iTRAQ-based comparative proteomic analysis reveals high temperature accelerated leaf senescence of tobacco (Nicotiana tabacum L.) during flue-curing

Tobacco (Nicotiana tabacum) is extensively cultivated all over the world for its economic value. During curing and storage, senescence occurs, which is associated with physiological and biochemical changes in postharvest plant organs. However, the molecular mechanisms involved in accelerated senescence due to high temperatures in tobacco leaves during curing need further elaboration. We studied molecular mechanisms of senescence in tobacco leaves exposed to high temperature during curing (Fresh, 38 °C and 42 °C), revealed by isobaric tags for relative and absolute quantification (iTRAQ) for the proteomic profiles of cultivar Bi’na1. In total, 8903 proteins were identified, and 2034 (1150 up-regulated and 1074 down-regulated) differentially abundant proteins (DAPs) were obtained from tobacco leaf samples. These DAPs were mainly involved in posttranslational modification, protein turnover, energy production and conversion. Sugar- and energy-related metabolic biological processes and pathways might be critical regulators of tobacco leaves exposed to high temperature during senescence. High-temperature stress accelerated tobacco leaf senescence mainly by down-regulating photosynthesis-related pathways and degrading cellular constituents to maintain cell viability and nutrient recycling. Our findings provide a valuable inventory of novel proteins involved in senescence physiology and elucidate the protein regulatory network in postharvest organs exposed to high temperatures during flue-curing.     

Tracking the Stability of Clinically Relevant Blood Plasma Proteins with Delta-S-Cys-Albumin—A Dilute-and-Shoot LC/MS-Based Marker of Specimen Exposure to Thawed Conditions

Biomolecular integrity can be compromised when blood plasma/serum (P/S) specimens are improperly handled. Compromised analytes can subsequently produce erroneous results—without any indication of having done so. We recently introduced an LC/MS-based marker of P/S exposure to thawed conditions called ΔS-Cys-Albumin which, aided by an established rate law, quantitatively tracks exposure of P/S to temperatures greater than their freezing point of ?30 °C. The purposes of this study were to (1) evaluate ΔS-Cys-Albumin baseline values in gastrointestinal cancer patients and cancer-free control donors, (2) empirically assess the kinetic profiles of ΔS-Cys-Albumin at 23 °C, 4 °C, and ?20 °C, and (3) empirically link ΔS-Cys-Albumin to the stability of clinically relevant proteins. ΔS-Cys-Albumin was measured at ≥ 9 different time points per exposure temperature in serum and K₂EDTA plasma samples from 24 separate donors in aliquots kept separately at 23 °C, 4 °C, and ?20 °C. Twenty-one clinically relevant plasma proteins were measured at four time points per temperature via a multiplexed immunoassay on the Luminex platform. Protein stability was assessed by mixed effects models. Coordinated shifts in stability between ΔS-Cys-Albumin and the unstable proteins were documented by repeated measures and Pearson correlations. Plasma ΔS-Cys-Albumin dropped from approximately 20% to under 5% within 96 h at 23 °C, 28 days at 4 °C, and 65 days at ?20 °C. On average, 22% of the 21 proteins significantly changed in apparent concentration at each exposure temperature (p < 0.0008 with >10% shift). A linear inverse relationship was found between the percentage of proteins destabilized and ΔS-Cys-Albumin (r = ?0.61; p < 0.0001)—regardless of the specific time/temperature of exposure. ΔS-Cys-Albumin tracks cumulative thawed-state exposure. These results now enable ΔS-Cys-Albumin to approximate the percentage of clinically relevant proteins that have been compromised by incidental plasma exposure to thawed-state conditions.     

Identification of acquired thermotolerance deficiency within the ditelosomic series of `Chinese Spring' wheat

The relative contribution of individual heat shock proteins to acquired thermotolerance was evaluated through analysis of chromosomal deletions in a ditelosomic series of the hexaploid wheat (Triticum aestivum L.) cultivar `Chinese Spring'. This study describes the identification of a line within this ditelosomic series that exhibited a reduced level of acquired thermotolerance. Changes in the temperature sensitivity of chlorophyll accumulation were used as an indicator of acquired thermotolerance. The temperature providing maximum chlorophyll accumulation was 30 °C in leaves under continuous light. A 30-min challenge temperature of 48 °C prior to the light exposure was shown to inhibit subsequent chlorophyll accumulation. Preincubation at 40 °C for 4 h before the 30-min 48 °C challenge triggered the acquired thermotolerance system of the plant resulting in chlorophyll accumulation upon exposure to light. Evaluation of the ditelosomic series revealed reductions in acquired thermotolerance levels in the DT7DS line relative to controls. Two-dimensional SDS polyacrylamide gel analysis was used to identify reduction in the level of two low molecular mass heat shock proteins in DT7DS.     

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5–6% dimethyl sulfoxide (Me₂SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at −80 °C. Alternatively, platelets were frozen with 5–6% Me₂SO at −30 °C or with 3% Me₂SO at −80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me₂SO (p = 0.0461) but not at −30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me₂SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at −30 °C or with 3% Me₂SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at −30 °C or with 3% Me₂SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.     

Effect of flowering stage and storage conditions on pollen quality of six male date palm genotypes

Availability of efficient male genotypes is critical for successful artificial pollination and regular bearing of female date palms. The effect of flowering stage and storage conditions on pollen quality of six male date palm genotypes encoded ‘ABD1′, ‘P4′, ‘P3′, ‘P8′, ‘P7′ and ‘P13′were evaluated. Pollen collected from spathes developed at the middle of flowering stage exhibited the best viability (90%) and germinability (85%) compared to other stages. Pollen viability was greater than 90%, except for ‘P8′ that exhibited 80%, while, germinability greatly varied among the genotypes. Pollen quality decreased during 4 months of storage upon genotype and temperature, with a minimum reduction at ?30 °C followed by 4 °C. Heat shock exposure (33 ± 2 °C) following storage revealed that pollen stored at ?30 °C or 4 °C should be used for pollination on the same day of take out to avoid dramatic quality loss. The ‘ABD1′, an early flowering genotype, proved highest pollen quality both at fresh stage and after storage. While, the ‘P3′, a late flowering genotype, retained its pollen quality during storage. However, the ‘P13′ genotype exhibited excellent pollen quality when fresh, but greatly loses germinability during storage.     

20. Electro-Extraction of Viruses from Infected Plant Tissue (Applied to Turnip Yellow Mosaic,Tobacco Mosaic,and Maize Streak Viruses)

ABSTRACT

An apparatus is described which was used for rapid extraction of viruses from frozen and thawed infected plant tissues. The novel principle is the establishment of a potential gradient of 15 to 20 volts/cm at approximately 90° across the leaves are surrounded by buffer of low molarity and of the appropriate hydrogen ion concentration. To keep the leaves in the correct orientation they were placed as single layers between coarse rigid plastic gauze. The method, termed electro-extraction, was used as the initial step in the purification of turnip yellow mosaic, tobacco mosaic and maize streak viruses. An electron micrograph of the purified maize streak virus is presented.     

Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

We have detected a plant β-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric β-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45°C. It is relatively resistant to proteases and insensitive to-SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by theEscherichia coli β-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay theE. coli β-glucuronidase in transgenic plants without interference from the endogenous plant activity.     

Effect of storage conditions on the LDL effectiveness in ovine sperm cryopreservation

The present study evaluated the effect of storage conditions on the LDL efficacy for cryopreserving ovine sperm. In this way, we compared egg yolk extender with three different forms for LDL storing, LDL diluted in Tris-glucose extender and stored in frozen (i) and freeze-dried (ii) states and LDL stored pure and added into the extender prior to use (iii). We also tested the effect of two storage temperatures (−20 and −80 °C) and three storage times (30, 60, 120 d). Frozen and freeze-dried extenders containing LDL, as well as LDL stored pure, improved post-thaw sperm quality. Storage temperatures did not influence negatively the cryoprotectiveness of LDL extenders. Furthermore, lyophilised LDL extenders stored at −20 °C were more effective in preserving sperm longevity than the other extenders stored at −20 °C. Finally, LDL extenders stored for 30 and 120 d were more efficient than 60 d in preserving ram sperm freezability.     

Assessment of the influence of different sample processing and cold storage duration on plant free proline content analyses

Introduction – A method which is widely accepted for the analysis of free proline content in plant tissues is based on the use of 3% sulfosalicylic acid as an extractant, followed by spectrophotometric quantification of a proline–ninhydrin complex in toluene. However, sample preparation and storage may influence the proline actually measured. This may give misleading or difficult to compare data. Objective and Methodology – To evaluate free proline levels fresh and frozen strawberry (Fragaria × ananassa Duch.) leaves and soybean [Glycine max (L.) Merr.] hypocotyl tissues were used. These were ground with or without liquid nitrogen and proline extracted with sulfosalicylic acid. A particular focus was the influence of plant sample cold storage duration (1, 4 and 12 weeks at ?20°C) on tissue proline levels measured. Results – The free proline content analyses, carried out in leaves of Fragaria × ananassa Duch. as well as in hypocotyls of Glycine max (L.) Merr., showed a significant influence of the sample preparation method and cold storage period. Long‐term storage of up to 12 weeks at ?20°C led to a significant increase in the measured proline in all samples analysed. Conclusion – The observed changes in proline content in plant tissue samples stored at ?20°C indicate the likelihood of the over‐estimation of the proline content if the proline analyses are delayed. Plant sample processing and cold storage duration seem to have an important influence on results of proline analyses. Therefore it is recommended that samples should be ground fresh and analysed immediately. Copyright © 2010 John Wiley & Sons, Ltd.     

A comparative study of the effects of freezing and frozen storage on intact and demembranated bull spermatozoa

The damage caused to bull sperm by freezing and thawing them without cryoprotectants was assessed in both intact and membrane-extracted cells. Preparations of membrane-extracted cells were produced by treating the sperm with 0.1% Triton X-100 and motility was restored with exogenously applied ATP and Mg²⁺. Motile demembranated sperm showed no detectable reduction in motility after freezing and thawing. In contrast, when intact cells where subjected to freezing and thawing they lost all motility. These damaged cells were also restored to motility when exogenous ATP and Mg²⁺ were added to the sperm mixture. Apparently freezing and thawing sperm cells causes damage to the plasma membrane which permits ATP and Mg²⁺ to freely enter or leave the cells, but does not damage the components of the sperm cell which generate motility.The effects of storage temperature on frozen demembranated sperm were also explored. Sperm held at ?20 °C showed marked structural changes and progressively decreased motility after prolonged storage. When sperm were frozen at ?20 °C the mitochondrial structures were completely lost after 48 to 72 hr and ATP caused the disintegration of the flagellum rather than initiating motility. Sperm which were frozen at ?76 °C retained motility after short periods of storage, but showed a significant decline in motility when thawed after 8 days. Demembranated sperm which were kept frozen at ?196 °C showed no significant loss of motility when thawed after 1 year of storage.     

Effects of lyophilization on the stability of bacteriophages against different serogroups of Shiga toxin-producing Escherichia coli

Lyophilization is commonly used to effectively preserve the stability of bacteriophages (phages) in long-term storage. However, information regarding the lyophilization of phages specific to Shiga toxin-producing Escherichia coli (STEC) strains is scarce. The objective of this study was to determine the effects of lyophilization with different cryoprotectants (sucrose and trehalose) and concentrations (0.1 M and 0.5 M) on the stability of seven lytic phages specific to STEC O157 and top 6 non-O157 strains during 6-month storage at −80 °C. The titers of lyophilized phages specific to STEC O26 (S1 O26) and STEC O121 (Pr121lvw) did not exhibit significant reduction after 6-month storage regardless of the use of cryoprotectants. Phages lytic against STEC O103 (Ro103C3lw) and STEC O145 (Ro145clw) with 0.1 M sucrose retained similar titers after lyophilization and frozen storage for 6 months (P > 0.05). Despite subtle differences, these results indicated that most of the selected phages had similar titer retention with the same cryoprotectants. Additionally, lytic activities of the phages against their primary hosts were not affected after lyophilization and 6-month frozen storage. Moreover, no detectable damage was observed on the lyophilized phage structures. These findings provide valuable insight into the use of lyophilization to preserve phages lytic against STEC strains.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 °C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

Studies on Destabilization of Caseinate Complex during Frozen Storage of Milk

Unpasteurized skim milk was storaged in a frozen state at ?7°C or ?20°C for up to several months. There was no increase of non casein and non protein nitrogens, but a slight increase of free tyrosine and a slight decrease of alkaline phosphatase activity were detected when storage period was prolonged. Destabilization occurred solely in caseinate complex, but non micellar casein appeared to be stable.

The contents of calcium and inorganic phosphate in the caseinate complex separated by ultracentrifugation were increased appreciably after frozen storage. The viscosity characteristics of frozen storaged skim milk was also investigated.

Caseinate complex was ultracentrifugally separated from skim milk before and after frozen storage, and then lyophilized. Skim milk itself was also lyophilized before and after frozen storage. Dispersibility was examined on the reconstituted suspension of the lyophilized samples.

The lyophilized sample from frozen storaged milk was much less dispersible than the lyophilized control sample prepared before frozen storage. However, when lyophilized samples were once resolved with reagents such as urea and potassium oxalate and then dialyzed against fresh milk, stable micelle resulted in both samples prepared before and after frozen storage.

Some reduction of dispersibility occurred during lyophilization and subsequent storage in a dried state in the caseinate complex prepared before frozen storage. This reduction was small when skim milk was lyophilized and stored.     

Overexpression of a multifunctional β-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter

The β-glucosidase, which hydrolyzes the β(1–4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of β-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including β-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2–4. We overexpressed the β-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with β-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of β-glucosidase in plants could further increase. The plant-expressed β-glucosidase had optimal activity at 80 °C and pH 5–6. In addition, the plant-expressed β-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with β-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial β-glucosidase gene as a reporter in plants based on alternative β-galactosidase activity.

     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells

Background  

The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots.     

Assessment of frozen larvae of Callosobruchus maculatus as hosts for rearing Pteromalus cerealellae (Ashmead) (Hymenoptera: Pteromalidae)

The suitability of frozen host larvae for rearing Pteromalus cerealellae (Ashmead) (Hymenoptera: Pteromalidae), an ectoparasitoid of Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) and other stored-product insects was investigated. The reproductive potential (number and sex ratio of progeny) of female P. cerealellae was compared on live (fresh) C. maculatus larvae (concealed within cowpea seeds) versus frozen larvae (obtained by freezing infested cowpea seeds at ?20 °C for 48 h) which were subsequently thawed and held at ambient conditions (～25 ± 1 °C, 50 ± 5% RH) for 4, 24, 48, 72, 96, and 120 h before exposure to female parasitoids. No significant differences were recorded in the numbers and sex ratios of the progeny produced by female P. cerealellae on live larvae compared to frozen host larvae that were thawed and held at ambient conditions for up to 96 h, suggesting that live and frozen larvae of C. maculatus are equally suitable for rearing P. cerealellae. However, the data showed that progeny production on frozen hosts gradually declined with thawing duration and was significantly reduced at the thawing duration of 120 h. When live and frozen host larvae were simultaneously presented together to female P. cerealellae at different exposure periods, relatively greater progeny production was recorded on live hosts than on frozen hosts at 12, 24, and 48 h of exposure. This may suggest preference of female P. cerealellae for live versus frozen host larvae. These results are discussed in relation to the life history strategy and host location behavior of P. cerealellae, and may have practical implications in the development of efficient mass rearing systems for the parasitoid.     

Eukaryotic protein production in designed storage organelles

Background  

Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.     

Fate of Escherichia coli O157:H7 in ground beef following high‐pressure processing and freezing

Aims: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. Methods and Results: Fresh ground beef was inoculated with a five‐strain cocktail of E. coli O157:H7 vacuum‐packaged, pressure‐treated at 400 MPa for 10 min at ?5 or 20°C and stored at ?20 or 4°C for 5–30 days. A 3‐log CFU g^?1 reduction of E. coli O157:H7 in the initial inoculum of 1 × 10⁶ CFU g^?1 was observed immediately after pressure treatment at 20°C. During frozen storage, levels of E. coli O157:H7 declined to <1 × 10² CFU g^?1 after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55–65°C. Conclusions: High‐pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g^?1 and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. Significance and Impact of the Study: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.