Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture.

Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro shoot culture that persists after tissue transfer to a medium without antibiotic. The effect is associated with an increase in oxidative stress injury in plant tissues. In this study, we assessed changes of reactive oxygen species accumulation, protein expression, and oxidative protein modification response associated with enduring timentin treatment-induced growth suppression in tobacco (Nicotiana tabacum L.) in vitro shoot culture. The study revealed a gradual 1.7 and 1.9-fold increase in superoxide (O2•-) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O2•- accumulation pattern was different. For PA shoots, the increase in O2•- concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O2•- concentration compared to TC during the period following the first week of cultivation. Although no protein expression differences were detectable between the TC and PA shoots by two-dimensional electrophoresis, the increase in O2•- concentration in PA shoots was associated with a 1.5-fold increase in protein carbonyl modification content after one week of cultivation, and protein carbonylation analysis revealed differential modification of 26 proteoforms involved in the biological processes of photosynthesis and glycolysis. The results imply that the timentin treatment-induced oxidative stress might be implicated in nontranslational cellular redox balance regulation, accelerates the development of senescence of the shoot culture, and contributes to the shoot growth-suppressing effect of antibiotic treatment.

Cold Plasma Treatment of Sunflower Seeds Modulates Plant-Associated Microbiome and Stimulates Root and Lateral Organ Growth.

Cold atmospheric pressure (CP) plasma irradiation of seeds has been shown to promote plant growth, but the molecular basis of this phenomenon is poorly understood. In our study, optimum irradiation of common sunflower seeds using a dielectric barrier discharge CP device stimulated growth of sunflower lateral organs and roots by 9-14% compared to the control. Metagenomic analysis revealed that the structure of plant-associated bacterial assembly was greatly modified upon CP treatment and could be attributed to the antimicrobial effect of CP-generated reactive species. The treatment resulted in the domination of spore forming Mycobacterium sp. in the above-ground tissues of the seedlings. While the overall bacterial diversity in the roots was barely affected, the CP-induced shift in microbial composition is the likely basis for the observed seedling root growth stimulation and the long-term effect on lateral organ growth and could be mediated by increase in water uptake and/or direct root signaling. Low amplitude protein abundance differences were detected in the roots of the emerging seedlings that are characteristic to low intensity stress stimuli response and could be linked to the changes in plant-associated microbiome upon CP treatment.

Identification of Echinacea Purpurea (L.) Moench Root LysM Lectin with Nephrotoxic Properties.

Echinacea purpurea (L.) Moench (EP) is a well-studied plant used for health benefits. Even though there are a lot of data on EP secondary metabolites, its active proteins are not studied well enough. The aim of our experiment was to purify lectin fraction from EP roots and evaluate its biological activity in vitro as well as its effect on kidney morphology in vivo. An EP root glycoprotein fraction was purified by affinity chromatography, identified by LC-MS/MS, and used for biological activity tests in vitro and in vivo. Identified glycoproteins were homologous with the LysM domain containing lectins from the Asteraceae plants Helianthus annuus L., Lactuca sativa L., Cynara cardunculus L. A purified fraction was tested by hemagglutination and hemagglutination inhibition (by carbohydrate reactions) in vitro. We purified the hemagglutinating active ~40 kDa size lactose, D-mannose, and D-galactose specific glycoproteins with two peptidoglycan binding LysM (lysine motif) domains. Purified LysM lectin was tested in vivo. Eight-week old Balb/C male mice (n = 15) were treated with 5 µg of the purified lectin. Injections were repeated four times per week. At the fifth experimental week, animals were sedated with carbon dioxide, then euthanized by cervical dislocation and their kidney samples were collected. Morphological changes were evaluated in hematoxylin and eosin stained kidney samples. The purified LysM lectin induced a statistically significant (p < 0.05) kidney glomerular vacuolization and kidney tubular necrosis (p < 0.001).

The Physiological Response of Lettuce to Red and Blue Light Dynamics Over Different Photoperiods.

This study aimed to evaluate the effect of dynamic red and blue light parameters on the physiological responses and key metabolites in lettuce and also the subsequent impact of varying light spectra on nutritive value. We explored the metabolic changes in carotenes, xanthophylls, soluble sugars, organic acids, and antioxidants; the response of photosynthetic indices [photosynthetic (Pr) and transpiration (Tr) rates]; and the intracellular to ambient CO2 concentration ratios (C i /C a ) in lettuce (Lactuca sativa L. "Lobjoits Green Cos"). They were cultivated under constant (con) or parabolic (dyn) blue (B, 452 nm) and/or red (R, 662 nm) light-emitting diode (LED) photosynthetic photon flux densities (PPFDs) at 12, 16, and 20 h photoperiods, maintaining consistent daily light integrals (DLIs) for each light component in all treatments, at 2.3 and 9.2 mol m-2 per day for blue and red light, respectively. The obtained results and principal component analysis (PCA) confirmed a significant impact of the light spectrum, photoperiod, and parabolic profiles of PPFD on the physiological response of lettuce. The 16 h photoperiod resulted in significantly higher content of xanthophylls (neoxanthin, violaxanthin, lutein, and zeaxanthin) in lettuce leaves under both constant and parabolic blue light treatments (BconRdyn 16 h and BdynRdyn 16 h, respectively). Lower PPFD levels under a 20 h photoperiod (BdynRdyn 20 h) as well as higher PPFD levels under a 12 h photoperiod (BdynRdyn 12 h) had a pronounced impact on leaf gas exchange indices (Pr, Tr, C i /C a ), xanthophylls, soluble sugar contents, and antioxidant properties of lettuce leaves. The parabolic PPFD lighting profile over a 16 h photoperiod (BdynRdyn 16 h) led to a significant decrease in C i /C a , which resulted in decreased Pr and Tr, compared with constant blue or red light treatments with the same photoperiod (BconRdyn and BdynRcon 16 h). Additionally, constant blue lighting produced higher α + ß-carotene and anthocyanin (ARI) content and increased carotenoid to chlorophyll ratio (CRI) but decreased biomass accumulation and antioxidant activity.

Photoresponse to different lighting strategies during red leaf lettuce growth.

The objective of the study was to investigate the effects of growth-stage specific lighting for the physiological homeostasis of red leaf lettuce (Lactuca sativa L. cv. Red Cos), by measuring the productivity of photosynthesis and primary metabolism. In the experiments, the main photosynthetic photon flux consisted of red (R) and blue (B) light, supplemented with blue, green (G) or UV-A wavelengths. Decrease of fructose, accompanied by significant decrease of stomatal conductance (gs), the ratio of intracellular to ambient CO2 concentration (Ci/Ca), photosynthetic rate (Pr), light adapted actual quantum yield of PSII photochemistry (ΦPSII), biomass formation and significant increase of transpiration rate (Tr) suggest that supplemental UV-A during maturity stage, after supplemental green irradiation during seedling stage (BRG to BRUV) was the least favourable condition for red leaf lettuce. However, constant irradiation with supplemental green (BRG) or supplemental green irradiation after increased blue exposure (B↑R to BRG) resulted in significant increase of Pr, gs, Ci/Ca, and light use efficiency(LUE), and decrease of Tr and Water use efficiency (WUE). Significant increase of leaf area was observed under supplemental green in both seedlings (BR; BRG) and matured plants (B↑R to BRG). Significant increase of specific leaf area was found under supplemental green (BRG) for seedlings and under increased blue (B↑R) for matured plants. Accordingly, the most favourable growth-stage specific lighting spectrum strategy for red leaf lettuce, based on photosynthetic and primary metabolite response, is supplemental green irradiation after increased blue exposure (B↑R to BRG), whereas, the most favourable condition for seedlings is BRG. According to the PCA correlation matrix, associations among the measured data indicate that WUE negatively correlated with gs and Ci/Ca, while LUE positively correlated with gs and Pr. However, weak correlations between ФPSII, LUE and photochemical reflectance index (PRI) suggest that selected light conditions were not optimal for red leaf lettuce.

Differential Chloroplast Proteomics of Temperature Adaptation in Apple (Malus x domestica Borkh.) Microshoots.

Temperature stress is one of the most common external factors that plants have to adapt to. Accordingly, plants have developed several adaptation mechanisms to deal with temperature stress. Chloroplasts are one of the organelles that are responsible for the sensing of the temperature signal and triggering a response. Here, chloroplasts are purified from low temperature (4° C), control (22° C) and high temperature (30° C) grown Malus x domestica microshoots. The purity of the chloroplast fractions is evaluated by marker proteins, as well as by using in silico subcellular localization predictions. The proteins are digested using filter-aided sample processing and analyzed using nano-LC MS/MS. 733 proteins are observed corresponding to published Malus x domestica gene models and 16 chloroplast genome -encoded proteins in the chloroplast preparates. In ANOVA, 56 proteins are found to be significantly differentially abundant (p < 0.01) between chloroplasts isolated from plants grown in different conditions. The differentially abundant proteins are involved in protein digestion, cytoskeleton structure, cellular redox state and photosynthesis, or have protective functions. Additionally, a putative chloroplastic aquaporin is observed. Data are available via ProteomeXchange with identifier PXD014212.

Treatment of Common Sunflower (Helianthus annus L.) Seeds with Radio-frequency Electromagnetic Field and Cold Plasma Induces Changes in Seed Phytohormone Balance, Seedling Development and Leaf Protein Expression.

Treatment of plant seeds with electromagnetic fields or non-thermal plasmas aims to take advantage of plant functional plasticity towards stimulation of plant agricultural performance. In this study, the effects of pre-sowing seed treatment using 200 Pa vacuum (7 min), 5.28 MHz radio-frequency cold plasma (CP -2, 5, and 7 min) and electromagnetic field (EMF -5, 10, 15 min) on seed germination kinetics, content of phytohormones, morphometric parameters of seedlings and leaf proteome were assessed. CP 7 min and EMF 15 min treatments caused 19-24% faster germination in vitro; germination in the substrate was accelerated by vacuum (9%) and EMF 15 min (17%). The stressors did not change the seed germination percentage, with exception of EMF 5 min treatment that caused a decrease by 7.5%. Meanwhile both CP 7 min and EMF 15 min treatments stimulated germination, but the EMF treatment resulted in higher weight of leaves. Stressor-specific changes in phytohormone balance were detected in seeds: vacuum treatment decreased zeatin amount by 39%; CP treatments substantially increased gibberellin content, but other effects strongly varied with the treatment duration; the abscisic acid content was reduced by 55-60% after the EMF treatment. Analysis of the proteome showed that short exposure of seeds to the EMF or CP induced a similar long-term effect on gene expression in leaves, mostly stimulating expression of proteins involved in photosynthetic processes and their regulation.

Endophytic Bacillus and Pseudomonas spp. Modulate Apple Shoot Growth, Cellular Redox Balance, and Protein Expression Under in Vitro Conditions.

Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro. Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant-endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H2DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant-endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype.

Patterns of low temperature induced accumulation of dehydrins in Rosaceae crops-Evidence for post-translational modification in apple.

Important crop plants of Rosaceae family are often damaged during winter due to the lack of acclimation and cold hardiness. One of the cellular responses of plants to cold stress is the accumulation of dehydrin proteins. We studied the expression of dehydrins in several Rosaceae species during low temperature treatment in vitro. Microshoots of Pyrus communis, Malus×domestica, Fragaria vesca, Fragaria×ananassa, Prunus cerasus and Prunus avium cultivars were grown in low temperature conditions. Genotype -specific accumulation of dehydrins was detected by immunoblot analysis of the extracted proteins. Untargeted difference gel electrophoresis of Malus x domestica microshoots revealed an extensive accumulation of three dehydrins. In a protein phosphatase assay, MdDHN2 and MdDHN4, but not MdDHN6 proteins were found to be extensively phosphorylated. In terms of the amount of protein synthesized, dehydrins are a major protein-level adaptation mechanism to low temperature in M. x domestica. In addition to dehydrins, the induction of proteins involved in the response for oxidative stress were observed. Additionally, a Xero2 -like dehydrin of F. vesca was detected by difference gel electrophoresis and identified by nano LC-MS/MS.

Application of multiplexed cysteine-labeled complex protein sample for 2D electrophoretic gel alignment.

The analysis of cellular subproteomes by 2DE is hampered by the difficulty of aligning gel images from samples that have very different protein composition. Here, we present a sensitive and cost-effective fluorescent labeling method for analyzing protein samples that is not dependent on their composition. The alignment is guided by inclusion of a complex mixture of proteins that is co-run with the sample. Maleimide-conjugated fluorescent dyes Dy-560 and Dy-635 are used to label the cysteine residues of the sample of interest and the alignment standard, respectively. The two differently labeled mixtures are then combined and separated on a 2D gel and, after selective fluorescence detection, an unsupervised image registration process is used to align the protein patters. In a pilot study, this protocol significantly improved the accuracy of alignment of nuclear proteins with total cellular proteins.

Substrate efflux propensity plays a key role in the specificity of secretory A-type phospholipases.

To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9-10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA(2)s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA(2)s are key players.

Instrument-independent software tools for the analysis of MS-MS and LC-MS lipidomics data.

Mass spectrometry (MS), particularly electrospray-MS, is the key tool in modern lipidomics. However, as even a modest scale experiment produces a great amount of data, data processing often becomes limiting. Notably, the software provided with MS instruments are not well suited for quantitative analysis of lipidomes because of the great variety of species present and complexities in response calibration. Here we describe the use of two recently introduced software tools: lipid mass spectrum analysis (LIMSA) and spectrum extraction from chromatographic data (SECD), which significantly increase the speed and reliability of mass spectrometric analysis of complex lipidomes. LIMSA is a Microsoft Excel add-on that (1) finds and integrates the peaks in an imported spectrum, (2) identifies the peaks, (3) corrects the peak areas for overlap by isotopic peaks of other species and (4) quantifies the identified species using included internal standards. LIMSA is instrument-independent because it processes text-format MS spectra. Typically, the analysis of one spectrum takes only a few seconds.The SECD software allows one to display MS chromatograms as two-dimensional maps, which is useful for visual inspection of the data. More importantly, however, SECD allows one to extract mass spectra from user-defined regions of the map for further analysis with, e.g., LIMSA. The use of select regions rather than simple time-range averaging significantly improves the signal-to-noise ratio as signals outside the region of interest are more efficiently excluded. LIMSA and SECD have proven to be robust and convenient tools and are available free of charge from the authors.

Linkage analysis of schizophrenia controlling for population substructure.

Etiological heterogeneity and complexity has hampered attempts to identify predisposing genes for schizophrenia. We sought to minimize the number of segregating genes involved by focusing on a population isolate with elevated disease prevalence. We exploited the well-established population history, and searched for disease susceptibility loci in families from two alternative founder lineages. We studied 28 schizophrenia pedigrees (123 nuclear families) from an outlying municipality on the eastern border of Finland. We divided the families based on their genealogy and defined two routes of immigration: southern and northern. We examined the kinship coefficients and allele frequency distributions within each group, and performed a linkage analysis based on 497 microsatellite markers across the genome. A high degree of historical relatedness was demonstrated by higher sharing of alleles than predicted by the relationships we identified within the previous four generations alone, as would be expected. Between the two subpopulations, allele frequencies were significantly different, consistent with their isolated genealogies. The southern families showed some evidence of linkage in a schizophrenia locus at 4q23 (Z = 3.3) near our previous finding with quantitative variation in verbal learning and memory [Paunio et al. (2004); Hum Mol Genet 13: 1693-1702], while the northern pedigrees gave most significant evidence on 10q21 (Z = 2.53). Joint analysis of families from both lineages suggested evidence of linkage only at 3p14 (Z = 3.18). Thus the detailed genealogical information led us to identification of distinct linkage signals for schizophrenia susceptibility loci between the three analyses we performed.

Seasonal acclimatization of brain lipidome in a eurythermal fish (Carassius carassius) is mainly determined by temperature.

Crucian carp (Carassius carassius) is an excellent vertebrate model for studies on temperature adaptation in biological excitable membranes, since the species can tolerate temperatures from 0 to +36 degrees C. To determine how temperature affects the lipid composition of brain, the fish were acclimated for 4 wk at +30, +16, or +4 degrees C in the laboratory, or seasonally acclimatized individuals were captured from the wild throughout the year (temperature = +1 to +23 degrees C), and the brain glycerophospholipid and sphingolipid compositions were analyzed in detail by electrospray-ionization mass spectrometry. Numerous significant temperature-related changes were found in the molecular species composition of the membrane lipids. The most notable and novel finding was a large (approximately 3-fold) increase of the di-22:6n-3 phosphatidylserine and phosphatidylethanolamine species in the cold. Since the increase of 22:6n-3 in the total fatty acyl pool of the brain was small, the formation of di-22:6n-3 aminophospholipid species appears to be a specific adaptation to low temperature. Such highly unsaturated species could be needed to maintain adequate membrane fluidity in the vicinity of transporters and other integral membrane proteins. Plasmalogens increased somewhat at higher temperatures, possibly to protect membranes against oxidation. The modifications of brain lipidome during the 4-wk laboratory acclimation were, in many respects, similar to those found in the wild, which indicates that the seasonal changes observed in the wild are temperature dependent rather than induced by other environmental factors.

Software tools for analysis of mass spectrometric lipidome data.

New software tools for quantitative analysis of mass spectrometric lipidome data have been developed. The LIMSA tool finds and integrates peaks in a mass spectrum, matches the peaks with a user-supplied list of expected lipids, corrects for overlap in their isotopic patterns, and quantifies the identified lipid species according to internal standards. Three different algorithms for isotopic correction (deconvolution) were implemented and compared. LIMSA has a convenient user interface and can be applied on any type of MS spectrum. Typically, analysis of one spectrum takes only a few seconds. The SECD tool, designed for analysis of LC-MS data sets, provides an intuitive and informative display of MS chromatograms as two-dimensional "maps" for visual inspection of the data and allows the user to extract mass spectra, to be further analyzed with LIMSA, from arbitrary regions of these maps. More reliable analysis of complex lipidome data with improved signal-to-noise ratio is obtained when compared to standard time-range averaged spectra. The functionality of these tools is demonstrated by analysis of standard mixtures as well as complex biological samples. The tools described here make accurate, high-throughput analysis of extensive sample sets feasible and are made available to the scientific community free of charge.

Efficient calculation of accurate masses of isotopic peaks.

This paper presents a new method for calculating accurate masses of isotopic peaks. It is based on breaking the calculation into a binary series of calculations. The molecule is built up by a series of such calculations. At each step the accurate masses are calculated as a probability weighted sum of the masses of the contributing peaks. The method is computationally efficient and accurate for both mass and relative abundance.