A proteomics approach for the analysis of hemolymph proteins involved in the immediate defense response of the soft tick, <Emphasis Type="Italic">Ornithodoros savignyi</Emphasis>, when challenged with <Emphasis Type="Italic">Candida albicans</Emphasis>

A proteomics approach was employed to identify proteins secreted into the hemolymph of Ornithodorus savignyi ticks 2 h after immune-challenge with the yeast, Candida albicans. Profiling of the proteins present in hemolymph of unchallenged ticks versus ticks challenged with heat-killed yeast revealed five proteins to be differentially expressed. The modulated protein spots were subjected to tandem mass spectrometry (MS/MS) analysis, but could not be positively identified. These proteins can be assigned to the immune response as they were not induced after aseptic injury. In an attempt to identify hemolymph proteins that recognize and bind to yeast cells, hemolymph obtained from both unchallenged and challenged ticks was incubated with C. albicans. Elution of the bound proteins followed by SDS–PAGE analysis indicated that three proteins (97, 88 and 26 kDa) present in both unchallenged and challenged hemolymph samples bind to yeast cells. The constant presence of these three proteins in tick hemolymph leads us to believe that they may be involved in non-self recognition and participate in yeast clearance from tick plasma. The analyzed yeast-binding proteins could also not be positively identified, suggesting that all the tick immune proteins investigated in this study are novel.     

Comparative proteomics of tuber induction, development and maturation reveal the complexity of tuberization process in potato (Solanum tuberosum L.)

Tuberization in potato ( Solanum tuberosum L.) is a developmental process that serves a double function, as a storage organ and as a vegetative propagation system. It is a multistep, complex process and the underlying mechanisms governing these overlapping steps are not fully understood. To understand the molecular basis of tuberization in potato, a comparative proteomic approach has been applied to monitor differentially expressed proteins at different development stages using two-dimensional gel electrophoresis (2-DE). The differentially displayed proteomes revealed 219 protein spots that change their intensities more than 2.5-fold. The LC-ES-MS/MS analyses led to the identification of 97 differentially regulated proteins that include predicted and novel tuber-specific proteins. Nonhierarchical clustering revealed coexpression patterns of functionally similar proteins. The expression of reactive oxygen species catabolizing enzymes, viz., superoxide dismutase, ascorbate peroxidase and catalase, were induced by more than 2-fold indicating their possible role during the developmental transition from stolons into tubers. We demonstrate that nearly 100 proteins, some presumably associated with tuber cell differentiation, regulate diverse functions like protein biogenesis and storage, bioenergy and metabolism, and cell defense and rescue impinge on the complexity of tuber development in potato.     

Plant‐extract‐induced changes in the proteome of the soil‐borne pathogenic fungus Thielaviopsis basicola

Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non‐host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2‐DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non‐host status. Analysis by LC‐MS/MS of unique and differentially expressed spots and identification using cross‐species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several “morpho” proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B₆ is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.     

Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.     

Proteomic Analysis of Interactions Between the Generalist Herbivore <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae) and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, comparative proteomics was used to investigate the interaction of Spodoptera exigua and Arabidopsis thaliana. By using 2-D electrophoresis of differentially expressed proteins, combined with high-throughput matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF/TOF MS, the changes in the abundance of proteins induced by insect feeding were studied in A. thaliana. More than 1,100 protein spots were reproducibly detected on each gel. The intensities of 30 protein spots in particular changed significantly, showing differences in volume of at least twofold. Among these, 17 protein spots were upregulated, and 13 were downregulated following an 8-h insect feeding period. Nineteen insect-feeding-responsive proteins were identified, all of which were involved in metabolic regulation, binding functions or cofactor requirement of protein, cell rescue, and defense and virulence, as assessed by Munich Information Center for Protein Sequences function category. About 50% of these were involved in metabolism, including transketolase, S-adenosylmethionine synthase 3, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, beta-ureidopropionase, GDP-d-mannose 3′,5′-epimerase, and fatty acid synthase. The identification of insect-feeding-responsive proteins on Arabidopsis provides not only new insights into insect stress but also a good start for further investigation of their functions. Understanding how the plant responses to insects in the proteomic level will provide tools for a better management of insect pest in the field.     

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.     

Proteomic approach to study leaf proteins in a fast-growing tree species, Gmelina arborea Linn. Roxb

Foliar proteome studies have become highly significant for a comprehensive understanding of complex processes associated with plant growth and development. In the present study, we present a proteomic approach to analyze leaf proteins in an important timber-yielding and fast-growing forest tree species, Gmelina arborea Linn. Roxb. (Verbanaceae). Foliar protein analysis involved protein extraction, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight (MALDI–TOF–TOF). From the 2-DE protein profile of Gmelina leaves, we identified and isolated 150 well-separated protein spots; among these, 64 protein spots were identified by mass spectrometric (MS/MS) analysis. These proteins were classified according to their involvement in basic biological functions, such as photosynthesis, amino acid metabolism, cytoskeleton, cell wall metabolism, stress-related proteins, redox maintenance, electron transport chain, phytohormone metabolism and protein translation and folding. Analytical variance was determined for the protein spots of samples from different plants. The present study is believed to provide a foundation for the use of leaf proteomics in addressing fundamental physiological and biochemical processes associated with growth and productivity of tree species such as Gmelina arborea.     

Variation in Endogenous Gibberellins,Abscisic Acid,and Carbohydrate Content During the Growth Cycle of Colored <Emphasis Type="Italic">Zantedeschia</Emphasis> spp., a Tuberous Geophyte

Phenologic changes and variation in the level of endogenous gibberellins (GAs), abscisic acid (ABA), carbohydrate content, and α-amylase activity were examined in colored Zantedeschia spp. cv. Cala Gold. These changes were examined in the primary bud tissues and in the attached tuber tissue during the growth cycle. Dormant tubers were dry-stored at 20°C for 3 months, planted in a phytotron, and grown under 22/16 ± 1°C. Plant development was monitored under continued irrigation until leaf senescence and tuber dormancy. GAs and ABA were extracted from the primary bud tissues, fractionated by HPLC, and analyzed using GC-SIM. Starch, glucose, soluble protein, and α-amylase activity were monitored in the tuber tissue attached to the primary bud. Endogenous changes in GAs and ABA in the primary bud were correlated with endogenous changes in carbohydrate content and α-amylase activity in the attached tuber tissue. These correlations were observed during the rest and the growth periods and were associated with developmental changes in the plant, that is, bud dormancy relaxation, bud growth, and inflorescence differentiation. ABA content decreased and a transient pulse of GA was measured in the primary bud concomitantly with the onset of shoot elongation in dry tubers during storage, before planting. The sharp increase of GAs in the bud preceded inflorescence differentiation as observed in dissected apices by about 15 days, as well as the increase in α-amylase activity in the attached tuber tissue. A steep decrease in starch level was measured in the tuber after planting, concomitantly with massive plant growth. These findings suggest a possible involvement of gibberellin in the initiation of α-amylase activity during dormancy relaxation in colored Zantedeschia and in the autonomous induction of flowering.     

A root proteomics-based insight reveals dynamic regulation of root proteins under progressive drought stress and recovery in <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek

To understand the complex drought response mechanism in crop plants, a systematic root proteomics approach was adopted to identify and analyze the expression patterns of differentially expressed major root proteins of Vigna radiata during short-term (3 days) and consecutive long-term water-deficit (6 days) as well as during recovery (6 days after re-watering). Photosynthetic gas exchange parameters of the plant were measured simultaneously during the stress treatment and recovery period. A total of 26 major protein spots were successfully identified by mass spectrometry, which were grouped according to their expression pattern during short-term stress as significantly up-regulated (9), down-regulated (10), highly down-regulated, beyond detection level of the software (2) and unchanged (5). The subsequent changes in the expression patterns of these proteins during long-term stress treatment and recovery period was analyzed to focus on the dynamic regulation of these functionally important proteins during progressive drought and recovery period. Cytoskeleton-related proteins were down-regulated initially (3d) but regained their expression levels during subsequent water-deficit (6d) while glycoprotein like lectins, which were primarily known to be involved in legume–rhizobia symbiosis, maintained their enhanced expression levels during both short and long-term drought treatment indicating their possible role in drought stress response of legumes. Oxidative stress-related proteins including Cu/Zn superoxide dismutase, oxidoreductase and aldehyde reductase were also up-regulated. The analyses of the dynamic regulation of these root proteins during short- and long-term water-deficit as well as recovery period may prove crucial for further understanding of drought response mechanisms in food legumes.     

花叶木薯变种和木薯栽培种ZM-Seaside叶片光合参数及蛋白组学分析

为研究木薯叶片光合效率对块根产量的影响,本研究利用蛋白质组学方法分析花叶木薯变种(低产种质)和栽培种ZM-Seaside(高产种质)光合作用能力的差异,揭示其鲜薯产量差异原因,为选育高产木薯品种提供基础数据。采用便携式LI-6400光合作用测定仪测定叶片净光合速率(Pn)、气孔导度(Cs)、胞间CO2浓度(Ci)和蒸腾速率(Tr),表明栽培种ZM-Seaside和花叶木薯变种叶片Cs、Ci和Tr没有显著差异,但ZM-Seaside的Pn显著高于花叶木薯变种;利用Western Blot技术分析叶片蛋白质表达水平,结果显示ZM-Seaside叶片与光合作用相关蛋白质Rubisco、OEC和PRXQ的表达水平显著高于花叶木薯变种;采用苯酚法提取叶片全蛋白质,并进行双向电泳分离,及Delta2D软件确定差异蛋白质点,以花叶木薯为对照,在ZM-Seaside叶片蛋白质双向电泳图谱上得到20个差异蛋白质点,其中上调表达15个,下调表达5个;通过MALDI-TOF-MS鉴定差异蛋白质,结合KEGG数据库将其按照功能进行分类,成功鉴定到其中16个涉及光合作用、碳和能量代谢、分子伴侣、结构蛋白、保护蛋白、解毒和抗氧化及未知功能蛋白质;利用String在线软件构建蛋白质互作网络,推测Ribulose-5-phosphate-3-epimerase和chloroplast latex aldolase-like protein是影响木薯叶片光合效率的关键蛋白质,由于它们的上调表达,对木薯块根产量提高有一定促进作用。     

Copper stress‐induced changes in leaf soluble proteome of Cu‐sensitive and tolerant Agrostis capillaris L. populations

Changes in leaf soluble proteome were explored in 3‐month‐old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1–50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO₄ spiked‐nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2‐DE (linear 4–7 pH gradient). Analysis of CCB‐stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC‐MS/MS. In both populations, Cu excess impacted both light‐dependent (OEE, cytochrome b6‐f complex, and chlorophyll a‐b binding protein), and ‐independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin‐NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S‐containing amino‐acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 ( http//proteomecentral.proteomexchange.org/dataset/PXD001930 ).     

Resting Spore Dormancy and Infectivity Characteristics of the Potato Powdery Scab Pathogen Spongospora subterranea

The soil‐borne potato pathogen Spongospora subterranea persists in soil as sporosori, which are aggregates of resting spores. Resting spores may germinate in the presence of plant or environmental stimuli, but direct evidence for resting spore dormancy is limited. A soilless tomato bait plant bioassay and microscopic examination were used to examine features of S. subterranea resting spore dormancy and infectivity. Dried sporosori inocula prepared from tuber lesions and root galls were infective after both short‐ and long‐term storage (1 week to 5 years for tuber lesions and 1 week to 1 year for root galls) with both young and mature root galls inocula showing infectivity. This demonstrated that a proportion of all S. subterranea resting spores regardless of maturity exhibit characteristics of stimuli‐responsive dormancy, germinating under the stimulatory conditions of the bait host plant bioassay. However, evidence for constitutive dormancy within the resting spore population was also provided as incubation of sporosorus inoculum in a germination‐stimulating environment did not fully exhaust germination potential even after 2.4 years. We conclude that S. subterranea sporosori contain both exogenous (stimuli‐responsive) and constitutively dormant resting spores, which enables successful host infection by germination in response to plant stimuli and long‐term persistence in the soil.