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Role of ptsP, orfT, and sss recombinase genes in root colonization by Pseudomonas fluorescens Q8r1-96.

Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.

Ascomycota↗

Increased low-density lipoprotein oxidation and impaired high-density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreases atherosclerosis.

BACKGROUND: Obesity-associated dyslipidemia in humans is associated with increased low-density lipoprotein (LDL) oxidation. Mice with combined leptin and LDL receptor deficiency are obese and show severe dyslipidemia and insulin resistance. We investigated the association between oxidation of apolipoprotein B-containing lipoproteins, high-density lipoprotein (HDL) antioxidant defense, and atherosclerosis in these mice. METHODS AND RESULTS: LDL receptor knockout (LDLR-/-), leptin-deficient (ob/ob), double-mutant (LDLR-/-;ob/ob), and C57BL6 mice were fed standard chow. Double-mutant mice had higher levels of non-HDL (P<0.001) and HDL (P<0.01) cholesterol and of triglycerides (P<0.001). They also had higher oxidative stress, evidenced by higher titers of autoantibodies against malondialdehyde-modified LDL (P<0.001). C57BL6 and ob/ob mice had no detectable lesions. Lesions covered 20% of total area of the thoracic abdominal aorta in double-mutant mice compared with 3.5% in LDLR-/- mice (P<0.01). Higher macrophage homing and accumulation of oxidized apolipoprotein B-100-containing lipoproteins were associated with larger plaque volumes in the aortic root of double-mutant mice (P<0.01). The activity of the HDL-associated antioxidant enzymes paraoxonase and lecithin:cholesterol acyltransferase (LCAT) (ANOVA; P<0.0001 for both) was lower in double-mutant mice. Adenovirus-mediated LCAT gene transfer in double-mutant mice increased plasma LCAT activity by 64% (P<0.01) and reduced the titer of autoantibodies by 40% (P<0.01) and plaque volume in the aortic root by 42% (P<0.05) at 6 weeks. CONCLUSIONS: Dyslipidemia and insulin resistance in obese LDL receptor-deficient mice are associated with increased oxidative stress and impaired HDL-associated antioxidant defense, evidenced by decreased paraoxonase and LCAT activity. Transient LCAT overexpression was associated with a reduction of oxidative stress and atherosclerosis.

Animals↗

Induction of hydrotropism in clinorotated seedling roots of Alaska pea, Pisum sativum L.

Roots of the agravitropic pea (Pisum sativum L.) mutant ageotropum show positive hydrotopism, whereas roots of Alaska peas are hydrotropically almost non-responsive. When the gravitropic response was nullified by rotation on clinostats, however, roots of Alaska peas showed unequivocal positive hydrotropism in response to a water potential gradient. These results suggest that roots of Alaska peas possess normal ability to respond hydrotropicallly and their weak hydrotropic response results from a counteracting effect of gravitropism.

Gravitation↗

Dual roles of the retinoblastoma protein in cell cycle regulation and neuron differentiation.

To assess the functions of the retinoblastoma protein (RB) during normal development, we have analyzed mouse embryos that lack a functional copy of the retinoblastoma gene (genotype: Rb-1 delta 20/Rb-1 delta 20). Our findings demonstrate that RB plays an important role in the regulation of the neuronal cell cycle. In mutant embryos, dividing cells are found well outside of the normal neurogenic regions in both the central and peripheral nervous systems. In addition to abnormal cell cycle regulation, however, the mutant embryos show two less expected phenotypes. First, many of the ectopically dividing cells die by apoptosis shortly after their entrance into S phase. In sensory ganglia, most nerve cells die by this process, beginning at about the same time as normal target-related neuronal death. Second, although the expression of certain differentiation markers such as N-CAM and Brn-3.0 appears to be near normal, nerve cells, especially in sensory ganglia, do not mature properly. Their morphology is stunted and expression of neuronal beta II tubulin is greatly reduced. Preferential reduction in the expression of TrkA, TrkB, and the low-affinity neurotrophin receptor p75LNGFR may be relevant to neuronal cell death and lack of neuronal differentiation seen in the mutant embryos. Primary cultures of dorsal root and trigeminal ganglion cells from later stage mutant embryos reveal a decrease in neuronal cell survival and in neurite outgrowth even in the presence of the appropriate neurotrophins. Taken together, these results suggest that the p110RB protein not only regulates progression through the cell cycle but is also important for cell survival and differentiation.

Animals↗

Development of Sulfate Uptake Capacity and ATP-Sulfurylase Activity during Root Elongation in Maize.

Sulfate uptake capacity and ATP-sulfurylase activity were determined in maize roots (Zea mays L. var. XL 363 and mutant XL 363 o2) at increasing root length. The pattern of uptake showed a close similarity to that of ATP-sulfurylase, both activities reaching the maximum level at 9 and 10 cm root length in the XL 363 and XL 363 o2 hybrids, respectively. In addition to the shift of the maximum, opaque-2 mutation caused an enhancement of the two activities at root length below and above the activity peak. The kinetic parameter of uptake, Km, showed a maximum at 3 to 4 and a minimum at 7 to 8 cm. The isoenzyme pattern of ATP-sulfurylase was the same in the two hybrids and did not change with root elongation. A common regulatory mechanism is postulated for uptake and activation of sulfate. The kinetic behavior is interpreted as an index of flexibility of the transport system toward different nutrient status of the environment.

Journal Article↗

Hormonal characterization of a nonrooting naphthalene-acetic Acid tolerant tobacco mutant by an immunoenzymic method.

The comparative analysis of plant hormones was undertaken on a 1-naphthaleneacetic acid tolerant mutant and normal tobacco (Nicotiana tabacum cv Xanthi) plantlets. The mutant plantlet was scrubby and impaired in its root morphogenesis. Degeneration of the root meristem was studied on tissue sections; it appeared very fast (as early as the 3rd or 4th day after sowing), after which the root was further transformed into a callus. Indoleacetic acid (IAA), abscisic acid (ABA), and the isopentenyladenine (iP)- and trans-zeatin(Z)-type cytokinin levels were measured in terminal buds and root tips 13 days after sowing, by enzyme linked immunosorbent assay of high performance liquid chromatography fractions. Some differences appeared between the apical buds of the two genotypes, but the mutant tobacco differed from the wild type mainly by the presence of higher levels of IAA, ABA, and iP + isopentenyladenosine (iPA) in its small root. Thus, the IAA, ABA, and iP + iPA contents were increased by a factor of 15, 7, and 24 times, respectively, in mutant root compared to wild-type tobacco roots. Previous work has shown that the mutation impairs membrane polarization effects induced by auxin at the cell level. The present results would favor the hypothesis that the mutation has also affected the control of growth regulator accumulation in tissues.

Journal Article↗

A proteomic analysis identifies glutathione S-transferase isoforms whose abundance is differentially regulated by ethylene during the formation of early root epidermis in Arabidopsis seedlings.

The plant hormone ethylene has been shown to play an important role in root hair development in Arabidopsis. With the aid of proteomic analysis, we identified three distinct glutathione S-transferase (GST) isoforms, AtGSTF2, AtGSTF8, and AtGSTU19, expressed early in root epidermal establishment in Arabidopsis seedlings. The AtGSTF2 protein was specifically up-regulated by ethylene. A subsequent RNA expression study revealed that the AtGSTF2 gene was highly sensitive to ethylene, whereas the transcripts for AtGSTF8 and AtGSTU19 were constitutively present in new root tissue of 4-day-old seedlings. The steady-state level of AtGSTF2 mRNA was greatly reduced in the roots of ethylene-insensitive mutants, while mutation at the CTR1 locus, which confers an ectopic root hair phenotype, resulted in a markedly elevated level of AtGSTF2 transcript in young root tissue. Although the physiological function of ethylene-induced AtGSTF2 is not yet clear, there are several possibilities for its role during early root development.

Amino Acids, Cyclic↗

A novel role for abscisic acid emerges from underground.

The continuous formation of lateral roots is a vital part of establishing a root system and enables plants to react with developmental plasticity to changing soil conditions. Evidence is accumulating that abscisic acid (ABA), which is known to be involved in stress responses, has an important role in lateral root formation. An ABA receptor mutant, fca-1, shows an altered response to ABA during lateral root formation. Interestingly, ABA seems to have distinct roles at different stages in lateral root development. The emerging role of ABA in lateral root development fits well with its general functional properties as a stress hormone, including its role in dormancy.

Abscisic Acid↗

ARR1 and ARR12 negatively regulate arsenic stress tolerance by controlling flavonoid metabolism in Arabidopsis.

ARR1/12-mediated cytokinin signaling negatively regulates the accumulation of glycosylated flavonoids, thereby increasing plant susceptibility to As(III) stress. Cytokinins negatively regulate arsenic stress tolerance in plants through cytokinin-signaling type-B Arabidopsis response regulators (B-ARRs), specifically ARR1 and ARR12. However, the mechanism by which cytokinin signaling regulates plant metabolite dynamics, particularly antioxidant flavonoids, in response to arsenic toxicity remains largely unknown. Here, we hypothesized that ARR1/12-mediated cytokinin signaling modulates flavonoid metabolism to regulate arsenite [As(III)] tolerance. By comparing the global metabolic changes in roots of the arr1 12 double mutant (rD) and wild-type (WT) plants, we found that As(III) stress globally reduced metabolite abundance in WT roots. Importantly, the rD mutant accumulated significantly more flavonoids, most in glycosylated forms, than WT under As(III) exposure, which was supported by the specific upregulation of UDP-glycosyltransferase genes involved in flavonoid glycosylation. Accordingly, exogenous application of the glycosylated quercitrin-enhanced As(III) tolerance in WT roots, strengthening that the increase of glycosylated flavonoids in rD roots was beneficial for plant survival under As(III) exposure. Our data collectively strongly support that the increased glycosylation of flavonoids in the rD mutant improves their antioxidant functionality, thereby enhancing the As(III) stress tolerance. This study provides a new insight into the negative role of cytokinin signaling in repressing glycosylated flavonoid accumulation, causing increased susceptibility of plants to As(III) stress. Manipulation of cytokinin signaling or flavonoid glycosylation is, therefore, a promising approach for heavy metal stress mitigation in crops.

Arabidopsis↗

Isolation and characterization of mutants of Arabidopsis thaliana with increased resistance to growth inhibition by indoleacetic acid-amino acid conjugates.

Two mutants of Arabidopsis thaliana that are resistant to growth inhibition by indole-3-acetic acid (IAA)-phenylalanine have been isolated. Both mutants were 2- to 3-fold more resistant than wild type to inhibition by IAA-phenylalanine, IAA-alanine, and IAA-glycine in root growth assays. The mutant icr1 (but not icr2) also shows some resistance to IAA-aspartate. Studies using 3H-labeled IAA-phenylalanine showed that the uptake of conjugate from the medium by icr1 was the same as wild type and was reduced by about 25% in icr2. No differences in hydrolysis of the exogenous conjugate were detected between the mutants and their wild-type parents. There was no significant metabolism of the IAA released from the [3H]IAA-phenylalanine, whereas exogenous [3H]IAA was rapidly metabolized to two unidentified products considerably more polar than IAA. Analysis of a cross between icr1 and icr2 indicated that these mutations were at distinct loci and that their effects were additive, and preliminary mapping data indicated that icr1 and icr2 were located at the top and bottom of chromosome V, respectively.

Amino Acids↗

Technical advance: an aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos.

A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high-resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild-type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three-dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short-root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.

Aniline Compounds↗

Ethylene and phosphorus availability have interacting yet distinct effects on root hair development.

The hypothesis that ethylene participates in the regulation of root hair development by phosphorus availability in Arabidopsis thaliana was tested by chemically manipulating ethylene synthesis and response and with ethylene-insensitive mutants. Low phosphorus-induced root hair development could be mimicked by adding the ethylene precursor, 1-aminocyclopropane-1-carboxylate (ACC), to high phosphorus media, and inhibited by adding ethylene inhibitors to low phosphorus media. Ethylene-insensitive mutants showed a reduced response to low phosphorus, indicating ethylene involvement in root hair responses to phosphorus deficiency. To dissect the nature of this involvement, the morphological and anatomical changes associated with increased root hair density were investigated. Growth in low phosphorus resulted in smaller, more numerous cortical cells, resulting in a larger number of root hair-bearing epidermal cell files. Cortical cell number was not affected by ethylene inhibitors, ACC, or mutations reducing ethylene sensitivity in roots grown with low phosphorus, indicating that ethylene does not participate in this response. The exception was the eir1 mutation, which strongly reduced this change in radial anatomy, supporting a role for polar auxin transport in this process. Trichoblast cell length was reduced by low phosphorus availability in all genotypes, but even more so for ein2-1 and ein4. The proportion of epidermal cells forming hairs and root hair length were reduced in ethylene-insensitive mutants, especially in the presence of low phosphorus. These results demonstrate multiple effects of low phosphorus from the earliest stages of root hair development, and cross-talk between ethylene and phosphorus in the control of a subset of the low phosphorus effects, concentrating on those later in development.

Amino Acids, Cyclic↗

Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene.

Leaf explants of Medicago truncatula were used to investigate the origins of auxin-induced root formation. On the application of auxin there is some callus formation (not the massive amount that occurs in response to auxin plus cytokinin) and roots appear shortly after the first visible callus. Histological examination reveals morphologically distinctive sheets of callus cells that emanate from the veins of the leaf explants and, within this cell type, root primordia are produced as well as some vascular tissue cells. What is suggested is that the vein-derived cells (VDCs) are procambial-like and function as pluripotent stem cells with a propensity to form root meristems or vascular tissues in response to added auxin. The development of root primordia from these pluripotent cells was clearly up-regulated by the use of the sickle (skl) mutant, which is a mutant impaired in ethylene signal transduction while the wild type and the sunn mutant, defective in auxin polar transport, produced similar numbers of roots. The skl mutant in generating many more roots concomitantly formed fewer vascular tissues. The root meristems differentiate similarly to normal roots producing a central cylinder of vascular tissue, which connects with the leaf explant veins. The VDCs appear to be derived from the cells of or near the phloem. The leaf observations suggest that a pool of stem cells exist in vascular tissue that, in combination with auxin and perhaps other factors, drive a diversity of plant development outcomes that is species specific. The way auxin interacts with other hormones is a key factor in determining the stem cell fate. The histological data in this study also assist in the interpretation of the molecular analysis of auxin-induced root formation in cultured leaves of M. truncatula.

Ethylenes↗

Identification of a jasmonate-regulated allene oxide synthase that metabolizes 9-hydroperoxides of linoleic and linolenic acids.

Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that catalyzes the first step in the conversion of 13-hydroperoxy linolenic acid to jasmonic acid and related signaling molecules in plants. Here, we report the molecular cloning and characterization of a novel AOS-encoding cDNA (LeAOS3) from Lycopersicon esculentum whose predicted amino acid sequence classifies it as a member of the CYP74C subfamily of enzymes that was hitherto not known to include AOSs. Recombinant LeAOS3 expressed in Escherichia coli showed spectral characteristics of a P-450. The enzyme transformed 9- and 13-hydroperoxides of linoleic and linolenic acid to alpha-ketol, gamma-ketol, and cyclopentenone compounds that arise from spontaneous hydrolysis of unstable allene oxides, indicating that the enzyme is an AOS. Kinetic assays demonstrated that LeAOS3 was approximately 10-fold more active against 9-hydroperoxides than the corresponding 13-isomers. LeAOS3 transcripts accumulated in roots, but were undetectable in aerial parts of mature plants. In contrast to wild-type plants, LeAOS3 expression was undetectable in roots of a tomato mutant that is defective in jasmonic acid signaling. These findings suggest that LeAOS3 plays a role in the metabolism of 9-lipoxygenase-derived hydroperoxides in roots, and that this branch of oxylipin biosynthesis is regulated by the jasmonate signaling cascade.

Amino Acid Sequence↗

Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis.

The plant root cap mediates the direction of root tip growth and protects internal cells. Root cap cells are continuously produced from distal stem cells, and the phytohormone auxin provides position information for root distal organization. Here, we identify the Arabidopsis thaliana auxin response factors ARF10 and ARF16, targeted by microRNA160 (miR160), as the controller of root cap cell formation. The Pro(35S):MIR160 plants, in which the expression of ARF10 and ARF16 is repressed, and the arf10-2 arf16-2 double mutants display the same root tip defect, with uncontrolled cell division and blocked cell differentiation in the root distal region and show a tumor-like root apex and loss of gravity-sensing. ARF10 and ARF16 play a role in restricting stem cell niche and promoting columella cell differentiation; although functionally redundant, the two ARFs are indispensable for root cap development, and the auxin signal cannot bypass them to initiate columella cell production. In root, auxin and miR160 regulate the expression of ARF10 and ARF16 genes independently, generating a pattern consistent with root cap development. We further demonstrate that miR160-uncoupled production of ARF16 exerts pleiotropic effects on plant phenotypes, and miR160 plays an essential role in regulating Arabidopsis development and growth.

Arabidopsis↗

High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1.

The NAR2 protein of Chlamydomonas reinhardtii has no known transport activity yet it is required for high-affinity nitrate uptake. Arabidopsis (Arabidopsis thaliana) possesses two genes, AtNRT3.1 and AtNRT3.2, that are similar to the C. reinhardtii NAR2 gene. AtNRT3.1 accounts for greater than 99% of NRT3 mRNA and is induced 6-fold by nitrate. AtNRT3.2 was expressed constitutively at a very low level and did not compensate for the loss of AtNRT3.1 in two Atnrt3.1 mutants. Nitrate uptake by roots and nitrate induction of gene expression were analyzed in two T-DNA mutants, Atnrt3.1-1 and Atnrt3.1-2, disrupted in the AtNRT3.1 promoter and coding regions, respectively, in 5-week-old plants. Nitrate induction of the nitrate transporter genes AtNRT1.1 and AtNRT2.1 was reduced in Atnrt3.1 mutant plants, and this reduced expression was correlated with reduced nitrate concentrations in the tissues. Constitutive high-affinity influx was reduced by 34% and 89%, respectively, in Atnrt3.1-1 and Atnrt3.1-2 mutant plants, while high-affinity nitrate-inducible influx was reduced by 92% and 96%, respectively, following induction with 1 mm KNO(3) after 7 d of nitrogen deprivation. By contrast, low-affinity influx appeared to be unaffected. Thus, the constitutive high-affinity influx and nitrate-inducible high-affinity influx (but not the low-affinity influx) of higher plant roots require a functional AtNRT3 (NAR2) gene.

Amino Acid Sequence↗

Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses.

Pyridoxine (vitamin B6) is a cofactor required by numerous enzymes in all cellular organisms. Plants are the major source of vitamin B6 for animals, yet the biosynthesis pathway and the function of vitamin B6 in plants are not well elucidated. In this study, an Arabidopsis pyridoxine synthase gene PDX1 was characterized and its in vivo functions were investigated. The PDX1 gene was expressed in all plant parts examined and its expression level was not significantly regulated by abiotic stress or the phytohormone abscisic acid. In roots, PDX1 was highly expressed in a defined region behind the root tips that undergoes rapid cell division. The PDX1 protein was mainly associated with the plasma membrane and endomembranes, implying a potential involvement of vitamin B6 in membrane function. To reveal the in vivo role of PDX1, Arabidopsis insertional mutants were isolated. Strikingly, the pdx1 knockout mutants were impaired in root growth and early seedling development. The stunted roots resulted from both reduced cell division and elongation. Supplementation of the growth media with pyridoxine or reintroduction of the wild-type PDX1 gene into the mutants completely restored the mutant growth, demonstrating that PDX1 is required for pyridoxine biosynthesis in planta. In addition to the developmental defects, pdx1 mutants are hypersensitive to osmotic stress and oxidative stress. These mutant seedlings had increased peroxidation of membrane lipids following UV treatment. Our study establishes a critical role of vitamin B6 in plant development and stress tolerance and suggests that vitamin B6 may represent a new class of antioxidant in plants.

Amino Acid Sequence↗

[Collection of Arabidopsis thaliana mutants with altered sensitivity to oxidative stress inductors].

Selective systems for screening Arabidopsis thaliana (L.) Heynh. mutants with altered sensitivity to the oxidative stress (OS) inductors norflurazon (NF), acifluorfen (AF), and plumbagin (PB) were developed and a collection of 28 mutants was obtained. Dwarf and necrotic forms predominated among the NF-tolerant mutants, while pigment mutants and those with changed root morphology prevailed among the AF-tolerant and PB-sensitive mutants, respectively. Genetic and biochemical analysis of certain mutants was performed; quantitative and qualitative changes in the content of superoxide dismutase and peroxidase isoforms have been revealed. These data, complemented by the data on the cross-tolerance (sensitivity) of the mutants to paraquat, indicate a correlation between tolerance to the OS inductors and the functions of antioxidant systems.

Arabidopsis↗