Characterization of a flower-specific gene encoding a putative myrosinase binding protein in Arabidopsis thaliana

A cDNA clone, 4B-1, previously isolated by differential screening is preferentially expressed in floral organs of Arabidopsis thaliana. Characterization of the full length cDNA and the genetic locus corresponding to 4B-1 cDNA revealed that it potentially encodes a myrosinase binding protein (MBP) which is presumably present in a large myrosinase complex. The deduced amino acid sequence of the polypeptide encoded by cDNA clone (designated f-AtMBP) appeared to consist of two parts: one region at the C-terminal half representing overall homology with AtMBP, an MBP homologue in A. thaliana, and the other at an extended N-terminal region of about 150 amino acids showing significant identity with the N-terminal region of the MBP-related protein reported in Brassica. Expression analysis by RNA blot and in situ hybridization showed that f-AtMBP was specifically expressed in floral meristems, pistils, stamens, petals, and ovules of immature flowers, but no expression was observed in the specialized cells called the myrosin cells in the hypocotyl and cotyledons of developing seeds where myrosinase enzymes are normally found. Although MBPs and MBP-related proteins are considered to be inducible by exogenous application of signal molecules and physical wounding, we found that f-AtMBP expression was not activated by such treatment, suggesting that f-AtMBP is a novel type of MBP specific to floral organs.     

Characterization of a ubiquitous expressed gene family encoding polygalacturonase in Arabidopsis thaliana

Pectin, as one of the major components of plant cell wall, has been implicated in many developmental processes occurring during plant growth. Among the different enzymes known to participate in the pectin structure modifications, polygalacturonase (PG) activity has been shown to be associated with fruit ripening, organ abscission and pollen grain development. Until now, sequence analyses of the deduced polypeptides of the plant PG genes allowed their grouping into three clades corresponding to genes involved in one of these three activities. In this study, we report the sequence of three genomic clones encoding PG in Arabidopsis thaliana. These genes, together with 16 other genes present in the databases form a large gene family, ubiquitously expressed, present on the five chromosomes with at least two gene clusters on chromosomes II and V, respectively. Phylogenetic analyses suggest that the A. thaliana gene family contains five classes of genes, with three of them corresponding to the previously defined clades. Comparison of positions and numbers of introns among the A. thaliana genes reveals structural conservation between genes belonging to the same class. The pattern of intron losses that could have given rise to the PG gene family is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a spliced mRNA. Following this event of intron loss, the acquisition of introns in novel positions is consistent with a mechanism of intron gain at proto-splice sites.     

Arabidopsis thaliana contains a single gene encoding squalene synthase

Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol, brassinosteroid, and triterpene biosynthesis. Arabidopsis thaliana contains two SQS-annotated genomic sequences, At4g34640 (SQS1) and At4g34650 (SQS2), organized in a tandem array. Here we report that the SQS1 gene is widely expressed in all tissues throughout plant development, whereas SQS2 is primarily expressed in the vascular tissue of leaf and cotyledon petioles, and the hypocotyl of seedlings. Neither the complete A. thaliana SQS2 protein nor the chimeric SQS resulting from the replacement of the 69 C-terminal residues of SQS2 by the 111 C-terminal residues of the Schizosaccharomyces pombe SQS were able to confer ergosterol prototrophy to a Saccharomyces cerevisiae erg9 mutant strain lacking SQS activity. A soluble form of SQS2 expressed in Escherichia coli and purified was unable to synthesize SQ from FPP in the presence of NADPH and either Mg²⁺ or Mn²⁺. These results demonstrated that SQS2 has no SQS activity, so that SQS1 is the only functional SQS in A. thaliana. Mutational studies revealed that the lack of SQS activity of SQS2 cannot be exclusively attributed to the presence of an unusual Ser replacing the highly conserved Phe at position 287. Expression of green fluorescent protein (GFP)-tagged versions of SQS1 in onion epidermal cells demonstrated that SQS1 is targeted to the endoplasmic reticulum (ER) membrane and that this location is exclusively dependent on the presence of the SQS1 C-terminal hydrophobic trans-membrane domain.     

AtZFP1, encoding Arabidopsis thaliana C2H2 zinc-finger protein 1, is expressed downstream of photomorphogenic activation

C₂H₂ zinc-finger proteins play important roles in plant development including floral organogenesis, leaf initiation, lateral shoot initiation, gametogenesis and seed development. The gene for one such protein from Arabidopsis, AtZFP1 (Arabidopsis thalianazinc-finger protein 1), is expressed at high levels in the shoot apex, including the apical meristem, developing leaves and the developing vascular system. In light-grown seedlings, AtZFP1 expression is induced about three days after germination, before the expansion of the true leaves. Dark-grown plants, in which photomorphogenesis is repressed, have no detectable AtZFP1 expression in the shoot apex. Under conditions which induce or mimic photomorphogenic development including growth in the light, shifting dark-grown plants to continuous light or growth on cytokinin in the dark, high levels of AtZFP1 expression are detected. Furthermore, AtZFP1 expression does not depend on active photosynthesis as shown by analysis of plants grown on the carotenoid biosynthetic inhibitor norflurazon. These results are discussed in relation to a possible role for AtZFP1 in shoot development, downstream of photomorphogenic activation.     

Characterization of katD, a kinesin-like protein gene specifically expressed in floral tissues of Arabidopsis thaliana

Kinesin and kinesin-like proteins (KLPs) are microtubule-based motor proteins that play important roles in organelle transport. Based on the homology to these proteins, a katD cDNA has now been isolated from a library prepared from flowers of Arabidopsis thaliana ecotype Columbia. Sequence analysis of the katD cDNA revealed an open reading frame of 2691bp [corrected], encoding a protein of 987 amino acids. Comparison of the nucleotide sequences of katD genomic and cDNA clones revealed the presence of 18 introns, 17 of which conform to the GU-AG rule. The central region of the KatD polypeptide exhibits substantial amino acid sequence homology to the motor domain of kinesin heavy chains, although the motor domain of KatD appears to be phylogenetically distant from those of other KLPs in plants. The amino-terminal region of KatD shares marked sequence similarity with the calponin homology domain, whereas the approximately 240-residue carboxyl-terminal region shows no significant homology to other known proteins. The predicted secondary structure of KatD revealed the lack of an alpha-helical coiled coil structure typical of kinesin heavy chains, suggesting that KatD may function as a monomeric motor. A recombinant truncated KatD protein containing the putative motor domain was shown both to bind to mammalian microtubules in a manner dependent on a non-hydrolyzable ATP analog, and to possess microtubule-dependent ATPase activity. Immunoblot and Northern blot analyses showed that both KatD protein and mRNA are expressed specifically in floral tissues. These results suggest that the structurally distinct KatD protein functions as a floral tissue-specific motor protein.     

Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana

A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron.     

A novel strategy for obtaining kanamycin resistance in Arabidopsis thaliana by silencing an endogenous gene encoding a putative chloroplast transporter

The use of bacterial antibiotic resistance markers in transgenic plants raises concerns about horizontal gene transfer to soil bacteria. We report here that kanamycin resistance in Arabidopsis thaliana can be achieved by silencing an endogenous gene encoding a putative chloroplast transporter, which presumably imports kanamycin into chloroplasts to interfere with ribosomal RNA. Homologs of the transporter exist in other plant species, suggesting this strategy may be generally useful for selecting transformed plant cells.     

The Arabidopsis thaliana PARTING DANCERS gene encoding a novel protein is required for normal meiotic homologous recombination

Recent studies of meiotic recombination in the budding yeast and the model plant Arabidopsis thaliana indicate that meiotic crossovers (COs) occur through two genetic pathways: the interference-sensitive pathway and the interference-insensitive pathway. However, few genes have been identified in either pathway. Here, we describe the identification of the PARTING DANCERS (PTD) gene, as a gene with an elevated expression level in meiocytes. Analysis of two independently generated transferred DNA insertional lines in PTD showed that the mutants had reduced fertility. Further cytological analysis of male meiosis in the ptd mutants revealed defects in meiosis, including reduced formation of chiasmata, the cytological appearance of COs. The residual chiasmata in the mutants were distributed randomly, indicating that the ptd mutants are defective for CO formation in the interference-sensitive pathway. In addition, transmission electron microscopic analysis of the mutants detected no obvious abnormality of synaptonemal complexes and apparently normal late recombination nodules at the pachytene stage, suggesting that the mutant's defects in bivalent formation were postsynaptic. Comparison to other genes with limited sequence similarity raises the possibility that PTD may present a previously unknown function conserved in divergent eukaryotic organisms.     

Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

A novel MYB-related gene from Arabidopsis thaliana expressed in developing anthers.

A novel MYB-like gene (AtMYB103) was isolated from a genomic library of Arabidopsis. Plants transgenic for chimeric AtMYB103 promoter/GUS genes expressed the enzyme in early anthers. In situ hybridization of flower sections showed a high level of AtMYB103 mRNA in the tapetum and middle layer of developing anthers.     

Ontogenetic phenotypic plasticity during the reproductive phase in Arabidopsis thaliana (Brassicaceae)

While phenotypic plasticity has been the focus of much research and debate in the recent ecological and evolutionary literature, the developmental nature of the phenomenon has been mostly overlooked. A developmental perspective must ultimately be an integral part of our understanding of how organisms cope with heterogeneous environments. In this paper I use the rapid cycling Arabidopsis thaliana to address the following questions concerning developmental plasticity. (1) Are there genetic and/or environmental differences in parameters describing ontogenetic trajectories? (2) Is ontogenetic variation produced by differences in genotypes and/or environments for two crucial traits of the reproductive phase of the life cycle, stem elongation and flower production? (3) Is there ontogenetic variability for the correlation between the two characters? I found genetic variation, plasticity, and variation for plasticity affecting at least some of the growth parameters, indicating potential for evolution via heterochronic shifts in ontogenetic trajectories. Within-population differences among families are determined before the onset of the reproductive phase, while among-population variation is the result of divergence during the reproductive phase of the ontogeny. Finally, the ontogenetic profiles of character correlations are very distinct between the ecologically meaningful categories of early- and late-flowering “ecotypes” in this species, and show susceptibility to environmental change.     

A novel blue light- and abscisic acid-inducible gene of Arabidopsis thaliana encoding an intrinsic membrane protein

Continuous irradiation with blue light (400–500 nm) induces flower formation in plantlets of Arabidopsis thaliana (C24) while red light (600–700 nm) is ineffective. This observation started a search for genes that are activated by blue light and initiate the morphogenic programme leading to flower formation. Several genes were identified via their cDNAs. From these clone AthH2, with an open reading frame for a hydrophobic 30.5 kDa polypeptide, was selected for further characterization of the corresponding gene. From a genomic library a DNA fragment of about 6.4 kb was isolated, comprising the coding region as well as 5-upstream and 3-downstream flanking segments. The coding region is composed of four exons, which specify a polypeptide of 286 amino acids. Several potential regulatory elements were found between position –670 and –1140 including GA and ABA sequence motifs. The latter could account for the observed induction of the AthH2 gene by ABA. Southern blot analysis of Arabidopsis genomic DNA suggests that the AthH2 gene is encoded by a single-copy gene. Hydropathy plots and secondary structure analysis of the putative polypeptide predict six membrane-spanning domains implicating a function as transmembrane channel protein. It displays significant homology with the proteins TR7a of pea (82%) and RD 28 of A. thaliana (68%).