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Higher activity of an aldehyde oxidase in the auxin-overproducing superroot1 mutant of Arabidopsis thaliana.

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1-3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.

Aldehyde Oxidase↗

Genetic analysis of indole-3-butyric acid responses in Arabidopsis thaliana reveals four mutant classes.

Indole-3-butyric acid (IBA) is widely used in agriculture because it induces rooting. To better understand the in vivo role of this endogenous auxin, we have identified 14 Arabidopsis mutants that are resistant to the inhibitory effects of IBA on root elongation, but that remain sensitive to the more abundant auxin indole-3-acetic acid (IAA). These mutants have defects in various IBA-mediated responses, which allowed us to group them into four phenotypic classes. Developmental defects in the absence of exogenous sucrose suggest that some of these mutants are impaired in peroxisomal fatty acid chain shortening, implying that the conversion of IBA to IAA is also disrupted. Other mutants appear to have normal peroxisomal function; some of these may be defective in IBA transport, signaling, or response. Recombination mapping indicates that these mutants represent at least nine novel loci in Arabidopsis. The gene defective in one of the mutants was identified using a positional approach and encodes PEX5, which acts in the import of most peroxisomal matrix proteins. These results indicate that in Arabidopsis thaliana, IBA acts, at least in part, via its conversion to IAA.

Amino Acid Sequence↗

Phytohormones, Rhizobium Mutants, and Nodulation in Legumes : III. Auxin Metabolism in Effective and Ineffective Pea Root Nodules.

High specific activity [(3)H]indole-3-acetic acid (IAA) was applied to the apical bud of intact pea (Pisum sativum L. cv Greenfeast) plants. Radioactivity was detected in all tissues after 24 hours. More radioactivity accumulated in the nodules than in the parent root on a fresh weight basis and more in effective (nitrogen-fixing) nodules than in ineffective nodules (which do not fix nitrogen).For most samples, thin layer chromatography revealed major peaks of radioactivity at the R(F) values of IAA and indole-3-acetylaspartic acid (IAAsp) and further evidence of the identity of these compounds was obtained by chromatography in other systems. Disintegrations per minute due to IAA per unit fresh weight were significantly greater for root than for nodule tissue, but were not significantly different for effective and ineffective nodules. Radioactivity due to IAAsp, expressed both on a percentage basis and per unit fresh weight, was significantly greater for nodule than for root tissue and significantly greater for the effective nodules than for the ineffective nodules. When [(3)H]IAA was applied to effective nodules, IAAsp was the dominant metabolite in the nodule. The data suggest that metabolism of auxins may be important for the persistence of a functional root nodule.

Journal Article↗

Phytohormones, Rhizobium mutants, and nodulation in legumes : v. Cytokinin metabolism in effective and ineffective pea root nodules.

[(3)H]Zeatin riboside was supplied to intact pea (Pisum sativum) plants either onto the leaves or onto the root nodules. When applied directly to nodules, approximately 70% of recovered radioactivity remained in the nodules, approximately 15% was detected in the root system, and 15% was in the shoot. However, when supplied to the leaves, little (3)H was transported, with approximately 0.05% of recovered radioactivity being found in the root system and nodules. On a fresh weight basis, nodules accumulated more (3)H than the parent root. In both types of studies, metabolites with an intact zeatin moiety were detected in root nodules.In all experiments, two-dimensional thin layer chromatography revealed that little (3)H remained as zeatin riboside in root or nodule tissue at the end of the labeling period. Nodules metabolized [(3)H]zeatin riboside to the following cytokinins/cytokinin metabolites: zeatin, adenosine, adenine, the O-glucosides of zeatin and zeatin riboside, lupinic acid, nucleotides of adenine and zeatin, and the dihydro derivatives of many of these compounds.Although a few small differences were observed, there were no major differences between root and nodule tissue in their metabolism of [(3)H] zeatin riboside. Furthermore, any differences between effective and ineffective nodules were generally minor.

Journal Article↗

Isolation and characterization of a rice mutant hypersensitive to Al.

Rice (Oryza sativa L.) is a highly Al-resistant species among small grain crops, but the mechanism responsible for the high Al resistance has not been elucidated. In this study, rice mutants sensitive to Al were isolated from M(3) lines derived from an Al-resistant cultivar, Koshihikari, irradiated with gamma-rays. Relative root elongation was used as a parameter for evaluating Al resistance. After initial screening plus two rounds of confirmatory testing, a mutant (als1) was isolated from a total of 560 lines. This mutant showed a phenotype similar to the wild-type plant in the absence of Al. However, in the presence of 10 microM Al, root elongation was inhibited 70% in the mutant, but only 8% in the wild-type plant. The mutant also showed poorer root growth in acid soil. The Al content of root apices (0-1 cm) was much lower in the wild-type plant. The sensitivity to other metals including Cd and La did not differ between the mutant and the wild-type plants. A small amount of citrate was secreted from the roots of the mutant in response to Al stress, but there was no difference from that secreted by the wild-type plant. Genetic analysis of F(2) populations between als1 and wild-type plants showed that the Al-resistant seedlings and Al-sensitive seedlings segregated at a 3 : 1 ratio, indicating that the high sensitivity to Al in als1 is controlled by a single recessive gene. The gene was mapped to the long arm of chromosome 6, flanked by InDel markers MaOs0619 and MaOs0615.

Aluminum↗

Root growth, secondary root formation and root gravitropism in carotenoid-deficient seedlings of Zea mays L.

The effect of ABA on root growth, secondary-root formation and root gravitropism in seedlings of Zea mays was investigated by using Fluridone-treated seedlings and a viviparous mutant, both of which lack carotenoids and ABA. Primary roots of seedlings grown in the presence of Fluridone grew significantly slower than those of control (i.e. untreated) roots. Elongation of Fluridone-treated roots was inhibited significantly by the exogenous application of 1 mM ABA. Exogenous application of 1 micromole and 1 nmole ABA had either no effect or only a slight stimulatory effect on root elongation, depending on the method of application. The absence of ABA in Fluridone-treated plants was not an important factor in secondary-root formation in seedlings less than 9-10 d old. However, ABA may suppress secondary-root formation in older seedlings, since 11-d-old control seedlings had significantly fewer secondary roots than Fluridone-treated seedlings. Roots of Fluridone-treated and control seedlings were graviresponsive. Similar data were obtained for vp-9 mutants of Z. mays, which are phenotypically identical to Fluridone-treated seedlings. These results indicate that ABA is necessary for neither secondary-root formation nor for positive gravitropism by primary roots.

Abscisic Acid↗

Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization.

In the embryo of Arabidopsis thaliana (L.) Heynh., formation of the hypocotyl/root axis is initiated at the early-globular stage, recognizable as oriented expansion of formerly isodiametric cells. The process depends on the activity of the gene MONOPTEROS (MP); mp mutant embryos fail to produce hypocotyl and radicle. We have analyzed the morphology and anatomy of mp mutant plants throughout the Arabidopsis life cycle. Mutants form largely normal rosettes and root systems, but inflorescences either fail to form lateral flowers or these flowers are greatly reduced. Furthermore, the auxin transport capacity of inflorescence axes is impaired and the vascular strands in all analyzed organs are distorted. These features of the mutant phenotype suggest that the MP gene promotes cell axialization and cell file formation at multiple stages of plant development.

Arabidopsis↗

Brassinosteroid signals control expression of the AXR3/IAA17 gene in the cross-talk point with auxin in root development.

Transgenic plants overexpressing AXR3/IAA17 were impaired in root growth. Specifically, they exhibited severe defects in lateral root and root hair development similar to the root phenotypes of epi-brassinolide (epiBL)-treated wild-type plants. Here, we investigated the involvement of AXR3/IAA17 gene expression in brassinosteroid (BR)-regulated root development. Exogenous epiBL application significantly induced expression of the AXR3/IAA17 gene as well as several Aux/IAA genes, such as AXR2/IAA7, SLR/IAA14, and IAA28. We analyzed the transcription levels of several Aux/IAA genes related to root development in the BR signaling mutant bri1 and the BR biosynthesis mutant det2. AXR3/IAA17 gene expression was significantly decreased in bri1 plants. In det2 plants, expression of AXR3/IAA17 slightly decreased. This in turn suggests that epiBL induced these Aux/IAA genes, and that these induced gene products might function as factors in root development. Furthermore, AXR3/IAA17 might be involved in the BR signaling pathway, suggesting an intersection node of BR-auxin signaling in root development.

Arabidopsis↗

Role of methylotrophy during symbiosis between Methylobacterium nodulans and Crotalaria podocarpa.

Some rare leguminous plants of the genus Crotalaria are specifically nodulated by the methylotrophic bacterium Methylobacterium nodulans. In this study, the expression and role of bacterial methylotrophy were investigated during symbiosis between M. nodulans, strain ORS 2060T, and its host legume, Crotalaria podocarpa. Using lacZ fusion to the mxaF gene, we showed that the methylotroph genes are expressed in the root nodules, suggesting methylotrophic activity during symbiosis. In addition, loss of the bacterial methylotrophic function significantly affected plant development. Indeed, inoculation of M. nodulans nonmethylotroph mutants in C. podocarpa decreased the total root nodule number per plant up to 60%, decreased the whole-plant nitrogen fixation capacity up to 42%, and reduced the total dry plant biomass up to 46% compared with the wild-type strain. In contrast, inoculation of the legume C. podocarpa with nonmethylotrophic mutants complemented with functional mxa genes restored the symbiotic wild phenotype. These results demonstrate the key role of methylotrophy during symbiosis between M. nodulans and C. podocarpa.

Crotalaria↗

The rhd6 Mutation of Arabidopsis thaliana Alters Root-Hair Initiation through an Auxin- and Ethylene-Associated Process.

Root-hair initiation in Arabidopsis thaliana provides a model for studying cell polarity and its role in plant morphogenesis. Root hairs normally emerge at the apical end of root epidermal cells, implying that these cells are polarized. We have identified a mutant, rhd6, that displays three defects: (a) a reduction in the number of root hairs, (b) an overall basal shift in the site of root-hair emergence, and (c) a relatively high frequency of epidermal cells with multiple root hairs. These defects implicate the RHD6 gene in root-hair initiation and indicate that RHD6 is normally associated with the establishment of, or response to, root epidermal cell polarity. Similar alterations in the site of root-hair emergence, although less extreme, were also discovered in roots of the auxin-, ethylene-, abscisic acid-resistant mutant axr2 and the ethylene-resistant mutant etr1. All three rhd6 mutant phenotypes were rescued when either auxin (indoleacetic acid) or an ethylene precursor (1-aminocyclopropane-1-carboxylic acid) was included in the growth medium. The rhd6 root phenotypes could be phenocopied by treating wild-type seedlings with an inhibitor of the ethylene pathway (aminoethoxyvinylglycine). These results indicate that RHD6 is normally involved in directing the selection or assembly of the root-hair initiation site through a process involving auxin and ethylene.

Journal Article↗

Concerted action of tenascin-C domains in cell adhesion, anti-adhesion and promotion of neurite outgrowth.

We used a new approach to identify domains of chicken tenascin-C required for interaction with cells. Instead of expressing the parts of interest, we deleted them from an otherwise intact tenascin-C molecule and scored for the concomitant change in activity. As a starting point for all mutant constructs we expressed the smallest naturally occurring tenascin-C splice variant in vertebrate cells. The tenascin-C mutants had either deletions of all EGF-like repeats, all fibronectin type III repeats or of the fibrinogen globe. In double mutants the fibronectin type III repeats were deleted together with either the EGF-like repeats or the fibrinogen globe, respectively. All tenascin-C variants assembled correctly to hexameric molecules of the expected molecular characteristics. Intact tenascin-C and the mutant missing the fibrinogen globe did not promote adhesion of chick embryo fibroblasts, whereas both, the hexamers containing solely the fibrinogen globe or the EGF-like repeats were adhesive substrates and even supported cell spreading. When tenascin-C was added to the medium of fibroblasts plated on fibronectin-coated wells, cell adhesion was blocked by intact tenascin-C, but not by mutants missing the fibrinogen globe. In neurite outgrowth assays using dorsal root ganglia, processes formed on all substrates except on the mutant missing only the fibrinogen globe, where the ganglia failed to adhere. The mutants missing the fibronectin type III repeats allowed more rapid neurite outgrowth than all other tenascin-C variants and the mutant consisting essentially of oligomerized EGF-like repeats was as active a substrate for neurite outgrowth as laminin. From the combined data, it is concluded that the activities of intact tenascin-C cannot be mimicked by investigating domain by domain, but the concerted action of several domains leads to the diverse cellular responses.

Animals↗

Type-B ARR transcription factors, ARR10 and ARR12, are implicated in cytokinin-mediated regulation of protoxylem differentiation in roots of Arabidopsis thaliana.

In the phosphorelay-mediated cytokinin signal transduction of Arabidopsis thaliana, certain members of the type-B authentic response regulator (ARR) family are implicated in the regulatory networks that are primarily propagated by the cytokinin-receptors [authentic histidine kinases (AHKs)]. Clarification of the involvement of each type-B ARR transcription factor in cytokinin-responsive phenomena is still at a very early stage. Here we analyzed the redundant function of two type-B ARR genes, ARR10 and ARR12, by constructing an arr10/arr12 double mutant. The resulting mutant plants showed stronger phenotypes with special reference to the cytokinin action in roots (e.g. inhibition of root elongation, green callus formation from root explants) than those for each single mutant, suggesting that ARR10 and ARR12 redundantly play an important role in the cytokinin signaling in roots. This idea was further supported by results from root-specific microarray analyses with the double mutant plant. We also showed that ARR10 and ARR12 are involved in the AHK-dependent signaling pathway that negatively regulates protoxylem specification in root vascular tissues. When the double mutant is combined with an arr1 allele, the resultant arr1/arr10/arr12 triple mutant showed phenotypes displaying a very poor growth, quite similar to those of the wooden leg (wol) mutant that virtually lacks cytokinin receptor activities in plants. In this triple arr mutant, the specification of root vascular tissues is also affected as severely as in wol. Taken together, we propose that ARR10 and ARR12, together with ARR1, redundantly play pivotal roles in the AHK-dependent phosphorelay signaling in response to cytokinin in roots.

Arabidopsis↗

Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake.

Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality. Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups. The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron. The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface. The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter. Here we show that ys1 is a membrane protein that mediates iron uptake. Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media. Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots. Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants.

Amino Acid Sequence↗

Cultivar-Specific Interactions of Soybean with Rhizobium fredii Are Regulated by the Genotype of the Root.

Rhizobium fredii USDA257 forms nitrogen-fixing nodules on soybean cultivar Peking, but not on cultivar McCall. This pattern of nodulation persists when McCall and Peking seedlings are cultivated together in plastic growth pouches. Reciprocal grafting experiments confirm that the root genotype, and not that of the shoot, regulates such cultivar specificity. When Peking roots are grafted onto McCall seedlings, the nodulation responses of roots similarly remain unaffected. Transposon-mutant 257DH4, which is derived from USDA257, can form nitrogen-fixing nodules on McCall. Such nodulation is blocked by the presence of USDA257 in the inoculum. Grafting experiments indicate that blocking is not due to a translocatable inhibitor produced by McCall roots or triggered by their interaction with USDA257. Thus, neither freely diffusible nor graft-transmissible substances are involved in cultivar-specific interactions of soybean with R. fredii and its derivatives.

Journal Article↗

The Arabidopsis COW1 gene encodes a phosphatidylinositol transfer protein essential for root hair tip growth.

Root hairs are a major site for the uptake of water and nutrients into plants, and they form an increasingly important model system for the study of development in higher plants. We now report on the molecular genetic analysis of the srh1 mutant in Arabidopsis thaliana impaired in root hair tip growth. We show that srh1 is a new allele of cow1 (can of worms1) and we identified the COW1 gene using a positional cloning strategy. The N-terminus of the COW1 protein is 32% identical to an essential phosphatidylinositol transfer protein (PITP), the yeast Sec14 protein (sec14p) while the C-terminus is 34.5% identical to a late nodulin of Lotus japonicus, Nlj16. We show that expression of the COW1 lipid-binding domain complements the growth defect associated with Sec14p dysfunction in yeast. In addition, we show that GFP fused to the COW1 protein specifically accumulates at the site of root hair outgrowth. We conclude that the COW1 protein is a PITP, essential for proper root hair growth.

Alleles↗

Mass spectrometric quantification of indole-3-acetic Acid in Rhizobium culture supernatants: relation to root hair curling and nodule initiation.

Indole-3-acetic acid (IAA) has been unequivocally identified in culture supernatants of Rhizobium strains by gas chromatography-mass spectrometry. A method for accurately quantitating IAA in bacterial culture supernatants, employing deuterium-labeled IAA as an internal standard, has been developed. Similar IAA concentrations were found in culture supernatants of chosen Rhizobium mutants (defective in nodule formation) and their corresponding parent strains. Since some of the mutants are known to adhere to root hairs, it can be concluded that root hair curling is not simply a consequence of IAA production by rhizobia.

Journal Article↗

AXR3 and SHY2 interact to regulate root hair development.

Signal transduction of the plant hormone auxin centres on the regulation of the abundance of members of the Aux/IAA family of transcriptional regulators, of which there are 29 in Arabidopsis. Auxin can influence Aux/IAA abundance by promoting the transcription of Aux/IAA genes and by reducing the half-life of Aux/IAA proteins. Stabilising mutations, which render Aux/IAA proteins resistant to auxin-mediated degradation, confer a wide range of phenotypes consistent with disruptions in auxin response. Interestingly, similar mutations in different family members can confer opposite phenotypic effects. To understand the molecular basis for this functional specificity in the Aux/IAA family, we have studied a pair of Aux/IAAs, which have contrasting roles in root hair development. We have found that stabilising mutations in AXR3/IAA17 blocks root hair initiation and elongation, whereas similar mutations in SHY2/IAA3 result in early initiation of root hair development and prolonged hair elongation, giving longer root hairs. The phenotypes resulting from double mutant combinations, the transient induction of expression of the proteins, and the pattern of transcription of the cognate genes suggest that root hair initiation is controlled by the relative abundance of SHY2 and AXR3 in a cell. These results suggest a general model for auxin signalling in which the modulation of the relative abundance of different Aux/IAA proteins can determine which down-stream responses are induced.

Arabidopsis↗