Differential proteomic responses to water stress induced by PEG in two creeping bentgrass cultivars differing in stress tolerance.

Protein metabolism and expression play important role in plant adaptation to water stress. The objectives of this study were to examine proteomic responses to water stress induced by polyethylene glycol (PEG) in creeping bentgrass (Agrostis stolonifera L.) leaves and to identify proteins associated with stress tolerance. Plants of two cultivars ('Penncross' and 'Penn-A4') differing in water stress tolerance were grown in sand irrigated daily with water (control) or PEG solution (osmotic potential of -0.66MPa) to induce water stress, for 28d in growth chambers. Shoot extension rate, relative water content and cell membrane stability were measured to compare drought tolerance between the two cultivars. All parameters maintained at a significantly higher level in 'Penn-A4' than in 'Penncross' under PEG treatment. After 28d of water stress, proteins were extracted from leaves and separated by difference gel electrophoresis. Among 56 stress-responsive protein spots, 46 were identified using mass spectrometry. Some proteins involved in primary nitrogen and carbon metabolism were down-regulated by PEG-induced water stress in both cultivars. The abundance of antioxidant enzyme proteins (ascorbate peroxidase, catalase and glutathione-S-transferase) increased under water stress, particularly ascorbate peroxidase in 'Penn-A4'. The abundance levels of actins, UDP-sulfoquinovose synthase and glucan exohydrolase were greater in 'Penn-A4' than in 'Penncross' under PEG treatment. Our results suggest that proteins involved in membrane synthesis, cell wall loosening, cell turgor maintenance, and antioxidant defense may play roles in perennial grass adaptation to PEG-induced water stress.

Reviewing the technical designs for experiments with ultraviolet-B radiation and impact on photosynthesis, DNA and secondary metabolism.

The ultraviolet-B (UV-B) portion of sunlight has received much attention in the last three decades, because radiation from this spectral region increases due to the stratospheric ozone depletion, which results from increases of chlorofluorocarbons in the atmosphere. Plant responses to UV-B exposure vary greatly and the interpretation of and comparison between studies is hindered, mainly by the contrasting experimental conditions used and interactive factors such as low light levels and possible artifacts due to the artificial experimental conditions. It seems likely that increases in solar UV-B radiation of the magnitude anticipated under current stratospheric ozone projections will not significantly inhibit photosynthesis and cause DNA damage in plants. This is in part due to the well-evolved protection mechanisms present in most plant species. One of the significant plant responses to UV-B is changes in foliar secondary chemistry, which could be translated into significant effects at higher trophic levels through plant-herbivore interactions and decomposition. Enhanced UV-B radiation due to stratospheric ozone depletion could also cause morphological changes that would affect competitive interactions, especially if contrasting UV-B sensitivity exists among the competitors.

Protein profile analysis of salt-responsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance.

Knowledge of stress-responsive proteins is critical for further understanding the molecular mechanisms of stress tolerance. The objectives of this study were to establish a proteomic map for a perennial grass species, creeping bentgrass (A. stolonifera L.), and to identify differentially expressed, salt-responsive proteins in two cultivars differing in salinity tolerance. Plants of two cultivars ('Penncross' and 'Penn-A4') were irrigated daily with water (control) or NaCl solution to induce salinity stress in a growth chamber. Salinity stress was obtained by adding NaCl solution of 2, 4, 6, and 8 dS m(-1) in the soil daily for 2-day intervals at each concentration, and then by watering soil with 10 dS m(-1) solution daily for 28 days. For proteomic map, using two-dimensional electrophoresis (2-DE), approximately 420 and 300 protein spots were detected in leaves and roots, respectively. A total of 148 leaf protein spots and 40 root protein spots were excised from the 2-DE gels and subjected to mass spectrometry analysis. In total, 106 leaf protein spots and 24 root protein spots were successfully identified. Leaves had more salt-responsive proteins than roots in both cultivars. The superior salt tolerance in 'Penn-A4', indicated by shoot extension rate, relative water content, and cell membrane stability during the 28-day salinity stress could be mainly associated with its higher level of vacuolar H(+)-ATPase in roots and UDP-sulfoquinovose synthase, methionine synthase, and glucan exohydrolase in leaves, as well as increased accumulation of catalase and glutathione S-transferase in leaves. Our results suggest that salinity tolerance in creeping bentgrass could be in part controlled by an alteration of ion transport through vacuolar H(+)-ATPase in roots, maintenance of the functionality and integrity of thylakoid membranes, sustained polyamine biosynthesis, and by the activation of cell wall loosening proteins and antioxidant defense mechanisms.

Differential proteomic response to heat stress in thermal Agrostis scabra and heat-sensitive Agrostis stolonifera.

Knowledge of heat-responsive proteins is critical for further understanding of the molecular mechanisms of heat tolerance. The objective of this study was to compare proteins differentially expressed in two C(3) grass species contrasting in heat tolerance, heat-tolerant thermal Agrostis scabra and heat-sensitive Agrostis stolonifera L., and to identify heat-responsive proteins for short- and long-term responses. Plants were exposed to 20/15 degrees C (day/night, control) or 40/35 degrees C (day/night, heat stress) in growth chambers. Leaves were harvested at 2 and 10 days after temperature treatment. Proteins were extracted and separated by fluorescence difference gel electrophoresis (DIGE). Thermal A. scabra had superior heat tolerance than A. stolonifera, as indicated by the maintenance of higher chlorophyll content and photochemical efficiency under heat stress. The two-dimensional difference electrophoresis detected 68 heat-responsive proteins in the two species. Thermal A. scabra had more protein spots either down- or up-regulated at 2 days of heat stress, but fewer protein spots were altered at 10 days of heat stress compared with A. stolonifera. Many protein spots exhibited transient down-regulation in thermal A. scabra (only at 2 days of heat treatment), whereas down-regulation of many proteins was also found at 10 days of heat treatment in A. stolonifera, which suggested that protein metabolism in thermal A. scabra might acclimate to heat stress more rapidly than those in A. stolonifera. The sequences of 56 differentially expressed protein spots were identified using mass spectrometry. The results suggest that the maintenance or less severe down-regulation of proteins during long-term (10 days) heat stress may contribute to the superior heat tolerance in thermal A. scabra, including those involved in photosynthesis [RuBisCo, RuBisCo activase, chloroplastic glyceraldehydes-3-phosphate dehydrogenase (GAPDH), chloroplastic aldolase, oxygen-evolving complex, photosystem I subunits], dark respiration (cytosolic GAPDH, cytoplasmic aldolase, malate dehydrogenase, hydroxypyruvate reductase, sedoheptulose-1,7-bisphosphatase), photorespiration [(hydroxypyruvate reductase, alanine aminotransferase (AlaAT), hydroxymethyltransferase (SHMT), glycine decarboxylase (GDC)], as well as heat and oxidative stress protection [heat shock cognate (HSC) 70 and FtsH-like protein].

Utility of proteomics techniques for assessing protein expression.

Proteomic technologies are currently used as an effective analytical tool for examining modifications in protein profiles. Understanding the natural variation of soybean seed proteins is necessary to evaluate potential unintended (collateral) effects due to transgenic modifications in genetically modified (GMO) soybeans. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to separate, identify and quantify the different classes of soybean seed proteins. Sixteen soybean genotypes, including four wild and twelve cultivated genotypes, belonging to four different subgroups were used as models for protein profile evaluation. Significant variations of allergen and anti-nutritional protein profiles were observed between two different groups, cultivated and wild soybean genotypes. However, only minor variations in protein profiles were observed within the soybean samples from the same group (cultivated or wild). These results may be useful to scientists needing to compare GMO and non-GMO soybeans once additional data are generated on additional soybean varieties and the same varieties grown at different geographical locations.

Incorporation of endothelial progenitor cells into the neovasculature of malignant glioma xenograft.

Endothelial progenitor cells (EPCs) are important initiators of vasculogenesis in the process of tumor neovascularization. However, it is unclear how circulating EPCs contribute to the formation of tumor microvessels. In this study, we isolated CD34(+)/CD133(+) cells from human umbilical cord blood (HUCB) and obtained EPCs with the capacities of forming colonies, uptaking acetylated low-density lipoprotein (ac-LDL), binding lectins and expressing vascular endothelial growth factor (VEGF) receptor 2 (VEGFR-2, KDR), CD31 and von Willebrand factor (vWF). These EPCs were actively proliferative and migratory, and could formed capillary-like tubules in response to VEGF. When injected into mice bearing subcutaneously implanted human malignant glioma, EPCs specifically accumulated at the sites of tumors and differentiated into mature endothelial cells (ECs), which accounted for 18% ECs of the tumor microvessels. The incorporation of circulating EPCs into tumor vessel walls significantly affected the morphology and structure of the vasculature. Our results suggest that circulating EPCs constitute important components of tumor microvessel network and contribute to tumor microvascular architecture phenotype heterogeneity.

Root proteomic responses to heat stress in two Agrostis grass species contrasting in heat tolerance.

Protein metabolism plays an important role in plant adaptation to heat stress. This study was designed to identify heat-responsive proteins in roots associated with thermotolerance for two C3 grass species contrasting in heat tolerance, thermal Agrostis scabra and heat-sensitive Agrostis stolonifera L. Plants were exposed to 20 degrees C (control), 30 C (moderate heat stress), or 40 degrees C (severe heat stress) in growth chambers. Roots were harvested at 2 d and 10 d after temperature treatment. Proteins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis. Seventy protein spots were regulated by heat stress in at least one species. Under both moderate and severe heat stress, more proteins were down-regulated than were up-regulated, and thermal A. scabra roots had more up-regulated proteins than A. stolonifera roots. The sequences of 66 differentially expressed protein spots were identified using mass spectrometry. The results suggested that the up-regulation of sucrose synthase, glutathione S-transferase, superoxide dismutase, and heat shock protein Sti (stress-inducible protein) may contribute to the superior root thermotolerance of A. scabra. In addition, phosphoproteomic analysis indicated that two isoforms of fructose-biphosphate aldolase were highly phosphorylated under heat stress, and thermal A. scabra had greater phosphorylation than A. stolonifera, suggesting that the aldolase phosphorylation might be involved in root thermotolerance.

Identification and characterization of proteins associated with plant tolerance to heat stress.

Heat stress is a major abiotic stress limiting plant growth and productivity in many areas of the world. Understanding mechanisms of plant adaptation to heat stress would facilitate the development of heat-tolerant cultivars for improving productivity in warm climatic regions. Protein metabolism involving protein synthesis and degradation is one of the most sensitive processes to heat stress. Changes in the level and expression pattern of some proteins may play an important role in plant adaptation to heat stress. The identification of stress-responsive proteins and pathways has been facilitated by an increasing number of tools and resources, including two-dimensional electrophoresis and mass spectrometry, and the rapidly expanding nucleotide and amino acid sequence databases. Heat stress may induce or enhance protein expression or cause protein degradation. The induction of heat-responsive proteins, particularly heat shock proteins (HSPs), plays a key role in plant tolerance to heat stress. Protein degradation involving various proteases is also important in regulating plant responses to heat stress. This review provides an overview of recent research on proteomic profiling for the identification of heat-responsive proteins associated with heat tolerance, heat induction and characteristics of HSPs, and protein degradation in relation to plant responses to heat stress.

Impact of solar ultraviolet-B on the proteome in soybean lines differing in flavonoid contents.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to systematically investigate the impact of solar ultraviolet-B (UV-B) radiation on the soybean leaf proteome. In order to investigate the protective role of flavonoids against UV-B, two isolines of the Clark cultivar (the standard line with moderate levels of flavonoids and the magenta line with reduced flavonoids) were grown in the field with or without natural levels of UV-B. The 12-day-old first trifoliates were harvested for proteomic analysis. More than 300 protein spots were reproducibly resolved and detected on each gel. Statistical analysis showed that 67 protein spots were significantly (P<0.05) affected by solar UV-B. Many more spots were altered by UV-B in the magenta line than in the standard line. Another 12 protein spots were not altered by UV-B but showed significantly (P<0.05) different accumulations between the two lines, and for most spots the line-specific differences were also observed under UV-B exclusion. Most of the differentially accumulated spots were identified by mass spectrometry. The proteins were quite diverse, and were involved in metabolism, energy, protein destination/storage, protein synthesis, disease/defense, transcription, and secondary metabolism. The results suggest that high levels of flavonoids lead to a reduction in UV-B sensitivity at the proteomic level.

Coupling short-term changes in ambient UV-B levels with induction of UV-screening compounds.

A substantial number of studies have been conducted over the last several decades to assess the potential impacts of long-term increases in ultraviolet-B radiation (UV-B between 280 and 320 nm) that will result from continued depletion of stratospheric ozone. However, seasonal changes, tropospheric chemistry and cloudiness are the dominant factors controlling ambient UV-B levels on a short-term or daily basis. The effects of short-term changes in UV-B on plant growth, phytochemistry and physiological processes have received relatively little attention. The USDA UV-B Monitoring and Research Program provides an excellent network of stations that provide an opportunity to monitor long-term changes in solar UV-B radiation and evaluate the responses of plants to short-term variation in UV-B levels on a near-real-time basis. In this study barley (Hordeum vulgare L.) and soybean (Glycine max [L] Merr.) were used as model systems. Emerging seedlings of these species were grown under either near-ambient levels of UV-B or under reduced levels (ca 90% reduction) in the field. Periodic measurements of foliar UV-screening compounds were made on separate groups of seedlings planted at intervals over the growing season during contrasting periods of ambient levels of UV radiation. The levels of UV-screening compounds correlated with UV-B levels in both species and with UV-A in soybean but the sensitivity of the response differed between the two species and among the soybean cultivars. Response differences among species may be related to unique secondary chemistry of each species, so one response estimate or action spectrum may not be appropriate for all species.

Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes.

We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.

Proteomic and genomic characterization of Kunitz trypsin inhibitors in wild and cultivated soybean genotypes.

In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.

Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis.

A combined proteomic approach was applied for the separation, identification, and comparison of two major storage proteins, beta-conglycinin and glycinin, in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with three different immobilized pH gradient (IPG) strips was an effective method to separate a large number of abundant and less-abundant storage proteins. Most of the subunits of beta-conglycinin were well-separated in the pH range 3.0-10.0, while acidic and basic glycinin polypeptides were well-separated in pH ranges 4.0-7.0 and 6.0-11.0, respectively. Although the overall distribution pattern of the protein spots was similar in both genotypes using pH 3.0-10.0, variations in number and intensity of protein spots were better resolved using a combination of pH 4.0-7.0 and pH 6.0-11.0. The total number of storage protein spots detected in wild and cultivated genotypes was approximately 44 and 34, respectively. This is the first study reporting the comparison of protein profiles of wild and cultivated genotypes of soybean seeds using proteomic tools.

Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins.

Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods-urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone-to determine their efficacy in separating soybean seed proteins by 2D-PAGE. In all four methods, seed storage proteins were well separated by 2D-PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin, and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were beta-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.