Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis

The mobilization of inorganic phosphate (Pi) in planta is a complex process regulated by a number of developmental and environmental cues. Plants possess many Pi transporters that acquire Pi from the rhizosphere and translocate it throughout the plant. A few members of the high-affinity Pht1 family of Pi transporters have been functionally characterized and, for the most part, have been shown to be involved in Pi acquisition. We recently demonstrated that the Arabidopsis Pi transporter, Pht1;5, plays a key role in translocating Pi between tissues. Loss-of-function pht1;5 mutant seedlings accumulated more P in shoots relative to wild type but less in roots. In contrast, overexpression of Pht1;5 resulted in a lower P shoot:root ratio compared with wild type. Also, the rosette leaves of Pht1;5-overexpression plants senesced early and contained less P, whereas reproductive organs accumulated more P than those of wild type. Herein we report the molecular response of disrupting Pht1;5 expression on other factors known to modulate P distribution. The results reveal reciprocal mis-regulation of PHO1, miR399d, and At4 in the pht1;5 mutant and Pht1;5-overexpressor, consistent with the corresponding changes in P distribution in these lines. Together our studies reveal a complex role for Pht1;5 in regulating Pi homeostasis.     

Variations in the Composition of Gelling Agents Affect Morphophysiological and Molecular Responses to Deficiencies of Phosphate and Other Nutrients

Low inorganic phosphate (Pi) availability triggers an array of spatiotemporal adaptive responses in Arabidopsis (Arabidopsis thaliana). There are several reports on the effects of Pi deprivation on the root system that have been attributed to different growth conditions and/or inherent genetic variability. Here we show that the gelling agents, largely treated as inert components, significantly affect morphophysiological and molecular responses of the seedlings to deficiencies of Pi and other nutrients. Inductively coupled plasma-mass spectroscopy analysis revealed variable levels of elemental contaminants not only in different types of agar but also in different batches of the same agar. Fluctuating levels of phosphorus (P) in different agar types affected the growth of the seedlings under Pi-deprivation condition. Since P interacts with other elements such as iron, potassium, and sulfur, contaminating effects of these elements in different agars were also evident in the Pi-deficiency-induced morphological and molecular responses. P by itself acted as a contaminant when studying the responses of Arabidopsis to micronutrient (iron and zinc) deficiencies. Together, these results highlighted the likelihood of erroneous interpretations that could be easily drawn from nutrition studies when different agars have been used. As an alternative, we demonstrate the efficacy of a sterile and contamination-free hydroponic system for dissecting morphophysiological and molecular responses of Arabidopsis to different nutrient deficiencies.Plant development is a dynamic and complex process often subject to biotic and abiotic stresses. On encountering nutrient stress, plants undergo an array of adaptive changes. Phosphorus (P) is an essential plant macronutrient, but low availability of soluble inorganic phosphate (Pi) is common in many natural and agricultural ecosystems (Marschner, 1995; Schachtman and Shin, 2007). Arabidopsis (Arabidopsis thaliana) is used as a model system to answer many of the fundamental questions related to responses of plants to Pi stress. To minimize variation caused by macroenvironmental and microenvironmental conditions, Pi-deficiency responses traditionally have been studied by growing Arabidopsis aseptically in continuously shaken liquid culture or on solid medium in petri plates. Although the use of liquid culture is a convenient technique for generating bulk tissue for assaying biochemical and molecular responses to Pi deprivation (Karthikeyan et al., 2002; Misson et al., 2005), continuous swirling to provide aeration makes the study of root hair development and root system architecture (RSA) all but impossible. Therefore, growth of seedlings on vertically oriented petri plates containing solidified nutrient medium with sufficient (1.0–2.5 mm) or limiting (0–10 μm) Pi would be ideal for morphophysiological and molecular studies (López-Bucio et al., 2002; Jain et al., 2007a).Agar and phytagel, extracted from red algae and bacteria, respectively, are commonly used gelling agents (http://www.sigmaaldrich.com/sigma/product). Ideally, a gelling agent would be an inert constituent of the plant growth medium. Studies show, however, that the agent itself causes variations in plant growth responses on otherwise identical nutrient media (Nowak and Asiedu, 1992; Scholten and Pierik, 1998a, 1998b; Beruto et al., 1999). Differences in the performance of gelling agents have been attributed to their variable physiochemical characteristics, such as nutrient diffusion rate, elemental and organic impurities, and gel strength (Debergh, 1983; Nairn et al., 1995). While the elemental contaminants in gelling agents used with nutrient-rich media may not significantly affect the growth of Arabidopsis seedlings, they could make a significant difference under nutrient-deficient conditions. It has been shown that a low concentration of Pi (25 μm) in the medium negated the inhibitory effect of Pi deprivation on shoot growth (López-Bucio et al., 2002). Furthermore, the Pi-deficiency response is also influenced by the interaction of Pi with other elements and compounds. For instance, primary roots of Pi-starved seedlings failed to enter the determinant growth phase, a hallmark of the Pi-deficiency response, when the medium was also deprived of iron (Fe; Sánchez-Calderón et al., 2005; Svistoonoff et al., 2007; Ward et al., 2008). Incidents of cross talk between P and other macroelements and microelements (potassium [K] and zinc [Zn]) have also been reported in crop species (Huang et al., 2000; Wang et al., 2002). Therefore, gel medium contaminants other than P could have a significant bearing on overall Pi-deficiency responses.Inherent genetic variability of Arabidopsis ecotypes is thought to be the likely cause of the large variations noted in morphological responses during Pi-deprivation studies (Chevalier et al., 2003). Some attribute the divergent reports to differences in experimental design (Al-Ghazi et al., 2003; Reymond et al., 2006). For example, photosynthetically active radiation ranging from 30 to 300 μmol m⁻² s⁻¹ has been used to grow Arabidopsis (Williamson et al., 2001; López-Bucio et al., 2002; Al-Ghazi et al., 2003; Chevalier et al., 2003; Jain et al., 2007a). Since photosynthates play a pivotal role in the regulation of the Pi-starvation responses (Liu et al., 2005; Jain et al., 2007a; Karthikeyan et al., 2007), variations in photosynthetically active radiation could be a contributory factor to these discrepancies.To further address disparities in Pi-starvation responses, we investigated the effects of the elemental composition of different batches and types of agar on morphophysiological and molecular responses of Pi-deprived Arabidopsis seedlings. The growth responses of Pi-deprived plants were correlated with P contamination levels of the gelling medium. Also, the effects of Fe contamination in agar were demonstrated under P−Fe− condition on morphological and molecular responses of the seedlings. This study also highlights the fact that P acts as a contaminant in influencing the responses of seedlings to micronutrient (Fe and Zn) deficiencies.     

Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics

The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of nonreceptor tyrosine kinases with high specificity and kinetic resolution.     

TSLP signaling network revealed by SILAC-based phosphoproteomics

Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets for TSLP/TSLPR-associated diseases in humans.     

Characterization of a cellulosome dockerin domain from the anaerobic fungus Piromyces equi

The recycling of photosynthetically fixed carbon in plant cell walls is a key microbial process. In anaerobes, the degradation is carried out by a high molecular weight multifunctional complex termed the cellulosome. This consists of a number of independent enzyme components, each of which contains a conserved dockerin domain, which functions to bind the enzyme to a cohesin domain within the protein scaffoldin protein. Here we describe the first three-dimensional structure of a fungal dockerin, the N-terminal dockerin of Cel45A from the anaerobic fungus Piromyces equi. The structure contains a novel fold of 42 residues. The ligand binding site consists of residues Trp 35, Tyr 8 and Asp 23, which are conserved in all fungal dockerins. The binding site is on the opposite side of the N- and C-termini of the molecule, implying that tandem dockerin domains, seen in the majority of anaerobic fungal plant cell wall degrading enzymes, could present multiple simultaneous binding sites and, therefore, permit tailoring of binding to catalytic demands.     

A functional annotation of subproteomes in human plasma

The data collected by Human Proteome Organization's Plasma Proteome Pilot project phase was analyzed by members of our working group. Accordingly, a functional annotation of the human plasma proteome was carried out. Here, we report the findings of our analyses. First, bioinformatic analyses were undertaken to determine the likely sources of plasma proteins and to develop a protein interaction network of proteins identified in this project. Second, annotation of these proteins was performed in the context of functional subproteomes involved in the coagulation pathway, the mononuclear phagocytic system, the inflammation pathway, the cardiovascular system, and the liver; as well as the subset of proteins associated with DNA binding activities. Our analyses contributed to the Plasma Proteome Database (http://www.plasmaproteomedatabase.org), an annotated database of plasma proteins identified by HPPP as well as from other published studies. In addition, we address several methodological considerations including the selective enrichment of post-translationally modified proteins by the use of multi-lectin chromatography as well as the use of peptidomic techniques to characterize the low molecular weight proteins in plasma. Furthermore, we have performed additional analyses of peptide identification data to annotate cleavage of signal peptides, sites of intra-membrane proteolysis and post-translational modifications. The HPPP-organized, multi-laboratory effort, as described herein, resulted in much synergy and was essential to the success of this project.     

Simultaneous observation of positive and negative nuclear Overhauser effects in oligopeptides due to segmental motion

Nuclear Overhauser effect (NOE) studies of the symmetrical cystine peptides (Formula: see text) (n = 1-3) in dimethylsulfoxide, have resulted in the simultaneous observation of both positive and negative NOEs. Positive NOEs are observed on the Trp C2H and C4H protons of the indole ring upon irradiation of Trp C alpha H and C beta H2 resonances in the peptides where n = 1 and 2. Negative NOEs are observed between backbone NH and C alpha H protons. The magnitudes of the observed NOEs are sensitive to changes in molecular size and solvent viscosity. The results demonstrate that NOEs may be a useful probe of sidechain segmental motion in oligopeptides.     

Solution structure of the CBM10 cellulose binding module from Pseudomonas xylanase A

Plant cell wall hydrolases generally have a modular structure consisting of a catalytic domain linked to one or more noncatalytic carbohydrate-binding modules (CBMs), whose common function is to attach the enzyme to the polymeric substrate. Xylanase A from Pseudomonas fluorescens subsp. cellulosa (Pf Xyn10A) consists of a family 10 catalytic domain, an N-terminal family IIa cellulose-binding module, and an internal family 10 cellulose-binding module. The structure of the 45-residue family 10 CBM has been determined in solution using NMR. It consists of two antiparallel beta-sheets, one with two strands and one with three, with a short alpha-helix across one face of the three-stranded sheet. There is a high density of aromatic residues on one side of the protein, including three aromatic residues (Tyr8, Trp22, and Trp24), which are exposed and form a flat surface on one face, in a classical polysaccharide-binding arrangement. The fold is closely similar to that of the oligonucleotide/oligosaccharide-binding (OB) fold, but appears to have arisen by convergent evolution, because there is no sequence similarity, and the presumed binding sites are on different faces.