Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis.

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-betaII isoenzyme involved in human insulin signaling.

Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis.

The cpd mutation localized by T-DNA tagging on Arabidopsis chromosome 5-14.3 inhibits cell elongation controlled by the ecdysone-like brassinosteroid hormone brassinolide. The cpd mutant displays de-etiolation and derepression of light-induced genes in the dark, as well as dwarfism, male sterility, and activation of stress-regulated genes in the light. The CPD gene encodes a cytochrome P450 (CYP90) sharing homologous domains with steroid hydroxylases. The phenotype of the cpd mutant is restored to wild type both by feeding with C23-hydroxylated brassinolide precursors and by ectopic overexpression of the CPD cDNA. Brassinosteroids also compensate for different cell elongation defects of Arabidopsis det, cop, fus, and axr2 mutants, indicating that these steroids play an essential role in the regulation of plant development.

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana.

A recessive pale mutation, designated as cs, was identified by transferred-DNA (T-DNA)-mediated insertional mutagenesis in Arabidopsis thaliana. The pale mutation, cosegregating with the hygromycin resistance marker of the T-DNA, was mapped to the position of the ch-42 (chlorata) locus on chromosome 4. Lack of genetic complementation between cs and ch-42 mutants indicated allelism. Plant boundaries of the T-DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full-length cDNA clones of the wild-type cs gene. Transformation of the pale mutant with T-DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T-DNA induced mutation. DNA sequence comparison of the cs mutant and its wild-type allele revealed that the T-DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild-type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark-light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.

High-frequency T-DNA-mediated gene tagging in plants.

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')II (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')II enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus- and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.

Aneuploidy detection with a short-term hexaploid wheat assay.

A new type of assay for the identification of agents causing aneuploidy is described. This assay takes advantage of allohexaploid wheat in which monosomic and nullisomic cell lineages can be genetically detected. The wheat strain used (Neatby's virescens) was homozygous for a pair of recessive alleles (v1) which in homozygous condition interfere with normal pigmentation of the leaves at low temperature whereas at higher temperature nearly normal green color formation is permitted. In a single dose this allele cannot suppress the formation of green color even at low temperature, i.e., it is hemizygous ineffective. This locus is in the short arm of chromosome 3B near the centromere. As a consequence of non-disjunction of this chromosome twin sectors may be detected, in which the monosomic cell lineages appear green whereas the trisomic sectors display with color on a cream-colored background at low temperature. This genetic system can also be used for the detection of deletions or duplications involving the short arm of chromosome 3B, and to some extent the A- and the D-genome homeologues. We have determined the pattern of differentiation of the shoot apex and on that basis we can separate the independent genetic events from reappearance of the sectors of common origin in the successive leaves. Such an understanding of development of the leaf sectors permits a quantitative estimation of the genetic response of the plants to mutagenic factors. We have found that X-rays, gamma-rays, p-fluorophenylalanine, 3-aminotriazole, caffeine, vinblastin sulfate, benzo[a]pyrene and auramine significantly increased aneuploidy, and diethylstilbestrol, sulfacetamide, safrole and dichlorvos caused some increase of sectoring. Cytological data on root tips of irradiated seeds support the interpretation of the mechanism of sector formation in the leaves. The test is simple, fast, inexpensive, and it does not require elaborate facilities or highly trained technicians. The trials were well reproducible during a period of 3 years in 2 laboratories. Therefore we consider the new assay a useful complement to other tests of chemicals or physical agents that may cause non-disjunction and other chromosomal aberrations in human populations.

Mutagen assay with Arabidopsis. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The small crucifer Arabidopsis is very economical for short-term yet complete assay for chemical mutagens. Thousands of gene loci can be simultaneously monitored for alterations within one culture by screening for segregation among the embryos (M2) developing on the M1 plants. This species is quite sensitive to a wide range of compounds and can activate promutagens to mutagens. There is a very high correlation between mutagenic responses of Arabidopsis and carcinogenicity in animals.

Gene locus specificity of the glucose effect in the thiamine pathway of the angiosperm, Arabidopsis.

All mutants at 3 loci in Arabidopsis thaliana (L.) Heynh., a higher plant, that are associated with the synthesis or coupling of the thiazole moiety of thiamine are susceptible to reversible glucose inhibition. In contrast, several different alleles involved in the synthesis of the pyrimidine moiety of the vitamin are insensitive to glucose. Glucose and maltose are equally effective inhibitors while fructose, lactose, ribose, and xylose are toxic. This toxicity is not released by added thiamine.

Direct evidence for models of heterosis provided by mutants of Arabidopsis blocked in the thiamine pathway.

Auxotrophic mutants genetically blocked at different steps of the thiamine pathway dramatically demonstrate the biochemical mechanism of hybrid vigor due to simple and perfect dominance at two unlinked loci. Heteroallelic hybrids of mutants requiring the pyrimidine moiety of thiamine display allelic complementation and thus furnish clear biochemical and genetic evidence for the superdominance hypothesis. Hybrids of low- and high-temperature-requiring leaky mutants demonstrate that heterozygosity at a single gene locus may confer developmental homeostasis on the heteroallelic combinations superior to that of the homoallelic parents. The results of this study on the autogamous plant, Arabidopsis, and of recent reports on the outbreeding species, Drosophila, render untenable the generalization that high versus low temperature dependent heterosis is determined by the breeding system.ZUSAMMENFASSUNG: Auxotrophe Arabidopsis-Mutanten, bei denen verschiedene Schritte der Thiaminsynthese genetisch blockiert sind, lassen deutlich den biochemischen Mechanismus der Heterosis erkennen, der auf einfacher und völliger Dominanz in zwei ungekoppelten Loci beruht. Heteroallele Hybriden von Mutanten, die den Pyrimidinanteil des Thiamins benötigen, zeigen allele Komplementation und liefern damit den klaren biochemischen und genetischen Beweis für die Superdominanz-Hypothese. Hybriden von leaky-Mutanten, die einen niedrigen bzw. hohen Temperaturbedarf haben, zeigen, daß Heterozygotie in einem einzelnen Genlocus den heteroallelen Kombinationen eine Entwicklungshomeostasie verleihen kann, die größer als die der homoallelen Eltern ist. Die Ergebnisse unserer Untersuchungen an der autogamen Pflanze Arabidopsis und neuere Ergebnisse bei Drosophila lassen die Verallgemeinerung, daß die Art der Temperaturabhängigkeit der Heterosis durch das Zuchtsystem bestimmt wird, nicht zu.

Early flowering in Arabidopsis induced by DNA base analogs.

Various isogenic lines of Arabidopsis differing in a single factor controlling flower initiation were cultured aseptically on media containing bromodeoxycytidine and bromodeoxyuridine (10(-5) M). The wild type under short-day illumination (8 hours daily) and the late mutant gi (2), in both continuous light and under short day, responded with dramatically earlier flower initiation. Another late mutant (ld) failed to respond. The effect of the DNA base analogs was nullified by the corresponding normal deoxyribonucleosides but not by the corresponding normal ribonucleosides.

Acceleration of flowering of the long-day plant Arabidopsis by 8-azaadenine.

The incorporation of 8-azaadenine (2×10(-5)M) to the aseptic culture medium reduced the time required for developing macroscopically visible flower primordia and the number of leaves appearing before the first flower buds to about half under 8 hrs daily illuumination in both wild type and a late monogenic mutant. In the presence of equimolar amount of adenine the flowering was not accelerated by the analog.