Quantitative proteomic analyses of crop seedlings subjected to stress conditions; a commentary

Quantitative proteomics is one of the analytical approaches used to clarify crop responses to stress conditions. Recent remarkable advances in proteomics technologies allow for the identification of a wider range of proteins than was previously possible. Current proteomic methods fall into roughly two categories: gel-based quantification methods, including conventional two-dimensional gel electrophoresis and two-dimensional fluorescence difference gel electrophoresis, and MS-based quantification methods consists of label-based and label-free protein quantification approaches. Although MS-based quantification methods have become mainstream in recent years, gel-based quantification methods are still useful for proteomic analyses. Previous studies examining crop responses to stress conditions reveal that each method has both advantages and disadvantages in regard to protein quantification in comparative proteomic analyses. Furthermore, one proteomics approach cannot be fully substituted by another technique. In this review, we discuss and highlight the basis and applications of quantitative proteomic analysis approaches in crop seedlings in response to flooding and osmotic stress as two environmental stresses.     

Applying the digital image correlation method to estimate the mechanical properties of bacterial biofilms subjected to a wall shear stress

A digital image correlation (DIC) method was applied to characterize the mechanical behavior of Pseudomonas aeruginosa biofilms in response to wall shear stress using digital video micrographs taken from biofilm flow cells. The appearance of the biofilm in the transmitted light photomicrographs presented a natural texture which was highly conducive to random encoding for DIC. The displacement fields were calculated for two biofilm specimens. The DIC method concurred with previous analysis showing that biofilms exhibit viscoelastic behavior, but had the advantage over simple length measurements of longitudinal strain that it could precisely measure local strains in length (x) and width (y) within biofilm clusters with a 2 μm resolution as a function of time and wall shear stress. It was concluded that DIC was more accurate at measuring elastic moduli than simple length measurements, but that time-lapse 3D images would enable even more accurate estimates to be performed.     

Natural distribution of uranium in skeletal bones and bone tissue fractions

It was shown that uranium is deposited in the mineral and the organic matrix of bones, and approximately 50% of uranium is bound with the latter. In the bone mineral, uranium is deposited not on the surface of hydroxyl apatite crystals but distributed uniformly within them.     

A repeated sampling bone chamber methodology for the evaluation of tissue differentiation and bone adaptation around titanium implants under controlled mechanical conditions

A repeated sampling bone chamber methodology was developed for the study of the influence of the mechanical environment on skeletal tissue differentiation and bone adaptation around titanium implants. Via perforations, bone grows into the implanted outer bone chamber, containing an inner bone chamber with a central test implant. An actuator—easily mounted on the outer bone chamber—allows a controlled mechanical stimulation of the test implant. After each experiment, the inner bone chamber—with its content—can be harvested and analysed. A new inner bone chamber with a central implant can be inserted consecutively in the outer bone chamber and a new experiment can start. Pilot studies led to a reliable surgical protocol and showed the applicability of the methodology, offering the possibility to study skeletal tissue differentiation and adaptation around implants under well-controlled mechanical conditions, and this protected from external loading. Repeated sampling of the bone chamber allows conducting several experiments within the same animal at the same site, thereby excluding subject- and site-dependent variability and reducing the amount of experimental animals.     

Calcium-dependent proteolysis in the myocardium of rats subjected to stress

Activity of a calcium-dependent neutral protease (calpain II) and its specific endogenous inhibitor was investigated in the myocardium of rats subjected to different stressors: cold, anaesthesia, 24 and 48 h starvation and food restriction for 7 and 14 days. Enzyme and inhibitor activities were determined in the 37,200 g supernatant of homogenates prepared from the free left ventricular wall of the heart. The specific activity of the myocardial calcium-dependent proteinase increased in all rats exposed to stressful stimuli, reaching maximum values in animals starved for 48 hours. Decrease in the specific activity of the inhibitor accompanied the changes in enzyme activity. Differences from normal control values were statistically significant in the starved animals and in animals fed a restricted diet for 7 or 14 days. These observations suggest that interaction between calpain II and its specific inhibitor plays a role in the regulation of the enzyme activity and furthermore, that stressful stimuli lead to increased calcium-dependent proteolysis in the myocardium.     

Fast-twitch skeletal muscles of dystrophic mouse pups are resistant to injury from acute mechanical stress

Loss of the dystrophin-glycoproteincomplex from muscle sarcolemma in Duchenne's muscular dystrophy (DMD)renders the membrane susceptible to mechanical injury, leaky toCa²⁺, and disrupts signaling, but the precise mechanism(s)leading to the onset of DMD remain unclear. To assess the role ofmechanical injury in the onset of DMD, extensor digitorum longus (EDL)muscles from C57 (control), mdx, andmdx-utrophin-deficient [mdx:utrn(/); dystrophic] pups aged 9-12 days were subjected to an acutestretch-injury or no-stretch protocol in vitro. Before the stretches,isometric stress was attenuated for mdx:utrn(/) comparedwith control muscles at all stimulation frequencies (P < 0.05). During the stretches, EDL muscles for each genotypedemonstrated similar mean stiffness values. After the stretches,isometric stress during a tetanus was decreased significantly for bothmdx and mdx:utrn(/) muscles compared withcontrol muscles (P < 0.05). Membrane injury assessedby uptake of procion orange dye was greater for dystrophic comparedwith control EDL (P < 0.05), but, within eachgenotype, the percentage of total cells taking up dye was not different for the no-stretch vs. stretch condition. These data suggest that thesarcolemma of maturing dystrophic EDL muscles are resistant to acutemechanical injury.

     

Quantitative evaluation of tryptophan oxidation in actin and troponin I from skeletal muscles using a rat model of acute oxidative stress

Increased levels of “ROS” cause oxidative stress and are believed to play a key role in the development of age‐related diseases and mammalian aging, e.g. through the oxidation of residues, at or close to, the protein surface. In this study, we have investigated the effects of ROS on tryptophan residues in alpha skeletal actin and troponin I (fast skeletal muscle isoform) using an established rat model of acute oxidative stress induced by X‐ray irradiation. In the control samples (no oxidative stress), the single Trp residue of troponin I (position 161) and the four tryptophan residues present in actin (positions 79, 86, 340, and 356) were only oxidized at very low levels. Post‐irradiation, the level of oxidized versions increased for most positions within 3 h. Tryptophan residues located inside the proteins, however, required longer time periods. Based on the increment masses of the tryptophan positions calculated from the b‐ and y‐ion series of the tandem mass spectra, the following oxidation products of tryptophan were detected: kynurenine; oxolactone; hydroxytryptophan or oxindolylalanine (isobaric); hydroxykynurenine; dioxindolylalanine, N‐formylkynurenine or dihydroxytryptophan (all three isobaric); and hydroxyl‐N‐formylkynurenine, with mass gains relative to tryptophan of 4, 14, 16, 20, 32, and 48 u, respectively. Despite a partial recovery after 24 h, the degree of oxidation of all oxidized versions was still higher than in the control samples.     

The mechanical behavior of isolated Avena coleoptile walls subjected to constant stress: properties and relation to cell elongation

In order to assess the role of the mechanical properties of the wall in auxin-induced cell elongation, a study has been made of the ability of isolated Avena coleoptile walls to extend (creep) when subjected to a constant applied stress. Creep occurs as a viscoelastic extension which has the following characteristics: the extension is proportional to log time and is partly reversible, and the extension rate has a Q₁₀ of about 1.05 and is markedly greater in auxin-pretreated walls. In nonconditioned walls the extension rate is proportional to applied stress, but pre-extension causes the appearance of an apparent yield strain. The similarity of creep and instantaneous plastic deformation in response to temperature or to pretreatment with auxin or KCN suggests that the instantaneous deformation is simply the viscoelastic extension which occurs at very short times. A comparison of these viscoelastic properties with the properties of auxin-induced cell elongation indicates that cell elongation requires more than just a physical extension of the wall. It is suggested that elongation occurs as a series of extension steps, each of which involves a viscoelastic extension preceded or accompanied by an auxin-dependent biochemical change in the wall properties.     

Quantitative changes in the degree of chromatin condensation during the cell cycle in differentiating Pisum sativum vascular tissue

Fixed tissue from the base of the fourth internode above the cotyledons of Pisum sativum plants were Feulgen-stained for DNA. In squash preparations three cell types: xylem vessel elements, phloem fibres and phloem sieve tube elements were identified. Measurement of Feulgen absorbance values in 0.5 X 0.5 microns measuring points across each nucleus were obtained with a scanning cytophotometer. Condensed chromatin was defined as any measuring point having an absorbance value greater than 0.32. Calculating 'percent condensed chromatin' as the percentage of the total nuclear DNA contained in those densely stained points, the pattern of change in this parameter during interphase in each cell type was observed. A generally similar pattern occurs in all three cell types. Percent condensed chromatin decreased from about 45% to 12% during G1, increased rapidly to the end of S, decreased during G2 and the following G1 to increase again during the subsequent S phase.     

The orientation of cell wall microtubules in wheat coleoptile segments subjected to phytohormone treatment

The effect of plant hormones was studied on the growth of excised coleoptile segments of wheat plantlets grown under daylight conditions. In addition to the change in growth, that in the orientation of microtubules and cellulose microfibrils was investigated in parenchyma cells. Following a 6-h treatment gibberellin, and still more kinetin, stímulated the thickening of segments, which became evident also in an altered orientation of microtubules. Whereas in the control the microtubules and wall microfibrils were oriented randomly, following gibberellin treatment they were all parallel and formed an acute angle with the longitudinal cell axis. A still more pronounced difference resulted after kinetin treatment, when microtubules were localized parallel with the longitudinal cell axis. Auxin had the opposite effect: it stimulated the elongation of the segments, which became evident in a transverse orientation of both wall microtubules and microfibrils.     

Quantitative evaluation of ultrastructural restructuring in the adrenal cortex of rats under stress

Quantitative and qualitative analysis has revealed that submicroscopic changes of the rat adrenal cortex in dynamics of the stress reaction examined at the level of "functional element" which combines basic structural components of the parenchyma and stroma are of the polyphase character and agree with data from the biochemical analysis of 11-OCS in blood plasma. At the stage of exhaustion of the stress reaction (72 hours of immobilization) the ultrastructure of the adrenal cortex testifies to the preservation of functional reserves in the secretory cells.