Analysis of Proteins Expressed by an Abiotic Stress Tolerant Pseudomonas putida (NBAII-RPF9) Isolate Under Saline and High Temperature Conditions

Pseudomonas putida (NBAII-RPF9) was identified as an abiotic stress tolerant bacterium capable of growing at 45 °C as well as in 1 M NaCl. The proteins expressed by this bacterium when subjected to these two stresses were analyzed by 2D gel and MALDI-TOF/MS. Two parameters viz., heat/saline shock (20 min at 45 °C/1 M solid NaCl added at mid log phase and incubated for 1 h) and heat/saline tolerance (24 h growth at 45 °C/in 1 M NaCl) were studied. Under heat shock 13 upregulated proteins and 1 downregulated protein were identified and under tolerance 6 upregulated proteins were identified. GroES and GroEL proteins were expressed under both tolerance and shock. Under saline shock 11 upregulated proteins were identified whereas under saline tolerance 6 upregulated proteins were identified and all these proteins had pI between 3 and 10 with molecular weights ranging from 14.3 to 97 kDa. Aspartate carbamoyltransferase was common under both the saline conditions studied. The analysis revealed involvement of heat stress responsive molecular chaperones and membrane proteins during heat stress. During salt stress, proteins involved in metabolic processes were found to be upregulated to favor growth and adaptation of the bacterium. Heat shock chaperones viz., DnaK and DnaJ were expressed under both saline and heat stress. This is the first report of protein profile obtained from a single bacterium under saline and heat stress and the studies reveal the complex mechanisms adapted by the organism to survive under high temperature or saline conditions.     

Proteomic Analysis of High Temperature Stress-Responsive Proteins in Chrysanthemum Leaves

Chrysanthemum is one of the most important ornamental flowers in the world, and temperature has a significant influence on its field production. In the present study, differentially expressed proteins were investigated in the leaves of Dendranthema grandiflorum ‘Jinba’ under high temperature stress using label-free quantitative proteomics techniques. The expressed proteins were comparatively identified and analyzed. A total of 1,463 heat-related, differentially expressed proteins were successfully identified by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS), and 1,463 heat-related, differentially expressed proteins were successfully identified by mass spectrometry after a high temperature treatment. Among these, 701 proteins were upregulated and 762 proteins were downregulated. The in-depth bioinformatics analysis of these differentially expressed proteins revealed that these were involved in energy metabolism pathways, protein metabolism, and heat shock. In the present study, the investigators determined the changes in the levels of some proteins, and their expression at the protein and molecular levels in chrysanthemum to help reveal the mechanism of heat resistance in chrysanthemum. Furthermore, the present study elucidated some of the proteins correlated to heat resistance in chrysanthemum, and their expression changes at the protein and molecular levels to help reveal the mechanism of heat resistance in this flower species. These results provide a theoretical basis for the selection of new heat resistant varieties of chrysanthemum in the field.     

LNA mediated in situ hybridization of miR171 and miR397a in leaf and ambient root tissues revealed expressional homogeneity in response to shoot heat shock in Arabidopsis thaliana

MicroRNAs (miRNAs) are endogenous non-protein coding RNA molecules of approximately 21 nucleotides in length capable of modulating gene expression in animals and plants. The role of miRNA based gene regulation has been proved in several pathways including in plant growth, development and stress response. In this study miR171 and miR397a were tested for their expression pattern under different heat shock regimes in shoot and root tissues of Arabidopsis thaliana using Locked Nucleic Acid (LNA) mediated in situ hybridization. With an increase in temperature across 35 °C, 40 °C and 45 °C there was a corresponding increased up-regulation of miR171 in leaf tissues compared to ambient temperature. Similarly, an unambiguous elevated expression of miR171 within increase in duration of exposure at each temperature regime across 1 h, 2 h and 3 h was noticed in comparison to ambient control leaf tissue. On the other hand, miR397a, which expressed at ambient control conditions, got down-regulated both with increase in heat and exposure regime in leaf tissues. Both miRNAs expressed in control ambient root tissues. Maintaining the root zone temperature at ambient conditions, upon imposing heat shock regime to shoot system, miR171 recorded corresponding increased up-regulation as indicated by the intensity of in situ hybridization, while miR397a got down-regulated. Given the differential homogeneity in expression pattern of both miRNA in leaf and root tissues experiencing heat shock regimes, possibilities of movement of heat shock induced signals to root tissues seem to be obvious.     

Comparative proteomic approach to identify proteins involved in flooding combined with salinity stress in soybean

Salinity together with waterlogging or flooding, a condition that occurs frequently in the field, can cause severe damage to crops. Combined flooding and salinity decreases the growth and survival of plants more than either stress alone. We report here the first proteomic analysis to investigate the global effects of saline flooding on multiple metabolic pathways. Soybean seedlings at the emergence (VE) stage were treated with 100 mM NaCl and flooded with water or 100 mM sodium chloride solution for 2 days. Proteins were extracted from hypocotyl and root samples and analyzed by two-dimensional gel electrophoresis followed by MALDI-TOF, MALDI-TOF/TOF mass spectrometry or immunoblotting. A total of 43 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue staining were identified by MALDI-TOF MS. Identities of several proteins were also validated by MS/MS analysis or immunoblot analysis. Twenty-nine proteins were upregulated, eight proteins were downregulated and six spots were newly induced. The identified proteins include well-known salt and flooding induced proteins as well as novel proteins expressed by the salinity-flooding combined stress. The comparative analysis identified changes at the proteome level that are both specific and part of a common or shared response. The identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to a combination of flooding and salinity.     

Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress

Rapeseed (Brassica napus L.), which is the third leading source of vegetable oil, is sensitive to drought stress during the early vegetative growth stage. To investigate the initial response of rapeseed to drought stress, changes in the protein expression profiles of drought-sensitive (RGS-003) and drought-tolerant lines (SLM-003), and their F1 hybrid, were analyzed using a proteomics approach. Seven-day-old rapeseed seedlings were treated with drought stress by restricting water for 7 days, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. In the sensitive rapeseed line, 35 protein spots were differentially expressed under drought stress, and proteins related to metabolism, energy, disease/defense, and transport were decreased. In the tolerant line, 32 protein spots were differentially expressed under drought stress, and proteins involved in metabolism, disease/defense, and transport were increased, while energy-related proteins were decreased. Six protein spots in F1 hybrid were common among expressed proteins in the drought-sensitive and -tolerant lines. Notably, tubulin beta-2 and heat shock protein 70 were decreased in the drought-sensitive line and hybrid F1 plants, while jasmonate-inducible protein and 20S proteasome subunit PAF1 were increased in the F1 hybrids and drought-tolerant line. These results indicate that (1) V-type H⁺ ATPase, plasma-membrane associated cation-binding protein, HSP 90, and elongation factor EF-2 have a role in the drought tolerance of rapeseed; (2) The decreased levels of heat shock protein 70 and tubulin beta-2 in the drought-sensitive and hybrid F1 lines might explain the reduced growth of these lines in drought conditions.     

Enhanced capacity of antigen presentation of HBc-VLP-pulsed RAW264.7 cells revealed by proteomics analysis

Many recent studies have indicated that virus-like particles (VLPs) have many potential applications in the fields of vaccine development and gene therapy. However, we still know little about the subtle mechanisms involved in the presentation of VLPs by antigen presenting cells (APCs). To illustrate the mechanisms, we utilized two-dimensional electrophoresis and tandem MS to compare and identify differentially expressed proteins between hepatitis B virus core antigen VLP (HBc-VLP)-pulsed and control RAW264.7 cells. Of the 25 spots identified as differentially expressed ( p < 0.05) between the two cell lines, 11 (corresponding to 11 unique proteins) were positively identified. Further analysis of two proteins, prohibitin and heat shock protein 70, confirmed that these proteins are expressed at higher levels in HBc-VLP-pulsed RAW264.7 cells compared with control cells. The proteins identified in this study will be useful in revealing the mechanisms that underlie VLP-APC interactions. Overall, this study also provides some useful suggestions for vaccine development and gene therapy.     

Comparative Proteomic Analysis of the Hepatic Response to Heat Stress in Muscovy and Pekin Ducks: Insight into Thermal Tolerance Related to Energy Metabolism

The Pekin duck, bred from the mallard (Anas platyrhynchos) in china, is one of the most famous meat duck species in the world. However, it is more sensitive to heat stress than Muscovy duck, which is believed to have originated in South America. With temperature raising, mortality, laying performance, and meat quality of the Pekin duck are severely affected. This study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach. Duck was cultured under 39°C ± 0.5°C for 1 h, and then immediately returned to 20°C for a 3 h recovery period, the liver proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, 61 differentially expressed proteins were detected, 54 were clearly identified by MALDI TOF/TOF MS. Of the 54 differentially expressed protein spots identified, 7 were found in both species, whereas 47 were species specific (25 in Muscovy duck and 22 in Pekin duck). As is well known, chaperone proteins, such as heat shock protein (HSP) 70 and HSP10, were abundantly up-regulated in both species in response to heat stress. However, we also found that several proteins, such as α-enolase, and S-adenosylmethionine synthetase, showed different expression patterns in the 2 duck species. The enriched biological processes were grouped into 3 main categories according to gene ontology analysis: cell death and apoptosis (20.93%), amino acid metabolism (13.95%) and oxidation reduction (20.93%). The mRNA levels of several differentially expressed protein were investigated by real-time RT-PCR. To our knowledge, this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals.     

Differential proteome analysis of mature and germinated seeds of Magnolia sieboldii K. Koch

Key message

This is the first reported proteomic analysis to study the dormancy breaking of Magnolia sieboldii seeds. Our results provide a fundamental reference for further studies on the regulation of protein expression during seed germination.

Abstract

Magnolia sieboldii K. Koch is an ornamental tree. The deep dormancy of its seeds hinders its cultivation for economic purposes. The biochemical basis of the regulation of seed germination remains poorly understood. The present study aimed to identify differentially expressed proteins in germinated seeds of M. sieboldii using polyethylene glycol fractionation. In total, 59 differentially expressed protein spots from two-dimensional gel maps were detected, 33 of which were identified by mass spectrometry. They were assigned to eight functional classes on the basis of their putative biological functions: photosynthesis (3 %), chaperonin/heat shock protein (9 %), protein and amino acid synthesis (9 %), stress/defense (18 %), cytoskeleton structure (3 %), metabolism (18 %), hormone and polyamine (9 %) and storage proteins (31 %). Among the other functions, the effects of plant hormones on seed germination may be one of the most important functions in plant growth. Gibberellins and ethylene positively regulate seed germination. The activities of several hormone-associated proteins possibly influencing seed germination were increased. The characterization of these proteins will be of great help in identifying the molecular mechanism underlying seed germination.     

Proteomic analysis in the induction of nodular cluster cultures in the bromeliad <Emphasis Type="Italic">Vriesea reitzii</Emphasis> Leme and Costa

Nodular cluster cultures (NCs) are globular organogenic clumps with a high regenerative potential applied to the large-scale micropropagation of bromeliads. In the present work, we identified differentially expressed proteins involved in the induction of NCs from seeds and leaf explants of the Brazilian native bromeliad Vriesea reitzii. Those explants were inoculated into Murashige and Skoog (MS) liquid medium free of plant growth regulators (PGR). To promote the induction of NCs, the seeds were grown in MS medium supplemented with 4 μM α-naphthaleneacetic acid (NAA), and the leaf segments in MS medium supplemented with 4 μM NAA and 2 μM 6-benzylaminopurine (BAP). After 21 days in culture, samples of each type of explant were collected for histological analysis and protein extraction. Proteomic analysis was performed by two-dimensional (2D) electrophoresis and protein identification by MALDI-TOF–TOF mass spectrometry. Enhanced protein content and number of detected spots on cultures supplemented with PGR were observed as compared to the cultures maintained in PGR-free MS culture medium. Five differentially expressed proteins were identified during the induction of NCs: heat shock 22 kDa, chaperone protein dnaJ 50, S-adenosylmethionine synthase 3, UDP-arabinopyranose mutase 1, and 14-3-3-like protein E. Such proteins are involved in stress response, cell metabolism, and cell division. The ability to regulate the effects of stress conditions in which the explants were subjected shows the presence of competent tissues for the acquisition of the morphogenic route associated to the induction of NCs.     

Transcript profiling and gene expression analysis under drought stress in <Emphasis Type="Italic">Ziziphus nummularia</Emphasis> (Burm.f.) Wright &amp; Arn.

Drought is one of the prime abiotic stresses responsible for limiting agricultural productivity. A number of drought responsive genes have been isolated and functionally characterized but these studies have been restricted to a few model plant systems. Very few drought responsive genes have been reported till date from non model drought tolerant plants. The present study aimed at identifying differentially expressed genes from a drought tolerant, non-model plant, Ziziphus nummularia (Burm.f.) Wight & Arn. One month old seedlings of Z. nummularia were subjected to drought stress by 30% Polyethylene glycol (PEG 6000) treatment for 6, 12, 24, 48 and 72 h. A significant reduction in RWC and increase in proline was observed at 24 h and 48 h of treatment. Suppression subtractive hybridization (SSH) library was constructed with drought stressed seedlings after 24 h and 48 h of PEG 6000 treatment. A total of 142 and 530 unigenes from 24 h and 48 h library were identified respectively. Gene ontology studies revealed that about 9.78% and 15.07% unigenes from 24 h and 48 h SSH libraries were expressed in “response to stress”. Fifteen putative drought responsive genes identified in SSH library were validated for drought responsive differential expression by RT-qPCR. Significant changes in fold expressions were observed with time in the treated samples compared to the control. A heat map revealing the expression profile of genes was constructed by hierarchical clustering. Various genes identified in SSH libraries can serve as a resource for marker discovery and selection of candidate genes to improve drought tolerance in other susceptible crops.     

Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure

Proteomes of heat tolerant (multivoltine) and heat susceptible (bivoltine) silkworms (Bombyx mori) in response to heat shock were studied. Detected proteins from fat body were identified by using MALDI-TOF/TOF spectrometer, MS/MS, and MS analysis. Eight proteins, including small heat shock proteins (sHSPs) and HSP70, were expressed similarly in both breeds, while 4 protein spots were expressed specifically in the bivoltine breed and 12 protein spots were expressed specifically in the multivoltine breed. In the present proteomics approach, 5 separate spots of sHSP proteins (HSP19.9, HSP20.1, HSP20.4, HSP20.8, and HSP21.4) were identified. Protein spot intensity of sHSPs was lower in the multivoltine breed than in the bivoltine breed after the 45°C heat shock treatment, while the difference between two breeds was not significant after the 41°C heat shock treatment. These results indicated that some other mechanisms might be engaged in thermal tolerance of multivotine breed except for the expression of sHSP and HSP70. There were visible differences in the intensity of heat shock protein expression between male and female, however, differences were not statistically significant.     

Comparative proteomic analysis of cold-induced sweetening in potato (Solanum tuberosum L.) tuber

Cold-induced sweetening is one of the major factors limiting the quality of fried potato products. To understand the mechanisms of protein regulation for cold-induced sweetening in potato tubers, a comparative proteomic approach was used to analyse the differentially expressed proteins both during control (25 °C, 30 days) and cold treatment (4 °C, 30 days) using two-dimensional gel electrophoresis. Quantitative image analyses indicated that there were 25 protein spots with their intensities significantly altered more than twofold. Of these proteins, 9 were up-regulated, 13 were down-regulated, 2 were absent, and 1 was induced in the cold-stored tubers. The MALDI-TOF/TOF MS analyses led to the identification of differentially expressed proteins that are involved in several processes and might work cooperatively to maintain metabolic homeostasis in tubers during low-temperature storage. The preponderance of metabolic proteins reflects the inhibition of starch re-synthesis and the accumulation of sugars in carbon fluxes, linking starch–sugar conversion. The respiration-related proteins suggest the transfer of respiratory activity from aerobic respiration to anaerobic respiration in the cold-stored tubers. The proteins associated with defence appear to protect the tuber cells from low-temperature stress. Some heat shock proteins that act as chaperones also displayed a differential expression pattern, suggesting a potentially important role in cold-stored tubers, although their exact contribution remains to be investigated. The proposed hypothetical model might explain the interaction of these differentially expressed proteins that are associated with cold-induced sweetening in tubers.     

Proteomic analysis of salt stress and recovery in leaves of Vigna unguiculata cultivars differing in salt tolerance

Key message

Cowpea cultivars differing in salt tolerance reveal differences in protein profiles and adopt different strategies to overcome salt stress. Salt-tolerant cultivar shows induction of proteins related to photosynthesis and energy metabolism.

Abstract

Salinity is a major abiotic stress affecting plant cultivation and productivity. The objective of this study was to examine differential proteomic responses to salt stress in leaves of the cowpea cultivars Pitiúba (salt tolerant) and TVu 2331 (salt sensitive). Plants of both cultivars were subjected to salt stress (75 mM NaCl) followed by a recovery period of 5 days. Proteins extracted from leaves of both cultivars were analyzed by two-dimensional electrophoresis (2-DE) under salt stress and after recovery. In total, 22 proteins differentially regulated by both salt and recovery were identified by LC–ESI–MS/MS. Our current proteome data revealed that cowpea cultivars adopted different strategies to overcome salt stress. For the salt-tolerant cultivar (Pitiúba), increase in abundance of proteins involved in photosynthesis and energy metabolism, such as rubisco activase, ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19), glycine decarboxylase (EC 1.4.4.2) and oxygen-evolving enhancer (OEE) protein 2, was observed. However, these vital metabolic processes were more profoundly affected in salt-sensitive cultivar (TVu), as indicated by the down-regulation of OEE protein 1, Mn-stabilizing protein-II, carbonic anhydrase (EC 4.2.1.1) and Rubisco (EC 4.1.1.39), leading to energy reduction and a decline in plant growth. Other proteins differentially regulated in both cultivars corresponded to different physiological responses. Overall, our results provide information that could lead to a better understanding of the molecular basis of salt tolerance and sensitivity in cowpea plants.