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.     

Analysis of transcriptional activity of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> ornithine-delta-aminotransferase gene promoter

Cloning of the Arabidopsis thaliana genomic DNA fragment presumably corresponding to the promoter region of the ornithine-delta-aminotransferase (OAT) gene is reported. The reporter-gene construct, containing the Escherichia coli beta-glucouronidase gene under control of the OAT gene promoter was generated. The Nicotian tabacum SR1 transformants carrying this construct were obtained. It was demonstrated that under normal conditions, expression of the reporter gene was associated with the meristems and the zones of intensive shoot growth. Possible role of the OAT gene in nitrogen metabolism and shoot development is discussed.     

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.     

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.     

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.     

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.     

Nutritional regulation of <Emphasis Type="Italic">ANR1</Emphasis> and other root-expressed MADS-box genes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The ANR1 MADS-box gene in Arabidopsis thaliana (L.) Heynh. has previously been identified as a key regulator of lateral root growth in response to signals from external nitrate (NO₃⁻). We have used quantitative real-time PCR to investigate the responsiveness of ANR1 and 11 other root-expressed MADS-box genes to fluctuations in the supply of N, P and S. ANR1 expression in roots of hydroponically grown Arabidopsis plants was specifically regulated by changes in the N supply, being induced by N deprivation and rapidly repressed by N re-supply. This pattern of N responsiveness differs from the NO₃⁻ -inducibility of ANR1 previously observed in Arabidopsis root cultures [H.M. Zhang and B.G. Forde (1998) Science 279:407–409]. Seven of the other MADS-box genes responded to N in a manner similar to ANR1, but less strongly, while four (AGL12, AGL17, AGL18 and AGL79) were unaffected. Six of the N-regulated genes (ANR1, AGL14, AGL16, AGL19, SOC1 and AGL21) belong to just two clades within the type II MADS-box lineage, while the other two (AGL26 and AGL56) belong to the poorly characterized type I lineage. Only SOC1 was additionally found to respond to changes in the P and S supply, suggesting a possible role in a general response to nutrient stress. Studies with an ANR1 transposon-insertion mutant provided no evidence for regulatory interactions between ANR1 and the other root-expressed MADS-box genes. The implications of the current data for our understanding of the role of ANR1 and other MADS box genes in the nutritional regulation of lateral root growth are discussed.     

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">MAX4</Emphasis> gene is involved in the regulation of low inorganic phosphate stress responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MAX4 gene has been shown to be involved in the regulation of shoot branching in Arabidopsis (Arabidopsis thaliana). However, little is known about the role of MAX4 gene in low inorganic phosphate (Pi) stress response in Arabidopsis. Here we showed that MAX4 gene is involved in the regulation of low Pi stress response in Arabidopsis. MAX4 gene was repressed by low Pi stress, and the max4 mutants showed lower anthocyanin content and longer primary root length. In addition, max4 mutant plants also displayed altered root architecture such as increased root-to-shoot ratio, lower lateral root number and root hair density compared with wild-type plants under low Pi stress. Higher total Pi contents were detected in shoots and roots of max4 plants than those of wild-type plants when subjected to low Pi stress, which was associated, at least in part, with increase in expression of WRKY75 as well as AtPT1 and AtPT2 genes encoding high-affinity Pi transporters. Taken together, all these results suggest that MAX4 gene mediates low Pi stress response, at least in part, by regulating the expression of WRKY75 as well as AtPT1 and AtPT2 genes.     

<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     

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.     

Genetic and environmental control of the <Emphasis Type="Italic">Verticillium</Emphasis> syndrome in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Verticillium spp. are major pathogens of dicotyledonous plants such as cotton, tomato, olive or oilseed rape. Verticillium symptoms are often ambiguous and influenced by development and environment. The aim of the present study was to define disease and resistance traits of the complex Verticillium longisporum syndrome in Arabidopsis thaliana (L.) Heynh. A genetic approach was used to determine genetic, developmental and environmental factors controlling specific disease and resistance traits and to study their interrelations.