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Jasmonate signaling mutants of Arabidopsis are susceptible to the soil fungus Pythium irregulare.

Jasmonic acid has properties of a plant hormone, including the induction of specific genes associated with plant defense. We previously described jar1-1, an Arabidopsis jasmonate response mutant that exhibits reduced sensitivity to methyl jasmonate. We have further characterized this mutant and two new alleles; jar1-2 from a gamma irradiated population, and jar1-4 from a T-DNA mutant population. Seedling root growth in jar1-1 was equally insensitive to methyl jasmonate and jasmonic acid, indicating that the defect was not in the conversion of methyl jasmonate to the acid. None of the jar1 mutants showed an altered sensitivity to auxin, cytokinin, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicating that the lesion does not affect the general uptake or transport of hormones. A soil fungus, Pythium irregulare, was found to blight jar1-1. Cultures of this organism caused the symptoms in all three jar1 mutants but not in wild type, indicating that increased susceptibility was due to the lesion in the JAR1 locus. A fatty acid desaturase triple mutant that is defective in the biosynthesis of jasmonic acid (J. Browse, Washington State University) was also susceptible, confirming that jasmonate is involved in resistance. The jar1-1 locus was mapped to the lower end of chromosome 2, about 11.4 cM from as1 and 1.6 cM from cer8. These results establish that jasmonate signaling plays an important role in resistance to soil micro-organisms in plants.

Alleles↗

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana.

The CURLY LEAF (CLF) gene in Arabidopsis thaliana (L). Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which tell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia.

Arabidopsis↗

Biological activity of Rhizobium sp. NGR234 Nod-factors on Macroptilium atropurpureum.

The broad host range of Rhizobium sp. NGR234 is based mainly on its ability to secrete a family of lipooligosaccharide Nod factors. To monitor Nod-factor purification, we used the small seeded legume Macroptilium atropurpureum, which responds evenly and consistently to Nod factors. At concentrations between approximately equal to 10(-11) M and 10(-9) M, this response takes the form of deformation of the root hairs. Higher concentrations (approximately equal to 10(-9) to 10(-7) M), provoked profound "shepherd's crook" type curling of the root hairs. Similar concentrations of Nod factors of Bradyrhizobium japonicum, Rhizobium leguminosarum, and R. meliloti also provoked marked curling of the root hairs, but the latter two species are unable to nodulate Macroptilium. On the other hand, plant hormones, hormone-like substances, inhibitors of hormone action, as well as substituents of Nod factors were without effect in this bioassay. We thus conclude that only Nod factors are capable of inducing shepherd's crook type curling of Macroptilium root hairs. Perturbations in the auxin-cytokinin balance induced "pseudo" nodulation on M. atropurpureum, as did NodNGR factors at concentrations between 10(-7) and 10(-6) M. Concomitant inoculation of Macroptilium with a NodABC- mutant of NGR234 and sulfated NodNGR factors (NodNGR[S]) gave rise to plants that slowly greened, showing that the NodNGR factors permitted entry of the Nod- mutant into the roots.

Bacterial Proteins↗

Differential ethylene sensitivity of epidermal cells is involved in the establishment of cell pattern in the Arabidopsis root.

Root hairs of Arabidopsis roots develop on trichoblasts located over the anticlinal (radial) walls of underlying cortical cells. Non-hair cells, on the other hand, develop on atrichoblasts overlying the periclinal (tangential) walls of cortical cells. Dark-grown wild-type seedlings, which produce little ethylene, are largely root hairless. Exogenous treatment of dark-grown plants with either ethylene or 1-aminocyclopropane-1-carboxylic acid (ACC) restores the development of root hairs in cells overlying the anticlinal cortical cell walls, indicating that cells in this position are more sensitive to ethylene than atrichoblasts. We used mutations in genes that overproduce ethylene (eto1, eto2, eto3 and eto4) to illustrate the positive regulatory role of ethylene. The preferential development of root hairs on epidermal cells overlying the cortical anticlinal cell walls in these mutants also illustrates that trichoblasts are more sensitive to ethylene than atrichoblasts. CTR1 is a negative regulator of the ethylene response and might, therefore, be a candidate regulator of differential sensitivity. CTR1 mRNA is expressed in all cell types in the root, suggesting that its transcriptional pattern alone cannot account for the differential sensitivity of epidermal cells to ethylene. Cellular mapping of wild-type and mutant roots supports previous findings indicating that ethylene acts after, and perhaps independently, of TTG during the establishment of cell fate in the root epidermis.

Amino Acids, Cyclic↗

The pattern of secondary root formation in curving roots of Arabidopsis thaliana (L.) Heynh.

A gravitational stimulus was used to induce the curvature of the main root of Arabidopsis thaliana. The number of secondary roots increased on the convex side and decreased on the concave side of any curved main root axes in comparison with straight roots used as the control. The same phenomenon was observed with the curved main roots of plants grown on a clinostat and of mutant plants exhibiting random root orientation. The data suggest that the pattern of lateral root formation is associated with curvature but is independent of the environmental stimuli used to induce curvature.

Arabidopsis↗

Slow degeneration of zebrafish Rohon-Beard neurons during programmed cell death.

Rohon-Beard cells are large, mechanosensory neurons located in the dorsal spinal cord of anamniote vertebrates. In most species studied to date, these cells die during development. We followed labeled Rohon-Beard cells in living zebrafish embryos and found that they degenerate slowly, over many days. During degeneration, the soma shrinks and finally disappears, and the processes become beady in appearance and finally break apart, but they do not retract. Zebrafish Rohon-Beard cells apparently fragment their DNA, as revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) labeling, before undergoing degenerative morphologic changes. We also followed the development of labeled dorsal root ganglion neurons, as they are developing at the same stages that Rohon-Beard cells are degenerating. We found that, although axons of both cell types extend into similar regions, Rohon-Beard cells degenerate normally in mutants lacking dorsal root ganglia, providing evidence that interactions between the two cell types are not responsible for Rohon-Beard cell degeneration. Developmental Dynamics 229:30-41,2004.

Animals↗

Severely reduced gravitropism in dark-grown hypocotyls of a starch-deficient mutant of Nicotiana sylvestris.

Gravitropism in dark-grown hypocotyls of the wild type was compared with a starch-deficient Nicotiana sylvestris mutant (NS 458) to test the effects of starch deficiency on gravity sensing. In a time course of curvature measured using infrared video, the response of the mutant was greatly reduced compared to the wild type; 72 hours after reorientation, curvature was about 10 degrees for NS 458 and about 70 degrees for wild type. In dishes maintained in a vertical orientation, wild-type hypocotyls were predominantly vertical, whereas NS 458 hypocotyls were severely disoriented with about 5 times more orientational variability than wild type. Since the growth rates were equal for both genotypes and phototropic curvature was only slightly inhibited in NS 458, the mutation probably affects gravity perception rather than differential growth. Our data suggest that starch deficiency reduces gravitropic sensitivity more in dark-grown hypocotyls than in dark- or light-grown roots in this mutant and support the hypothesis that amyloplasts function as statoliths in shoots as well as roots.

Darkness↗

Agrobacterium rhizogenes mutants that fail to bind to plant cells.

Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria to bind to carrot cells. The parent strain bound to carrot cells in greatest numbers in low-ionic-strength media such as 4% sucrose but still showed significant binding in Murashige-Skoog tissue culture medium. All of the mutants showed reduced binding in 4% sucrose after 2 h of incubation with carrot cells. One mutant was delayed in binding in 4% sucrose. This mutant and one other mutant also showed reduced binding to carrot cells in Murashige-Skoog medium. To determine whether the Tn5 insertion was responsible for the mutant phenotype, DNA containing the Tn5 insertion was cloned from the mutant bacteria and used to introduce Tn5 into the parent strain in the same location as in the original mutant by marker exchange. The resulting transconjugants had the same avirulent, nonattaching phenotype as the original mutants, suggesting that the mutant phenotype was due to the Tn5 insertion. The cloned DNA containing the Tn5 insertion was also tested for homology to DNA of known genes that affect attachment of Agrobacterium tumefaciens to plant cells by DNA hybridization. No homology to chv, att, or pscA clones was observed. In addition, cloned chv, att, and pscA genes from A. tumefaciens were unable to complement the attachment-minus A. rhizogenes mutants. Thus, the A. rhizogenes nonattaching mutants appear to be different from the previously described A. tumefaciens mutants.

Bacterial Adhesion↗

Loss of CLE40, a protein functionally equivalent to the stem cell restricting signal CLV3, enhances root waving in Arabidopsis.

Continuous growth and development of plants is controlled by meristems that harbour stem cell pools. Division of stem cells and differentiation of their progeny are coordinated by intercellular signaling. In Arabidopsis, stem cells in shoot and floral meristems secrete CLAVATA3, a member of the CLE protein family that activates the CLV1/CLV2 receptor complex in underlying cells to restrict the size of the stem cell population. We found that CLE40 encodes a potentially secreted protein that is distantly related to CLV3. While CLV3 transcripts are confined to stem cells of the shoot system, CLE40 is expressed at low levels in all tissues, including roots. Misexpression and promoter swap experiments show that CLE40 can fully substitute for CLV3 to activate CLV signalling in the shoot, indicating that CLV3 and CLE40 are functionally equivalent proteins that differ mainly in their expression patterns. Analysis of cle40 mutants shows that wild-type expression levels of CLE40 are insufficient to contribute to CLV signalling. High level expression of CLV3 or CLE40 results in a premature loss of root meristem activity, indicating that activation of a CLV-like signaling pathway may restrict cell fate also in roots. The cellular organization of cle40 root meristems is normal, but mutant roots grow in a strongly waving pattern, suggesting a role for CLE40 in a signaling pathway that controls movement of the root tip.

Amino Acid Sequence↗

The diageotropica mutant of tomato lacks high specific activity auxin binding sites.

Tomato plants homozygous for the diageotropica (dgt) mutation exhibit morphological and physiological abnormalities which suggest that they are unable to respond to the plant growth hormone auxin (indole-3-acetic acid). The photoaffinity auxin analog [3H]5N3-IAA specifically labels a polypeptide doublet of 40 and 42 kilodaltons in membrane preparations from stems of the parental variety, VFN8, but not from stems of plants containing the dgt mutation. In roots of the mutant plants, however, labeling is indistinguishable from that in VFN8. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system, which is altered in a tissue-specific manner in the mutant.

Affinity Labels↗

Temperature-sensitive mutations that arrest Arabidopsis shoot development.

To identify genes involved in meristem function we have designed a screen for temperature-sensitive mutations that cause a conditional arrest of early shoot development in Arabidopsis. We describe the characterization of three mutations, arrested development (add) 1, 2 and 3. At the restrictive temperature the add1 and add2 mutations disrupt apical meristem function as assayed by leaf initiation. Furthermore, add1 and add2 plants exhibit defects in leaf morphogenesis following upshift from permissive to restrictive temperature. This result suggests that proximity to a functional meristem is required for the completion of normal leaf morphogenesis. The add3 mutation does not have a dramatic effect on the production of leaves by the apical meristem; however, add3 prevents the expansion of leaf blades at high temperature. Thus, in this mutant the temperature-dependent arrest of epicotyl development is due to a failure of normal leaf development rather than new leaf initiation. While all add mutants have a reduced rate of root growth in comparison to wild-type plants, the mutants do not display a temperature-dependent arrest of root development. All add mutants display some developmental defects at low temperature, suggesting that these mutations affect genes involved in inherently temperature-sensitive developmental processes.

Arabidopsis↗

Nutritionally relevant parameters in low-phytate barley (hordeumvulgare L.) grain mutants.

Nutritionally relevant parameters in barley low-phytate mutant grains were analyzed in order to assess the potential value of these lines for future feeding trials. Phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate) levels in grains from A- and B-type low-phytate mutants corresponded to 25% and 66% of those of the parent line content, respectively. These relative decreases in phytate were accompanied by proportional increases of inorganic phosphate amounts. Apart from phytate, A-type grains also contained substantial quantities of myo-inositol 1,3,4,5-tetrakisphosphate. Phytate levels in straw and root material from mutants were similar to parent line controls, indicating that low-phytate mutations were grain specific. Analysis of K, Mg, Ca, and Zn revealed normal or slightly increased mineral cation levels in grains from all low-phytate lines, suggesting that mutationally impaired phytate accumulation did not affect mineral storage capacity. Other nutritionally important parameters such as starch and protein contents were similar to parent line controls. Finally, dynamic changes in the phosphorus composition during kernel development suggested that A-type mutations directly affected phytate synthesis, whereas B-type mutations seemed to act on regulation of synthesis.

6-Phytase↗

Cloning and characterization of a Nicotiana tabacum methylputrescine oxidase transcript.

The oxidative deamination of N-methylputrescine is an essential step in both pyridine and tropane alkaloid biosynthesis. Reverse genetic approaches have not resulted in the cloning of a methylputrescine oxidase gene (MPO). However, we have used a homology-based approach to clone a full-length tobacco MPO1 cDNA. The MPO1 gene is part of a small multigene family comprised of approximately six members. MPO1-like transcript levels increased in roots that were either deprived of auxin or treated with methyl jasmonic acid. Similar to other known nicotine biosynthetic genes in domesticated tobacco, MPO1-like mRNA levels were lower in roots with the mutant a and b alleles. The MPO1 protein was expressed in bacteria as a recombinant Thioredoxin-His(6)-MPO1 fusion protein. The recombinant MPO1 protein utilized N-methylputrescine more efficiently than other diamines. Therefore, the kinetic properties of the MPO1 enzyme may play an important role in determining the pyridine alkaloid profiles observed in tobacco roots.

Amino Acid Sequence↗

Selenate-resistant mutants of Arabidopsis thaliana identify Sultr1;2, a sulfate transporter required for efficient transport of sulfate into roots.

To investigate how plants acquire and assimilate sulfur from their environment, we isolated and characterized two mutants of Arabidopsis thaliana deficient in sulfate transport. The mutants are resistant to selenate, a toxic analogue of sulfate. They are allelic to each other and to the previously isolated sel1 (selenate-resistant) mutants, and have been designated sel1-8 and sel1-9. Root elongation in these mutants is less sensitive to selenate than in wild-type plants. Sulfate uptake into the roots is impaired in the mutants under both sulfur-sufficient and sulfur-deficient conditions, but transport of sulfate to the shoot is not affected. The sel1 mutants contain lesions in the sulfate transporter gene Sultr1;2 located on the lower arm of chromosome 1. The sel1-1, sel1-3 and sel1-8 mutants contain point mutations in the coding sequences of Sultr1;2, while the sel1-9 mutant has a T-DNA insertion in the Sultr1;2 promoter. The Sultr1;2 cDNA derived from wild-type plants is able to complement Saccharomyces cerevisiae mutants defective in sulfate transport, but the Sultr1;2 cDNA from sel1-8 is not. The Sultr1;2 gene is expressed mainly in roots, and accumulation of transcripts increases during sulfate deprivation. Examination of transgenic plants containing the Sultr1;2 promoter fused to the GUS-reporter gene indicates that Sultr1;2 is expressed mainly in the root cortex, the root tip and lateral roots. Weaker expression of the reporter gene was observed in hydathodes, guard cells and auxiliary buds of leaves, and in anthers and the basal parts of flowers. The results indicate that Sultr1;2 is primarily involved in importing sulfate from the environment into the root.

Alleles↗

Myelin formation by Schwann cells in the absence of beta4 integrin.

The interaction of the Schwann cell with its basal lamina has been hypothesized to be an important prerequisite for the formation of a myelin sheath in the peripheral nervous system. One possible player in this interaction is beta4 integrin; it is up-regulated during myelin formation and, in association with alpha6 integrin, can interact with particular components of the Schwann cell basal lamina. In order to characterize the functional roles of beta4 integrin during myelination, we investigated myelination in the absence of beta4 integrin, i.e., in peripheral nerve tissue from beta4 integrin-deficient mice. Because the mutants die within several hours after birth, we cultured dorsal root ganglia from neonatal mutants under conditions that promote myelination, quantified the myelin segments by immunofluorescence, and investigated the ultrastructure of the cultured myelin sheaths. In another approach, we quantified the few myelin sheaths that are detectable in femoral nerves of newborn animals. Based on both approaches, we conclude that myelination by Schwann cells can occur in the absence of beta4 integrin demonstrating that this Schwann cell component is dispensable for myelin formation in peripheral nerves.

Animals↗

Root graviresponsiveness and columella cell structure in carotenoid-deficient seedlings of Zea mays.

Root graviresponsiveness in normal and carotenoid-deficient mutant seedlings of Zea mays was not significantly different. Columella cells in roots of mutant seedlings were characterized by fewer, smaller, and a reduced relative volume of plastids as compared to columella cells of normal seedlings. Plastids in columella cells of mutant seedlings possessed reduced amounts of starch. Although approximately 10 per cent of the columella cells in mutant seedlings lacked starch, their plastids were located at the bottom of the cell. These results suggest that (i) carotenoids are not necessary for root gravitropism, (ii) graviresponsiveness is not necessarily proportional to the size, number, or relative volume of plastids in columella cells, and (iii) sedimentation of plastids in columella cells may not result directly from their increased density due to starch content. Plastids in columella cells of normal and mutant seedlings were associated with bands of microtubule-like structures, suggesting that these structures may be involved in 'positioning' plastids in the cell.

Carotenoids↗

Molecular genetic analysis of plant gravitropism.

The analysis of mutants is a powerful approach for elucidating the components of complex biological processes. A growing number of mutants have been isolated which affect plant gravitropism and the classes of mutants found thus far provide important information about the gravity response mechanism. The wide variety of mutants isolated, especially in Arabidopsis, indicates that gravitropism is a complex, multi-step process. The existence of mutants altered in either root gravitropism alone, shoot gravitropism alone, or both indicates that the root and shoot gravitropic mechanisms have both separate and common steps. Reduced starch mutants have confirmed the role of amyloplasts in sensing the gravity signal. The hormone auxin is thought to act as the transducing signal between the sites of gravity perception (the starch parenchyma cells surrounding the vascular tissue in shoots and the columella cells of root caps) and asymmetric growth (the epidermal cells of the elongation zone(s) of each organ). To date, all mutants that are resistant to high concentrations of auxin have also been found to exhibit a reduced gravitropic response, thus supporting the role of auxin. Not all gravitropic mutants are auxin-resistant, however, indicating that there are additional steps which do not involve auxin. Studies with mutants of tomato which exhibit either reduced or reversed gravitropic responses further support the role of auxin redistribution in gravitropism and suggest that both red light and cytokinin interact with gravitropism through controlling lateral auxin transport. Plant responses to gravity thus likely involve changes in both auxin transport and sensitivity.

Arabidopsis↗

Rescue of defective auxin-mediated gene expression and root meristem function by inhibition of ethylene signalling in sterol biosynthesis mutants of Arabidopsis.

The roles of sterols in plant development are not well understood, but evidence is emerging that they are required for cell division, polarity and patterning by mechanisms that are independent of brassinosteroids, of which they are precursors. Previous evidence shows that two sterol-defective mutants of Arabidopsis thaliana (L.) Heynh., hyd1 and fk(hyd2), are defective in root development. Here we show that the HYD1 gene, like the FK gene, is transcriptionally active in both primary and lateral root meristems, though not in the shoot apical meristem. The patterns of cell division during early stages of lateral root initiation in the hyd1 and fk(hyd2) mutants appear normal. Previous evidence also suggests that auxin and ethylene signalling is defective in the mutants. Here we show that the cytokinin- and ethylene-responsive ACS1::GUS reporter in the fk(hyd2) mutant responds to exogenous cytokinins but not to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicative of normal cytokinin signalling but supporting the hypothesis that ethylene signalling is defective. The defective root meristem cell division activity and expression patterns of the auxin-regulated DR5::GUS and IAA2::GUS reporters can be rescued to a significant extent by the pharmacological or genetic inhibition of ethylene signalling, but not by treatment with aminoethoxyvinylglycine, an inhibitor of ethylene synthesis. This supports the emerging view that the hyd1 and fk(hyd2) mutants exhibit an enhanced and unregulated ethylene signalling activity, which accounts for at least part of the observed mutant phenotypes, including disrupted auxin signalling. The possible relationship between ethylene signalling, membrane sterols and meristem function is discussed.

Arabidopsis↗