The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis.

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Identificació de QTLs implicats en l'adaptació a canvis de temperatura en la planta model Arabidopsis thaliana

Estudi realitzat a partir d’una estada al Max Planck Institute for Plant Breeding Research, a Alemanya, entre 2006 i 2008. En aquest treball s´ha identificat una nove interacció epistàtica entre l'ecotip Landsberg, originari d'Europa del nord, i Kashmir-2 o Kondara, ambdós originaris d'Àsia Central. Els anàlisi de QTLs en poblacions recombinants Ler x Kas-2 i Ler x Kond indica el requeriment de 3 loci en Ler x Kas-2 i 2 loci en Ler x Kond. Els híbrids incompatibles crescuts a temperatures baixes (16°C) mostren seriosos defectes en el desenvolupament, mort cel.lular espontània i resistència a Hyaloperonospora parasitica. Aquests fenotips es suprimeixen a elevades temperatures o per mutació d'EDS1, o depleció dels nivells d'àcid sal.licílic per transformació amb salicil.lat hidroxilasa (NahG). El grau de severitat en els fenotips observats correlaciona amb els nivells d'àcid sal.licílic, indicant que aquesta molècula és essencial per a la senyalització d'incompatibilitats genètiques en Arabidopsis.

Competitive ability not kinship affects growth of Arabidopsis thaliana accessions.

In many organisms, individuals behave more altruistically towards relatives than towards unrelated individuals. Here, we conducted a study to determine if the performance of Arabidopsis thaliana is influenced by whether individuals are in competition with kin or non-kin. We selected seven pairs of genetically distinct accessions that originated from local populations throughout Europe. We measured the biomass of one focal plant surrounded by six kin or non-kin neighbours in in vitro growth experiments and counted the number of siliques produced per pot by one focal plant surrounded by four kin or non-kin neighbours. The biomass and number of siliques of a focal plant were not affected by the relatedness of the neighbour. Depending on the accession, a plant performed better or worse in a pure stand than when surrounded by non-kin plants. In addition, whole-genome microarray analyses revealed that there were no genes differentially expressed between kin and non-kin conditions. In conclusion, our study does not provide any evidence for a differential response to kin vs non-kin in A. thaliana. Rather, the outcome of the interaction between kin and non-kin seems to depend on the strength of the competitive abilities of the accessions.

Peroxisomal polyhydroxyalkanoate biosynthesis is a promising strategy for bioplastic production in high biomass crops.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers with diverse plastic-like properties. PHA biosynthesis in transgenic plants is being developed as a way to reduce the cost and increase the sustainability of industrial PHA production. The homopolymer polyhydroxybutyrate (PHB) is the simplest form of these biodegradable polyesters. Plant peroxisomes contain the substrate molecules and necessary reducing power for PHB biosynthesis, but peroxisomal PHB production has not been explored in whole soil-grown transgenic plants to date. We generated transgenic sugarcane (Saccharum sp.) with the three-enzyme Ralstonia eutropha PHA biosynthetic pathway targeted to peroxisomes. We also introduced the pathway into Arabidopsis thaliana, as a model system for studying and manipulating peroxisomal PHB production. PHB, at levels up to 1.6%-1.8% dry weight, accumulated in sugarcane leaves and A. thaliana seedlings, respectively. In sugarcane, PHB accumulated throughout most leaf cell types in both peroxisomes and vacuoles. A small percentage of total polymer was also identified as the copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in both plant species. No obvious deleterious effect was observed on plant growth because of peroxisomal PHA biosynthesis at these levels. This study highlights how using peroxisomal metabolism for PHA biosynthesis could significantly contribute to reaching commercial production levels of PHAs in crop plants.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.

Fatty acid signaling in Arabidopsis.

Many organisms use fatty acid derivatives as biological regulators. In plants, for example, fatty acid-derived signals have established roles in the regulation of developmental and defense gene expression. Growing numbers of these compounds, mostly derived from fatty acid hydroperoxides, are being characterized. The model plant Arabidopsis thaliana is serving a vital role in the discovery of fatty acid-derived signal molecules and the genetic analysis of their synthesis and action. The Arabidopsis genome sequencing project, the availability of large numbers of mutants in fatty acid biosynthesis and signal transduction, as well as excellent pathosystems, make this plant a tremendously useful model for research in fatty acid signaling. This review summarizes recent progress in understanding fatty acid signaling in A. thaliana and highlights areas of research where progress is rapid. Particular attention is paid to the growing literature on the jasmonate family of regulators and their role in defense against insects and microbial pathogens.

Defining the site of light perception and initiation of phototropism in Arabidopsis.

Phototropism is an adaptive response allowing plants to optimize photosynthetic light capture. This is achieved by asymmetric growth between the shaded and lit sides of the stimulated organ. In grass seedlings, the site of phototropin-mediated light perception is distinct from the site of bending; however, in dicotyledonous plants (e.g., Arabidopsis), spatial aspects of perception remain debatable. We use morphological studies and genetics to show that phototropism can occur in the absence of the root, lower hypocotyl, hypocotyl apex, and cotyledons. Tissue-specific expression of the phototropin1 (phot1) photoreceptor demonstrates that light sensing occurs in the upper hypocotyl and that expression of phot1 in the hypocotyl elongation zone is sufficient to enable a normal phototropic response. Moreover, we show that efficient phototropism occurs when phot1 is expressed from endodermal, cortical, or epidermal cells and that its local activation rapidly leads to a global response throughout the seedling. We propose that spatial aspects in the steps leading from light perception to growth reorientation during phototropism differ between grasses and dicots. These results are important to properly interpret genetic experiments and establish a model connecting light perception to the growth response, including cellular and morphological aspects.

BRX promotes Arabidopsis shoot growth.

? BREVIS RADIX (BRX) has been identified through a loss-of-function allele in the Umkirch-1 accession in a natural variation screen for Arabidopsis root growth vigor. Physiological and gene expression analyses have suggested that BRX is rate limiting for auxin-responsive gene expression by mediating cross-talk with the brassinosteroid pathway, as impaired root growth and reduced auxin perception of brx can be (partially) rescued by external brassinosteroid application. ? Using genetic tools, we show that brx mutants also display significantly reduced cotyledon and leaf growth. ? Similar to the root, the amplitude and penetrance of this phenotype depends on genetic background and shares the physiological features, reduced auxin perception and brassinosteroid rescue. Furthermore, reciprocal grafting experiments between mutant and complemented brx shoot scions and root stocks suggest that the shoot phenotypes are not an indirect consequence of the root phenotype. Finally, BRX gain-of-function lines display epinastic leaf growth and, in the case of dominant negative interference, increased epidermal cell size. Consistent with an impact of BRX on brassinosteroid biosynthesis, this phenotype is accompanied by increased brassinosteroid levels. ? In summary, our results demonstrate a ubiquitous, although quantitatively variable role of BRX in modulating the growth rate in both the root and shoot.

Myotonic dystrophy transgenic mice exhibit pathologic abnormalities in diaphragm neuromuscular junctions and phrenic nerves

Myotonic dystrophy Type 1 (DM-1) is caused by abnormal expansion of a (CTG) repeat located in the DM protein kinase gene. Respiratory problems have long been recognized to be a major feature of this disorder. Because respiratory failure can be associated with dysfunction of phrenic nerves and diaphragm muscle, we examined the diaphragm and respiratory neural network in transgenic mice carrying the human genomic DM-1 region with expanded repeats of more than 300 CTG, a valid model of the human disease. Morphologic and morphometric analyses revealed distal denervation of diaphragm neuromuscular junctions in DM-1 transgenic mice indicated by a decrease in the size and shape complexity of end-plates and a reduction in the concentration of acetyl choline receptors on the postsynaptic membrane. More importantly, there was a significant reduction in numbers of unmyelinated, but not of myelinated, fibers in DM-1 phrenic nerves; no morphologic alternations of the nerves or loss of neuronal cells were detected in medullary respiratory centers or cervical phrenic motor neurons. Because neuromuscular junctions are involved in action potential transmission and the afferent phrenic unmyelinated fibers control the inspiratory activity, our results suggest that the respiratory impairment associated with DM-1 may be partially due to pathologic alterations in neuromuscular junctions and phrenic nerves.

The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses.

Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo- and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JA-regulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response.

Low-grade and high-grade mammary carcinomas in WAP-T transgenic mice are independent entities distinguished by Met expression.

Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.