A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology

A root gravitropism mutant was isolated from the DuPont Arabidopsis thaliana T-DNA insertional mutagenesis collection. This mutant has reduced root gravitropism, hence the name rgrl. Roots of rgrl are shorter than those of wild-type, and they have reduced lateral root formation. In addition, roots of rgrl coil clockwise on inclined agar plates, unlike wild-type roots which grow in a wavy pattern. The rgrl mutant has increased resistance, as measured by root elongation, to exogenously applied auxins (6-fold to indole-3-acetic acid, 3-fold to 2,4-dichlorophenoxyacetic acid, and 2-fold to napthyleneacetic acid). It is also resistant to polar auxin transport inhibitors (2-fold to triiodobenzoic acid and 3- to 5-fold lo napthyleneacetic acid). The rgrl mutant does not appear to be resistant to other plant hormone classes. When grown in the presence of 10^?2 M 2.4-dichlorophenoxyacetic acid, rgrl roots have fewer root hairs than wild type. All these rgrl phenotypes are Mendelian recessives. Complementation tests indicate that rgrl is not allelic to previously characterized agravitropic or auxin-resistant mutants. The rgrl locus was mapped using visible markers to 1.4 ± 0.6 map units from the CHI locus at 1–65.4. The rgrl mutation and the T-DNA cosegregate, suggesting that rgrl was caused by insertional gene inactivation.     

A double mutant allele,csr1-4, of Arabidopsis thaliana encodes an acetolactate synthase with altered kinetics

A comparison is made of the kinetic characteristics of acetolactate synthase (EC 4.1.3.18) in extracts from Columbia wild type and four near-isogenic, herbicide-resistant mutants of Arobidopsis thaliana (L.) Heynh. The mutants used were the chlorsulfuron-resistant GH50 (csr1-1), the imazapyr-resistant GH90 (csr1-2), the triazolopyrimidine-resistant Tzp5 (csr1-3) and the multiherbicide-resistant, double mutant GM4.8 (csr1-4), derived from csr1-1 and csr1-2 by intragenic recombination (G. Mourad et al. 1994, Mol. Gen. Genet. 243, 178–184). and V _max values for the substrate pyruvate were unaffected by any of the mutations giving rise to herbicide resistance. Feedback inhibition by L-valine (L-Val), L-leucine (L-Leu) and L-isoleucine (L-Ile) of acetolactate synthase extracted from wild type and mutants fitted a mixed competitive pattern most closely. K_i values for L-Val, L-Leu and L-Ile inhibition were not significantly different from wild type in extracts from csr1-1, csr1-2, and csr1-3. K _i values were significantly higher than wild type by two- and five-fold, respectively, for csr1-4 with L-Val and L-Leu but not L-Ile. GM4.8 (csr1-4) plants were also highly resistant in their growth to added L-Val and L-Leu. The data suggest that (i) single mutational changes occurred that affected the binding of herbicides to the acetolactate synthase molecule without influencing the binding of substrates and feedback inhibitors (e.g. csr1-1, csr1-2 and csr1-3) and (ii) bringing two of these single mutations (csr1-1 and csr1-2) together in a double mutant (csr1-4) gave rise to an enzyme with altered characteristics as well as plants with changed growth in response to added L-Val and L-Leu. The implications of these conclusions for genetic transformation using these herbicide-resistant genes are discussed.Abbreviations ALS acetolactate synthase - L-Val L-valine - L-Leu L-leucine - L-Ile L-isoleucine This work was supported in part by a grant-in-aid of research from the Natural Sciences and Engineering Research Council of Canada to J.K. and by a research grant award from Purdue University to G.M.     

Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

Molecular basis of alpha-methyltryptophan resistance in amt-1, a mutant of Arabidopsis thaliana with altered tryptophan metabolism.

A mutant of Arabidopsis thaliana, amt-1, was previously selected for resistance to growth inhibition by the tryptophan analog alpha-methyltryptophan. This mutant had elevated tryptophan levels and exhibited higher anthranilate synthase (AS) activity that showed increased resistance to feedback inhibition by tryptophan. In this study, extracts of the mutant callus exhibited higher AS activity than wild-type callus when assayed with either glutamine or ammonium sulfate as amino donor, thus suggesting that elevated AS activity in the mutant was due to an alteration in the alpha subunit of the enzyme. The mutant also showed cross-resistance to 5-methylanthranilate and 6-methylanthranilate and mapped to chromosome V at or close to ASA1 (a gene encoding the AS alpha subunit). ASA1 mRNA and protein levels were similar in mutant and wild-type leaf extracts. Levels of ASA1 mRNA and protein were also similar in callus cultures of mutant and wild type, although the levels in callus were higher than in leaf tissue. Sequencing of the ASA1 gene from amt-1 revealed a G to A transition relative to the wild-type gene that would result in the substitution of an asparagine residue in place of aspartic acid at position 341 in the predicted amino acid sequence of the ASA1 protein. The mutant allele in strain amt-1 has been renamed trp5-1.     

A mutant of Arabidopsis thaliana displaying altered patterns of cellulose deposition

A homozygous recessive mutant of Arabidopsis thaliana has been selected which displays altered patterns of cellulose deposition. The mutant was selected because leaf and stem trichomes lacked the strong birefringence under polarized light which is characteristic of plant cells which contain highly ordered cellulose in their secondary cell walls. Compared with wild-type A. thaliana, this mutant (designated tbr for trichome birefringence) also displays reduced birefringence in the xylem of the leaf. Direct chemical analyses of root, stem, and leaf tissues, including isolated leaf trichomes, support the conclusion that tbr is impaired in its ability to deposit secondary wall cellulose in specific cell types, most notably in trichomes where the secondary wall appears to be totally absent. Altered patterns of wound-induced callose deposition in trichomes and surrounding cells is another trait which also co-segregates with the tbr mutation.     

The altered responses of a new mutant long life span 1 to cytokinin in Arabidopsis thaliana

Cytokinins are a class of essential plant hormones regulating plant growth and development.Although the two-component phosphorelay pathway of cytokinin has been well characterized,the intact cytokinin responses regulation picture still needs to be fully depicted.Here we report a new mutant,long life span 1(lls1),which displays dwarf stature,curled leaves,numerous axillary branches and nearly 5-month life span.Exogenous cytokinin could not recover the phenotypes of the mutant.Moreover,mutation in lls1 suppressed the cytokinin-responsive phenotypes,including root and hypocotyl growth inhibition,anthocyanin accumulation,metaxylem promotion in primary root development.The induction of cytokinin-responsive genes,ARR5,AHP5,and CKX3,was also suppressed in lls1.According to quantitative RT-PCR(qRT-PCR) and microarray results,the basal expression of positive factors AHP5,ARR1,and ARR10 were down-regulated,while the negative factors ARR4 and ARR5 were up-regulated.Our results suggested that LLS1 gene might be involved in the regulation of cytokinin signaling.It was mapped to chromosome 4 where no other cytokinin relevant gene has been reported.     

Mutants of Arabidopsis thaliana with altered phototropism

Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and second positive phototropism. However, while the amplitude for first positive phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in first positive phototropism are shifted to higher fluence by a factor of 20–30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 mol·m^-2 to 2700 mol·m^-2, but shows normal gravitropism. Strain JK345 shows no first positive phototropism, and reduced gravitropism and second positive phototropism. Strain JK229 shows no measurable first positive phototropism, but normal gravitropism and second positive phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. first positive and second positive phototropism contain at least one common element; and 3. first positive phototropism can be substantially altered without any apparent alteration of second positive phototropism.Abbreviation WT wild-type     

Phenotypic diversity and altered environmental plasticity in Arabidopsis thaliana with reduced Hsp90 levels

The molecular chaperone HSP90 aids the maturation of a diverse but select set of metastable protein clients, many of which are key to a variety of signal transduction pathways. HSP90 function has been best investigated in animal and fungal systems, where inhibition of the chaperone has exceptionally diverse effects, ranging from reversing oncogenic transformation to preventing the acquisition of drug resistance. Inhibition of HSP90 in the model plant Arabidopsis thaliana uncovers novel morphologies dependent on normally cryptic genetic variation and increases stochastic variation inherent to developmental processes. The biochemical activity of HSP90 is strictly conserved between animals and plants. However, the substrates and pathways dependent on HSP90 in plants are poorly understood. Progress has been impeded by the necessity of reliance on light-sensitive HSP90 inhibitors due to redundancy in the A. thaliana HSP90 gene family. Here we present phenotypic and genome-wide expression analyses of A. thaliana with constitutively reduced HSP90 levels achieved by RNAi targeting. HSP90 reduction affects a variety of quantitative life-history traits, including flowering time and total seed set, increases morphological diversity, and decreases the developmental stability of repeated characters. Several morphologies are synergistically affected by HSP90 and growth temperature. Genome-wide expression analyses also suggest a central role for HSP90 in the genesis and maintenance of plastic responses. The expression results are substantiated by examination of the response of HSP90-reduced plants to attack by caterpillars of the generalist herbivore Trichoplusia ni. HSP90 reduction potentiates a more robust herbivore defense response. In sum, we propose that HSP90 exerts global effects on the environmental responsiveness of plants to many different stimuli. The comprehensive set of HSP90-reduced lines described here is a vital instrument to further examine the role of HSP90 as a central interface between organism, development, and environment.     

Nucleotide sequence variation at two genes of the phenylpropanoid pathway, the FAH1 and F3H genes, in Arabidopsis thaliana

The FAH1 and F3H genes encode ferulate-5-hydroxylase and flavanone-3-hydroxylase, which are enzymes in the pathways leading to the synthesis of sinapic acid esters and flavonoids, respectively. Nucleotide variation at these genes was surveyed by sequencing a sample of 20 worldwide Arabidopsis thaliana ecotypes and one Arabidopsis lyrata spp. petraea stock. In contrast with most previously studied genes, the percentage of singletons was rather low in both the FAH1 and the F3H gene regions. There was, therefore, no footprint of a recent species expansion in the pattern of nucleotide variation in these regions. In both FAH1 and F3H, nucleotide variation was structured into two major highly differentiated haplotypes. In both genes, there was a peak of silent polymorphism in the 5' part of the coding region without a parallel increase in silent divergence. In FAH1, the peak was centered at the beginning of the second exon. In F3H, nucleotide diversity was highest at the beginning of the gene. The observed pattern of variation in both FAH1 and F3H, although suggestive of balancing selection, was compatible with a neutral model with no recombination.     

Disruption of NAP1 genes in Arabidopsis thaliana suppresses the fas1 mutant phenotype,enhances genome stability and changes chromatin compaction

Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.     

bor1-1, an Arabidopsis thaliana mutant that requires a high level of boron.

bor1-1 (high boron requiring), an Arabidopsis thaliana mutant that requires a high level of B, was isolated. When the B concentration in the medium was reduced to 3 microM, the expansion of rosette leaves was severely affected in bor1-1 but not in wild-type plants. In a medium containing 30 microM B the mutant grew normally but showed female sterility, whereas the wild type was able to set seeds. These defects of the bor1-1 mutant were not detected with supplementation of 100 microM B. In vivo concentrations of B in bor1-1 mutants were lower than those of the wild type, especially in the inflorescence stems. Tracer experiments using 10B suggested that the mutant has defects in uptake and/or translocation of B. The mutation was mapped on the lower arm of chromosome 2.     

Flower development in the organ number mutant clavata1-1 of Arabidopsis thaliana (Brassicaceae)

Flowers of the organ number (meristic) mutant clavata1-1 of Arabidopsis thaliana (Brassicaceae) were studied to examine timing and patterns of floral organogenesis as compared to the wild type. All clavata1-1 flowers examined had four- instead of two-loculed gynoecia; half showed increased numbers of stamens; and 10% formed increased numbers of sepals. An inflorescence plastochron index was used to establish the timing of developmental events during flower organogenesis. clavata1-1 flowers initiate faster but grow more slowly than in the wild type. The stages of sepal and stamen initiation were prolonged compared to those of the wild type. Although gynoecial initiation was not prolonged, the preceding stage was and it was characterized by a proliferation of meristematic cells above the initiating stamens. The clavata1-1 flower apex did not become wider than that of the wild type until after the establishment of the gynoecium. We propose that clavata1-1 is a heterochronic mutant, where flower organ number increases are due partly to prolongation of organ initiation stages.     

Wilt-induced ABA biosynthesis, gene expression and down-regulation of rbcS mRNA levels in Arabidopsis thaliana

The kinetics of wilt-induced abscisic acid (ABA) biosynthesis were investigated in shoots of Arabidopsis thaliana (L.) Heynh Landsberg erecta. ABA concentrations were measured using a radioimmunoassay (RIA) based on the monoclonal antibody MAC 252, and the RIA validated by comparison with combined gas chromatography-mass spectrometry using a [²H₃] labelled internal standard. The basal ABA content of Arabidopsis shoots was ca 10 ng g^?1 fresh weight; the concentrations had increased ca 4-fold within 30 min of the initiation of wilting, increased ca 8-fold after 4 h and 11-fold after 8 h. This stress-induced ABA production was dependent on de novo gene expression; pre-treatment of leaves and shoots with the metabolic inhibitors cordycepin and cycloheximide reduced the rate of subsequent stress-induced ABA biosynthesis from 12.5 ng g^?1 h^?1 to 1 ng g^?1 h^?1 and 0 ng g^?1 h^?1, respectively. In vitro translation of mRNA isolated from shoots subjected to wilting or ABA treatment followed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed only minor changes. The effects of wilting and ABA on the content of total ribulose 1,5-bisphosphate carboxylase/oxygenase small sub-unit (rbcS) mRNA were also determined. Both wilting and exogenous ABA resulted in a substantial reduction in the amount of rbcS mRNA, an effect readily reversed by rehydration of wilted shoots. However, the effects of wilting were not mediated solely by newly-synthesised endogenous ABA, as wilting also reduced rbcS mRNA levels in the ABA-deficient aba-1 mutant, which did not produce ABA in response to loss of turgor. The amount of rbcS mRNA was higher in aba-1 shoots, suggesting that cellular rbcS mRNA levels are normally down-regulated by ABA. Cold treatment induced ABA production in wild type shoots only, but resulted in an increased rbcS mRNA content of both wild type and aba-1 shoots.     

Molecular characterization of two highly homologous receptor-like kinase genes, RLK902 and RKL1, in Arabidopsis thaliana

Receptor-like kinases (RLKs) constitute a large family of signal perception molecules. We characterized two highly homologous RLK genes, RLK902 and RKL1, in Arabidopsis. RLK902 and RKL1 showed a 75% amino acid sequence identity over their entire regions. In the RLK902 pro::GUS transgenic lines, GUS activity was strong in the root tips, lateral root primordia, stipules, and floral organ abscission zones, while the RKL1 promoter activity was dominant in the stomata cells, hydathodes and trichomes of young rosette leaves, and floral organ abscission zones. Neither the rlk902 mutant line, rkl1 mutant line nor rlk902/rkl1 double-knockout mutant line showed any significant phenotypes under normal growth conditions. These results suggest that RLK902 and RKL1 might mediate the signal transduction pathway in which at least one other complementary signaling pathway to these two RLKs might exist.     

A novel male-sterile mutant of Arabidopsis thaliana,faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine

A mutant exhibiting conditional male sterility, in which fertility was restored under conditions of high humidity, was identified in T-DNA tagged lines of Arabidopsis thaliana. Scanning electron microscopy (SEM) demonstrated that the pollen surface was almost smooth and the reticulate pattern not prominent. Thus, the mutant was named faceless pollen-1 (flp1). Transmission electron microscopy (TEM) revealed that the smooth appearance was due to tryphine filling in the exine cavities and covering the pollen surface. The lipid droplets in the tryphine of mutant pollen were smaller and more numerous than those of the wild type. SEM analysis also demonstrated that pollen exine was easily damaged by acetolysis, suggesting that a component of exine, sporopollenin, was defective in the mutant. In addition, the stems and siliques had reduced amounts of wax crystals. A predicted amino acid sequence of the cDNA that corresponded to the tagged gene, fip1, showed sequence similarity to proteins involved in wax biosynthesis. The FLP1 protein is likely to play a role in the synthesis of the components of tryphine, sporopollenin of exine and the wax of stems and siliques.