A network-based comparative framework to study conservation and divergence of proteomes in plant phylogenies

Comparative functional genomics offers a powerful approach to study species evolution. To date, the majority of these studies have focused on the transcriptome in mammalian and yeast phylogenies. Here, we present a novel multi-species proteomic dataset and a computational pipeline to systematically compare the protein levels across multiple plant species. Globally we find that protein levels diverge according to phylogenetic distance but is more constrained than the mRNA level. Module-level comparative analysis of groups of proteins shows that proteins that are more highly expressed tend to be more conserved. To interpret the evolutionary patterns of conservation and divergence, we develop a novel network-based integrative analysis pipeline that combines publicly available transcriptomic datasets to define co-expression modules. Our analysis pipeline can be used to relate the changes in protein levels to different species-specific phenotypic traits. We present a case study with the rhizobia-legume symbiosis process that supports the role of autophagy in this symbiotic association

Comparison of Vacuum MALDI and AP-MALDI Platforms for the Mass Spectrometry Imaging of Metabolites Involved in Salt Stress in Medicago truncatula

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is routinely used to determine the spatial distributions of various biomolecules in tissues. Recently, there has been an increased interest in creating higher resolution images using sources with more focused beams. One such source, an atmospheric pressure (AP) MALDI source from MassTech, has a laser capable of reaching spatial resolutions of 10 μm. Here, the AP-MALDI source coupled with a Q Exactive HF Orbitrap platform is compared to the commercial MALDI LTQ Orbitrap XL system using Medicago truncatula root nodules. AP-MALDI parameters, such as the S-lens value, capillary temperature, and spray voltage, were optimized on the Q Exactive-HF platform for optimal detection of plant metabolites. The performance of the two systems was evaluated for sensitivity, spatial resolution, and overall ability to detect plant metabolites. The commercial MALDI LTQ Orbitrap XL was superior regarding the number of compounds detected, as at least two times more m/z were detected compared to the AP-MALDI system. However, although the AP-MALDI source requires a spatial resolution higher than 10 μm to get the best signal, the spatial resolution at 30 μm is still superior compared to the 75 μm spatial resolution achieved on the MALDI platform. The AP-MALDI system was also used to investigate the metabolites present in M. truncatula roots and root nodules under high salt and low salt conditions. A discriminative analysis with SCiLS software revealed m/z ions specific to the control and salt conditions. This analysis revealed 44 m/z ions present at relatively higher abundances in the control samples, and 77 m/z enriched in the salt samples. Liquid chromatography-tandem MS was performed to determine the putative molecular identities of some of the mass ions enriched in each sample, including, asparagine, adenosine, and nicotianamine in the control samples, and arginine and soyasaponin I in the salt treated samples

Intersection Of Salt Signaling With Nodule Initiation And Maturation During Medicago Truncatula-Sinorhizobium Meliloti Symbiosis.

Legumes contribute significantly to sustainable agriculture because of their high protein content. This intrinsic nitrogen is the product of a mutualistic association between their roots, and a group of soil bacteria, collectively known as rhizobia. The rhizobium-legume symbiosis is a complex developmental process that involves rhizobial entry and differentiation, coupled in space and time, with the development of a root organ (the nodule) and is subject to modulation by environmental factors. Salt stress is a strong negative regulator of rhizobium-legume symbiosis, but despite its agricultural impact, the mechanism of salt regulation of rhizobium-legume symbiosis is poorly understood. This dissertation addresses this issue with focus on early rhizobium-legume signaling and maturation of nodules, using the model legume Medicago truncatula. The rhizobium-legume symbiosis is initiated with a molecular dialogue between the symbiotic partners, followed by the entry of rhizobia inside the host. These processes are under tight genetic regulation and involve the symbiotic induction of several host genes. I used a candidate gene-approach to locate the temporal intersection of salt and rhizobium-legume signaling and found that the ionic component of salinity hyperinduces Early Nodulin 11 (ENOD11) and ERF Required for Nodulation 1 (ERN1). I also found that the hyperinduction of ENOD11 requires two symbiotic rhizobial signals- Nod factors (NF) and succinoglycan and the host transcription factor Nodulation Signaling Pathway 2 (NSP2). In order to explore the possibility of an extensive transcriptional re-programming during rhizobium-legume signaling in the presence of salinity, I profiled the host root transcriptome and discovered strong transcriptional upregulation by salt, of several host genes associated with NF perception, rhizobial infection and ubiquitination. Curiously, I found that the hyperinduction of these genes correlate with an inhibition of rhizobial entry into the host. These findings highlight that early rhizobium-legume signaling and rhizobial colonization of the host are under tight transcriptional regulation of the host. Despite the early inhibition of infection, few nodules develop under salt stress. However, these nodules are morphologically abnormal, indicating that salinity must interfere with nodule maturation, a prerequisite for nitrogen fixation. Consistent with the theme of early symbiotic signaling, I found host genes involved during the early stages of nodule differentiation to remain highly expressed in nodules that developed under salt stress, suggesting a delay in maturation. This inference was further corroborated when I found that genes associated with the later stages of nodule development and nitrogen fixation showed decreased expression in the presence of salt. Additionally, I observed that this developmental shift of nodules under salinity was associated with differential accumulation of osmolytes, such as, proline betaine and homostachydrine in the salt-stressed nodules. Together, my work provides a mechanistic understanding of the intersection between nodulation and salt signaling, a question of major agricultural impact

Room temperature synthesis of nanocrystalline SnO through sonochemical route

Nanocrystalline tin monoxide (SnO) has been synthesized through sonication-assisted precipitation technique at room temperature. The key in obtaining phase pure SnO at room temperature lies in exploiting high-power ultrasound (450W). The physical phenomenon responsible for the sonochemical process is acoustic cavitation. A mixed phase is formed on decreasing the ultrasonic power.-The heat generated during sonication does not explain the difference in phase formation under varying ultrasonic power as the difference in temperatures of the solutions during sonication under different power ratings was within 10-15 degrees C. SEM and XRD analysis were carried out for the investigation of powder morphology and crystalline structure of the material. (C) 2007 Published by Elsevier B.V

Purification and characterization of a gelatinolytic serine protease from the seeds of ash gourd <em>Benincasa hispida</em> (Thunb.) Cogn.

77-87In Ayurveda, Benincasa hispida (Thunb.) Cogn. (Ash gourd) was recommended for management of diabetes, peptic ulcer, and other diseases. This plant is rich in proteolytic enzymes and proteases have wide application in food and laundry industry. Therefore, the search for new potential plant proteases continues. A soluble gelatinolytic plant serine protease (AG2) had been purified from the seeds of Benincasa hispida. The molecular mass of the monomer was estimated to be about 11 kDa by SDS-PAGE and 11211.1 Da by MALDI-TOF. The protease activity was strongly inhibited by PMSF only but not at all by soyabean trypsin inhibitor and resists autodigestion. Thus AG2 belongs to subtilisin family. The optimum pH and temperature are 10.0 and 30°C respectively. This protease was quite stable in presence of a cationic surfactant, an oxidizing agent and in basic pH medium. The protease AG2 can hydrolyze casein, azoalbumin and TAME but it was inert towards BAPNA. The kinetic parameters Km and Vmax were 0.117 ± 0.00067 mM and 470.592 ± 0.631 unit mg-1 min-1 respectively using casein as substrate. The CD spectrum showed it as a typical α/β class of protease. The N-terminal sequence of first 17 amino acid residues (MQQFFNEPSSLLIVVVR) is unique in nature

Temperature dependent photoemission spectroscopy on lightly-doped sodium tungsten bronze

Temperature dependent photoemission studies on lightly doped (x = 0.025) sodium tungsten bronzes, NaxWO3 have been investigated by high-resolution photoemission spectroscopy. The experimental results show evidence for polaron formation at the valence band edge and the photoemission spectra taken in different modes of the electron analyzer suggest that the density of states at the valence band edge gradually moves to other k-points in the Brillouin zone with increasing temperature and explain the dynamics of polarons in the insulating disordered sodium tungsten bronzes. (C) 2012 Elsevier Ltd. All rights reserved

Photoelectron spectromicroscopy study of metal-insulator transition in NaxWO3

We have investigated the validity of percolation model, which is quite often invoked to explain the metal-insulator transition in sodium tungsten bronzes, NaxWO(3) by photoelectron spectromicroscopy. The spatially resolved direct spectromicroscopic probing on both the insulating and metallic phases of high quality single crystals of NaxWO(3) reveals the absence of any microscopic inhomogeneities embedded in the system within the experimental limit. Neither any metallic domains in the insulating host nor any insulating domains in the metallic host have been found to support the validity of percolation model to explain the metal-insulator transition in NaxWO(3). The possible origin of insulating phase in NaxWO(3) is due to the Anderson localization of all the states near E-F. The localization occurs because of the strong disorder arising from random distribution of Na+ ions in the WO3 lattice

The putative transporter MtUMAMIT14 participates in nodule formation in Medicago truncatula

Abstract Transport systems are crucial in many plant processes, including plant–microbe interactions. Nodule formation and function in legumes involve the expression and regulation of multiple transport proteins, and many are still uncharacterized, particularly for nitrogen transport. Amino acids originating from the nitrogen-fixing process are an essential form of nitrogen for legumes. This work evaluates the role of MtN21 (henceforth MtUMAMIT14), a putative transport system from the MtN21/EamA-like/UMAMIT family, in nodule formation and nitrogen fixation in Medicago truncatula. To dissect this transporter’s role, we assessed the expression of MtUMAMIT14 using GUS staining, localized the corresponding protein in M. truncatula root and tobacco leaf cells, and investigated two independent MtUMAMIT14 mutant lines. Our results indicate that MtUMAMIT14 is localized in endosomal structures and is expressed in both the infection zone and interzone of nodules. Comparison of mutant and wild-type M. truncatula indicates MtUMAMIT14, the expression of which is dependent on the presence of NIN, DNF1, and DNF2, plays a role in nodule formation and nitrogen-fixation. While the function of the transporter is still unclear, our results connect root nodule nitrogen fixation in legumes with the UMAMIT family

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade

: Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root-nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula