Genomic analysis of the terpenoid synthase (<Emphasis Type="Italic">AtTPS</Emphasis>) gene family of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

A family of 40 terpenoid synthase genes ( AtTPS) was discovered by genome sequence analysis in Arabidopsis thaliana. This is the largest and most diverse group of TPS genes currently known for any species. AtTPS genes cluster into five phylogenetic subfamilies of the plant TPS superfamily. Surprisingly, thirty AtTPS closely resemble, in all aspects of gene architecture, sequence relatedness and phylogenetic placement, the genes for plant monoterpene synthases, sesquiterpene synthases or diterpene synthases of secondary metabolism. Rapid evolution of these AtTPS resulted from repeated gene duplication and sequence divergence with minor changes in gene architecture. In contrast, only two AtTPS genes have known functions in basic (primary) metabolism, namely gibberellin biosynthesis. This striking difference in rates of gene diversification in primary and secondary metabolism is relevant for an understanding of the evolution of terpenoid natural product diversity. Eight AtTPS genes are interrupted and are likely to be inactive pseudogenes. The localization of AtTPS genes on all five chromosomes reflects the dynamics of the Arabidopsis genome; however, several AtTPS genes are clustered and organized in tandem repeats. Furthermore, some AtTPS genes are localized with prenyltransferase genes ( AtGGPPS, geranylgeranyl diphosphate synthase) in contiguous genomic clusters encoding consecutive steps in terpenoid biosynthesis. The clustered organization may have implications for TPS gene evolution and the evolution of pathway segments for the synthesis of terpenoid natural products. Phylogenetic analyses highlight events in the divergence of the TPS paralogs and suggest orthologous genes and a model for the evolution of the TPS gene family.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

WRKY gene family evolution in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The Arabidopsis thaliana WRKY proteins are characterized by a sequence of 60 amino acids including WRKY domain. It is well established that these proteins are involved in the regulation of various physiological programs unique to plants including pathogen defense, senescence and response to environmental stresses, which attracts attention of the scientific community as to how this family might have evolved. We tried to satisfy this curiosity and analyze reasons for duplications of these gene sequences leading to their diversified gene actions. The WRKY sequences available in Arabidopsis thaliana were used to evaluate selection pressure following duplication events. A phylogenetic tree was constructed and the WRKY family was divided into five sub-families. After that, tests were conducted to decide whether positive or purified selection played key role in these events. Our results suggest that purifying selection played major role during the evolution of this family. Some amino acid changes were also detected in specific branches of phylogeny suggesting that relaxed constraints might also have contributed to functional divergence among sub-families. Sites relaxed from purifying selection were identified and mapped onto the structural and functional regions of the WRKY1 protein. These analyses will enhance our understanding of the precise role played by natural selection to create functional diversity in WRKY family.     

Molecular analysis of the<Emphasis Type="Italic"> CRINKLY4</Emphasis> gene family in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The maize (Zea mays L.) CRINKLY4 (CR4) gene encodes a serine/threonine receptor-like kinase that controls an array of developmental processes in the plant and endosperm. The Arabidopsis thaliana (L.) Heynh. genome encodes an ortholog of CR4, ACR4, and four CRINKLY4-RELATED (CRR) proteins: AtCRR1, AtCRR2, AtCRR3 and AtCRK1. The available genome sequence of rice (Oryza sativa L.) encodes a CR4 ortholog, OsCR4, and four CRR proteins: OsCRR1, OsCRR2, OsCRR3 and OsCRR4, not necessarily orthologous to the Arabidopsis CRRs. A phylogenetic study showed that AtCRR1 and AtCRR2 form a clade closest to the CR4 group while all the other CRRs form a separate cluster. The five Arabidopsis genes are differentially expressed in various tissues. A construct formed by fusion of the ACR4 promoter and the GUS reporter, ACR4::GUS, is expressed primarily in developing tissues of the shoot. The ACR4 cytoplasmic domain functions in vitro as a serine/threonine kinase, while the AtCRR1 and AtCRR2 kinases are not active. The ability of ACR4 to phosphorylate AtCRR2 suggests that they might function in the same signal transduction pathway. T-DNA insertions were obtained in ACR4, AtCRR1, AtCRR2, AtCRR3 and AtCRK1. Mutations in acr4 show a phenotype restricted to the integuments and seed coat, suggesting that Arabidopsis might contain a redundant function that is lacking in maize. The lack of obvious mutant phenotypes in the crr mutants indicates they are not required for the hypothetical redundant function.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

An expression atlas of miRNAs in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. MiRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

Exploitation of colinear relationships between the genomes of<Emphasis Type="Italic"> Lotus japonicus</Emphasis>,<Emphasis Type="Italic"> Pisum sativum</Emphasis> and<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>, for positional cloning of a legume symbiosis gene

The Lotus japonicus LjSYM2 gene, and the Pisum sativum orthologue PsSYM19, are required for the formation of nitrogen-fixing root nodules and arbuscular mycorrhiza. Here we describe the map-based cloning procedure leading to the isolation of both genes. Marker information from a classical AFLP marker-screen in Lotus was integrated with a comparative genomics approach, utilizing Arabidopsis genome sequence information and the pea genetic map. A network of gene-based markers linked in all three species was identified, suggesting local colinearity in the region around LjSYM2/PsSYM19. The closest AFLP marker was located just over 200 kb from the LjSYM2 gene, the marker SHMT, which was converted from a marker on the pea map, was only 7.9 kb away. The LjSYM2/PsSYM19 region corresponds to two duplicated segments of the Arabidopsis chromosomes AtII and AtIV. Lotus homologues of Arabidopsis genes within these segments were mapped to three clusters on LjI, LjII and LjVI, suggesting that during evolution the genomic segment surrounding LjSYM2 has been subjected to duplication events. However, one marker, AUX-1, was identified based on colinearity between Lotus and Arabidopsis that mapped in physical proximity of the LjSym2 gene.Communicated by J.S. Heslop-Harrison     

A meiotic time-course for<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

A meiotic time-course for Arabidopsis pollen mother cells has been established based on BrdU pulse-labelling of nuclear DNA in the meiotic S-phase. Labelled flower buds were sampled at intervals and the progress of labelled cells through meiosis assessed by anti-BrdU antibody detection. The overall duration of meiosis from the end of meiotic S-phase to the tetrad stage, at 18.5°C, was 33 h, which is about three times longer than the mitotic cell cycle in seedlings. The onset of leptotene was defined by reference to the loading of the axis-associated protein Asy1, and this permitted the detection of a definite G2 stage, having a maximum duration of 9 h. It is likely, from two independent sources of evidence, that the meiotic S-phase has a duration similar to that of G2. The durations of leptotene and zygotene/pachytene are 6 h and 15.3 h, respectively, but the remaining meiotic division stages are completed very rapidly, within 3 h. The establishment of a meiotic time-course provides a framework for determining the relative timing and durations of key molecular events of meiosis in Arabidopsis in relation to cytologically defined landmarks. In addition, it will be important in a broader developmental context for determining the timing of epigenetic mechanisms that are known or suspected to occur during meiosis.     

Expression of<Emphasis Type="Italic"> FoxE</Emphasis> and<Emphasis Type="Italic"> FoxQ</Emphasis> genes in the endostyle of<Emphasis Type="Italic"> Ciona intestinalis</Emphasis>

We have analyzed the expression patterns of two Fox genes, FoxE and FoxQ, in the ascidian Ciona intestinalis. Expression of Ci-FoxE was specific to the endostyle of adults, being prominent in the thyroid-equivalent region of zone 7. Ci-FoxQ was expressed in several endodermal organs of adult ascidians, such as the endostyle, branchial sac and esophagus. In the endostyle, the pattern of Ci-FoxQ expression was similar to that of CiTTF-1, being prominent in the thyroid-equivalent regions of zones 7 and 8. Therefore, these Fox genes may perform thyroid-equivalent functions in the ascidian endostyle.Edited by N. Satoh     

<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.     

Complementary expression of<Emphasis Type="Italic"> AP-2</Emphasis> and<Emphasis Type="Italic"> AP-2rep</Emphasis> in ectodermal derivatives of<Emphasis Type="Italic"> Xenopus</Emphasis> embryos

In an attempt to define the pattern of developmental expression of AP-2rep and AP-2 in Xenopus embryos, we cloned a Xenopus AP-2rep cDNA. The AP-2rep message was localized in the organizer region at the gastrula stage whereas AP-2 was expressed ventro-laterally in the animal hemisphere. Later, AP-2rep was expressed in the entire neural tissue at the neurula stage while AP-2 was predominantly expressed in the cranial neural crest areas. The endogenous expression of AP-2 in the neural crest area was diminished by ectopic injection of AP-2rep RNA, suggesting a role for AP-2rep in the differentiation of neural tissues by restricting the expression of AP-2 in the Xenopus embryo.     

Endogenous protein mono-ADP-ribosylation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Protein mono-ADP-ribosylation post-translationally transfers the ADP-ribose moiety from the β-NAD⁺ donor to various protein acceptors. This type of modification has been widely characterized and shown to regulate protein activities in animals, yeast and prokaryotes, but has never been reported in plants. In this study, using [³²P]NAD⁺ as the substrate, ADP-ribosylated proteins in Arabidopsis were investigated. One protein substrate of 32 kDa in adult rosette leaves was found to be radiolabeled. Heat treatment, protease sensitivity and nucleotide derivative competition assays suggested a covalent reaction of NAD⁺ with the 32 kDa protein. [carbonyl-¹⁴C]NAD⁺ could not label the 32 kDa protein, confirming that the modification was ADP-ribosylation. Poly (ADP-ribose) polymerase inhibitor failed to suppress the reaction, but chemicals that destroy mono-ADP-ribosylation on specific amino acid residues could break up the linkage, suggesting that the reaction was not a poly-ADP-ribosylation but rather a mono-ADP-ribosylation. This modification mainly existed in leaves and was enhanced by oxidative stresses. In young seedlings, two more protein substrates with the size of 45 kDa and over 130 kDa, respectively, were observed in addition to the 32 kDa protein, indicating that different proteins were modified at different developmental stages. Although the substrate proteins remain to be identified, this is the first report on the characterization of endogenously mono-ADP-ribosylated proteins in plants.     

Genome-wide comparative analysis of the <Emphasis Type="Italic">IQD</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Background  

Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.