<Emphasis Type="Italic">pgd1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant,is defective in pollen germination

An Arabidopsis deletion mutant was fortuitously identified from the alpha population of T-DNA insertional mutants generated at the University of Wisconsin Arabidopsis Knockout Facility. Segregation and reciprocal crosses indicated that the mutant was a gametophytic pollen sterile mutant. Pollen carrying the mutation has the unusual phenotype that it is viable, but cannot germinate. Thus, the mutant was named pollen germination defective mutant 1 (pgd1), based on the pollen phenotype. Flanking sequences of the T-DNA insertion in the pgd1 mutant were identified by thermal asymmetric interlaced (TAIL) PCR. Sequencing of bands from TAIL PCR revealed that the T-DNA was linked to the gene XLG1, At2g23460, at its downstream end, while directly upstream of the T-DNA was a region between At2g22830 and At2g22840, which was 65 genes upstream of XLG1. Southern blotting and genomic PCR confirmed that the 65 genes plus part of XLG1 were deleted in the pgd1 mutant. A 9,177 bp genomic sequence containing the XLG1 gene and upstream and downstream intergenic regions could not rescue the pgd1 pollen phenotype. One or more genes from the deleted region were presumably responsible for the pollen germination defect observed in the pgd1 mutant. Because relatively few mutations have been identified that affect pollen germination independent of any effect on pollen viability, this mutant line provides a new tool for identification of genes specifically involved in this phase of the reproductive cycle.     

A new <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant <Emphasis Type="Italic">apetala1-20</Emphasis>

A new deletion allele of the APETALA1 (AP1) gene encoding a type II MADS-box protein with the key role in the initiation of flowering and development of perianth organs has been identified in A. thaliana. The deletion of seven amino acids in the conserved region of the K domain in the ap1-20 mutant considerably delayed flowering and led to a less pronounced abnormality in the corolla development compared to the weak ap1-3 and intermediate ap1-6 alleles. At the same time, a considerable stamen reduction has been revealed in ap1-20 as distinct from ap1-3 and ap1-6 alleles. These data indicate that the K domain of AP1 can be crucial for the initiation of flowering and expression regulation of B-class genes controlling stamen development.     

Two WD-repeat genes from cotton are functional homologues of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis><Emphasis Type="Italic">TRANSPARENT TESTA GLABRA1</Emphasis> (<Emphasis Type="Italic">TTG1</Emphasis>) gene

Cotton fibres are single, highly elongated cells derived from the outer epidermis of ovules, and are developmentally similar to the trichomes of Arabidopsis thaliana. To identify genes involved in the molecular control of cotton fibre initiation, we isolated four putative homologues of the Arabidopsis trichome-associated gene TRANSPARENT TESTA GLABRA1 (TTG1). All four WD-repeat genes are derived from the ancestral D diploid genome of tetraploid cotton and are expressed in many tissues throughout the plant, including ovules and growing fibres. Two of the cotton genes were able to restore trichome formation in ttg1 mutant Arabidopsis plants. Both these genes also complemented the anthocyanin defect in a white-flowered Matthiola incana ttg1 mutant. These results demonstrate parallels in differentiation between trichomes in cotton and Arabidopsis, and indicate that these cotton genes may be functional homologues of AtTTG1.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants overexpressing <Emphasis Type="Italic">Ramosa1</Emphasis> maize gene show an increase in organ size due to cell expansion

The structure of the plant inflorescence and flower is an important agronomic and ornamental trait studied for its potential economic applications. In particular, the capacity to modify flower size has always been a breeder’s goal. Genetic and molecular studies have shown that the Zea mays gene Ramosa1 (Ra1) is involved in inflorescence branching regulation. In fact the ra1 loss of function mutation causes extra branching of the inflorescence. In this work we suggest a possible utilization of the Ramosa1 maize gene as a tool to modify inflorescence architecture and flower size in transgenic plants. In fact overexpression of this gene in Arabidopsis plants promotes an increase in reproductive organ size. Pollen, seeds, cotyledons, leaves and roots are also larger than those of the wild type. Analysis of organs from transformants showed that cell expansion was increased without apparently affecting cell division. These results suggest that the RA1 protein is able to up-regulate cell expansion in all organs of Arabidopsis plants.     

An <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> tubulin mutant with conditional root-skewing phenotype

Low doses of microtubule-interacting drugs cause wild-type Arabidopsis thaliana seedling roots to twist in a left-handed helical direction. We here report molecular characterization of an A. thaliana tubulin mutant whose roots twist in a right-handed direction and have shallow left-handed cortical microtubule arrays when challenged with low doses of microtubule drugs. In the absence of the drug, growth and development of the mutant was apparently normal. In this conditional twisting mutant, Cys213 of α-tubulin6 was exchanged with Tyr. The mutant tubulin was incorporated into the microtubule polymer with wild-type tubulins, and thus acted as a dominant-negative mutation. These results suggest that compromised microtubules in wild-type and mutant roots are qualitatively distinct and affect skewing direction differently.     

The Carboxylesterase Gene Family from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger / hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.     

Both caffeoyl Coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 and caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 are involved in redundant functions for lignin,flavonoids and sinapoyl malate biosynthesis in Arabidopsis

Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Orientation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> KAT1 Channel in the Plasma Membrane

The Arabidopsis thaliana KAT1, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K⁺ channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K⁺ channel, we have inserted a flag epitope in the NH₂ terminus or the S3–S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to the Shaker K⁺ channel.     

Simple method of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> cultivation in liquid nutrient medium

Simple method of Arabidopsis thaliana w.t. cv. Columbia (L.) Heynh. cultivation in liquid nutrient medium is presented. After 5 weeks of growth in soil, the plants were transferred to modified Hoagland nutrient medium. This allowed us to cultivate Arabidopsis in conditions comparable to all other hydroponically grown higher plants used in plant physiology and plant stress physiology experiments. Absence of agar in growth medium and free access to whole root system makes this method useful also in experiments concerning root physiology.     

Overexpression of Organellar and Cytosolic <Emphasis Type="Italic">AtHSP90</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Impairs Plant Tolerance to Oxidative Stress

Three AtHSP90 isoforms, cytosol-localized AtHSP90.2, chloroplast-localized AtHSP90.5, and endoplasmic reticulum (ER)-localized AtHSP90.7 genes, were constitutively overexpressed in Arabidopsis thaliana to study their functional mechanisms under oxidative stress. Overexpression of AtHSP90 genes reduced germination of transgenic seeds under oxidative stress. When exposed to 10 mM H₂O₂, AtHSP90 transgenic seedlings displayed lower activities of superoxide dismutase, catalase, and peroxidase; higher content of malondialdehyde; and higher levels of protein damage than detected in the wild type. This indicated that overexpression of AtHSP90.2, AtHSP90.5, and AtHSP90.7 in Arabidopsis impaired plant tolerance to oxidative stress. Moreover, overexpression of chloroplast- and ER-localized AtHSP90 resulted in lower resistance to oxidative stress than that of cytosolic AtHSP90. This suggested that HSP90.2, HSP90.5, and HSP90.7 localized in different cellular compartments were involved in different functional mechanisms during oxidative stress.     

Nutritional suitability and ecological relevance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis> as foodplants for the cabbage butterfly, <Emphasis Type="Italic">Pieris rapae</Emphasis>

The thale cress, Arabidopsis thaliana, is considered to be an important model species in studying a suite of evolutionary processes. However, the species has been criticized on the basis of its comparatively small size at maturity (and consequent limitations in the amount of available biomass for herbivores) and on the duration and timing of its life cycle in nature. In the laboratory, we studied interactions between A. thaliana and the cabbage butterfly, Pieris rapae, in order to determine if plants are able to support the complete development of the herbivore. Plants were grown in pots from seedlings in densities of one, two, or four per pot. In each treatment, one, two, or five newly hatched larvae of P. rapae were placed on fully developed rosettes of A. thaliana. In a separate experiment, the same densities of P. rapae larvae were reared from hatching on single mature cabbage (Brassica oleracea) plants. Pupal fresh mass and survival of P. rapae declined with larval density when reared on A. thaliana but not on B. oleracea. However, irrespective of larval density and plant number, some P. rapae were always able to complete development on A. thaliana plants. A comparison of the dry mass of plants in different treatments with controls (= no larvae) revealed that A. thaliana partially compensated for plant damage when larval densities of P. rapae were low. By contrast, single cress plants with 5 larvae generally suffered extensive damage, whereas damage to B. oleracea plants was negligible. Rosettes of plants that were monitored in spring, when A. thaliana naturally grows, were not attacked by any insect herbivores, but there was often extensive damage from pulmonates (slugs and snails). Heavily damaged plants flowered less successfully than lightly damaged plants. Small numbers of generalist plant-parasitic nematodes were also recovered in roots and root soil. By contrast, plants monitored in a sewn summer plot were heavily attacked by insect herbivores, primarily flea beetles (Phyllotreta spp.). These results reveal that, in natural populations of A. thaliana, there is a strong phenological mismatch between the plant and most of its potential specialist insect herbivores (and their natural enemies). However, as the plant is clearly susceptible to attack from non-insect generalist invertebrate herbivores early in the season, these may be much more suitable for studies on direct defense strategies in A. thaliana.