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     

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.     

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⁺, ch¹ und ch² des ch-Locus zugeordnet. ch¹/ch¹ blockiert die Chlorophyll b-Synthese vollständig, ch²/ch² nur etwa zu 90% gegenüber dem Wildtyp und in der Heterozygote ch²/ch¹ wird offenbar die Blockade durch komplementative Effekte noch stärker aufgehoben (Superdominanz des ch²- über das ch¹-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 ch¹/ch¹ und ch²/ch² 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.

---

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⁺, ch¹ and ch²) 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 ch¹/ch¹ the synthesis of chlorophyll b is completely blocked, in ch²/ch² only about 10 percent of the wild-type pigment is present, and in the heterozygote ch²/ch¹ a complementation effect is observed (i. e. superdominance of ch² over ch¹) 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 ch¹/ch¹ and ch²/ch² 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.

---

---

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

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     

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.     

Polyamine content in Arabidopsis thaliana (L.) Heynh during recovery after low and high temperature treatments

Comparative studies on the effect of temperature treatment on the endogenous polyamine content in wild type and the ethylene insensitive mutant eti5 of Arabidopsis thaliana (L.) Heynh were performed. The levels of free and conjugated putrescine, spermidine and spermine were measured in rosette leaves of 38-day-old plants subjected to low and high temperature for 24 h in darkness. Data for fractions measured in treated wild type plants during recovery suggest that alterations in polyamine levels may be a consequence of the conversion of the supernatant-bound into free form and vice versa, while in treated eti5 plants de novo synthesis of spermidine and spermine could not be excluded. It was found that high temperature provoked more significant changes in polyamine levels than low temperature. The results suggest that the eti5 mutant showed a better ability to recover after the temperature treatments than wild type partly as a consequence of changes in polyamine content.     

A Direct Screening Procedure for Gravitropism Mutants in Arabidopsis thaliana (L.) Heynh.

In order to isolate gravitropism mutants of Arabidopsis thaliana (L.) Heynh. var Estland for the genetic dissection of the gravitropism pathway, a direct screening procedure has been developed in which mutants are selected on the basis of their gravitropic response. Variability in hypocotyl curvature was dependent on the germination time of each seed stock, resulting in the incorrect identification of several lines as gravitropism mutants when a standard protocol for the potentiation of germination was used. When the protocol was adjusted to allow for differences in germination time, these lines were eliminated from the collection. Out of the 60,000 M2 seedlings screened, 0.3 to 0.4% exhibited altered gravitropism. In approximately 40% of these mutant lines, only gravitropism by the root or the hypocotyl was altered, while the response of the other organ was unaffected. These data support the hypothesis that root and hypocotyl gravitropism are genetically separable.     

Endogenous gibberellin levels influence in-vitro shoot regeneration in Arabidopsis thaliana (L.) Heynh.

The regeneration of shoot buds from callus cells in vitro is an important technique in modern plant genetic manipulation. Whilst it is clear that genetic factors play a major role in determining the ability of callus cells to become organized into regenerating shoot buds, the precise nature of these factors remains unknown. Here we show that callus derived from mutants of Arabidopsis thaliana which have reduced levels of endogenous bioactive gibberellins (GAs), or reduced responsivity to GAs, regenerates shoot buds more readily than does callus derived from wild-type controls. In addition, exogenous GA reduces, and exogenous paclobutrazol (a GA-biosynthesis inhibitor) increases, the frequency of shoot bud regeneration from wild-type callus. These results show that GA levels play a role in regulating shoot bud regeneration from callus, and suggest that variation in endogenous GA levels or responsivity may account for a major component of the genetic variation in shoot bud regeneration frequency described in other species.     

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.     

Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh.

The catalase multigene family in Arabidopsis includes three genes encoding individual subunits that associate to form at least six isozymes that are readily resolved by nondenaturing gel electrophoresis. CAT1 and CAT3 map to chromosome 1, and CAT2 maps to chromosome 4. The nucleotide sequences of the three coding regions are 70 to 72% identical. The amino acid sequences of the three catalase subunits are 75 to 84% identical and 87 to 94% similar, considering conservative substitutions. Both the individual isozymes and the individual subunit mRNAs show distinct patterns of spatial (organ-specific) expression. Six isozymes are detected in flowers and leaves and two are seen in roots. Similarly, mRNA abundance of the three genes varies among organs. All three mRNAs are highly expressed in bolts, and CAT2 and CAT3 are highly expressed in leaves.     

The use of telotrisomics for centromere mapping in Arabidopsis thaliana (L.) Heynh.

For 4 different telotrisomics of Arabidopsis thaliana segregation ratios are given for a number of genes located on the extra telocentric chromosome. These data are discussed in relation to the meiotic behaviour expected with telotrisomics. By relating the frequencies of recombinants in the progeny of heterozygous telotrisomics with the map position of the loci, the centromere position may be determined (not only in respect to order, but also in respect to distance). Chromosome arms may differ with respect to univalent loss and the amount of recombination suppression near the centromere in telotrisomics.     

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 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).