Stress-induced changes in protease composition are determined by nitrogen supply in non-nodulating white clover

An inbreeding line of white clover has been identified which remains non-nodulated under appropriate physiological conditions and so the nitrogen concentration of the plant can be manipulated by altering the nitrate supply to the roots. Non-nodulating plants were used to test the hypothesis that acclimation to nitrogen limitation in white clover involves changes in protease activity and composition. These results indicate that acclimation to nitrogen limitation involves the realignment of constituent proteases without necessarily incurring significant changes in total protease activity. Plants grown at 2.5, 5.0, 7.5, and 10 mM nitrate showed a positive correlation between nitrate supply and foliar protein concentration. Protein profiles, revealed by Coomassie-stained SDS-PAGE, were unchanged between treatments for a given amount of protein. Serine, aspartate/metalloprotease, and two cysteine proteases were identified in the leaves. Although total protease activity per gram fresh weight was unchanged between treatments, the relative contributions of these four proteases was determined by nitrate supply. When plants were stressed further by withholding nitrate there was an increase in cysteine protease activity, but a senescence-related aspartate/metalloprotease was not visible. Hence, while protease expression in white clover leaves responded to the current and past nitrogen status of the plant, the proteases involved in remobilization during nutrient limitation were distinct from those involved during the main senescence period. It is suggested that nitrogen limitation induced an early, reversible stage of senescence in which perturbations in protease activity facilitated the degradation of non-essential proteins in order to increase the chances of plant survival or seed set.     

Changes in lysosomal cathepsins during liver regeneration

The effect if $\text{2}\text{3}$ hepatectomy in the adult rat on the specific and total activities of two lysosomal endopeptidases, cathepsins B₁ and D, has been examined. The specific activity of both enzymes fell rapidly following hepatectomy when compared to paired, sham-operated controls. When changes in total protein were compared to changes in cathepsins B₁ and D, all three decreased in a parallel fashion for the first 18 h. At 24 h, total liver protein increased rapidly while cathepsins B₁ and D continued to decrease. Cathepsin B₁ fell to a level of 12% of non-operated control levels at 36 h, while total protein was already back to 40% of control levels. In contrast to the decreases in the activities of the cathepsins, there was an increase in the activities of lactate dehydrogenase and malate dehydrogenase during the first two days of regeneration. The clear lag in replacement of the cathepsins relative to other liver proteins following partial hepatectomy suggests that cathepsin activity is selectively controlled and that lowering the levels of cathepsins B₁ and D may play an important role in the decreased degradation of protein seen during the early phases of liver regeneration.     

Expression profiles of Na+,K+-ATPase during acute and chronic hypo-osmotic stress in the blue crab Callinectes sapidus

During acclimation to dilute seawater, the specific activity of Na+,K+-ATPase increases substantially in the posterior gills of the blue crab Callinectes sapidus. To determine whether this increase occurs through regulation of pre-existing enzyme or synthesis of new enzyme, mRNA and protein levels were measured over short (<24 h) and long (18 days) time courses. Na+,K+-ATPase expression, both mRNA and protein, did not change during the initial 24-h exposure to dilute seawater (10 ppt salinity). Thus, osmoregulation in C. sapidus during acute exposure to low salinity likely involves either modulation of existing enzyme or mechanisms other than an increase in the amount of Na+,K+-ATPase enzyme. However, crabs exposed to dilute seawater over 18 days showed a 300% increase in Na+,K+-ATPase specific activity as well as a 200% increase in Na+,K+-ATPase protein levels. Thus, it appears that the increase in Na+,K+-ATPase activity during chronic exposure results from the synthesis of new enzyme. The relative amounts of mRNA for the alpha-subunit increased substantially (by 150%) during the acclimation process, but once the crabs had fully acclimated to low salinity, the mRNA levels had decreased and were not different from levels in crabs fully acclimated to high salinity. Thus, there is transient induction of the Na+,K+-ATPase mRNA levels during acclimation to dilute seawater.     

Physiological behaviour of Arthrospira (Spirulina) maxima during acclimation to changes in irradiance

The physiological behaviour of Arthrospira (Spirulina) maxima during acclimation to sudden changes in irradiance from high (HL) to low light (LL) and vice versa was studied by following parameters concerning growth rate, pigment, carbohydrate and protein cell contents. Applying first order kinetics, the specific acclimation rates for the parameters considered were calculated. During HL to LL shift, pigments increased to compensate for a reduction in growth irradiance in order to maintain relatively high growth rates, whereas carbohydrates decreased at the highest rate. The synthesis of phycobiliproteins proceeded at a rate similar or little higher than that of chlorophyll a, indicating their importance in the light harvesting at low irradiance. During LL to HL shift, carbohydrate biosynthesis was increased, whereas pigment and protein cell contents decreased. The kinetic analysis suggested that the pigment decrease could be accounted for both by dilution through growth and in vivo degradation. During this transition, the initially high cell pigment content gave rise to a very heavy carbohydrate synthesis, which for a short time, after the shift to HL conditions, overshot the final steady-state. In the same period the specific growth rate also increased notably, overshooting the μmax. The acclimation rates of the measured parameters were faster during LL to HL transition then during the reverse. The physiological response of A. maxima during the acclimation to sudden irradiance shifts points out the ability of this cyanobacterium to alter light harvesting and highlights again the key role of carbohydrates when the cells underwent an energy crisis during down-shift. This revised version was published online in August 2006 with corrections to the Cover Date.     

Prostaglandins E2 and F alpha levels in white and brown adipose tissues of cold acclimated rats as measured by a new micromethod.

A new micro-method was used to determine the effects of cold acclimation of rats on the levels of prostaglandin E2 and F alpha in both white and brown adipose tissues. Whereas PGF alpha levels were significantly higher than PGE2 levels in white fat, no difference between the amounts of the two prostaglandins was observed in brown fat. In both tissues, cold acclimation did not induce any change in prostaglandin levels.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Plasma opioid peptide responses during heat acclimation in humans

Plasma beta-endorphin, Met-enkephalin and Peptide F immunoreactivity (ir) were measured at rest and following exercise on three days (days 1, 4, 8) of an eight day heat acclimation regime. Fourteen male subjects demonstrated physiological heat acclimation adaptations. Our data demonstrated a differential response of peripheral plasma levels of endogenous opioid peptides (EOP) to exercise in the heat. In addition, EOP did not follow the same time-course of other physiological adaptations as no differences (day 1 vs. 4 vs. 8) in resting or exercise levels were observed over the eight day heat acclimation regime. Significant increases in beta-endorphin ir (pre- to post-exercise) appear to reflect concomitant exercise-heat related changes. The increased peripheral levels of beta-endorphin were correlated to plasma levels of cortisol. Heat and exercise stress may result in a reduction of Met-enkephalin ir observed in peripheral plasma and might be due to degradation or a decrease in processing from the larger precursors. The differential responses of EOP suggest the possibility of separate physiological roles for these peptides during exercise in the heat but peripheral plasma levels of EOP do not appear to reflect acute heat acclimation changes.     

An Early Stage of Cold Acclimation with Four Phases of Protein Synthesis in Crowns of Winter Wheat

During an early stage of cold acclimation, prominent changes in protein-synthetic activities were found to occur in the crown, which is the part where the stem joins the root of winter wheat (Triticum aestivum L. cv. Horoshirikomugi). This stage was complete within a week of cold treatment, and from the protein-synthetic activities, this stage of cold acclimation could be divided into four phases. First, when the plant seedlings were placed at 0°C, there was a lag period of 1d and no newly inducible proteins were formed during this time. During the second phase (1 to 2d), as the first response to cold, 16 new proteins were synthesized and the active synthesis of 6 preexisting proteins was reinitiated, while syntheses of at least 5 preexisting proteins were depressed. During the third phase (2 to 5d), the levels of most of the cold-inducible proteins reached a maximum, but synthesis of at least 6 preexisting proteins started to decrease. During the fourth phase (after 5d), the synthetic activities of the 6 proteins returned to the original levels and synthesis of another set of 3 new proteins started. During this phase, the synthesis of both protein fractions, the cold-inducible and the preexisting proteins, reached a steady state. After this period, no major changes in the protein profile could be detected. During the third phase, the most active synthesis of the cold-inducible proteins, in particular, proteins designated C10 (M_r 53k), C12a (M_r 46k), and C12b (M_r 46k), occurred, concurrent with the abrupt and transient decrease in the synthetic activities of a set of 6 preexisting proteins. These results suggest that, in addition to the induction of a set of new proteins, the preferential or selective synthesis of proteins required for accommodation to the cold environment takes place at an early stage of acclimation.     

Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii

Information is limited on sulfur (S)-sources inside plant cells for synthesis of the proteins for acclimation to S-starvation. We found that a green alga, Chlamydomonas reinhardtii, when transferred to S-starved conditions, degrades 85% of a chloroplast membrane lipid, sulfoquinovosyl diacylglycerol (SQDG), to redistribute its S to a large part of protein fraction as early as by 6h. Furthermore, the degradation of SQDG preceded that of proteins such as ribulose bisphosphate carboxylase/oxygenase, the candidates of internal S-sources. SQDG was thus demonstrated to yield a major internal S-source for protein synthesis during the early phase of acclimation process to S-starvation.     

Down-regulation of activity and expression of three transport-related proteins in the gills of the euryhaline green crab, Carcinus maenas, in response to high salinity acclimation

The euryhaline green crab, Carcinus maenas, undergoes an annual cycle of salinity exposure, having to adapt to low salinity during its annual spring migration into estuaries, and then having to re-adapt to high salinity when it moves off-shore at the end of summer. Most studies have focused on low salinity acclimation, the activation of osmoregulatory mechanisms, and the induction of transport protein and transport-related enzyme activity and gene expression. In this study we followed the changes in hemolymph osmolality, carbonic anhydrase activity, and mRNA expression of three proteins through a complete cycle of low (15 ppt) and high (32 ppt) salinity acclimation. One week of low salinity acclimation resulted in hemolymph osmoregulation and a four-fold induction of branchial carbonic anhydrase activity. Relative mRNA expression increased for two CA isoforms (CAc 100-fold, and CAg 7-fold) and the α-subunit of the Na/K-ATPase (8-fold). Upon re-exposure to high salinity, hemolymph osmolality increased to 32 ppt acclimated levels by 6 h, and mRNA levels returned to high salinity, baseline levels within 1 week. However, CA activity remained unchanged in response to high salinity exposure for the first week and then gradually declined to baseline levels over 4 weeks. The relative timing of these changes suggests that while whole-organism physiological adaptations and regulation at the gene level can be very rapid, changes at the level of protein expression and turnover are much slower. It is possible that the high metabolic cost of protein synthesis and/or processing could be the underlying reason for long biological life spans of physiologically important proteins.     

Pair-wise multicomparison and OPLS analyses of cold-acclimation phases in Siberian spruce

Analysis of metabolomics data often goes beyond the task of discovering biomarkers and can be aimed at recovering other important characteristics of observed metabolomic changes. In this paper we explore different methods to detect the presence of distinctive phases in seasonal-responsive changes of metabolomic patterns of Siberian spruce (Picea obovata) during cold acclimation occurred in the period from mid-August to January. Multivariate analysis, specifically orthogonal projection to latent structures discriminant analysis (OPLS-DA), identified time points where the metabolomic patterns underwent substantial modifications as a whole, revealing four distinctive phases during acclimation. This conclusion was re-examined by a univariate analysis consisting of multiple pair-wise comparisons to identify homogeneity intervals for each metabolite. These tests complemented OPLS-DA, clarifying biological interpretation of the classification: about 60% of metabolites found responsive to the cold stress indeed changed at one or more of the time points predicted by OPLS-DA. However, the univariate approach did not support the proposed division of the acclimation period into four phases: less than 10% of metabolites altered during the acclimation had homogeneous levels predicted by OPLS-DA. This demonstrates that coupling the classification found by OPLS-DA and the analysis of dynamics of individual metabolites obtained by pair-wise multicomparisons reveals a more correct characterization of biochemical processes in freezing tolerant trees and leads to interpretations that cannot be deduced by either method alone. The combined analysis can be used in other ‘omics’-studies, where response factors have a causal dependence (like the time in the present work) and pair-wise multicomparisons are not conservative.     

Light-induced changes in the amounts of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase and its mRNA in barley plants grown under a diurnal light/dark cycle

Seedlings of barley were grown either in continuous darkness or under a diurnal 12 h light/12 h dark cycle and the effects on NADPH-protochlorophyllide oxidoreductase were followed at two different levels. Firstly, the relative content of the mRNA encoding the NADPH-protochlorophyllide oxidoreductase was measured by dot-blot hybridization. Secondly, changes in the enzyme polypeptide were monitored either by the method of immunoblotting or by immunogold labelling of ultrathin sections of Lowicryl-embedded leaf tissue. Our results demonstrate that drastic diurnal changes in the level of mRNA sequences and the enzyme protein are unlikely to occur in plants which have been grown under natural light/dark conditions. In the dark, protein and mRNA accumulation occurs at an early developmental stage. These results are difficult to reconcile with the suggestion that the massive accumulation of mRNA and enzyme protein in dark-grown seedlings is primarily the consequence of an artificially extended darkperiod. In addition to the plastid-specific NADPH-protochlorophyllide oxidoreductase a closely related polypeptide has been detected outside the plastid in the surrounding cytoplasm (Dehseh et al. 1986b, Planta 169, 172–183). During the diurnal light/dark treatment of seedlings the concentrations of the two protein populations did not show any variation indicative of an exchange between the two protein populations across the plastid envelope.Abbreviation poly(A)⁺RNA polyadenylated RNA     

Pair-wise multicomparison and OPLS analyses of cold-acclimation phases in Siberian spruce

Analysis of metabolomics data often goes beyond the task of discovering biomarkers and can be aimed at recovering other important characteristics of observed metabolomic changes. In this paper we explore different methods to detect the presence of distinctive phases in seasonal-responsive changes of metabolomic patterns of Siberian spruce (Picea obovata) during cold acclimation occurred in the period from mid-August to January. Multivariate analysis, specifically orthogonal projection to latent structures discriminant analysis (OPLS-DA), identified time points where the metabolomic patterns underwent substantial modifications as a whole, revealing four distinctive phases during acclimation. This conclusion was re-examined by a univariate analysis consisting of multiple pair-wise comparisons to identify homogeneity intervals for each metabolite. These tests complemented OPLS-DA, clarifying biological interpretation of the classification: about 60% of metabolites found responsive to the cold stress indeed changed at one or more of the time points predicted by OPLS-DA. However, the univariate approach did not support the proposed division of the acclimation period into four phases: less than 10% of metabolites altered during the acclimation had homogeneous levels predicted by OPLS-DA. This demonstrates that coupling the classification found by OPLS-DA and the analysis of dynamics of individual metabolites obtained by pair-wise multicomparisons reveals a more correct characterization of biochemical processes in freezing tolerant trees and leads to interpretations that cannot be deduced by either method alone. The combined analysis can be used in other ‘omics’-studies, where response factors have a causal dependence (like the time in the present work) and pair-wise multicomparisons are not conservative.