Linkage relationships of recessive chlorophyll mutations inArabidopsis thaliana (L.)Heynh

A new method enabling to localize recessive alleles controling lethal embryonal or chlorophyll mutations in linkage groups has been devised and verified. The information on the linkage was obtained in B₁ in repulsion after the crosses with recessive visible markers representing the individual linkage groups. The distinction of four B₁ genotypes was achieved by means of Müller's embryotest. Altogether eight mutant alleles were localized. The allelesch 2411, ch 4062 andX 3 are carried by the first linkage group, the allelesM 33 andM 25 by the third and the allelech 1378 by the fourth linkage group. The mutant allelesch 42 andM 4–6–18 showed the linkage with the markers of the fifth and the sixth linkage groups simultaneously. The possibilities of further development and use of this method are discussed.     

Genetic diversity of natural populations of Arabidopsis thaliana (L.) Heynh. in Karelia

The genetic structure of ten natural populations of Arabidopsis thaliana (L.) Heynh. at eight isozyme loci was studied. The populations were located in the northern part of the species range, 200 km from the north to the south along the Onega Lake coast in Karelia. Considerable genetic diversity (P99% = 43.7, Hobs = 0.003) was revealed that is not typical of populations of self-pollinating plant species. A direct correlation between the proportion of polymorphic loci and geographical latitude was shown (r = 0.68; P < 0.05). It is suggested that a high polymorphism level in Karelian Arabidopsis thaliana (L.) populations increasing from the south to the north is due to extreme environmental conditions in the northern part of the species range. The distribution of genetic diversity within and between populations is typical of self-pollinating species: the larger part of the total diversity resides among populations (GST = 0.583).     

Ultrastructure of microsporogenesis and microgametogenesis inArabidopsis thaliana (L.) Heynh. ecotype Wassilewskija (Brassicaceae)

Summary The process of microsporogenesis and microgametogenesis was studied at the ultrastructural level in wild-typeArabidopsis thaliana ecotype Wassilewskija to provide a basis for comparison with nuclear male-sterile mutants of the same ecotype. From the earliest stage studied to mature pollen just prior to anther dehiscence, microsporocyte/microspore/pollen development follows the general pattern seen in most angiosperms. The tapetum is of the secretory type with loss of the tapetal cell walls beginning at about the time of microsporocyte meiosis. Wall loss exhibits polarity with the tapetal protoplasts becoming located at a distance from the inner tangential walls first, followed by an increase in distance from the radial walls beginning at the interior edge and progressing outward. The inner tangential and radial tapetal walls are completely degenerated by the microspore tetrad stage. Unlike other members of the Brassicaceae that have been studied, the tapetal cells ofA. thaliana Wassilewskija also lose their outer tangential walls, and secretion occurs from all sides of the cells. Exine wall precursors are secreted from the tapetal cells in a process that appears to involve dilation of individual endoplasmic reticulum cisternae that fuse with the tapetal cell membrane and release their contents into the locule. Following completion of the exine, the tapetal cell plastids develop membranebound inclusions with osmiophilic and electron-transparent regions. The plastids undergo ultrastructural changes that suggest breakdown of the inclusion membranes followed by release of their contents into the locule prior to the complete degeneration of the tapetal cells.     

The microtubular cytoskeleton during development of the zygote,proembryo and free-nuclear endosperm inArabidopsis thaliana (L.) Heynh

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm inA. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.Abbreviations DIC differential interference contrast optics - MT microtubule - PPB preprophase band of microtubule We thank Ms. Margaret Travers for her helpful English translation of Yakovlev and Alimova (1976) and Mr. James Whitehead for preparation of Fig. 11. M.C.W. was supported by an Australian Postgraduate Research Award.     

Effect of chronic irradiation on the genetic structure of natural populations of Arabidopsis thaliana (L.) Heynh

The genetic structure of Arabidopsis thaliana populations from in the Chernobyl zone of radioactive contamination was studied. The dynamics of genotypic and allele frequencies of eight polymorphic loci were described. Analysis of the results obtained and earlier data revealed selection for loci Per1 and Acp2. Sharp fluctuations of allele frequencies were observed in consecutive generations of chronically irradiated A. thaliana populations. These fluctuations led to replacement of the most frequent alleles, including those close to fixation.     

The estimation of heritability of some developmental characters in natural populations ofArabidopsis thaliana (L.)Heynh

The investigation concerns to what extent the phenotypic variability of some characters connected with the developmental rate inArabidopsis thaliana (L.)Heynh. is of genetic nature. For this reason, in a variable natural populationSt? the values of coefficients of heritability were estimated by means of offspring-parent regression coefficients and of intraclass correlation coefficients for the three following characters: “number of days to appearance of the flower primordia”, “number of rosette leaves” and “number of rosette leaves per day”. The regression coefficients were calculated (1) by using simple progeny means, (2) the maternal data were repeated with each individual offspring value, and (3) the weighted regression coefficients were used. It has been found that about a half of phenotypic differences in developmental characters studied is determined genetically. The question is discussed whether such heritability estimates can be influenced by dominance or interactions, as it results from considerations about the population structure inArabidopsis. There are no substantial differences between the methods used, and in the given case, the estimation of heritability with the aid of the intraclass correlation coefficient appears to be very advantageous.     

Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) Heynh

An unforeseen side-effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol-1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. 'Columbia' was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol-1) with 0.35 mmol mol-1 CO2 in N2. Ethylene in full-sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol-1 O2. At the same time, silique ACC oxidase mRNA increased three-fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis.     

Heteroblasty in Arabidopsis thaliana (L.) Heynh

 Heteroblasty in Arabidopsis thaliana was analyzed in a variety of plants with mutations in leaf morphology using a tissue-specific β-glucuronidase gene marker. Some mutants exhibited their mutant phenotypes specifically in foliage leaves. The phenotypes associated with the foliage-leaf-specific mutations were also found to be induced ectopically in cotyledons in the presence of the lec1 mutation. Moreover, the features of an emf1lec1 double mutant showed that cotyledons can be partially converted into carpelloids. When heteroblastic traits were examined in foliage leaves in the presence of certain mutations or natural deviations by histochemical analysis of the expression of the tissue-specific marker gene, it was found that ectopic expression of the developmental program for the first foliage leaves in lec1 cotyledons seemed to affect the heteroblastic features of the first set of foliage leaves, while foliage leaves beyond the third position appeared normal. Similarly, in wild-type plants, discrepancies in heteroblastic features, relative to standard features, of foliage leaves at early positions seemed to be eliminated in foliage leaves at later positions. These results suggest that heteroblasty in foliage leaves might be affected in part by the heteroblastic stage of the preceding foliage leaves but is finally controlled autonomously at each leaf position. Received: 9 July 1999 / Accepted: 17 August 1999     

Diversity of Arabidobsis thaliana (L.) Heynh. natural populations at the northern limits of the species range: RAPD analysis

In natural populations of Arabidobsis thaliana (L.) Heynh., occupying northern limits of the species range (Karelia), the level of genetic diversity was evaluated. In two insular and one mainland population variability at 82 RAPD loci was tested. Considerable genetic diversity revealed (P = 4.5%; Hexp = 0.177) was not typical of self-pollinating plant species. It was demonstrated that genetic differentiation among the populations (G(ST)= 0.680) was rather high, pointing to the low level of gene flow in the isolated insular populations. It was suggested that the high level of Arabidopsis population polymorphism in Karelia could be associated with extreme growing conditions at the northern limits of the species range.     

Diversity of Arabidobsis thaliana (L.) Heynh. natural populations at the northern limits of the species range: RAPD analysis

In natural populations of Arabidobsis thaliana (L.) Heynh., occupying northern limits of the species range (Karelia), the level of genetic diversity was evaluated. In two insular and one mainland population variability at 82 RAPD loci was tested. Considerable genetic diversity revealed (P = 4.5%; H _exp = 0.177) was not typical of self-pollinating plant species. It was demonstrated that genetic differentiation among the populations (G _ST = 0.680) was rather high, pointing to the low level of gene flow in the isolated insular populations. It was suggested that the high level of Arabidopsis population polymorphism in Karelia could be associated with extreme growing conditions at the northern limits of the species range.     

Organogenetic capacity of mutant lines and their hybrids ofArabidopsis thaliana (L.) Heynh

An experiment was performed to reveal the organogenetic capacity of some mutant lines ofArabidopsis thaliana (L.) Heynh. and their hybrids. The mutant lines have lower shoot- and root-forming capacity than the normal line, but their hybrids recovered the capacity to the level of the normal parent line, and in some cases they showed hybrid vigor. Leaf segments showed both the shoot- and root-forming capacity in the normal line and in hybrids. Stem segments showed high shoot-forming and low root-forming capacity in all the lines and hybrids. Calluses showed root-forming capacity but scarcely formed shoots in both the parent lines and hybrids.     

The dependence of the frequency of recessive lethal and vital mutations inArabidopsis thaliana (L.)Heynh. on the concentration of N-nitroso-N-methylurea

The dependence of the frequency of recessive lethal (two groups), chlorophyll and morphological mutations on the mutagen concentration was determined in M₂ after subjection to N-nitroso-N-methylurea applied to seeds ofArabidopsis in three concentrations (0·05, 0·10 and 0·20mm). The observed frequencies were compared with the theoretically expected ones for the linear and for two exponential types of dependence, by using the t-test, according to the formulas m=k. C, m=k. C^3/2, m=k. C². No satisfactory agreement with any expected type of dependence was found when directly observed frequencies were used. Since a considerable deficit of mutation frequency was observed in high concentrations, the correction of frequency values was done with respect to the probability of occurence of double mutations. After such a correction, a clear exponential relation was found in both types of lethals and a linear one in chlorophyll and morphological mutations. The probable occurence of multiple mutations should be, therefore, taken into account if the dependence of mutation frequency on the concentration of mutagen is discussed.     

Genphysiologie quantitativer Merkmale bei Arabidopsis thaliana (L.) Heynh.

Zusammenfassung In biometrischen Untersuchungen zur Genetik quantitativer Merkmale wird bisher weitgehend das Problem der Genregulation übersehen. Chlorophyll b-Defektmutanten der Crucifere Arabidopsis thaliana (L.) Heynh. erlauben eine direkte biochemische und physiologische Prüfung ihrer quantitativen Genwirkungen in der Biosynthesekette der Plastidenpigmente. Auf Grund bestimmter Testkreuzungen zwischen zwei Mutanten und der Normalform sowie eines kombinierten Verfahrens zur phänotypischen Klassifizierung bestehend aus präparativer Dünnschichtchromatographie aller Plastidenpigmente, visueller Blattfarbenbonitur, papierchromatographischem Einzelpflanzentest und quantitativer Pigmentmessung der Linien werden in Übereinstimmung mit anderen Autoren die nachweisbaren Genotypen einer Allelenreihe ch ⁺, ch ¹ und ch ² des ch-Locus zugeordnet. ch ¹/ch ¹ blockiert die Chlorophyll b-Synthese vollständig, ch ²/ch ² nur etwa zu 90% gegenüber dem Wildtyp und in der Heterozygote ch ²/ch ¹ wird offenbar die Blockade durch komplementative Effekte noch stärker aufgehoben (Superdominanz des ch ²- über das ch ¹-Allel). Die in den Spaltungsgenerationen erhöhten Chlorophyll- und Carotinoid-Varianzen aller Genotypen gegenüber den Eltern weisen auf einen evtl. polygenen Hintergrund der Gesamtpigmentbildung hin, dessen physiologischer Wirkungsbereich aber relativ zu dem des Hauptgens ch begrenzt ist. Orientierende In-vitro-Versuche ergeben, daß im plastidenfreien Blattgewebe-Extrakt des Wildtyps ch ⁺/ch ⁺ ein Wirkstoffprinzip vorhanden ist, das belichtete Chloroplasten der in vivo pigmentdefekten Genotypen ch ¹/ch ¹ und ch ²/ch ² zur Chlorophyll b-Synthese befähigt. Weiterführende Aussagen über Art, Umfang und Zeitpunkt der hierbei angenommenen Enzymaktivitäts- bzw. Substratsänderungen werden von Versuchen mit variierten Strahlungsbedingungen und verbesserten Nachweismethoden der Pigmentvorstufen erwartet.

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Physiological genetics of quantitative characters in Arabidopsis thaliana (L.) Heynh.Part 1: segregation and biosynthesis of pigments in chlorophyll-b defect mutants

Summary In biometric studies on the genetics of quantitative characters the problem of regulation of the activity of genes is rarely considered. Mutants of Arabidopsis thaliana (L.) Heynh. (Cruciferae), defective for chlorophyll b, permit a direct biochemical and physiological determination of their quantitative gene effects in the biosynthetic pathway of the plastid pigments. In agreement with other authors, the detectable genotypes are found to be based on multiple alleles (ch ⁺, ch ¹ and ch ²) at the ch locus. This evidence was obtained by test crosses of two mutants with the wild type and by a phaenotypic classification of the progeny on the basis of preparatory thin layer chromatography, paper chromatography tests of single plants, and quantitative spectrophotometry of the lines. In ch ¹/ch ¹ the synthesis of chlorophyll b is completely blocked, in ch ²/ch ² only about 10 percent of the wild-type pigment is present, and in the heterozygote ch ²/ch ¹ a complementation effect is observed (i. e. superdominance of ch ² over ch ¹) resulting in still more pigment production. In the segregating generations the variances for chlorophyll and carotinoids of all the genotypes are higher than in the parents and therefore suggest a possible polygenic background for pigment development. However, the physiological effect of the genetic background is slight as compared with the effect of the major gene ch. Preliminary experiments show that in tissue extracts of wild type leaves (ch ⁺/ch ⁺) there is an effective agent which enables irradiated chloroplasts of the defective ch ¹/ch ¹ and ch ²/ch ² genotypes to synthesize chlorophyll b in vitro. Further information on the mechanism, extent and the time of action of the active agent should be obtained by varying the conditions of irradiation and by refining the tests for pigment precursors.

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Die pigmentphysiologischen Untersuchungen wurden durch eine. Sachbeihilfe der Deutschen Forschungsgemeinschaft unterstützt.     

Isolation and biochemical characterization of fusicoccin-insensitive variant lines of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H⁺-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a ³H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.Abbreviations A_o binding site concentration - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-nor-8-hydroxyfusicoccin - K_D dissociation constant of the FCBP-radioligand complex We are grateful to Iris Sandorf and Gudrun Henrichs for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany and by Fonds der Chemischen Industrie (literature provision).     

Isolation of mutations affecting the development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

We screened for mutations deleterious to the freezing tolerance of Arabidopsis thaliana (L.) Heynh. ecotype Columbia. Tolerance was assayed by the vigor and regrowth of intact plants after cold acclimation and freezing. From a chemically mutagenized population, we obtained 13 lines of mutants with highly penetrant phenotypes. In 5 of these, freezing sensitivity was attributable to chilling injury sustained during cold acclimation, but in the remaining 8 lines, the absence of injury prior to freezing suggested that they were affected specifically in the development of freezing tolerance. In backcrosses, freezing sensitivity from each line segregated as a single nuclear mutation. Complementation tests indicated that the 8 lines contained mutations in 7 different genes. The mutants' freezing sensitivity was also detectable in the leakage of electrolytes from frozen leaves. However, 1 mutant line that displayed a strong phenotype at the whole-plant level showed a relatively weak phenotype by the electrolyte leakage assay.     

Gravitropism in leaves of Arabidopsis thaliana (L.) Heynh

In higher plants, stems and roots show negative and positive gravitropism, respectively. However, current knowledge on the graviresponse of leaves is lacking. In this study, we analyzed the positioning and movement of rosette leaves of Arabidopsis thaliana under light and dark conditions. We found that the radial positioning of rosette leaves was not affected by the direction of gravity under continuous white light. In contrast, when plants were shifted to darkness, the leaves moved upwards, suggesting negative gravitropism. Analysis of the phosphoglucomutase and shoot gravitropism 2-1 mutants revealed that the sedimenting amyloplasts in the leaf petiole are important for gravity perception, as is the case in stems and roots. In addition, our detailed physiological analyses revealed a unique feature of leaf movement after the shift to darkness, i.e. movement could be divided into negative gravitropism and nastic movement. The orientation of rosette leaves is ascribed to a combination of these movements.     

Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

Summary Abscisic acid (ABA) has been implicated as a regulatory factor in plant cold acclimation. In the present work, the cold-acclimation properties of an ABA-deficient mutant (aba) of Arabidopsis thaliana (L.) Heynh. were analyzed. The mutant had apparently lost its capability to cold acclimate: the freezing tolerance of the mutant was not increased by low temperature treatment but stayed at the level of the nonacclimated wild type. The mutational defect could be complemented by the addition of exogenous ABA to the growth medium, restoring freezing tolerance close to the wild-type level. This suggests that ABA might have a central regulatory function in the development of freezing tolerance in plants. Cold acclimation has been previously correlated to the induction of a specific set of proteins that have been suggested to have a role in freezing tolerance. However, these proteins were also induced in the aba mutant by low temperature treatment.