Physiological and Proteomic Responses of Contrasting Alfalfa (Medicago sativa L.) Varieties to High Temperature Stress

High temperature (HT) is an important factor for limiting global plant distribution and agricultural production. As the global temperature continues to rise, it is essential to clarify the physiological and molecular mechanisms of alfalfa responding the high temperature, which will contribute to the improvement of heat resistance in leguminous crops. In this study, the physiological and proteomic responses of two alfalfa (Medicago sativa L.) varieties contrasting in heat tolerance, MS30 (heat-tolerant) and MS37 (heat-sensitive), were comparatively analyzed under the treatments of continuously rising temperatures for 42 days. The results showed that under the HT stress, the chlorophyll content and the chlorophyll fluorescence parameter (Fv/Fm) of alfalfa were significant reduced and some key photosynthesis-related proteins showed a down-regulated trend. Moreover, the content of Malondialdehyde (MDA) and the electrolyte leakage (EL) of alfalfa showed an upward trend, which indicates both alfalfa varieties were damaged under HT stress. However, because the antioxidation-reduction and osmotic adjustment ability of MS30 were significantly stronger than MS37, the damage degree of the photosynthetic system and membrane system of MS30 is significantly lower than that of MS37. On this basis, the global proteomics analysis was undertaken by tandem mass tags (TMT) technique, a total of 6,704 proteins were identified and quantified. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that a series of key pathways including photosynthesis, metabolism, adjustment and repair were affected by HT stress. Through analyzing Venn diagrams of two alfalfa varieties, 160 and 213 differentially expressed proteins (DEPs) that had dynamic changes under HT stress were identified from MS30 and MS37, respectively. Among these DEPs, we screened out some key DEPs, such as ATP-dependent zinc metalloprotease FTSH protein, vitamin K epoxide reductase family protein, ClpB3, etc., which plays important functions in response to HT stress. In conclusion, the stronger heat-tolerance of MS30 was attributed to its higher adjustment and repair ability, which could cause the metabolic process of MS30 is more conducive to maintaining its survival and growth than MS37, especially at the later period of HT stress. This study provides a useful catalog of the Medicago sativa L. proteomes with the insight into its future genetic improvement of heat-resistance.

Comparative Physiological, Biochemical, and Proteomic Responses of Photooxidation-Prone Rice Mutant 812HS under High Light Conditions

Photosynthetic efficiency decreases as light energy surpasses the photosynthesis capacity. This study was designed to investigate the potential effects of high-intensity light on the photooxidation-prone mutant 812HS of rice and its wild-type 812S during yellow and recovering stages. Results showed that in the yellowing stage, light oxidation occurs due to the exposure of mutant 812HS leaves to the high sunlight, which causes yellowing of the leaves, leading to a reduction in the photochemical activities, physiological mechanisms, and protein contents in mutant 812HS. In the recovery stage, mutant 812HS leaves were exposed to the maximum high brightness, the mutant’s leaves were draped with a dark cover to decrease the exposure of leaves of the plants from direct sunlight, which leads to the restoration of the green color again to the mutant 812HS leaves, leading to improving the performance of the photochemical activities, physiological mechanisms, and protein contents in mutant 812HS. Exposing leaves of mutant 812HS to high light at the yellow stage also resulted in a decrease in the net photosynthetic rate (Pn) in carotenoids content and chlorophyll a and b. Similarly, chlorophyll fluorescence of mutant 812HS decreased in (O-I-J-I-P) curves, and the ATP content, Mg2+-ATPase, and Ca2+-ATPase activities also decreased. An increase in energy dissipation was observed, while ABS/RC, DI0/RC, and TR0/RC values in mutant 812HS at the yellow stage increased. During photooxidation, an increase in O2•– and H2O2 contents was observed in mutant 812HS. While O2•– and H2O2 contents were decreased in mutant 812HS at the recovery stage. The rate of thylakoid membrane protein content was significantly decreased in mutant 812HS at the yellow stage, while at the recovery stage, there was no significant decrease. Our findings showed that photooxidation prompted oxidative damages and lipid peroxidation that caused severe damages to the membranes of the cell, photosynthetic pigments degradation, protein levels, and photosynthesis inhibition in mutant 812HS.

Potential Probiotic Pediococcus pentosaceus M41 Modulates Its Proteome Differentially for Tolerances Against Heat, Cold, Acid, and Bile Stresses

Probiotics containing functional food confer health benefits in addition to their nutritional properties. In this study, we have evaluated the differential proteomic responses of a potential novel probiotic Pediococcus pentosaceus M41 under heat, cold, acid, and bile stress conditions. We identified stress response proteins that could provide tolerances against these stresses and could be used as probiotic markers for evaluating stress tolerance. Pediococcus pentosaceus M41 was exposed for 2 h to each condition: 50°C (heat stress), 4°C (cold stress), pH 3.0 (acid stress) and 0.05% bile (bile stress). Proteomic analysis was carried out using 2D-IEF SDS PAGE and LC-MS/MS. Out of 60 identified proteins, 14 upregulated and 6 downregulated proteins were common among all the stress conditions. These proteins were involved in different biological functions such as translation-related proteins, carbohydrate metabolism (phosphoenolpyruvate phosphotransferase), histidine biosynthesis (imidazole glycerol phosphate synthase) and cell wall synthesis (tyrosine-protein kinase CapB). Proteins such as polysaccharide deacetylase, lactate oxidase, transcription repressor NrdR, dihydroxyacetone kinase were upregulated under three out of the four stress conditions. The differential expression of these proteins might be responsible for tolerance and protection of P. pentosaceus M41 against different stress conditions.

Cell Wall Proteomics Reveal Phenotypic Adaption of Drug-Resistant Mycobacterium smegmatis to Subinhibitory Rifampicin Exposure

Despite the availability of effective drug treatment, Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, kills ~1. 5 million people annually, and the rising prevalence of drug resistance increasingly threatens to worsen this plight. We previously showed that sublethal exposure to the frontline anti-TB drug, rifampicin, resulted in substantial adaptive remodeling of the proteome of the model organism, Mycobacterium smegmatis, in the drug-sensitive mc2155 strain [wild type (WT)]. In this study, we investigate whether these responses are conserved in an engineered, isogenic mutant harboring the clinically relevant S531L rifampicin resistance-conferring mutation (SL) and distinguish the responses that are specific to RNA polymerase β subunit- (RpoB-) binding activity of rifampicin from those that are dependent on the presence of rifampicin alone. We verified the drug resistance status of this strain and observed no phenotypic indications of rifampicin-induced stress upon treatment with the same concentration as used in WT (2.5 μg/ml). Thereafter, we used a cell wall-enrichment strategy to focus attention on the cell wall proteome and observed 253 proteins to be dysregulated in SL bacteria in comparison with 716 proteins in WT. We observed that decreased abundance of ATP-binding cassette (ABC) transporters and increased abundance of ribosomal machinery were conserved in the SL strain, whereas the upregulation of transcriptional machinery and the downregulation of numerous two-component systems were not. We conclude that the drug-resistant M. smegmatis strain displays some of the same proteomic responses observed in WT and suggest that this evidence supports the hypothesis that rifampicin exercises effects beyond RpoB-interaction alone and that mycobacteria recognise rifampicin as a signaling molecule in an RpoB-independent manner at sublethal doses. Taken together, our data indicates mixed RpoB-independent and RpoB-dependent proteomic remodeling in WT mycobacteria, with evidence for RpoB-independent ABC transporter downregulation, but drug activity-based transcriptional upregulation and two-component system downregulation.

Multicenter reverse-phase protein array data integration

Among the technologies available for protein biomarker discovery and validation, reverse-phase protein array (RPPA) benefits from unequalled sample throughput. Panels of high-quality antibodies enable the quantification by RPPA of protein abundance and posttranslational modifications in biological specimens with high precision and sensitivity. Incorporation of RPPA technology into clinical and drug development pipelines requires robust assays that generate reproducible results across multiple laboratories. We implemented the first international multicenter pilot study to investigate RPPA workflow variability. We characterized the proteomic responses of a series of breast cancer cells to two cancer drugs. This analysis quantified 86,832 sample spots, representing 108 biological samples, arrayed at three independent RPPA platforms. This unique integrated set of data is publicly available as a resource to the proteomic and cancer research communities to catalyse further analysis and investigation. We anticipate that this dataset will form a reference for the comparison of RPPA workflows and reagents, which can be expanded in the future, and will aid the identification of platform-robust treatment-marker antigens in breast cancer cells.

Physiological and Proteomic Responses of Pitaya to PEG-Induced Drought Stress

Pitaya (Hylocereus polyrhizus L.) is highly tolerant to drought stress. Elucidating the response mechanism of pitaya to drought will substantially contribute to improving crop drought tolerance. In the present study, the physiological and proteomic responses of the pitaya cultivar ‘Zihonglong’ were compared between control seedlings and seedlings exposed to drought stress (−4.9 MPa) induced by polyethylene glycol for 7 days. Drought stress obviously enhanced osmolyte accumulation, lipid peroxidation, and antioxidant enzyme activities. Proteomic data revealed drought stress activated several pathways in pitaya, including carbohydrate and energy metabolism at two drought stress treatment time-points (6 h and 3 days). Other metabolic pathways, including those related to aspartate, glutamate, glutathione, and secondary metabolites, were induced more at 3 days than at 6 h, whereas photosynthesis and arginine metabolism were induced exclusively at 6 h. Overall, protein expression changes were consistent with the physiological responses, although there were some differences in the timing. The increases in soluble sugar contents mainly resulted from the degradation and transformation of insoluble carbohydrates. Differentially accumulated proteins in amino acid metabolism may be important for the conversion and accumulation of amino acids. GSH and AsA metabolism and secondary metabolism may play important roles in pitaya as enzymatic and nonenzymatic antioxidant systems. The enhanced carbohydrate and energy metabolism may provide the energy necessary for initiating the above metabolic pathways. The current study provided the first proteome profile of this species exposed to drought stress, and may clarify the mechanisms underlying the considerable tolerance of pitaya to drought stress.

Physiological and Proteomic Responses to Drought in Leaves of Amygdalus mira (Koehne) Yü et Lu

Various environmental stresses strongly influence plant development. Among these stresses is drought, which is a serious threat that can reduce agricultural productivity and obstruct plant growth. Although the mechanism of plants in response to drought has been studied extensively, the adaptive strategies of Amygdalus mira (Koehne) Yü et Lu grown in drought and rewatered habitats remain undefined. Amygdalus mira from the Tibetan Plateau has outstanding nutritional and medicinal values and can thrive in extreme drought. In this study, the physiological and proteomic responses in leaves of A. mira were investigated during drought and recovery period. The changes in plant growth, photosynthesis, enzymes, and non-enzymatic antioxidant under drought and rewatering were also analyzed in leaves. Compared with controls, A. mira showed stronger adaptive and resistant characteristics to drought. In addition, the proteomic technique was also used to study drought tolerance mechanisms in A. mira leaves. Differentially expressed proteins were identified using mass spectrometry. Accordingly, 103 proteins involved in 10 functional categories: cytoskeleton dynamics, energy metabolism, carbohydrate metabolism, photosynthesis, transcription and translation, transport, stress and defense, molecular chaperones, other materials metabolism, and unknown function were identified. These results showed that an increase of stress-defense-related proteins in leaves after drought treatment contributed to coping with drought. Importantly, A. mira developed an adaptive mechanism to scavenge reactive oxygen species (ROS), including enhancing antioxidant enzyme activities and non-enzymatic antioxidant contents, reducing energy, and adjusting the efficiency of gas exchanges. These results may help to understand the acclimation of A. mira to drought.