PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism.

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.

Alterations of the 5'untranslated region of SLC16A12 lead to age-related cataract.

PURPOSE. Knowledge of genetic factors predisposing to age-related cataract is very limited. The aim of this study was to identify DNA sequences that either lead to or predispose for this disease. METHODS. The candidate gene SLC16A12, which encodes a solute carrier of the monocarboxylate transporter family, was sequenced in 484 patients with cataract (134 with juvenile cataract, 350 with age-related cataract) and 190 control subjects. Expression studies included luciferase reporter assay and RT-PCR experiments. RESULTS. One patient with age-related cataract showed a novel heterozygous mutation (c.-17A>G) in the 5'untranslated region (5'UTR). This mutation is in cis with the minor G-allele of the single nucleotide polymorphism (SNP) rs3740030 (c.-42T/G), also within the 5'UTR. Using a luciferase reporter assay system, a construct with the patient's haplotype caused a significant upregulation of luciferase activity. In comparison, the SNP G-allele alone promoted less activity, but that amount was still significantly higher than the amount of the common T-allele. Analysis of SLC16A12 transcripts in surrogate tissue demonstrated striking allele-specific differences causing 5'UTR heterogeneity with respect to sequence and quantity. These differences in gene expression were mirrored in an allele-specific predisposition to age-related cataract, as determined in a Swiss population (odds ratio approximately 2.2; confidence intervals, 1.23-4.3). CONCLUSIONS. The monocarboxylate transporter SLC16A12 may contribute to age-related cataract. Sequences within the 5'UTR modulate translational efficiency with pathogenic consequences.

A conserved transcript pattern in response to a specialist and a generalist herbivore.

Transcript patterns elicited in response to attack reveal, at the molecular level, how plants respond to aggressors. These patterns are fashioned both by inflicted physical damage as well as by biological components displayed or released by the attacker. Different types of attacking organisms might therefore be expected to elicit different transcription programs in the host. Using a large-scale DNA microarray, we characterized gene expression in damaged as well as in distal Arabidopsis thaliana leaves in response to the specialist insect, Pieris rapae. More than 100 insect-responsive genes potentially involved in defense were identified, including genes involved in pathogenesis, indole glucosinolate metabolism, detoxification and cell survival, and signal transduction. Of these 114 genes, 111 were induced in Pieris feeding, and only three were repressed. Expression patterns in distal leaves were markedly similar to those of local leaves. Analysis of wild-type and jasmonate mutant plants, coupled with jasmonate treatment, showed that between 67 and 84% of Pieris-regulated gene expression was controlled, totally or in part, by the jasmonate pathway. This was correlated with increased larval performance on the coronatine insensitive1 glabrous1 (coi1-1 gl1) mutant. Independent mutations in COI1 and GL1 led to a faster larval weight gain, but the gl1 mutation had relatively little effect on the expression of the insect-responsive genes examined. Finally, we compared transcript patterns in Arabidopis in response to larvae of the specialist P. rapae and to a generalist insect, Spodoptera littoralis. Surprisingly, given the complex nature of insect salivary components and reported differences between species, almost identical transcript profiles were observed. This study also provides a robustly characterized gene set for the further investigation of plant-insect interaction.

Stochastic models and numerical algorithms for a class of regulatory gene networks.

Regulatory gene networks contain generic modules, like those involving feedback loops, which are essential for the regulation of many biological functions (Guido et al. in Nature 439:856-860, 2006). We consider a class of self-regulated genes which are the building blocks of many regulatory gene networks, and study the steady-state distribution of the associated Gillespie algorithm by providing efficient numerical algorithms. We also study a regulatory gene network of interest in gene therapy, using mean-field models with time delays. Convergence of the related time-nonhomogeneous Markov chain is established for a class of linear catalytic networks with feedback loops.

Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways.

The PHO1 family comprises 11 members in Arabidopsis thaliana. In order to decipher the role of these genes in inorganic phosphate (Pi) transport and homeostasis, complementation of the pho1 mutant, deficient in loading Pi to the root xylem, was determined by the expression of the PHO1 homologous genes under the control of the PHO1 promoter. Only PHO1 and the homologue PHO1;H1 could complement pho1. The PHO1;H1 promoter was active in the vascular cylinder of roots and shoots. Expression of PHO1;H1 was very low in Pi-sufficient plants, but was strongly induced under Pi-deficient conditions. T-DNA knock-out mutants of PHO1;H1 neither showed growth defects nor alteration in Pi transport dynamics, or Pi content, compared with wild type. However, the double mutant pho1/pho1;h1 showed a strong reduction in growth and in the capacity to transfer Pi from the root to the shoot compared with pho1. Grafting experiments revealed that phenotypes associated with the pho1 and pho1/pho1;h1 mutants were linked to the lack of gene expression in the root. The increased expression of PHO1;H1 under Pi deficiency was largely controlled by the transcription factor PHR1 and was suppressed by the phosphate analogue phosphite, whereas the increase of PHO1 expression was independent of PHR1 and was not influenced by phosphite. Together, these data reveal that although transfer of Pi to the root xylem vessel is primarily mediated by PHO1, the homologue PHO1;H1 also contributes to Pi loading to the xylem, and that the two corresponding genes are regulated by Pi deficiency by distinct signal transduction pathways.

Reprogramming plant cells for endosymbiosis.

The establishment of arbuscular mycorrhizal (AM) symbioses, formed by most flowering plants in association with glomeromycotan fungi, and the root-nodule (RN) symbiosis, formed by legume plants and rhizobial bacteria, requires an ongoing molecular dialogue that underpins the reprogramming of root cells for compatibility. In both endosymbioses, there are distinct phases to the interaction, including a presymbiotic anticipation phase and, subsequently, an intraradical accommodation of the microsymbiont. Maintenance of the endosymbiosis then depends on reciprocal nutrient exchange with the microsymbiont-obtaining plant photosynthates in exchange for mineral nutrients: enhanced phosphate and nitrogen uptake from AM fungi and fixed nitrogen from rhizobia. Despite the taxonomically distinct groups of symbionts, commonalities are observed in the signaling components and the modulation of host cell responses in both AM and RN symbioses, reflecting common mechanisms for plant cell reprogramming during endosymbiosis.

Adding injury to insult: pathogen detection and responses.

Genomic approaches to the study of the expression of plant genes induced in response to disease and attack are now showing that there is an intimate association between pathogen perception and general stress detection.

A role for the alpha-chain connecting peptide motif in mediating TCR-CD8 cooperation.

To generate peripheral T cells that are both self-MHC restricted and self-MHC tolerant, thymocytes are subjected to positive and negative selection. How the TCR discriminates between positive and negative selection ligands is not well understood, although there is substantial evidence that the CD4 and CD8 coreceptors play an important role in this cell fate decision. We have previously identified an evolutionarily conserved motif in the TCR, the alpha-chain connecting peptide motif (alpha-CPM), which allows the TCR to deliver positive selection signals. Thymocytes expressing alpha-CPM-deficient receptors do not undergo positive selection, whereas their negative selection is not impaired. In this work we studied the ligand binding and receptor function of alpha-CPM-deficient TCRs by generating T cell hybridomas expressing wild-type or alpha-CPM-deficient forms of the T1 TCR. This K(d)-restricted TCR is specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide(252-260) IASA-YIPSAEK(ABA)I and is therefore amenable to TCR photoaffinity labeling. The experiments presented in this work show that alpha-CPM-deficient TCRs fail to cooperate with CD8 to enhance ligand binding and functional responses.

Probing the interaction between feline immunodeficiency virus and CD134 by using the novel monoclonal antibody 7D6 and the CD134 (Ox40) ligand.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+ T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+ T cells, with weaker expression on CD8+ T cells, concordant with the expansion of FIV into CD8+ T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

A downstream mediator in the growth repression limb of the jasmonate pathway.

Wounding plant tissues initiates large-scale changes in transcription coupled to growth arrest, allowing resource diversion for defense. These processes are mediated in large part by the potent lipid regulator jasmonic acid (JA). Genes selected from a list of wound-inducible transcripts regulated by the jasmonate pathway were overexpressed in Arabidopsis thaliana, and the transgenic plants were then assayed for sensitivity to methyl jasmonate (MeJA). When grown in the presence of MeJA, the roots of plants overexpressing a gene of unknown function were longer than those of wild-type plants. When transcript levels for this gene, which we named JASMONATE-ASSOCIATED1 (JAS1), were reduced by RNA interference, the plants showed increased sensitivity to MeJA and growth was inhibited. These gain- and loss-of-function assays suggest that this gene acts as a repressor of JA-inhibited growth. An alternative transcript from the gene encoding a second protein isoform with a longer C terminus failed to repress jasmonate sensitivity. This identified a conserved C-terminal sequence in JAS1 and related genes, all of which also contain Zim motifs and many of which are jasmonate-regulated. Both forms of JAS1 were found to localize to the nucleus in transient expression assays. Physiological tests of growth responses after wounding were consistent with the fact that JAS1 is a repressor of JA-regulated growth retardation.

The peroxisome proliferator-activated receptors at the cross-road of diet and hormonal signalling.

The peroxisome proliferator-activated receptors (PPARs) are members of the steroid/thyroid nuclear receptor superfamily of ligand-activated transcription factors. To date, three isotypes have been identified, alpha, beta and gamma, encoded by three different genes. The alpha isotype is expressed at high levels in the liver where it has a role in lipid oxidation. Its expression and activity follow a diurnal rhythm that parallels the circulating levels of corticosterone in the bloodstream. The gamma isotype on the other hand, is mainly expressed in adipose tissue and has a critical role in adipocyte differentiation and lipid storage. The function of the ubiquitously expressed isotype, PPAR beta, remains to be determined. Besides fulfilling different roles in lipid metabolism, the different PPAR isotypes also have different ligand specificities. A new approach to identify ligands was developed based on the ligand-dependent interaction of PPAR with the recently characterized co-activator SRC-1. This so-called CARLA assay has allowed the identification of fatty acids and eicosanoids as PPAR ligands. Although the evidence clearly links PPAR isotypes to distinct functions, the molecular basis for this isotype-specificity is still unclear. All three isotypes are able to bind the same consensus response element, formed by a direct repeat of two AGGTCA hexamers separated by one base, though with different affinities. We recently demonstrated that besides the core DR-1 element, the 5' flanking sequence should be included in the definition of a PPRE. Interestingly, the presence of this flanking sequence is of particular importance in the context of PPAR alpha binding. Moreover, it reflects the polarity of the PPAR-RXR heterodimer on DNA, with PPAR binding to the 5' half-site and RXR binding to the 3' half-site. This unusual polarity may confer unique properties to the bound heterodimer with respect to ligand binding and interaction with co-activators and corepressors.

Comparative functional analysis of two fibroblast growth factor receptor 1 (FGFR1) mutations affecting the same residue (R254W and R254Q) in isolated hypogonadotropic hypogonadism (IHH).

FGFR1 mutations have been identified in both Kallmann syndrome and normosmic HH (nIHH). To date, few mutations in the FGFR1 gene have been structurally or functionally characterized in vitro to identify molecular mechanisms that contribute to the disease pathogenesis. We attempted to define the in vitro functionality of two FGFR1 mutants (R254W and R254Q), resulting from two different amino acid substitutions of the same residue, and to correlate the in vitro findings to the patient phenotypes. Two unrelated GnRH deficient probands were found to harbor mutations in FGFR1 (R254W and R254Q). Mutant signaling activity and expression levels were evaluated in vitro and compared to a wild type (WT) receptor. Signaling activity was determined by a FGF2/FGFR1 dependent transcription reporter assay. Receptor total expression levels were assessed by Western blot and cell surface expression was measured by a radiolabeled antibody binding assay. The R254W maximal receptor signaling capacity was reduced by 45% (p<0.01) while R254Q activity was not different from WT. However, both mutants displayed diminished total protein expression levels (40 and 30% reduction relative to WT, respectively), while protein maturation was unaffected. Accordingly, cell surface expression levels of the mutant receptors were also significantly reduced (35% p<0.01 and 15% p<0.05, respectively). The p.R254W and p.R254Q are both loss-of-function mutations as demonstrated by their reduced overall and cell surface expression levels suggesting a deleterious effect on receptor folding and stability. It appears that a tryptophan substitution at R254 is more disruptive to receptor structure than the more conserved glutamine substitution. No clear correlation between the severity of in vitro loss-of-function and phenotypic presentation could be assigned.

ppGpp controlled by the Gac/Rsm regulatory pathway sustains biocontrol activity in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.