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.     

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).     

Genetic base of Arabidopsis thaliana (L.) Heynh.: Fitness of plants for extreme conditions in northern margins of species range

Flowering time and vernalization requirement were studied in eight natural Karelian populations (KPs) of Arabidopsis thaliana. These KPs consisted of late-flowering plants with elevated expression of flowering repressor FLC and a reduced expression level of flowering activator SOC1 compared to the early-flowering ecotypes Dijon-M and Cvi-0. Despite variations in flowering time and the vernalization requirement among the KPs, two-week-old seedlings showed no changes in either the nucleotide sequence of the FRI gene or the relative expression levels of FRI and its target gene FLC that would be responsible for this variation. An analysis of abscisic acid (ABA) biosynthesis and catabolism genes (NCED3 and CYP707A2) did not show significant differences between late-flowering KPs and the early-flowering ecotypes Dijon-M and Cvi-0. Cold treatment (4°C for 24 h) induced the expression of not only NCED3, but also RD29B, a gene involved in the ABA-dependent cold-response pathway. The relative levels of cold activation of these genes were nearly equal in all genotypes under study. Thus, the ABA-dependent cold response pathway does not depend on FLC expression. The lack of significant differences between northern populations, as well as the ecotypes Dijon- M (Europe) and Cvi-0 (Cape Verde Islands), indicates that this pathway is not crucial for fitness to the northern environment.     

Genotypic control of the speed of development inArabidopsis thaliana (L.)Heynh. lines obtained from natural populations

Sixteen early, medium late, and late homozygote lines ofArabidopsis thaliana, obtained from three natural populations, were crossed with a common check variety S 96. The speed of development was expressed by the value log (x+y), wherex stands for the number of days after which flower primordia appear, andy for the number of leaves in the rosette. The experiments were caried out under conditions which emphasized the differences in the speed of development. We assessed the degree of dominancy, heritability on the basis of F₁ and F₂ variance, and analyzed F₂ distributions. It was established that some of the investigated lines differ from the variety S 96 in one pair of dominant genes or genes with incomplete dominancy for late flowering, other in two pairs of genes with complementary effects, and that the variety S 96 probably also contains other alleles causing late flowering inhibited by some type of epistasis. Even though the most important part is always played by one or two pairs of genes with high dfficiency, genes with lower efficiency often take part in the process; in some lines the genes with high efficiency could not be established.     

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.     

Genetic Diversity in Natural Populations of Arabidopsis thaliana (L.) Heynh. from 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 (P _99% = 43.7, H _obs = 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 (G _ST = 0.583).     

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     

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.     

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.     

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT

Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996     

pho3: a phosphorus-deficient mutant of Arabidopsis thaliana (L.) Heynh

A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component involved in regulating P nutrition. Received: 21 March 2000 / Accepted: 15 August 2000     

Hormone-response mutants of Arabidopsis thaliana (L.) Heynh. impaired in somatic embryogenesis

Plant hormones are considered to be the key factors involved in triggering in vitro induced plant morphogenesis, including somatic embryogenesis (SE). Mutants affected in SE and altered in hormonal response therefore provide valuable material for genetic research on in vitro induced plant embryogenesis. The capacity for SE was studied in 27 mutants with defects in response to different plant hormones: auxin, ABA, gibberellin and cytokinin, and evaluated in 2-week-old mutant and wild-type cultures in terms of their efficiency and productivity. SE was induced in vitro via a direct morphogenic pathway, through the culture of immature zygotic embryos on standard solid medium with 5 μM 2,4-D. The majority of the analyzed mutants displayed a significantly impaired capacity for SE; and those affected belonged to several different hormone-defective groups, including forms affected in auxin (axr4), gibberellin (ga) and ABA (abi, hyl1, cpb20, abh1) response. These mutants showed a significant decrease in embryogenic response as manifested by a low efficiency and/or productivity of SE. Additionally, SE efficiency was analyzed for axr4-1 mutant on media supplemented with different auxins while GA₃ and inhibitors of gibberellins (uniconazol P and paclobutrazol), were applied for pkl1-1-mutant. The selected mutants provide a valuable research tool for studying the molecular mechanisms determining the induction of embryogenesis in cultures of somatic tissues. Their usefulness in further studies is discussed.     

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.