<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.     

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.     

<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     

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.     

Opposing effects of reduced DNA methylation on flowering time in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Demethylation of DNA promotes flowering in plants from the vernalization-responsive ecotype C24 of Arabidopsis thaliana (L.) Heynh., but delays flowering in the ecotype Landsberg erecta which is not responsive to vernalization. To investigate these contrasting effects of low methylation we have monitored flowering times and expression of two repressors of flowering, FLC and FWA, in low-methylation plants from three late-flowering mutants in the ecotype Landsberg erecta. Demethylation of DNA decreased FLC expression in the vernalization-responsive mutants, but was not associated with a promotion of flowering; rather, in some lines, demethylation delayed flowering. The opposing effects of demethylation could be explained by its differential effect on the expression of two repressors of flowering. FLC was down-regulated in plants with low methylation, promoting flowering, while FWA was activated in response to demethylation, which probably delays the transition to flowering. Expression of the FWA gene did not delay flowering in plants of ecotype C24; our data suggest that the FWA protein of C24 may be non-functional.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a susceptible host plant for the holoparasite <Emphasis Type="Italic">Cuscuta </Emphasis>spec

Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.     

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.     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

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.     

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.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.