Fluorescent labeling of the root cap cells with the bioactive NBD-S chemical probe based on the cellulose biosynthesis inhibition herbicides.

Development of the methods to examine the molecular targets of biologically active compounds is one of the most important subjects in experimental biology/biochemistry. To evaluate the usability of the (7-nitro-2,1,3-benzoxadiazole)-thioether (NBD-S) probe for this purpose, bioactive chemical probe (1) as the cellulose biosynthesis (CB) inhibitor was synthesized and tested. As a result, a variety of fluorescently-labeled particles and organelles were found in the columella root cap cells of radish plants. Of note, well-defined cellular organelles were clearly recognized in the detaching root cap cells (border-like cells). These results imply that the bioactive NBD-S chemical probe could be a valuable direct-labeling reagent. Analysis of these fluorescent substances would be helpful in providing new information on defined molecular targets and events.

Retraction notice to "Proteomic studies on lactic acid bacteria: A review" [Biochem. Biophys. Rep. 14 (2019) 100689].

[This retracts the article DOI: 10.1016/j.bbrep.2018.04.009.].

Proteomic studies on lactic acid bacteria: A review.

Probiotics are amongst the most common microbes in the gastro-intestinal tract of humans and other animals. Prominent among probiotics are Lactobacillus and Bifidobacterium. They offer wide-ranging health promoting benefits to the host which include reduction in pathological alterations, stimulation of mucosal immunity and interaction with mediators of inflammation among others. Proteomics plays a vital role in understanding biological functions of a cell. Proteomics is also slowly and steadily adding to the existing knowledge on role of probiotics. In this paper, the proteomics of probiotics, with special reference to lactic acid bacteria is reviewed with a view to understand i) proteome map, ii) mechanism of adaptation to harsh gut environment such as low pH and bile acid, iii) role of cell surface proteins in adhering to intestinal epithelial cells, and iv) as a tool to answer basic cell functions. We have also reviewed various analytical methods used to carry out proteome analysis, in which 2D-MS and LC-MS/MS approaches were found to be versatile methods to perform high-throughput sample analyses even for a complex gut samples. Further, we present future road map of understanding gut microbes combining meta-proteomics, meta-genomics, meta-transcriptomics and -metabolomics.

Common bean proteomics: Present status and future strategies.

Common bean (Phaseolus vulgaris L.) is a legume of appreciable importance and usefulness worldwide to the human population providing food and feed. It is rich in high-quality protein, energy, fiber and micronutrients especially iron, zinc, and pro-vitamin A; and possesses potentially disease-preventing and health-promoting compounds. The recently published genome sequence of common bean is an important landmark in common bean research, opening new avenues for understanding its genetics in depth. This legume crop is affected by diverse biotic and abiotic stresses severely limiting its productivity. Looking at the trend of increasing world population and the need for food crops best suited to the health of humankind, the legumes will be in great demand, including the common bean mostly for its nutritive values. Hence the need for new research in understanding the biology of this crop brings us to utilize and apply high-throughput omics approaches. In this mini-review our focus will be on the need for proteomics studies in common bean, potential of proteomics for understanding genetic regulation under abiotic and biotic stresses and how proteogenomics will lead to nutritional improvement. We will also discuss future proteomics-based strategies that must be adopted to mine new genomic resources by identifying molecular switches regulating various biological processes. SIGNIFICANCE: Common bean is regarded as "grain of hope" for the poor, being rich in high-quality protein, energy, fiber and micronutrients (iron, zinc, pro-vitamin A); and possesses potentially disease-preventing and health-promoting compounds. Increasing world population and the need for food crops best suited to the health of humankind, puts legumes into great demand, which includes the common bean mostly. An important landmark in common bean research was the recent publication of its genome sequence, opening new avenues for understanding its genetics in depth. This legume crop is affected by diverse biotic and abiotic stresses severely limiting its productivity. Therefore, the need for new research in understanding the biology of this crop brings us to utilize and apply high-throughput omics approaches. Proteomics can be used to track all the candidate proteins/genes responsible for a biological process under specific conditions in a particular tissue. The potential of proteomics will not only help in determining the functions of a large number of genes in a single experiment but will also be a useful tool to mine new genes that can provide solution to various problems (abiotic stress, biotic stress, nutritional improvement, etc). We believe that a combined approach including breeding along with omics tools will lead towards attaining sustainability in legumes, including common bean.

Methyl jasmonate elicits the biotransformation of geraniol stored as its glucose conjugate into methyl geranate in Achyranthes bidentata plant.

To investigate the biotransformation pathway of airborne geraniol by Achyranthes bidentata (A. bidentata), deuterium labeled geraniol was applied with or without methyl jasmonate (MeJA), and the biosynthesized metabolites were analyzed. In A. bidentata leaves, geraniol was conjugated with glucose. The conjugate was then metabolized to afford methyl geranate only under MeJA elicitation. MeJA elicits the biotransformation of geraniol into methyl geranate by inducing the conversion of the intermediate, glucose conjugate of geraniol.

An Integrated Biochemical, Proteomics, and Metabolomics Approach for Supporting Medicinal Value of Panax ginseng Fruits.

Panax ginseng roots are well known for their medicinal properties and have been used in Korean and Chinese traditional medicines for 1000s of years. However, the medicinal value of P. ginseng fruits remain poorly characterized. In this study, we used an integrated biochemical, proteomics, and metabolomics approach to look into the medicinal properties of ginseng fruits. DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)] assays showed higher antioxidant activities in ginseng fruits than leaves or roots. Two-dimensional gel electrophoresis (2-DE) profiling of ginseng fruit proteins (cv. Cheongsun) showed more than 400 spots wherein a total of 81 protein spots were identified by mass spectrometry using NCBInr, UniRef, and an in-house developed RNAseq (59,251 protein sequences)-based databases. Gene ontology analysis showed that most of the identified proteins were related to the hydrolase (18%), oxidoreductase (16%), and ATP binding (15%) activities. Further, a comparative proteome analysis of four cultivars of ginseng fruits (cvs. Yunpoong, Gumpoong, Chunpoong, and Cheongsun) led to the identification of 22 differentially modulated protein spots. Using gas chromatography-time of flight mass spectrometry (GC-TOF MS), 66 metabolites including amino acids, sugars, organic acids, phenolic acids, phytosterols, tocopherols, and policosanols were identified and quantified. Some of these are well known medicinal compounds and were not previously identified in ginseng. Interestingly, the concentration of almost all metabolites was higher in the Chunpoong and Gumpoong cultivars. Parallel comparison of the four cultivars also revealed higher amounts of the medicinal metabolites in Chunpoong and Gumpoong cultivars. Taken together, our results demonstrate that ginseng fruits are a rich source of medicinal compounds with potential beneficial health effects.

Expect the Unexpected Enrichment of "Hidden Proteome" of Seeds and Tubers by Depletion of Storage Proteins.

Dynamic resolution of seed and tuber protein samples is highly limited due to the presence of high-abundance storage proteins (SPs). These proteins inevitably obscure the low-abundance proteins (LAPs) impeding their identification and characterization. To facilitate the detection of LAPs, several methods have been developed during the past decade, enriching the proteome with extreme proteins. Most of these methods, if not all, are based on the specific removal of SPs which ultimately magnify the proteome coverage. In this mini-review, we summarize the available methods that have been developed over the years for the enrichment of LAPs either from seeds or tubers. Incorporation of these methods during the protein extraction step will be helpful in understanding the seed/tuber biology in greater detail.

Understanding the plant-pathogen interactions in the context of proteomics-generated apoplastic proteins inventory.

The extracellular space between cell wall and plasma membrane acts as the first battle field between plants and pathogens. Bacteria, fungi, and oomycetes that colonize the living plant tissues are encased in this narrow region in the initial step of infection. Therefore, the apoplastic region is believed to be an interface which mediates the first crosstalk between host and pathogen. The secreted proteins and other metabolites, derived from both host and pathogen, interact in this apoplastic region and govern the final relationship between them. Hence, investigation of protein secretion and apoplastic interaction could provide a better understanding of plant-microbe interaction. Here, we are briefly discussing the methods available for the isolation and normalization of the apoplastic proteins, as well as the current state of secretome studies focused on the in-planta interaction between the host and the pathogen.

Moving forward in plant food safety and security through NanoBioSensors: Adopt or adapt biomedical technologies?

Plant-based foods are integral part of our day-to-day diet. Increasing world population has put forth an ever increasing demand for plant-based foods, and food security remains a major concern. Similarly, biological, chemical, and physical threats to our food and increasing regulatory demands to control the presence of foreign species in food products have made food safety a growing issue. Nanotechnology has already established its roots in diverse disciplines. However, the food industry is yet to harness the full potential of the unique capabilities offered by this next-generation technology. While there might be safety concerns in regards to integration of nanoproducts with our food products, an aspect of nanotechnology that can make remarkable contribution to different elements of the food chain is the use of nanobiosensors and diagnostic platforms for monitoring food traceability, quality, safety, and nutritional value. This brings us to an important question that whether existing diagnostic platforms that have already been well developed for biomedical and clinical application are suitable for food industry or whether the demands of the food industry are altogether different that may not allow adoption/adaptation of the existing technology. This review is an effort to raise this important "uncomfortable" yet "timely" question.

Methyl jasmonate elicits the production of methyl (E)-2-hexenoate from (Z)-2-hexenol via (Z)-2-hexenal in Achyranthes bidentata plant.

The medicinal herbal plant Achyranthes bidentata (A. bidentata) produces the sweet-odor ester - methyl (E)-2-hexenoate (1) as the major volatile in response to methyl jasmonate (MeJA). Here, we investigated the biosynthetic pathway of methyl (E)-2-hexenoate (1). The common plant precursor (Z)-3-hexenal was only slightly metabolized into methyl (E)-2-hexenoate (1), and its application scarcely enhanced the production of this ester. By contrast, a structurally related alcohol, (Z)-2-hexenol, as well as a deuteride derivative thereof could be efficiently metabolized into methyl (E)-2-hexenoate (1). Thus, we hypothesize that A. bidentata possess a specific pathway for the production of methyl (E)-2-hexenoate (1) from (Z)-2-hexenol in response to MeJA.

The 14-3-3 isoforms chi and epsilon differentially bind client proteins from developing Arabidopsis seed.

The 14-3-3-protein family is prominently expressed during seed filling and modulates protein interactions and enzymatic activities, in a phosphorylation-dependent manner. To investigate the role(s) of 14-3-3 proteins in oilseed development, we have begun to characterize the Arabidopsis thaliana 14-3-3 "interactome" for two phylogenetically distinct isoforms. Proteins from developing Arabidopsis seed were incubated with a Sepharose affinity matrix containing covalently bound recombinant Arabidopsis 14-3-3 isoforms chi (χ) or epsilon (ε). Eluted proteins were quantitatively identified using GeLC-MS/MS coupled to spectral counting. Analysis of nine biological replicates revealed a total of 104 putative 14-3-3 binding proteins eluted from this affinity matrix compared to controls. Interestingly, these results imply that χ and ε could have distinct preferences for client proteins. Both isoforms interact with client proteins involved in various metabolic pathways, including glycolysis and de novo fatty acid synthesis. These results suggest 14-3-3 proteins interact with multiple biochemical processes of Arabidopsis seed development. Furthermore, these data suggest isoform specificity of client proteins and possibly even functional specialization between the 14-3-3 isoforms χ and ε in Arabidopsis seed development.

Tropospheric ozone and plants: absorption, responses, and consequences.

Ozone is now considered to be the second most important gaseous pollutant in our environment. The phytotoxic potential of O3 was first observed on grape foliage by B.L. Richards and coworkers in 1958 (Richards et al. 1958). To date, unsustainable resource utilization has turned this secondary pollutant into a major component of global climate change and a prime threat to agricultural production. The projected levels to which O3 will increase are critically alarming and have become a major issue of concern for agriculturalists, biologists, environmentalists and others plants are soft targets for O3. Ozone enters plants through stomata, where it disolves in the apoplastic fluid. O3 has several potential effects on plants: direct reaction with cell membranes; conversion into ROS and H2O2 (which alters cellular function by causing cell death); induction of premature senescence; and induction of and up- or down-regulation of responsive components such as genes , proteins and metabolites. In this review we attempt to present an overview picture of plant O3 interactions. We summarize the vast number of available reports on plant responses to O3 at the morphological, physiological, cellular, biochemical levels, and address effects on crop yield, and on genes, proteins and metabolites. it is now clear that the machinery of photosynthesis, thereby decreasing the economic yield of most plants and inducing a common morphological symptom, called the "foliar injury". The "foliar injury" symptoms can be authentically utilized for biomonitoring of O3 under natural conditions. Elevated O3 stress has been convincingly demonstrated to trigger an antioxidative defense system in plants. The past several years have seen the development and application of high-throughput omics technologies (transcriptomics, proteomics, and metabolomics) that are capable of identifying and prolifiling the O3-responsive components in model and nonmodel plants. Such studies have been carried out ans have generated an inventory of O3-Responsive components--a great resource to the scientific community. Recently, it has been shown that certain organic chemicals ans elevated CO2 levels are effective in ameliorating O3-generated stress. Both targeted and highthroughput approaches have advanced our knowledge concerning what O3-triggerred signaling and metabolic pathways exist in plants. Moreover, recently generated information, and several biomarkers for O3, may, in the future, be exploited to better screen and develop O3-tolerant plants.

Systems analysis of seed filling in Arabidopsis: using general linear modeling to assess concordance of transcript and protein expression.

Previous systems analyses in plants have focused on a single developmental stage or time point, although it is often important to additionally consider time-index changes. During seed development a cascade of events occurs within a relatively brief time scale. We have collected protein and transcript expression data from five sequential stages of Arabidopsis (Arabidopsis thaliana) seed development encompassing the period of reserve polymer accumulation. Protein expression profiling employed two-dimensional gel electrophoresis coupled with tandem mass spectrometry, while transcript profiling used oligonucleotide microarrays. Analyses in biological triplicate yielded robust expression information for 523 proteins and 22,746 genes across the five developmental stages, and established 319 protein/transcript pairs for subsequent pattern analysis. General linear modeling was used to evaluate the protein/transcript expression patterns. Overall, application of this statistical assessment technique showed concurrence for a slight majority (56%) of expression pairs. Many specific examples of discordant protein/transcript expression patterns were detected, suggesting that this approach will be useful in revealing examples of posttranscriptional regulation.

Rice OsACDR1 (Oryza sativa accelerated cell death and resistance 1) is a potential positive regulator of fungal disease resistance.

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-offunction and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

A high-resolution two dimensional Gel- and Pro-Q DPS-based proteomics workflow for phosphoprotein identification and quantitative profiling.

The two-dimensional (2-D) gel-based proteomics platform remains the workhorse for proteomics and is fueled by a number of key improvements, including fluorescence-based stains for detection and quantification of proteins and phosphoproteins with high sensitivity and linear dynamic ranges. One such stain is Pro-Q diamond phosphoprotein stain (Pro-Q DPS), which binds to the phosphate moiety of phospho-proteins irrespective of the phosphoamino acid. We recently introduced a modified Pro-Q DPS protocol to detect phosphoprotein spots on 2-D gels with very low background addressing some prime concerns, including high cost and reproducibility of Pro-Q DPS. The major modifications were a threefold dilution of Pro-Q DPS and the use of threefold less volume of the diluted staining solution. In this chapter, use of the modified Pro-Q DPS protocol along with the 2-D gel-based proteomics for phosphoprotein detection and quantification is described in detail. This 2-D gel- and Pro-Q DPS-based proteomics workflow has seven major steps: preparation of total protein, separation of proteins by 2-D gel electrophoresis, detection of phosphoprotein and total protein, image analysis and quantitative expression profiling, excision of 2-D spots, mass spectrometry analysis, and data processing and organization. Involvement of the modified Pro-Q DPS protocol in this proteomics workflow alone reduces the overall cost by at least ninefold for conducting phospho-proteomics analysis on a global scale, thereby making this entire process economically attractive to the scientific community.