of available iron despite its abundance. To take up apoplasmic precipitated iron, plants secrete phenolics such as protocatechuic acid (PCA) and caffeic acid. The molecular pathways and genes of iron uptake strategies are already characterized, whereas the molecular mechanisms of phenolics synthesis and secretion have not been clarified, and no phenolics efflux transporters have been identified in plants yet. Here we describe the identification of a phenolics efflux transporter in rice. We identified a cadmium-accumulating rice mutant in which the amount of PCA and caffeic acid in the xylem sap was dramatically reduced and hence named it phenolics efflux zero 1 (pez1). PEZ1 localized to the plasma membrane and transported PCA when expressed in Xenopus laevis oocytes. PEZ1 localized mainly in the stele of roots. In the roots of pez1, precipitated apoplasmic iron increased. The growth of PEZ1 overexpression lines was severely restricted, and these lines accumulated more iron as a result of the high solubilization of precipitated apoplasmic iron in the stele. We show that PEZ1 is responsible for an increase of PCA concentration in the xylem sap and is essential for the utilization of apoplasmic precipitated iron in the stele.     

The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains

Key message

Rice OsYSL9 is a novel transporter for Fe(II)-nicotianamine and Fe(III)-deoxymugineic acid that is responsible for internal iron transport, especially from endosperm to embryo in developing seeds.

Abstract

Metal chelators are essential for safe and efficient metal translocation in plants. Graminaceous plants utilize specific ferric iron chelators, mugineic acid family phytosiderophores, to take up sparingly soluble iron from the soil. Yellow Stripe 1-Like (YSL) family transporters are responsible for transport of metal-phytosiderophores and structurally similar metal-nicotianamine complexes. Among the rice YSL family members (OsYSL) whose functions have not yet been clarified, OsYSL9 belongs to an uncharacterized subgroup containing highly conserved homologs in graminaceous species. In the present report, we showed that OsYSL9 localizes mainly to the plasma membrane and transports both iron(II)-nicotianamine and iron(III)-deoxymugineic acid into the cell. Expression of OsYSL9 was induced in the roots but repressed in the nonjuvenile leaves in response to iron deficiency. In iron-deficient roots, OsYSL9 was induced in the vascular cylinder but not in epidermal cells. Although OsYSL9-knockdown plants did not show a growth defect under iron-sufficient conditions, these plants were more sensitive to iron deficiency in the nonjuvenile stage compared with non-transgenic plants. At the grain-filling stage, OsYSL9 expression was strongly and transiently induced in the scutellum of the embryo and in endosperm cells surrounding the embryo. The iron concentration was decreased in embryos of OsYSL9-knockdown plants but was increased in residual parts of brown seeds. These results suggested that OsYSL9 is involved in iron translocation within plant parts and particularly iron translocation from endosperm to embryo in developing seeds.

     

The OsNRAMP1 iron transporter is involved in Cd accumulation in rice

Cadmium (Cd) is a heavy metal toxic to humans and the accumulation of Cd in the rice grain is a major agricultural problem, particularly in Asia. The role of the iron transporter OsNRAMP1 in Cd uptake and transport in rice was investigated here. An OsNRAMP1:GFP fusion protein was localized to the plasma membrane in onion epidermal cells. The growth of yeast expressing OsNRAMP1 was impaired in the presence of Cd compared with yeast transformed with an empty vector. Moreover, the Cd content of OsNRAMP1-expressing yeast exceeded that of the vector control. The expression of OsNRAMP1 in the roots was higher in a high Cd-accumulating cultivar (Habataki) than a low Cd-accumulating cultivar (Sasanishiki) regardless of the presence of Cd, and the amino acid sequence of OsNRAMP1 showed 100% identity between Sasanishiki and Habataki. Over-expression of OsNRAMP1 in rice increased Cd accumulation in the leaves. These results suggest that OsNRAMP1 participates in cellular Cd uptake and Cd transport within plants, and the higher expression of OsNRAMP1 in the roots could lead to an increase in Cd accumulation in the shoots. Our results indicated that OsNRAMP1 is an important protein in high-level Cd accumulation in rice.     

The role of the ABCA1 transporter and cholesterol efflux in familial hypoalphalipoproteinemia

Defects in the gene encoding for the ATP binding cassette (ABC) transporter A1 (ABCA1) were shown to be one of the genetic causes for familial hypoalphalipoproteinemia (FHA). We investigated the role of ABCA1-mediated cholesterol efflux in Dutch subjects suffering from FHA. Eighty-eight subjects (mean HDL cholesterol levels 0.63 +/- 0.21 mmol/l) were enrolled. Fibroblasts were cultured and loaded with [3H]cholesterol. ABCA1 and non-ABCA1-mediated efflux was studied by using apolipoprotein A-I (apoA-I), HDL, and methyl-beta-cyclodextrin as acceptors. Efflux to apoA-I was decreased in four patients (4/88, 4.5%), and in all cases, a mutation in the ABCA1 gene was found. In the remaining 84 subjects, no correlation between efflux and apoA-I or HDL cholesterol was found. Efflux to both HDL and cyclodextrin, in contrast, did correlate with HDL cholesterol plasma levels (r = 0.34, P = 0.01; and r = 0.27, P = 0.008, respectively). The prevalence of defects in ABCA1-dependent cholesterol efflux in Dutch FHA patients is low. The significant correlation between plasma HDL cholesterol levels and methyl-beta-cyclodextrin-mediated efflux in the FHA patients with normal ABCA1 function suggests that non-ABCA1-mediated efflux might also be important for plasma HDL cholesterol levels in these individuals.     

The role of different regions of ATP-binding cassette transporter A1 in cholesterol efflux

ABCA1 is a key element of cholesterol efflux, but the mechanism of ABCA1-dependent cholesterol efflux is still unclear. Monoclonal antibodies against ABCA1 were used to map functional domains of ABCA1. Two antibodies were directed against a fragment of the first extracellular loop of ABCA1, and the third antibody was directed against a fragment of the fourth extracellular loop. One antibody against the first loop inhibited cholesterol efflux from human macrophages without inhibiting apolipoprotein A-I (apoA-I) binding and internalization. Another antibody against the first loop inhibited apoA-I binding and internalization without inhibiting cholesterol efflux. The antibody against the fourth loop inhibited apoA-I binding to ABCA1 but enhanced cholesterol efflux from macrophages and reduced intracellular cholesterol content. This antibody also increased cholesterol efflux from HeLa cells transfected with ABCA1 but not from cells with DeltaPEST-ABCA1. The mechanism of the stimulating effect of this antibody on cholesterol efflux was found to be stabilization of ABCA1 leading to the increase in abundance of cell surface ABCA1. We conclude that a site on the first extracellular loop is required for cholesterol efflux, whereas a site on the fourth extracellular loop may be responsible for ABCA1 stability.     

The iron transporter DMT1

Divalent metal transporter 1 (DMT1) is the first mammalian transmembrane iron transporter to be identified. In 1997, parallel experiments from two groups provided compelling evidence of its function. Fleming and colleagues identified mutations in DMT1 (formerly known as Nramp2 and DCT1) in mice and rats with defects in intestinal iron absorption and red blood cell iron utilization. Gunshin and co-workers (H Gunshin, B MacKenzie, UV Berger, Y Gunshin, MF Romero, WF Boron, S. Nussberger, JL Gollan, MA Hediger, Cloning and characterization of a mammalian proton-coupled metal-ion transporter, Nature 388 (1997) 482-488.) isolated DMT1 through an expression cloning strategy looking for mRNAs that stimulated iron uptake by Xenopus oocytes. Taken together, these data indicate that the twelve transmembrane domain protein DMT1 transfers iron across the apical surface of intestinal cells and out of transferrin cycle endosomes. Human DMT1 may be a good target for pharmacological intervention in patients with iron overload disorders attributable to increased iron absorption.     

Role of the iron transporter OsNRAMP1 in cadmium uptake and accumulation in rice

The heavy metal cadmium (Cd) is toxic to humans, and its accumulation in rice grains is a major agricultural problem. Rice has seven putative metal transporter NRAMP genes, but microarray analysis showed that only OsNRAMP1 is highly up-regulated by iron (Fe) deficiency. OsNRAMP1 localized to the plasma membrane and transported Cd as well as Fe. OsNRAMP1 expression was observed mainly in roots and was higher in the roots of a high-Cd-accumulating cultivar (Habataki) than in those of a low-Cd-accumulating cultivar (Sasanishiki). The amino acid sequence of OsNRAMP1 in the Sasanishiki and Habataki cultivars was found to be 100% identical. These results suggest that OsNRAMP1 participates in cellular Cd uptake and that the differences observed in Cd accumulation among cultivars are because of differences in OsNRAMP1 expression levels in roots.     

The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1

Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.     

A multidrug efflux transporter in Listeria monocytogenes

A chromosomal gene (mdrL) was found in Listeria monocytogenes L028, showing a high degree of similarity with multidrug efflux transporters of the major facilitator superfamily (family 2). An allele-substituted mutant of this gene failed to pump out ethidium bromide and presented lower minimal inhibitory concentrations of macrolides, cefotaxime and heavy metals. This is the first multidrug efflux pump described in Listeria.     

The role of active oxygen in iron tolerance of rice (Oryza sauva L.)

Summary Iron tolerance of rice (Oryza sativa L.) was investigated using an oxygen depleted hydroculture system. Treatment with high concentrations of Fe²⁺ induced yellowing and bronzing symptoms as well as iron coatings at the root surface. Root and shoot growth were inhibited by increasing iron concentration in the medium. All symptoms were more pronounced in an iron sensitive cultivar (IR 64) compared to an iron tolerant one (IR 9764-45-2). Superoxide dismutase and peroxidase activity of root extracts of IR 97 were about twice that of IR 64 in untreated control plants. No significant increase of peroxidase activity was detected with increasing iron concentration in the medium. Catalase activity of IR 64 was slightly higher than that of IR 97, independent of iron concentration.Abbreviations SOD Superoxide dismutase (EC 1.15.1.1) - POD peroxidase (EC 1.11.1.7) - EDTA ethylenediamintetraacetic acid - fwt fresh weight - Hepes (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) - BSA bovine serum albumin - IR 97 IR 9764-45-2 an iron tolerant rice cultivar - IR 64 iron sensitive rice cultivar - PM plasma membrane     

Pharmacokinetics of quinacrine efflux from mouse brain via the P-glycoprotein efflux transporter

The lipophilic cationic compound quinacrine has been used as an antimalarial drug for over 75 years but its pharmacokinetic profile is limited. Here, we report on the pharmacokinetic properties of quinacrine in mice. Following an oral dose of 40 mg/kg/day for 30 days, quinacrine concentration in the brain of wild-type mice was maintained at a concentration of ～1 μM. As a substrate of the P-glycoprotein (P-gp) efflux transporter, quinacrine is actively exported from the brain, preventing its accumulation to levels that may show efficacy in some disease models. In the brains of P-gp-deficient Mdr1(0/0) mice, we found quinacrine reached concentrations of ～80 μM without any signs of acute toxicity. Additionally, we examined the distribution and metabolism of quinacrine in the wild-type and Mdr1(0/0) brains. In wild-type mice, the co-administration of cyclosporin A, a known P-gp inhibitor, resulted in a 6-fold increase in the accumulation of quinacrine in the brain. Our findings argue that the inhibition of the P-gp efflux transporter should improve the poor pharmacokinetic properties of quinacrine in the CNS.     

The role of phenolics in goldenrod ball gall resistance and formation

Some genotypes of Solidago altissima have been shown to exhibit a hypersensitive response to the tephritid ball gallmaker Eurosta solidaginis. When the gallmaker attempts to stimulate gall formation in these genotypes, necrotic tissue surrounds the larva and apparently causes larval death [Anderson, S. S., et al. (1989) Ecology 70, 1048]. Other genotypes have varying degrees of resistance or susceptibility [McCrea, K. D. and Abrahamson, W. G. (1987) Ecology 68, 822]. Studies of other host plants have shown that phenolic compounds can play an important role in this defensive reaction. To determine the role of phenolics in the resistance of S. altissima to ball gallmakers, weekly tissue samples were collected from two resistant and two susceptible clones, both with and without Eurosta attack. These samples were analysed for total phenolics using the Folin-Ciocalteau procedure. Phenolic content increased over time in all clones. In unattacked ramets, phenolic levels were higher in susceptible clones than in resistant clones. No evidence of an increase in phenolics was seen with a hypersensitive response, but a significantly higher concentration was found in plants that had initiated normal gall development. A second study of rapidly growing galls in three additional susceptible clones confirmed this increase in phenolics, demontrated that the higher phenolic concentrations were restricted to gall tissue, and showed that phenolic levels increase as much as five-fold in galls near their peak growth period. Increases in total phenolic levels were clearly not responsible for the hypersensitive reaction for resistance, but phenols could potentially play a role in gall formation by influencing the hormonal control of growth and/or in protection of the gallmaker from its natural enemies.     

Reduction and release of ferritin iron by plant phenolics

The reductive release of ferritin iron by several naturally occurring o-diphenols was studied. The initial rate of iron release was quantified by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex, which absorbs maximally at 562 nm. The initial rate of iron release was dependent upon o-diphenol concentration, but not on the concentration of the chromophoric chelating agent, ferrozine, Stoichiometric measurements resulted in a ratio of 2Fe(II) released per molecule of o-diphenol. The series of o-diphenols studied included, caffeic acid, chlorogenic acid, dihydrocaffeic acid, 3,4-dihydroxybenzoic acid, and several analogs. These reductants represent an oxidation reduction potential range of 0.38 volts. A direct correlation between reducing power of the o-diphenols and rate of ferritin iron release was observed. Superoxide dismutase, catalase, mannitol, or general radical traps had no effect on the rate of iron removal; however, EDTA and oxalate inhibited iron release. A mechanism for ferritin iron reduction and release by o-diphenols consistent with the experimental observations is discussed.     

三磷酸腺苷结合盒转运体和细胞胆固醇外排

胆固醇是细胞质膜的重要组成成分。然而,过多的胆固醇累积可导致细胞中毒。异常的胆固醇胞内迁移与蓄积是造成许多心血管疾病如动脉粥样硬化的分子基础。细胞内胆固醇稳态由胆固醇的吸收、合成及外排等一系列过程调控。在哺乳动物细胞中,调节胆固醇合成、吸收和外排是维持体内胆固醇平衡的必要生理过程。本综述着重概述了三磷酸腺苷结合盒转运体(ABC)家族,如ABCA1、ABCG1、ABCG5和ABCG8的细胞功能及生理作用,以及这些转运体在调控胆固醇胞外转运中的分子机制。