Flat-Surface grafting inArabidopsis thaliana

We have developed an efficient method of grafting inArabidopsis thaliana that uses flat-surface cutting and a capillary tube for support of the graft junction. Approximately 40% of grafts resulted in healthy plants that produced amounts of seed comparable to ungrafted plants.     

Interaction ofFLC and late-flowering mutations inArabidopsis thaliana

The phenotype caused by mutations that affect the timing of flowering inArabidopsis thaliana has been most extensively analyzed in the Landsbergerecta (Ler) genetic background. In Ler, the late-flowering phenotype ofFRIGIDA and mutations inLUMINIDEPENDENS is suppressed by the Ler allele ofFLC. In this study, the interactions of nine mutations conferring late flowering with theFLC allele of the Columbia ecotype (FLC-Col), which does not suppress late flowering, were examined. The effect on flowering time of combining six of the mutations withFLC-Col was additive; plants containingFLC-Col withfd, gi, fwa, fha, ft, andfe flowered slightly later than plants containing these mutations with theLer allele ofFLC. In contrast, a synergistic effect was observed betweenFLC-Col and three mutations;fca, fpa, andfve plants became extremely late flowering when combined withFLC-Col. Maximum delay in flowering for the majority of the mutant strains requiredFLC-Col in a homozygous state, although forfpa andfe a single copy ofFLC-Col allowed maximum lateness. In addition, thefd andfe mutations became more dominant in the presence ofFLC-Col.     

Screening for mutations affecting sexual reproduction after activation tagging inArabidopsis thaliana

In this work, a seed-set-based screening was performed on 70 lines of Arabidopsis thaliana after activation tagging mutagenesis to identify mutations in reproductive mechanisms. Five mutants showed significantly lower seed set than the wild type and confirmed the phenotype in the progeny. This phenotype was linked with the marker gene bar carried by T-DNA conferring glufosinate resistance. Genetic analysis revealed that the mutation inheritance was sporophytic in 3 mutants and gametophytic in 2 mutants. In addition, 2 mutants had an extra T-DNA copy. Thus activation tagging can be an effective strategy to identify new mutations affecting sporogenesis or gametogenesis.     

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.     

Development of inflorescences inArabidopsis thaliana

Those mutants were studied whose defects resulted in the morphological changes of inflorescences inArabidopsis thaliana. We characterized newly isolatedcorymbosa mutants andacaulis5 mutants. Thecorymbosa1-1 mutation was caused by the defects in the elongation of pedicels and the previously identifiederecta mutation belonged to this class. Thecorymbosa2-1 mutation was caused mainly by the increase of the number of the floral buds in the inflorescence. The expression of theERECTA gene whose defect resulted to the corymbose inflorescence was analyzed. TheERECTA gene was expressed in subsets of cells in both the peripheral zone and central zone and was thought to have important role for the development of inflorescences. The phenotypes of theacaulis5 mutation was pronounced just after the transition from the vegetative to reproductive growth phase. We found that the expressions of the genes for EXGT-A1 and γ-TIP were drastically reduced in theacaulis5 mutants. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Isolation of nutritional mutants inArabidopsis thaliana

An accumulation method was devised for the isolation of nutritional mutants of the CruciferArabidopsis thaliana. Six mutants were obtained, in all of which the synthesis of thiamine was blocked. The mutants showed chlorophyll deficiencies and were more or less depressed in growth. Thiamine, when added to the substrate, fully restored normal development. Three of the mutants also grew on a substrate containing the pyrimidine moiety of the vitamin molecule, one on a medium with the thiazole part, one on a substrate containing a mixture of both compounds, whereas the sixth mutant required the complete thiamine molecule for normal growth. Two of the three pyrimidine-less mutants were allelic, the third, when crossed with each of the other two, yielded F₁'s showing wild type growth in their youth but deficiency symptoms in later stages. In each of the other mutants different loci were involved. The relation between the method employed and the type of mutant isolated is discussed.     

Genes conferring late flowering inArabidopsis thaliana

Three naturally occurring late flowering, vernalization responsive ecotypes ofArabidopsis thaliana, Pitztal, Innsbruck and Kiruna-2, were each crossed with the early flowering ecotypes of Landsbergerecta, Columbia and Niederzenz. Analysis of the subsequent generations suggested that late flowering in Kiruna-2 is recessive and mainly determined by a single, late flowering gene. This late flowering gene is not, however, the same as that in any of the late flowering mutants generated in the Landsbergerecta background. Both Pitztal and Innsbruck appear to contain the same dominant gene which confers late flowering to these ecotypes. The early flowering parents Niederzenz and Landsberg both contain genes which modify the phenotype of this dominant late flowering locus, causing F₁ plants to flower either earlier (Landsberg) or later (Niederzenz) than the late parent. Mapping of the dominant late flowering locus from Pitztal demonstrated that late flowering co-segregated with an RFLP marker from one end of chromosome 4. This is a similar position to that ofFLA, the gene responsible for late flowering of theArabidopsis ecotypes Sf-2 and Le-O.     

Genetic pathways controlling carpel development inArabidopsis thaliana

Carpel development inArabidopsis is known to be controlled by the organ identity geneAGAMOUS. However, even in the absence of AGAMOUS function, many carpel properties can arise suggesting that other genes are also involved. Two new carpel genes,CRABS CLAW andSPATULA, have been recognised by their specific disruptions to carpel development in mutant plants. These disruptions suggest thatCRABS CLAW normally plays a role in promoting the growth of specific regions of the carpel wall, whereasSPATULA apparently has a primary function in promoting development of the transmitting tract. When the function of these genes is also compromised along with that ofAGAMOUS in multiply mutant plants, carpelloid properties vanish. ThusAGAMOUS, CRABS CLAW andSPATULA act together in specifying carpel development, although none can do this alone. BecauseSPATULA mutants are epistatic to mutants of another carpel development gene,ETTIN, the latter may normally act by suppressing the action ofSPATULA in specific regions of the developing gynoecium. There is indirect evidence thatETTIN, and another morphogenetic gene,PINOID, act through regulating auxin-induced growth in specific regions of the developing flower, but it is not yet known how this could result in the suppression of SPATULA function. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

QTL analysis of flowering time inArabidopsis thaliana

Quantitative trait loci (QTL) analyses based on restriction fragment length polymorphism maps have been used to resolve the genetic control of flowering time in a cross between twoArabidopsis thaliana ecotypes H51 and Landsbergerecta, differing widely in flowering time. Five quantitative trait loci affecting flowering time were identified in this cross (RLN1-5), four of which are located in regions containing mutations or loci previously identified as conferring a late-flowering phenotype. One of these loci is coincident with theFRI locus identified as the major determinant for late flowering and vernalization responsiveness in theArabidopsis ecotype Stockholm.RLN5, which maps to the lower half of chromosome five (between markers mi69 and m233), only affected flowering time significantly under short day conditions following a vernalization period. The late-flowering phenotype of H51 compared to Landsbergerecta was due to alleles conferring late flowering at only two of the five loci. At the three other loci, H51 possessed alleles conferring early flowering in comparison to those of Landsbergerecta. Combinations of alleles conferring early and late flowering from both parents accounted for the transgressive segregation of flowering time observed within the F₂ population. Three QTL,RLN1,RLN2 andRLN3 displayed significant genotype-by-environment interactions for flowering time. A significant interaction between alleles atRLN3 andRLN4 was detected.     

Experimental and genetic analysis of root development inArabidopsis thaliana

The cellular organisation of theArabidopsis thaliana root is remarkably regular. A fate map of the primary root and root meristem that predicts the developmental destinies of cells within the embryonic root primordium has been constructed. Nevertheless, laser ablation experiments demonstrate that root meristem cells develop according to position and not according to lineage. Mutational analysis has identified genes required for cell specification in the radial as well as in the apical-basal dimension. The corresponding gene functions appear to be necessary during embryogenesis for the formation of a correctly patterned primary root. H Lambers Section editor     

Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase inArabidopsis thaliana

Summary NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD-dependent GAPDH) was purified to homogeneity and injected into a rabbit to induce a polyclonal antibody. The antibody was judged to be of high specificity and high affinity. This antibody was used to probe sections ofArabidopsis leaf, stem or roots which were fixed using either paraformaldehyde or a high-pressure freezing method. Our results show that the NAD-dependent GAPDH localizes in the nucleus as well as in the cytosol. In phloem tissue, the NAD-dependent GAPDH was found in companion cells but not in the sieve element.     

Trisomics inArabidopsis thaliana and the location of linkage groups

Different populations of the grasshopper Arcyptera fusca located through a small valley of the Pyrenees present an unstable B-chromosome system. Frequencies of individuals carrying Bs ranged from 11% to 50%. In the testes of these males the number of Bs varied among the different follicles ranging from 0 to 4 with 2 being the number most commonly found. The variation in the number of supernumeraries probably resulted from their preferential non-disjunction in the carly mitosis prior to the differentiation of the follicles. The meiotic behaviour of Bs depends on their number within cach follicle. When two or more Bs are present they usually pair and segregate regularly; B univalents divide in anaphase I and segregate without further division in anaphase II in 75% of the cells observed. The presence of Bs does not affect the chiasma frequency, however, the males with Bs had fewer follicles in their testes; this event could be related with the non-existence of follicles with more than 4 Bs.     

Improved efficiency for T-DNA-mediated transformation and plasmid rescue inArabidopsis thaliana

A vector was constructed for the isolation of gene fusions to thelacZ reporter gene following T-DNA integration into the genome ofArabidopsis thaliana. To facilitate the generation of taggedA. thaliana plants, we established a modified method for high-frequency transformation ofA. thaliana byAgrobacterium tumefaciens. The main modification required was to inhibit the methylation of T-DNA in the transformed calli. Apparently, cytosine residues of thenos-nptII gene used as a selectable marker were methylated, and the expression of this gene was suppressed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regeneration of transformed (kanamycin-resistant) shoots. A total of 150 transgenic plants were isolated, and in 17 of these expression of thelacZ reporter was detected byin situ staining. The T-DNA insert together with flanking plant DNA sequences was cloned intoEscherichia coli by plasmid rescue from some of the T₃ transformants that harbored one copy of the integrated T-DNA. Comparison of the rescued DNA with the corresponding DNA of the transgenic plant showed that most of the rescued plasmids had undergone rearrangements. These rearrangements could be totally avoided if anmcrAB (modified cytosine restriction) mutant ofE. coli was used as the recipient in plasmid rescue.     

Cell biology and genetics of root hair formation inArabidopsis thaliana

Summary In this review we integrate the information available on the cell biology of root hair formation with recent findings from the analysis of root hair mutants ofArabidopsis thaliana. The mature Arabidopsis root epidermis consists of root-hair-producing cells and non-root-hair-producing cells. Root hair growth begins with a swelling of the outer epidermal wall. It has been postulated that this is due to a pH-mediated localised cell wall loosening. From the bulge a single root hair emerges which grows by tip growth. The root hair tip consists of a vesicle-rich zone and an organelle-rich subapical zone. The vesicles supply new plasma membrane and cell wall material for elongation. The cytoskeleton and its associated regulatory proteins such as profilin and spectrin are proposed to be involved in the targeting of vesicles. Ca²⁺ influxes and gradients are present in hair tips, but their function is still unclear. Mutants have been isolated with lesions in various parts of the root hair developmental pathway from bulge identity and initiation, to control of tip diameter and extent and polarity of elongation.Abbreviations [Ca²⁺]_c cytosolic calcium concentration - MT microtubule - PM plasma membrane - VRZ vesicle-rich zone - WT wild type Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Chilling,oxidative stress and antioxidant responses inArabidopsis thaliana callus

Chilling ofArabidopsis thaliana (L.) Heynh. callus tissue to 4 °C led to conditions of oxidative stress, as indicated by increased levels of the products of peroxidative damage to cell membranes. Cellular H₂O₂ was also observed to increase initially upon chilling but by day 8 cellular levels had declined to below control levels. Although levels of catalase activity remained similar to those in unchilled tissue, activity of ascorbate peroxidase increased between days 4 and 8 of chilling to 4 °C. In callus held at 23 °C, levels of reduced glutathione remained static whereas they rose in callus held at 4 °C. Levels of oxidised glutathione were initially low but increased significantly by day 4 in the chilled callus. At 23 °C, however, levels of oxidised glutathione remained low. Between days 1 and 3 at 4 °C, levels of glutathione reductase activity increased but by day 8 glutathione reductase activity was similar to that in cells held at 23 °C. Exposure of callus to abscisic acid at 23 °C also led to increased activities of ascorbate peroxidase and glutathione reductase.Abbreviations ABA abscisic acid - GSH reduced glutathione - GSSG oxidised glutathione - TTC 2,35-triphenyltetrazolium chloride This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

Root apical organization inArabidopsis thaliana 1. Root cap and protoderm

Summary We investigated the development of the root cap and protoderm inArabidopsis thaliana root tips.A. Thaliana roots have closed apical organization with the peripheral root cap, columella root cap and protoderm developing from the dermatogen/calyptrogen histogen. The columella root cap arises from columella initials. The initials for the peripheral root cap and protoderm are arranged in a collar and the initiation event for these cells occurs in a sequential pattern that is coordinated with the columella initials. The resulting root cap appears as a series of interconnected spiraling cones. The protoderm, in three-dimensions, is a cylinder composed of cell files made up of packets of cells. The number of cell files within the protoderm cylinder increases as the root ages from one to two weeks. The coordinated division sequence of the dermatogen/calyptrogen and the increase in the number of protoderm cell files are both features of post-embryonic development within the primary root meristem.Abbreviations RCP root cap/protoderm - CI columella initial - PI protoderm initial     

The very low mutagenic activity of sodium azide inArabidopsis thaliana

Sodium azide, reported to be a strong mutagen in barley, revealed a very weak mutagenic activity inArabidopsis.