Salt-induced and Salt-suppressed Proteins in Tomato Leaves

Tomato (Solanum lycopersicum cv. Money Maker) seedlings at the two-leaf stage were grown in one-half strength Hoagland solution supplemented with 50 mm NaCl for 4 days, with 100 mm NaCl for 4 days, with 150 mm NaCl for 4 days, and with a final concentration 200 mm NaCl for 2 days. Solutions were refreshed every 2 days for treated and untreated seedlings. Non-treated plants were grown in nonamended one-half strength Hoagland solution. Three biological replicates (BR) were included for treated and control experiments. At the end of treatments, the uppermost three newly expanded leaves from all 12 plants in each BR were collected and bulked to extract total protein. Proteomic analysis resulted in the identification of several salt-induced and salt-suppressed proteins. Salt-induced proteins were: vacuolar H+-ATPase A1 subunit isoform (1.6-fold), germin-like protein (1.5-fold), ferredoxin-NADP (+) reductase (1.2-fold), quinone oxidoreductase-like protein (4.4-fold), heat-shock protein (4.9-fold), and pyrophosphorylase (1.7-fold). Salt-suppressed proteins were: ATPase alpha subunit (−1.5-fold) and rubisco activase (−1.4-fold). Proteins identified in this study affect cellular activities for antioxidant, stress protection, carbon fixation, and carbohydrate partitioning in young tomato leaves under salt stress

The Al-induced proteomes of epidermal and outer cortical cells in root apex of cherry tomato \u27LA 2710\u27

This paper reports a laser capture microdissection-tandem mass tag-quantitative proteomics analysis of Al-sensitive cells in root tips. Cherry tomato (Solanum lycopersicum var. cerasiforme ‘LA2710’) seedlings were treated under 15 μM Al3+ activity for 13 d. Root-tip longitudinal fresh frozen tissue sections of 10 μm thickness were prepared. The Al-sensitive root zone and cells were determined using histochemical analysis of root-tips and micro-sections. A procedure for collecting the Al-sensitive cells using laser capture microdissection-protein extraction-tandem mass tag-proteomics analysis was developed. Proteomics analysis of 18 μg protein/sample with three biological replicates per treatment condition identified 3879 quantifiable proteins each associated with two or more unique peptides. Quantified proteins constituted a broad range of Kyoto Encyclopedia of Genes and Genomes pathways when searched in the annotated tomato genome. Differentially expressed proteins between the Al-treated and non-Al treated control conditions were identified, including 128 Al-up-regulated and 32 Al-down-regulated proteins. Analysis of functional pathways and protein-protein interaction networks showed that the Al-down-regulated proteins are involved in transcription and translation, and the Al-up-regulated proteins are associated with antioxidant and detoxification and protein quality control processes. The proteomics data are available via ProteomeXchange with identifier PXD010459 under project title ‘LCM-quantitative proteomics analysis of Al-sensitive tomato root cells’. Significance This paper presents an efficient laser capture microdissection-tandem mass tag-quantitative proteomics analysis platform for the analysis of Al sensitive root cells. The analytical procedure has a broad application for proteomics analysis of spatially separated cells from complex tissues. This study has provided a comprehensive proteomics dataset expressed in the epidermal and outer-cortical cells at root-tip transition zone of Al-treated tomato seedlings. The proteomes from the Al-sensitive root cells are valuable resources for understanding and improving Al tolerance in plants

Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots

Single-cell-type proteomics provides the capability to revealing the genomic and proteomics information at cell-level resolution. However, the methodology for this type of research has not been well-developed. This paper reports developing a workflow of laser capture microdissection (LCM) followed by gel-liquid chromatography-tandem mass spectrometry (GeLC-MS/MS)-based proteomics analysis for the identification of proteomes contained in individual cell layers of tomato roots. Thin-sections (~10-μm thick, 10 sections per root tip) were prepared for root tips of tomato germinating seedlings. Epidermal and cortical cells (5000–7000 cells per tissue type) were isolated under a LCM microscope. Proteins were isolated and then separated by SDS–polyacrylamide gel electrophoresis followed by in-gel-tryptic digestion. The MS and MS/MS spectra generated using nanoLC-MS/MS analysis of the tryptic peptides were searched against ITAG2.4 tomato protein database to identify proteins contained in each single-cell-type sample. Based on the biological functions, proteins with proven functions in root hair development were identified in epidermal cells but not in the cortical cells. Several of these proteins were found in Al-treated roots only. The results demonstrated that the cell-type-specific proteome is relevant for tissue-specific functions in tomato roots. Increasing the coverage of proteomes and reducing the inevitable cross-contamination from adjacent cell layers, in both vertical and cross directions when cells are isolated from slides prepared using intact root tips, are the major challenges using the technology in proteomics analysis of plant roots

Differential Root Proteome Expression in Tomato Genotypes with Contrasting Drought Tolerance Exposed to Dehydration

A comparative proteomics study using isobaric tags for relative and absolute quantitation (iTRAQ) was performed on a mesophytic tomato (Solanum lycopersicum) cultivar and a dehydration-resistant wild species (Solanum chilense) to identify proteins that play key roles in tolerance to water deficit stress. In tomato ‘Walter’ LA3465, 130 proteins were identified, of which 104 (80%) were repressed and 26 (20%) were induced. In S. chilense LA1958, a total of 170 proteins were identified with 106 (62%) repressed and 64 (38%) induced. According to their putative molecular functions, the differentially expressed proteins belong to the following subgroups: stress proteins, gene expression, nascent protein processing, protein folding, protein degradation, carbohydrate metabolism, amino acid and nucleotide metabolism, lipid metabolism, signal transduction, and cell cycle regulation. Based on changes in protein abundance induced by the dehydration treatment, cellular metabolic activities and protein biosynthesis were suppressed by the stress. In S. chilense, dehydration treatment led to elevated accumulation of proteins involved in post-transcriptional gene regulation and fidelity in protein translation including prefoldin, which promotes protein folding without the use of adenosine-5′-triphosphate (ATP), several hydrophilic proteins, and calmodulin in the calcium signal transduction pathway. Those protein changes were not found in the susceptible tomato, ‘Walter’. Within each functional protein group, proteins showing opposite changes (dehydration induced vs. repressed) in the two species were identified and roles of those proteins in conferring tolerance to water deficit stress are discussed. Information provided in this report will be useful for selection of proteins or genes in analyzing or improving dehydration tolerance in tomato cultivars

Proteome profile changes during poly-hydroxybutyrate intracellular mobilization in gram positive Bacillus cereus tsu1

Bacillus cereus is a bacterial species which grows efficiently on a wide range of carbon sources and accumulates biopolymer poly-hydroxybutyrate (PHB) up to 80% cell dry weight. PHB is an aliphatic polymer produced and stored intracellularly as a reservoir of carbon and energy, its mobilization is a key biological process for sporulation in Bacillus spp. Previously, B. cereus tsu1 was isolated and cultured on rapeseed cake substrate (RCS), with maximum of PHB accumulation reached within 12 h, and depleted after 48 h. Fore-spore and spore structure were observed after 24 h culture

Comparative Proteomics of Root Apex and Root Elongation Zones Provides Insights into Molecular Mechanisms for Drought Stress and Recovery Adjustment in Switchgrass

Switchgrass plants were grown in a Sandwich tube system to induce gradual drought stress by withholding watering. After 29 days, the leaf photosynthetic rate decreased significantly, compared to the control plants which were watered regularly. The drought-treated plants recovered to the same leaf water content after three days of re-watering. The root tip (1cm basal fragment, designated as RT1 hereafter) and the elongation/maturation zone (the next upper 1 cm tissue, designated as RT2 hereafter) tissues were collected at the 29th day of drought stress treatment, (named SDT for severe drought treated), after one (D1W) and three days (D3W) of re-watering. The tandem mass tags mass spectrometry-based quantitative proteomics analysis was performed to identify the proteomes, and drought-induced differentially accumulated proteins (DAPs). From RT1 tissues, 6156, 7687, and 7699 proteins were quantified, and 296, 535, and 384 DAPs were identified in the SDT, D1W, and D3W samples, respectively. From RT2 tissues, 7382, 7255, and 6883 proteins were quantified, and 393, 587, and 321 proteins DAPs were identified in the SDT, D1W, and D3W samples. Between RT1 and RT2 tissues, very few DAPs overlapped at SDT, but the number of such proteins increased during the recovery phase. A large number of hydrophilic proteins and stress-responsive proteins were induced during SDT and remained at a higher level during the recovery stages. A large number of DAPs in RT1 tissues maintained the same expression pattern throughout drought treatment and the recovery phases. The DAPs in RT1 tissues were classified in cell proliferation, mitotic cell division, and chromatin modification, and those in RT2 were placed in cell wall remodeling and cell expansion processes. This study provided information pertaining to root zone-specific proteome changes during drought and recover phases, which will allow us to select proteins (genes) as better defined targets for developing drought tolerant plants. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD017441

Identification of Proteins for Salt Tolerance Using a Comparative Proteomics Analysis of Tomato Accessions with Contrasting Salt Tolerance

Tomato (Solanum lycopersicum) has a wide variety of genotypes differing in their responses to salinity. This study was performed to identify salt-induced changes in proteomes that are distinguishable among tomatoes with contrasting salt tolerance. Tomato accessions [LA4133 (a salt-tolerant cherry tomato accession) and ‘Walter’ LA3465 (a salt-susceptible accession)] were subjected to salt treatment (200 mm NaCl) in hydroponic culture. Salt-induced changes in the root proteomes of each tomato accession were identified using the isobaric tags for relative and absolute quantitation (iTRAQ) method. In LA4133, 178 proteins showed significant differences between salt-treated and non-treated control root tissues (P ≤ 0.05); 169 proteins were induced (1.3- to 5.1-fold) and nine repressed (–1.7- to –1.3-fold). In LA3465, 115 proteins were induced (1.3- to 6.4-fold) and 23 repressed (–2.5- to –1.3-fold). Salt-responsive proteins from the two tomato accessions were involved in the following biological processes: root system development and structural integrity; carbohydrate metabolism; adenosine-5′-triphosphate regeneration and consumption; amino acid metabolism; fatty acid metabolism; signal transduction; cellular detoxification; protein turnover and intracellular trafficking; and molecular activities for regulating gene transcription, protein translation, and post-translational modification. Proteins affecting diverse cellular activities were identified, which include chaperonins and cochaperonins, heat-shock proteins, antioxidant enzymes, and stress proteins. Proteins exhibiting different salt-induced changes between the tolerant and susceptible tomato accessions were identified, and these proteins were divided into two groups: 1) proteins with quantitative differences because they were induced or repressed by salt stress in both accessions but at different fold levels; and 2) proteins showing qualitative differences, where proteins were induced in one vs. repressed or not changed in the other accession. Candidate proteins for tolerance to salt and secondary cellular stresses (such as hypo-osmotic stress and dehydration) were proposed based on findings from the current and previous studies on tomato and by the use of the Arabidopsis thaliana protein database. Information provided in this report will be very useful for evaluating and breeding for plant tolerance to salt and/or water deficit stresses

Heat-induced Proteome Changes in Tomato Leaves

Three tomato (Solanum lycopersicum) cultivars [Walter LA3465 (heat-tolerant), Edkawi LA 2711 (unknown heat tolerance, salt-tolerant), and LA1310 (cherry tomato)] were compared for changes in leaf proteomes after heat treatment. Seedlings with four fully expanded leaves were subjected to heat treatment of 39/25 °C at a 16:8 h light–dark cycle for 7 days. Leaves were collected at 1200 hr, 4 h after the light cycle started. For ‘Walter’ LA3465, heat-suppressed proteins were geranylgeranyl reductase, ferredoxin-NADP (+) reductase, Rubisco activase, transketolase, phosphoglycerate kinase precursor, fructose–bisphosphate aldolase, glyoxisomal malate dehydrogenase, catalase, S-adenosyl-L-homocysteine hydrolase, and methionine synthase. Two enzymes were induced, cytosolic NADP-malic enzyme and superoxide dismutase. For ‘Edkawi’ LA2711, nine enzymes were suppressed: ferredoxin-NADP (+) reductase, Rubisco activase, S-adenosylmethionine synthetase, methioine synthase, glyoxisomal malate dehydrogenase, enolase, flavonol synthase, M1 family peptidase, and dihydrolipoamide dehydrogenase. Heat-induced proteins were cyclophilin, fructose-1,6-bisphosphate aldolase, transketolase, phosphoglycolate phosphatase, ATPase, photosystem II oxygen-evolving complex 23, and NAD-dependent epimerase/dehydratase. For cherry tomato LA1310, heat-suppressed proteins were aminotransferase, S-adenosyl-L-homocysteine hydrolase, L-ascorbate peroxidase, lactoylglutathione lyase, and Rubisco activase. Heat-induced enzymes were glyoxisomal malate dehydrogenase, phosphoribulokinasee, and ATP synthase. This research resulted in the identification of proteins that were induced/repressed in all tomato cultivars evaluated (e.g., Rubisco activase, methionine synthase, adenosyl-L-homocysteine hydrolase, and others) and those differentially expressed (e.g., transketolase)

Drought-Induced Leaf Proteome Changes in Switchgrass Seedlings

Switchgrass (Panicum virgatum) is a perennial crop producing deep roots and thus highly tolerant to soil water deficit conditions. However, seedling establishment in the field is very susceptible to prolonged and periodic drought stress. In this study, a “sandwich” system simulating a gradual water deletion process was developed. Switchgrass seedlings were subjected to a 20-day gradual drought treatment process when soil water tension was increased to 0.05 MPa (moderate drought stress) and leaf physiological properties had expressed significant alteration. Drought-induced changes in leaf proteomes were identified using the isobaric tags for relative and absolute quantitation (iTRAQ) labeling method followed by nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS) analysis. Additionally, total leaf proteins were processed using a combinatorial library of peptide ligands to enrich for lower abundance proteins. Both total proteins and those enriched samples were analyzed to increase the coverage of the quantitative proteomics analysis. A total of 7006 leaf proteins were identified, and 257 (4% of the leaf proteome) expressed a significant difference (p \u3c 0.05, fold change \u3c0.6 or \u3e1.7) from the non-treated control to drought-treated conditions. These proteins are involved in the regulation of transcription and translation, cell division, cell wall modification, phyto-hormone metabolism and signaling transduction pathways, and metabolic pathways of carbohydrates, amino acids, and fatty acids. A scheme of abscisic acid (ABA)-biosynthesis and ABA responsive signal transduction pathway was reconstructed using these drought-induced significant proteins, showing systemic regulation at protein level to deploy the respective mechanism. Results from this study, in addition to revealing molecular responses to drought stress, provide a large number of proteins (candidate genes) that can be employed to improve switchgrass seedling growth and establishment under soil drought conditions (Data are available via ProteomeXchange with identifier PXD004675)

Effect of continuous white light illumination on glucosinolate metabolism during postharvest storage of broccoli

Broccoli is a vegetable consumed globally due to its important nutritional properties, including high concentrations of glucosinolates. Light treatment can be an important tool to delay postharvest senescence. In this work it was evaluated the effect of postharvest continuous white light illumination on glucosinolate metabolism of broccoli heads. Five glucosinolates were identified, one aliphatic (glucoraphanin) and four indolics (glucobrassicin, neoglucobrassicin, 4-methoxyglucobrassicin and 4-hydroxyglucobrassicin). Level of total glucosinolates decreased from 10.1 μmol/g dry tissue to 1.4 μmol/g dry tissue in control samples after five days of storage, while the decrement was only until 3.0 μmol/g dry tissue in treated samples. The expression of genes associated with glucosinolate metabolism decreased during the first three days but this decrease was greater in illuminated samples. After five days, treated samples showed a higher expression (more than twice) in most of these genes with respect to the controls, coinciding with the higher glucosinolate content. Storage of broccoli heads under continuous white light allows to keep higher values of glucosinolate contents while maintaining at the same time the visual quality.Fil: Casajús, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Civello, Pedro Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Martinez, Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Howe, Kevin. Cornell University. Department of Food Science & Technology; Estados UnidosFil: Fish, Tara. Cornell University. Department of Food Science & Technology; Estados UnidosFil: Yang, Yong. Cornell University. Department of Food Science & Technology; Estados UnidosFil: Thannhauser, Theodore. Cornell University. Department of Food Science & Technology; Estados UnidosFil: Li, Li. Cornell University. Department of Food Science & Technology; Estados UnidosFil: Gómez Lobato, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentin