Analysis of mutations induced by carbon ions in Arabidopsis thaliana

To investigate the nature of mutations induced by accelerated ions in higher plants, the effects of carbon-ion-irradiation were compared with those of electron-irradiation in Arabidopsis thaliana. Point-like mutations and rearrangements were induced at a similar frequency after carbon-ion-irradiation, whereas point-like mutations were more frequently induced after electron-irradiation. Sequence analysis revealed that carbon-ion-induced point-like mutations were mostly short deletions. In the case of rearrangements, deletions, inversions, insertions, and translocations were found. The estimated frequency of deletion induction was comparable to that of fast neutrons. Analysis of chromosome breakpoints revealed that carbon ions frequently deleted small regions around the breakpoints, whereas electron-irradiation often duplicated these regions. Moreover, for both types of radiation, broken ends with microhomologies were frequently rejoined. Results of the breakpoint and broken end analyses suggest that non-homologous end-joining (NHEJ) leads to the rejoining of double strand breaks (dsbs) after cells are exposed to both types of radiation, but the type of NHEJ that occurs as a result of damage is different. The results indicated that carbon-ion-induced mutations are most likely nulls and that the induced rearrangements may arise through a unique mechanism. These findings indicate that accelerated ions are a useful mutagen for both forward and reverse genetics for plants.     

Molecular nature of mutations induced by high-LET irradiation with argon and carbon ions in Arabidopsis thaliana

Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101-124keVμm(-1). In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290keVμm(-1). We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M(2) pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3-5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keVμm(-1) radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5-30.0keVμm(-1) and 27% for 101-124keVμm(-1)). Therefore, the 290keVμm(-1) heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.     

Mutation rate and novel tt mutants of Arabidopsis thaliana induced by carbon ions

Irradiation of Arabidopsis thaliana by carbon ions was carried out to investigate the mutational effect of ion particles in higher plants. Frequencies of embryonic lethals and chlorophyll-deficient mutants were found to be significantly higher after carbon-ion irradiation than after electron irradiation (11-fold and 7.8-fold per unit dose, respectively). To estimate the mutation rate of carbon ions, mutants with no pigments on leaves and stems (tt) and no trichomes on leaves (gl) were isolated at the M2 generation and subjected to analysis. Averaged segregation rate of the backcrossed mutants was 0.25, which suggested that large deletions reducing the viability of the gametophytes were not transmitted, if generated, in most cases. During the isolation of mutants, two new classes of flavonoid mutants (tt18, tt19) were isolated from carbon-ion-mutagenized M2 plants. From PCR and sequence analysis, two of the three tt18 mutant alleles were found to have a small deletion within the LDOX gene and the other was revealed to contain a rearrangement. Using the segregation rates, the mutation rate of carbon ions was estimated to be 17-fold higher than that of electrons. The isolation of novel mutants and the high mutation rate suggest that ion particles can be used as a valuable mutagen for plant genetics.     

Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.     

Comprehensive identification of mutations induced by heavy‐ion beam irradiation in Arabidopsis thaliana

Heavy‐ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy‐ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole‐genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array‐CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy‐ion beams. Array‐CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar‐ion and Fe‐ion irradiation, respectively, with deletion sizes ranging from 149 to 602 180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar‐ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy‐ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy‐ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.     

Specificity of mutations induced by carbon ions in budding yeast Saccharomyces cerevisiae

To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of γ-ray irradiation in the budding yeast Saccharomyces cerevisiae.

The mutational effect and specificity of carbon-ion beams were studied in the URA3 gene of the yeast. Our experiments showed that the carbon ions generated more than 10 times the number of mutations induced by γ-rays, and that the types of base changes induced by carbon ions include transversions (68.7%), transitions (13.7%) and deletions/insertions (17.6%). The transversions were mainly G:C → T:A, and all the transitions were G:C → A:T. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5′-AC(A/T)-3′ sequence were found to be easily changed. Large deletions and duplications were not observed, whereas ion-induced mutations in Arabidopsis thaliana were mainly short deletions and rearrangements. The remarkable feature of yeast mutations induced by carbon ions was that the mutation sites were localized near the linker regions of nucleosomes, whereas mutations induced by γ-ray irradiation were located uniformly throughout the gene.     

Saturating the genetic map of Arabidopsis thaliana with embryonic mutations

One goal of the Arabidopsis genome project is to identify every gene with an essential function in growth and development. Towards that end, the results are reported here of a mapping project designed to enhance the linkage map of Arabidopsis and establish a valuable resource of mutations in essential genes with known map locations. Embryo-defective (emb) mutations were chosen because they represent the most common heritable defect identified following mutagenesis in Arabidopsis. Multiple marker lines with easily scored phenotypes were constructed to facilitate mapping efforts. Recombination data were obtained for 169 mutants defective in embryo-genesis. The chromosomal locations of 110 emb genes are presented in this report. Twenty-six of these genes are tagged with T-DNA. Nine other mutants isolated following seed transformation appear to contain chromosomal translocations. Another 31 mutant genes in the collectiohave been assigned to a linkage group but not yet placed on the map. Nineteen examples of duplicate alleles have also been found. This is consistent with the estimate that approximately 500 genes readily mutate to give an embryo-defective phenotype in Arabidopsis. With continued progress, it may therefore be possible to approach saturation for this important class of mutations. Molecular cloning of these genes should be facilitated by identifying cDNAs and genomic sequences that map to similar locations.     

Leaf-variegated mutations and their responsible genes in Arabidopsis thaliana

Leaf variegation has long been known as a recessive genetic trait in higher plants. Unlike albino mutants, leaf-variegated mutants are non-lethal and thus enable us to study a novel mechanism of plastid development and maintenance. Variegation results from a defect that makes chloroplast development unstable, since at least part of the tissues gives rise to normal chloroplasts. Despite the fact that leaf-variegated mutants have contributed to the findings of maternal inheritance or have been used as genetic markers, these mutations and the responsible loci have been poorly understood at the molecular level. A comprehensive study of the leaf-variegated mutants is possible in Arabidopsis, since such mutants have been known and the cloning can be at relative ease as a model plant. Here I summarize recent progress on characterization of the Arabidopsis leaf-variegated mutants. Detailed analysis of the responsible loci revealed that variegation is caused by a defect in various metabolic pathways related to organelle functions. Thus, studies on these genes provide us with novel redundant mechanisms by which heteroplasmic organelles such as plastids and mitochondria can survive from an environmental stress.     

Cytogenetic effects induced by accelerated carbon ions with shielding

Our work aims to understand the effects of shielding on the induction of biological damage by heavy charged particles and to compare the shielding effects of different materials at the same LET from two aspects: the biological effectiveness including or not including secondary particles emitted at large angles and the biological effectiveness at different angles with respect to the beam direction. We designed and conducted biological experiments to determine the biological effectiveness of 200 MeV/u carbon ions after traversing different shielding materials (Lucite and aluminium). Whole blood samples, which were either attached to the shielding material (48 mm Lucite or 29 mm aluminium)or positioned at 300 cm away from it at different angles with respect to the beam axis, were exposed to carbon ion beams. For comparison, whole blood samples were exposed directly to 200 MeV/u carbon ions. Chromosomal aberrations in lymphocytes were scored. The results indicated that the biological effectiveness per unit dose was not significantly changed by 48 mm Lucite or 29 mm aluminium, and no significant differences were observed in lymphocytes attached to the target and in lymphocytes positioned at a distance of 300 cm away from the target, at 0o angle of the beam axis. However, when plotted as a function of the number of ions hitting the shielding target, the curves are separated and the shield increases the effectiveness per unit ion. The frequency of chromosomal aberrations at tilted angles behind 29 mm Al and 48 mm Lucite was almost the same. These lesions were considered to be caused by secondary particles due to the passage of particles through the shielding materials.     

Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana

Using the cryo-fixation/freeze-substitution method, we studied the ultrastructural changes and behavior of vacuoles and related organelles (rER and Golgi bodies) during microspore and pollen development, and pollen maturation of Arabidopsis thaliana. In young microspores forming exine (pollen outer cell wall), vacuoles looked like those of somatic cells. In microspores during the formation of intine (inner cell wall), a large vacuole appeared which was made by fusion of pre-existing vacuoles and probably absorption of solutions. In the young pollen grain after the first mitosis, a large vacuole was divided into small vacuoles. The manner of division was not by binary fission and centripetally, but by the invagination of tonoplasts from one side to the opposite side of a vacuole. After the second mitosis, somatic type vacuoles disappeared. In mature pollen grains just before germination, membrane-bound structures containing fine fibrillar substances (MBFs) appeared. The MBFs were considered to be storage vacuoles. In pollen grains from flowers in bloom, MBFs changed to lysosomal structures with acid phosphatases (lytic vacuole). They gradually increased in number and volume, and decomposed the cytoplasm. The autolysis of pollen grains is the first finding in this study, which may contribute to the loss of ability of pollen germination after anthesis.     

Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana

Phytosulfokine (PSK) is a peptide growth factor that requires tyrosine sulfation carried out by tyrosylprotein sulfotransferase (TPST) for its activity. PSK is processed from precursor proteins encoded by five genes in Arabidopsis thaliana and perceived by receptor kinases encoded by two genes in Arabidopsis. pskr1‐3 pskr2‐1 and tpst‐1 knockout mutants displayed reduced seed production, indicative of a requirement for PSK peptide signaling in sexual plant reproduction. Expression analysis revealed PSK precursor and PSK receptor gene activity in reproductive organs with strong expression of PSK2 in pollen. In support of a role for PSK signaling in pollen, in vitro pollen tube (PT) growth was enhanced by exogenously added PSK while PTs of pskr1‐3 pskr2‐1 and of tpst‐1 were shorter. In planta, growth of wild‐type pollen in pskr1‐3 pskr2‐1 and tpst‐1 flowers appeared slower than growth in wild‐type flowers. But PTs did eventually reach the base of the style, suggesting that PT elongation rate may not be responsible for the reduced fertility. Detailed analysis of anthers, style and ovules did not reveal obvious developmental defects. By contrast, a high percentage of unfertilized ovules in pskr1‐3 pskr2‐1 and in tpst‐1 siliques displayed loss of funicular PT guidance, suggesting that PSK signaling is required to guide the PT from the transmitting tract to the embryo sac. Cross‐pollination experiments with wild‐type, pskr1‐3 pskr2‐1 and tpst‐1 male and female parents revealed that both the PT and the female sporophytic tissue and/or female gametophyte contribute to successful PT guidance via PSK signaling and to fertilization success.     

Minichromosome stability induced by partial genome duplication in Arabidopsis thaliana

Two partially reconstructed karyotypes (RK1 and RK2) of Arabidopsis thaliana have been established from a transformant, in which four structurally changed chromosomes (α, β, γ, and δ) were involved. Both karyotypes are composed of 12 chromosomes, 2n = 1￠￠+ 3￠￠+ 4￠￠+ 5￠￠+ a￠￠+ g￠￠ = 12 {2}n = {1}\prime \prime + {3}\prime \prime + {4}\prime \prime + {5}\prime \prime + \alpha \prime \prime + \gamma \prime \prime = {12} for RK1 and 2n = 3￠￠+ 4￠￠+ 5￠￠+ a￠￠+ b￠￠+ g￠￠ = 12 {2}n = {3}\prime \prime + {4}\prime \prime + {5}\prime \prime + \alpha \prime \prime + \beta \prime \prime + \gamma \prime \prime = {12} for RK2, and these chromosome constitutions were relatively stable at least for three generations. Pairing at meiosis was limited to the homologues (1, 3, 4, 5, α, β, or γ), and no pairing occurred among non-homologous chromosomes in both karyotypes. For minichromosome α (mini α), precocious separation at metaphase I was frequently observed in RK2, as found for other minichromosomes, but was rare in RK1. This stable paring of mini α was possibly caused by duplication of the terminal tip of chromosome 1 that is characteristic of RK1.     

Analysis of oxidative signalling induced by ozone in Arabidopsis thaliana

We are using acute ozone as an elicitor of endogenous reactive oxygen species (ROS) to understand oxidative signalling in Arabidopsis. Temporal patterns of ROS following a 6 h exposure to 300 nL L(-1) of ozone in ozone-sensitive Wassilewskija (Ws-0) ecotype showed a biphasic ROS burst with a smaller peak at 4 h and a larger peak at 16 h. This was accompanied by a nitric oxide (NO) burst that peaked at 9 h. An analysis of antioxidant levels showed that both ascorbate (AsA) and glutathione (GSH) were at their lowest levels, when ROS levels were high in ozone-stressed plants. Whole genome expression profiling analysis at 1, 4, 8, 12 and 24 h after initiation of ozone treatment identified 371 differentially expressed genes. Early induction of proteolysis and hormone-responsive genes indicated that an oxidative cell death pathway was triggered rapidly. Down-regulation of genes involved in carbon utilization, energy pathways and signalling suggested an inefficient defense response. Comparisons with other large-scale expression profiling studies indicated some overlap between genes induced by ethylene and ozone, and a significant overlap between genes repressed by ozone and methyl jasmonate treatment. Further, analysis of cis elements in the promoters of ozone-responsive genes also supports the view that phytohormones play a significant role in ozone-induced cell death.     

Transmissible and nontransmissible components of anthropometric variation in the Alexanderwohl Mennonites: II. Resolution by path analysis

A study of the intergenerational transmissibilities of 34 anthropometric measures from the Alexanderwohl Mennonite congregations of Kansas and Nebraska is presented. Results presented tend to confirm the suggestion made previously by us (Devor et al., 1985) that patterns of transmissibility conform to a concept of "functional multifactorial complexes" operating in the body in a way analogous to the "field" concept of dental morphology.     

Inhibition of dimethylnitrosamine-induced mutagenesis in Arabidopsis thaliana by diethyldithiocarbamate and carbon monoxide

Diethyldithiocarbamate and carbon monoxide markedly inhibited the frequency of embryonic and chlorophyll mutations induced by the metabolism-requiring mutagen dimethylnitrosamine in the higher plant Arabidopsis thaliana. In contrast, the monoamine oxidase substrates, tryptamine, benzylamine and 2-phenylethylamine, had no such effect. The mutagenicity of a direct-acting mutagen, N-methyl-N-nitrosourea, was not altered by these inhibitors or substrates.     

Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil

Plant sexual reproduction involves the growth of tip-polarized pollen tubes through the female tissues in order to deliver the sperm nuclei to the egg cells. Despite the importance of this crucial step, little is known about the molecular mechanisms involved in this spatial and temporal control of the tube growth. In order to study this process and to characterize the structural composition of the extracellular matrix of the male gametophyte, immunocytochemical and biochemical analyses of Arabidopsis pollen tube wall have been carried out. Results showed a well-defined localization of cell wall epitopes with highly esterified homogalacturonan and arabinogalactan-protein mainly in the tip region, weakly methylesterified homogalacturonan back from the tip and xyloglucan and (1→5)-α-L-arabinan all along the tube. Here, we present complementary data regarding (1) the ultrastructure of the pollen tube cell wall and (2) the immunolocalization of homogalacturonan and arabinan epitopes in 16-h-old pollen tubes and in the stigma and the transmitting tract of the female organ. Discussion regarding the pattern of the distribution of the cell wall epitopes and the possible mechanisms of cell adhesion between the pollen tubes and the female tissues is provided.Key words: arabinan, cell adhesion, cell wall, homogalacturonan, pistil, pollen tube growth, transmitting tractFertilization of flowering plants requires the delivery of the two sperm cells, carried by the fast growing tip-polarized pollen tube, to the egg cell. At every stage of the pollen tube development within the stigma, style and ovary, pollen tubes are guided to the ovules via multiple signals that need to pass through the cell wall of the pollen tube to reach their targets.^1–6The analysis of Arabidopsis pollen tube cell wall has recently been reported.⁷ Results showed a well-defined localization of cell wall epitopes with highly methylesterified homogalacturonan (HG) and arabinogalactan-protein (AGP) mainly in the tip region, weakly methylesterified HG back from the tip and xyloglucan and arabinan all along the tube. In addition, according to the one letter nomenclature of xyloglucan,⁸ the main motif of Arabidopsis pollen tube xyloglucan was XXFG harboring one O-acetyl group. In order to bring new information regarding the possible interaction between the pollen tubes and the female tissues, the ultrastructural organization of the pollen tube cell wall, the cytological staining and immunolocalization of the cell wall epitopes of the pistil and especially the transmitting tract (TT), a specialized tissue where pollen tubes grow, were carried out.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

Misregulation of phosphoinositides in Arabidopsis thaliana decreases pollen hydration and maternal fertility

Phosphoinositides are important lipids involved in membrane identity, vesicle trafficking, and intracellular signaling. In recent years, phosphoinositides have been shown to play a critical role in polarized secretion in plants, as perturbations of phosphoinositide metabolism, through loss of function mutants, result in defects in root hair elongation and pollen tube growth, where polarized secretion occurs rapidly. In the Brassicaceae, responses of stigmatic papillae to compatible pollen are also thought to involve highly regulated secretory events to facilitate pollen hydration and penetration of the pollen tube through the stigmatic surface. We therefore sought to analyze the female sporophyte fertility of the root hair defective4-1 mutant and the PI 4-kinase β1/β2 double mutant, which differentially affect phosphatidylinositol-4-phosphate (PI4P) levels. Stigmas from both mutants supported slower rates of pollen grain hydration, and the fecundity of these mutants was also diminished as a result of failed pollination events. This study therefore concludes that PI4P is integral to appropriate pistil responses to compatible pollen.     

The effect of induced mutations on quantitative traits in Arabidopsis thaliana: Natural versus artificial conditions

Mutations are the ultimate source of all genetic variations. New mutations are expected to affect quantitative traits differently depending on the extent to which traits contribute to fitness and the environment in which they are tested. The dogma is that the preponderance of mutations affecting fitness will be skewed toward deleterious while their effects on nonfitness traits will be bidirectionally distributed. There are mixed views on the role of stress in modulating these effects. We quantify mutation effects by inducing mutations in Arabidopsis thaliana (Columbia accession) using the chemical ethylmethane sulfonate. We measured the effects of new mutations relative to a premutation founder for fitness components under both natural (field) and artificial (growth room) conditions. Additionally, we measured three other quantitative traits, not expected to contribute directly to fitness, under artificial conditions. We found that induced mutations were equally as likely to increase as decrease a trait when that trait was not closely related to fitness (traits that were neither survivorship nor reproduction). We also found that new mutations were more likely to decrease fitness or fitness‐related traits under more stressful field conditions than under relatively benign artificial conditions. In the benign condition, the effect of new mutations on fitness components was similar to traits not as closely related to fitness. These results highlight the importance of measuring the effects of new mutations on fitness and other traits under a range of conditions.     

Reduction of cell size induced by enod40 in Arabidopsis thaliana

An extensive analysis of organ and cell size was performed in three different Arabidopsis lines transformed with the early nodulin gene enod40 under control of the CaMV35S promoter. All three transgenic lines presented a significant decrease in the mean size of both epidermal internode and leaf mesophyll cells. Flow cytometric and image analysis of enod40-transfected protoplasts prepared from wild-type Arabidopsis cell suspensions showed that transient expression of the gene resulted in reduced forward light scattering (a factor correlated with particle size) and cell size. The direct administration of ENOD40 peptide to fresh protoplasts also resulted in reduced forward scattering with respect to the control and to the administration of unrelated peptides. As far as is known this is the first report documenting a biological effect of enod40 at the cellular level in non-legume plants.