Identification of calmodulin binding proteins in the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Calmodulin (CaM) is a primary Ca²⁺ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.     

Chloroplast ribonucleoprotein-like proteins of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis> are not involved in RNA stability and RNA editing

Many RNA recognition motif (RRM)-containing proteins are known to exist in chloroplasts. Major members of the RRM protein family, which are chloroplast ribonucleoproteins (cpRNPs), have been investigated in seed plants, including tobacco and Arabidopsis thaliana, but never in early land plants, such as bryophytes. In this study, we surveyed RRM proteins encoded in the moss Physcomitrella patens genome and predicted 25 putative chloroplast RRM proteins. Among them, two RRM-containing proteins, PpRBP2a and PpRBP2b, resembled cpRNPs and were thus referred to as cpRNP-like proteins. However, knockout mutants of either one or two PpRBP2 genes exhibited a wild type-like phenotype. Unlike Arabidopsis cpRNPs, the levels of mRNA accumulation in chloroplasts were not affected in the PpRBP2 knockout mutants. In addition, the efficiency of RNA editing was also not altered in the mutants. This suggests that PpRBP2a and 2b may be functionally distinct from Arabidopsis cpRNPs.     

Comparative proteomic analysis of leaf tissue from tomato plants colonized with <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis>

A comparative proteomic approach was performed to analyze the differential accumulation of leaf proteins in response to the symbiosis between Solanum lycopersicum and the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis. Protein profiling was examined in leaves from tomato plants colonized with AMF (M), as well as non-colonized plants fertilized with low phosphate (20 μM P; NM-LP) and non-colonized plants fertilized with regular phosphate Hoagland’s solution (200 μM P; NM-RP). Comparisons were made between these groups, and 2D-SDS-PAGE revealed that 27 spots were differentially accumulated in M vs. NM-LP. Twenty-three out of the 27 spots were successfully identified by mass spectrometry. Two of these proteins, 2-methylene-furan-3-one reductase and auxin-binding protein ABP19a, were up-accumulated in M plants. The down-accumulated proteins in M plants were associated mainly with photosynthesis, redox, and other molecular functions. Superoxide dismutase, harpin binding protein, and thioredoxin peroxidase were down-accumulated in leaves of M tomato plants when compared to NM-LP and NM-RP, indicating that these proteins are responsive to AMF colonization independently of the phosphate regime under which they were grown. 14-3-3 protein was up-accumulated in NM-RP vs. NM-LP plants, whereas it was down-accumulated in M vs. NM-LP and M vs. NM-RP, regardless of their phosphate nutrition. This suggests a possible regulation by P nutrition and AMF colonization. Our results demonstrate AMF-induced systemic changes in the expression of tomato leaf proteins, including the down-accumulation of proteins related to photosynthesis and redox function.     

Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea

Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein–protein interaction.     

Comparative protein profiles of <Emphasis Type="Italic">Butea superba</Emphasis> tubers under seasonal changes

Seasonal changes are major factors affecting environmental conditions which induce multiple stresses in plants, leading to changes in protein relative abundance in the complex cellular plant metabolic pathways. Proteomics was applied to study variations in proteome composition of Butea. superba tubers during winter, summer and rainy season throughout the year using two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight tandem mass spectrometry. A total of 191 protein spots were identified and also classified into 12 functional groups. The majority of these were mainly involved in carbohydrate and energy metabolism (30.37 %) and defense and stress (18.32 %). The results exhibited the highest numbers of identified proteins in winter-harvested samples. Forty-five differential proteins were found in different seasons, involving important metabolic pathways. Further analysis indicated that changes in the protein levels were due mainly to temperature stress during summer and to water stress during winter, which affected cellular structure, photosynthesis, signal transduction and homeostasis, amino-acid biosynthesis, protein destination and storage, protein biosynthesis and stimulated defense and stress mechanisms involving glycolytic enzymes and relative oxygen species catabolizing enzymes. The proteins with differential relative abundances might induce an altered physiological status within plant tubers for survival. The work provided new insights into the better understanding of the molecular basis of plant proteomes and stress tolerance mechanisms, especially during seasonal changes. The finding suggested proteins that might potentially be used as protein markers in differing seasons in other plants and aid in selecting B. superba tubers with the most suitable medicinal properties in the future.     

Proteomic analysis of sensitive and multi drug resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Multidrug-resistant tuberculosis (MDR-TB) is caused by bacteria that are resistant to the most effective anti TB drugs (Isoniazid and Rifampicin) with or without resistance to other drugs. Novel intervention strategies to eliminate this disease based on finding proteins can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profile of MDR-TB with sensitive isolates. Two-dimensional gel electrophoresis (2DE) along with mass spectrometry is a powerful and effective tool to identification and characterization of Mycobacterium tuberculosis. Two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for diagnosis and comparison of proteins. We identified 14 protein spots in MDR-TB isolates that 2DE analysis showed these spots absent in M. tuberculosis sensitive isolates (Rv1876, Rv0379, Rv0147, Rv2031c, Rv3597c, Rv1886c, MT0493, Rv0440, Rv3614c, Rv1626, Rv0443, Rv0475, Rv3057 and unknown protein. The results showed 22 protein spots which were up regulated (or expressed) by the MDR-TB isolates, (Rv1240, Rv3028c, Rv2971, Rv2114c, Rv3311, Rv3699, Rv1023, Rv1308, Rv3774, Rv0831c, Rv2890c, Rv1392, Rv0719, Rv0054, Rv3418c, Rv0462, Rv2215, Rv2986c, Rv3248c and Rv1908c)). Two up regulated protein spots were identified in sensitive isolate (Rv1133c and Rv0685). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug resistant and sensitive of M. tuberculosis.     

Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety

Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) from wheat affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions of specific proteins. The objective of this study was to link LMW-GS genes and haplotypes characterized using well known Glu-A3, Glu-B3, and Glu-D3 gene-specific primers to their protein products in a single wheat variety. A total of 36 LMW-GS genes and pseudogenes were amplified from the Korean cultivar Keumkang. These include 11 Glu-3 gene haplotypes, two from the Glu-A3 locus, two from the Glu-B3 locus, and seven from the Glu-D3 locus. To establish relationships between gene haplotypes and their protein products, a glutenin protein fraction was separated by two-dimensional gel electrophoresis (2-DGE) and 17 protein spots were analyzed by N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS). LMW-GS proteins were identified that corresponded to all Glu-3 gene haplotypes except the pseudogenes. This is the first report of the comprehensive characterization of LMW-GS genes and their corresponding proteins in a single wheat cultivar. Our approach will be useful to understand the contributions of individual LMW-GS to the end-use quality of flour.     

Phosphoproteomic Analysis in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Roots Treated with <Emphasis Type="Italic">Rhizobium etli</Emphasis> Nodulation Factors

Legumes are unique in their ability to establish symbiotic interactions with rhizobacteria, providing a source of assimilable nitrogen; this symbiosis is regulated by complex signaling process between the plant and the bacteria. The participation of specific protein kinases during the initial steps of the nodulation process has been established. However, their substrates or the signaling networks implicated are not fully understood. Herein, a phosphoproteomic analysis of Phaseolus vulgaris roots treated for 24 h with specific Nod factors was performed using an immobilized metal ion affinity chromatography enrichment and two-dimensional gel electrophoresis approach with mass spectrometry identification. A total of 33 protein spots showing more than 1.5-fold shift were identified (17 protein spots in which the relative abundance increased and 16 that decreased). The majority of the identified root phosphoproteins displaying an increased relative abundance are presumed to have functions related to the biosynthesis and folding of proteins, energy metabolism, or cytoskeleton rearrangements, which reflect the metabolic status of the roots as being part of the developmental processes leading to nodule initiation and the importance of cytoskeleton rearrangement in the P. vulgaris–rhizobia symbiosis. The proteins in which relative abundance decreased are associated with defense and oxido-reduction processes, which could indicate a suppression of plant defense responses during the establishment of the rhizobia–legume interaction and an increase of reactive oxygen species production.     

The transcriptome of flower development provides insight into floral scent formation in <Emphasis Type="Italic">Freesia hybrida</Emphasis>

Freesia hybrida is a popular cut flower that is globally cultivated. Despite fragrance being an important floral trait, little is known of its associated molecular mechanisms in F. hybrida. In this study, cDNA libraries were constructed for three floral developmental stages in F. hybrida. A total of 74,660 unigenes were obtained from RNA sequencing and de novo assembly, of which 72.20% were annotated by seven public protein databases. Approximately 12,420 differentially expressed genes were identified during flower development. Gas chromatography–mass spectrometry analysis detected a total of 34 floral volatile compounds, primarily volatile monoterpenes, which accounted for approximately 90% of the volatiles. Using a system analysis-based approach, 36 candidate genes related to volatile terpenes were identified, in which the majority of up-regulated genes associated with the biosynthesis of monoterpenes and the majority of down-regulated genes associated with the biosynthesis of sesquiterpenes. Of these, FhDXS2A, FhGPPs and FhTPSs were considered to be important in floral scent formation. This study provides an in-depth assessment of floral scent and a reference for the future molecular breeding of floral scent in ornamental plants.     

Constitutive Expression of a Tomato Small Heat Shock Protein Gene <Emphasis Type="Italic">LeHSP21</Emphasis> Improves Tolerance to High-Temperature Stress by Enhancing Antioxidation Capacity in Tobacco

It is well established that small heat shock proteins (sHSPs) play an important role in thermotolerance in various organisms due to their abundance and diversity. In the present study, a chloroplast small heat shock protein gene (LeHSP21) from tomato (Lycopersicon esculentum cv PKM-1) was constitutively expressed in tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants via Agrobacterium-mediated transformation. When compared to wild-type control plants, transgenic tobacco plants constitutively expressing LeHSP21, driven by the cauliflower mosaic virus 35S promoter, exhibited improved tolerance to both high temperature and oxidative stress. Furthermore, when the seedlings were subjected to high temperature treatment, the activities of anti-oxidative enzymes and the content of proline were significantly higher in transgenic plants than those in the wild-type plants. Our results presented here demonstrate the feasibility of improving high temperature and oxidative stress tolerance in plants through the expression of LeHSP21 gene.     

Identification and Molecular Characterization of HOS15-interacting Proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Chromatin modification is a key mechanism of gene expression in eukaryotes, and involve interactions among several proteins. Recently, we reported that HOS15, a cullin-based E3 ligase receptor, is involved in chromatin remodeling, and regulates gene expression and cold tolerance in Arabidopsis thaliana. To identify the protein complexes that function in conjunction with HOS15, we performed FLAGtag affinity purification using transgenic Arabidopsis plants expressing HOS15-FLAG, and isolated HOS15-interacting proteins. To identify these proteins, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis was conducted, and 16 proteins were identified. Database searches revealed that these proteins were histone variants, histone deacetylases, mRNA splicing regulators, a protein kinase, and proteins of unknown function. The ability of these proteins binding to HOS15 was confirmed using yeast two-hybrid, co-immunoprecipitation (Co-IP), and luciferase complementation imaging (LCI) assays. Our data suggest that specific interactions between HOS15 and those proteins involve in chromatin remodeling and RNA processing regulates plant development and abiotic stress in Arabidopsis.     

Overexpression of Tomato Homolog of Glycolate/Glycerate Transporter Gene <Emphasis Type="Italic">PLGG1/AtLrgB</Emphasis> Leads to Reduced Chlorophyll Biosynthesis

LrgA and LrgB genes have been identified as new components in regulation of programmed cell death (PCD) in bacteria. While in Arabidopsis, it has been documented that AtLrgB plays a crucial role in chloroplast development and photorespiration by acting as a glycolate/glycerate translocator (PLGG1) in the chloroplast inner membrane. However, little is known about LrgB homologs in other plant species, especially those with fleshy fruits. In this study, a homologous gene of AtLrgB, here designated SlLrgB, was identified in tomato. Similar to AtLrgB, structure analysis suggests that the LrgA and LrgB genes have evolved into two domains of the SlLrgB protein. Expression pattern analysis showed that SlLrgB accumulated mainly in green tissues and could be regulated by light, hormone, and abiotic stress treatments. Compared to wild-type plants, parts of SlLrgB overexpression plants displayed etiolated leaves and a growth retardation phenotype, with significantly reduced chlorophyll content both in leaves and fruits. The qPCR results revealed that the SGR gene, which was associated with chlorophyll degradation, was severely repressed. Two key genes in the chlorophyll biosynthesis pathway, CAO and POR, were also suppressed in the SlLrgB overexpression plants. Taken together, we suggest that SlLrgB may play important roles in the regulation of chlorophyll metabolism pathways in tomato.     

Artificial targeting of a nucleus-encoded RNA-binding protein AtRZ1a to chloroplasts affects flowering and ABA response of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the fact that many nucleus-encoded RNA-binding proteins (RBPs) are targeted to chloroplasts and play essential roles in RNA metabolism in chloroplasts, the question of whether artificial targeting of a nucleus-encoded RBP to chloroplasts affects chloroplast function and plant growth has never been addressed. In this study, a nuclear zinc finger-containing Arabidopsis RBP, designated AtRZ1a, which was previously shown to play a role in stress response, was artificially targeted to chloroplasts, and the growth and stress response of the transgenic plants were evaluated. Confocal analysis of the cellular localization of the cTP_AtRZ1a protein containing the N-terminal chloroplast transit peptide (cTP) from rubisco small subunit revealed that the cTP_AtRZ1a fusion protein is successfully targeted to chloroplasts. When grown under normal conditions, flowering of the transgenic plants was delayed, and the FLC expression was significantly upregulated in the transgenic plants. Artificial targeting of AtRZ1a to chloroplasts severely inhibited seedling growth of the plants in the presence of ABA by upregulating expression of ABA signaling-related genes ABI3 and ABI4. Taken together, these results suggest that artificial targeting of a nucleus-encoded AtRZ1a to chloroplasts affects the growth and development of Arabidopsis under normal or ABA treated conditions.     

Dissecting Pistil Responses to Incompatible and Compatible Pollen in Self-Incompatibility <Emphasis Type="Italic">Brassica oleracea</Emphasis> Using Comparative Proteomics

Angiosperms have developed self-incompatibility (SI) systems to reject self-pollen, thereby promoting outcrossing. The Brassicaceae belongs to typical sporophytic system, having a single S-locus controlled SI response, and was chosen as a model system to study SI-related intercellular signal transduction. In this regard, the downstream factor of EXO70A1 was unknown. Here, protein two-dimensional electrophoresis (2-DE) method and coupled with matrix-assisted laser desorption ionization/time of flight of flight mass spectrometry (MALDI-TOF -MS) and peptide mass fingerprinting (PMF) was used to further explore the mechanism of SI responses in Brassica oleracea L. var. capitata L. at protein level. To further confirm the time point of protein profile change, total proteins were collected from B. oleracea pistils at 0 min, 1 h, and 2 h after self-pollination. In total 902, 1088 and 1023 protein spots were separated in 0 min, 1 h and 2 h 2-DE maps, respectively. Our analyses of self-pollination profiles indicated that proteins mainly changed at 1 h post-pollination in B. oleracea. Moreover, 1077 protein spots were separated in cross-pollinated 1 h (CP) pistil 2-DE map. MALDI-TOF-MS and PMF successfully identified 34 differentially-expressed proteins (DEPs) in SP and CP 1 h 2-DE maps. Gene ontology and KEGG analysis revealed an array of proteins grouped in the following categories: stress and defense response (35%), protein metabolism (18%), carbohydrate and energy metabolism (12%), regulation of translation (9%), pollen tube development (12%), transport (9%) and cytoskeletal (6%). Sets of DEPs identified specifically in SP or only up-regulated expressed in CP pistils were chosen for funther investigating in floral organs and during the process of self- and cross-pollination. The function of these DEPs in terms of their potential involvement in SI in B. oleracea is discussed.     

Proteomic analysis of cold stress-responsive proteins in <Emphasis Type="Italic">Thellungiella</Emphasis> rosette leaves

Low temperature is one of the most severe environmental factors that impair plant growth and agricultural production. To investigate how Thellungiella halophila, an Arabidopsis-like extremophile, adapts to cold stress, a comparative proteomic approach based on two-dimensional electrophoresis was adopted to identify proteins that changed in abundance in Thellungiella rosette leaves during short term (6 h, 2 and 5 days) and long term (24 days) exposure to cold stress. Sixty-six protein spots exhibited significant change at least at one time point and maximal cold stress induced-proteome change was found in long-term cold stress group while the minimal change was found in 6-h cold treatment group. Fifty protein spots were identified by mass spectrometry analysis. The identified proteins mainly participate in photosynthesis, RNA metabolism, defense response, energy pathway, protein synthesis, folding and degradation, cell wall and cytoskeleton and signal transduction. These proteins might work cooperatively to establish a new homeostasis under cold stress. Nearly half of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology suggesting that the cold stress tolerance of T. halophila is achieved, at least partly, by regulation of chloroplast function. All protein spots involved in RNA metabolism, defense response, protein synthesis, folding and degradation were found to be upregulated markedly by cold treatment, indicating enhanced RNA metabolism, defense and protein metabolism may play crucial roles in cold tolerance mechanism in T. halophila.