| 1. |
- Alsterfjord, Magnus, et al.
(författare)
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Plasma membrane H+-ATPase and 14-3-3 Isoforms of Arabidopsis leaves: Evidence for isoform specificity in the 14-3-3/H+-ATPase interaction
- 2004
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Ingår i: Plant and Cell Physiology. - : Oxford University Press (OUP). - 1471-9053 .- 0032-0781. ; 45:9, s. 1202-1210
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane H+-ATPase is activated by binding of 14-3-3 protein to the phosphorylated C terminus. Considering the large number of 14-3-3 and H+-ATPase isoforms in Arabidopsis (13 and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H+-ATPase isoforms. We now show that the H'-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 istiforms tested bind to the H+-ATPase in vitro. Using specific antibodies for nine of the 14-3-3 isoforms, we show that GF14epsilon, mu, lambda, omega, chi, phi, nu, and upsilon are present in leaves, but that isolated plasma membranes lack GF14chi, phi and upsilon. Northern blots using isoform-specific probes for all 14-3-3 and H+-ATPase isoforms showed that transcripts were present for most of the isoforms. Based on mRNA levels, GF14epsilon, mu, lambda and chi are highly expressed 14-3-3 isoforms, and AHA1, 3, and 11 highly expressed H+-ATPase isoforms in leaves. However, mass peptide fingerprinting identified AHA1 and 2 with the highest score, and their presence could be confirmed by MS/MS. It may be calculated that under 'unstressed' conditions less than one percent of total 14-3-3 is attached to the H+-ATPase. However, during a condition requiring full activation of H+ pumping, as induced here by the presence of the fungal toxin fusicoccin, several percent of total 14-3-3 may be engaged in activation of the H+-ATPase.
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| 2. |
- Baureus Koch, Catrin, et al.
(författare)
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Interaction between weak low frequency magnetic fields and cell membranes.
- 2003
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Ingår i: Bioelectromagnetics. - : Wiley. - 0197-8462. ; 24:6, s. 395-402
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Tidskriftsartikel (refereegranskat)abstract
- The question of whether very weak low frequency magnetic fields can affect biological systems, has attracted attention by many research groups for quite some time. Still, today, the theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely low frequency (ELF) magnetic fields on the transport of Ca2+ was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested two quantum mechanical theoretical models that assume that biologically active ions can be bound to a channel protein and influence the opening state of the channel. Vesicles were exposed for 30 min at 32 °C and the calcium efflux was studied using radioactive 45Ca as a tracer. Static magnetic fields ranging from 27 to 37 T and time varying magnetic fields with frequencies between 7 and 72 Hz and amplitudes between 13 and 114 T (peak) were used. We show that suitable combinations of static and time varying magnetic fields directly interact with the Ca2+ channel protein in the cell membrane, and we could quantitatively confirm the model proposed by Blanchard. Bioelectromagnetics 24:395-402, 2003. © 2003 Wiley-Liss, Inc.
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| 3. |
- Christensen, Anna, et al.
(författare)
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Functional characterization of Arabidopsis calreticulin1a : a key alleviator of endoplasmic reticulum stress
- 2008
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Ingår i: Plant and Cell Physiology. - : Oxford University Press. - 0032-0781 .- 1471-9053. ; 49:6, s. 912-924
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Tidskriftsartikel (refereegranskat)abstract
- The chaperone calreticulin plays important roles in a variety of processes in the endoplasmic reticulum (ER) of animal cells, such as Ca2+ signaling and protein folding. Although the functions of calreticulin are well characterized in animals, only indirect evidence is available for plants. To increase our understanding of plant calreticulins we introduced one of the Arabidopsis isoforms, AtCRT1a, into calreticulin-deficient (crt–/–) mouse embryonic fibroblasts. As a result of calreticulin deficiency, the mouse crt–/– fibroblasts have decreased levels of Ca2+ in the ER and impaired protein folding abilities. Expression of the AtCRT1a in mouse crt–/– fibroblasts rescued these phenotypes, i.e. AtCRT1a restored the Ca2+-holding capacity and chaperone functions in the ER of the mouse crt–/– fibroblasts, demonstrating that the animal sorting machinery was also functional for a plant protein, and that basic calreticulin functions are conserved across the Kingdoms. Expression analyses using a β-glucuronidase (GUS)–AtCRT1a promoter construct revealed high expression of CRT1a in root tips, floral tissues and in association with vascular bundles. To assess the impact of AtCRT1a in planta, we generated Atcrt1a mutant plants. The Atcrt1a mutants exhibited increased sensitivity to the drug tunicamycin, an inducer of the unfolded protein response. We therefore conclude that AtCRT1a is an alleviator of the tunicamycin-induced unfolded protein response, and propose that the use of the mouse crt–/– fibroblasts as a calreticulin expression system may prove useful to assess functionalities of calreticulins from different species.
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| 4. |
- Christensen, Anna, et al.
(författare)
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Higher plant calreticulins have acquired specialized functions in arabidopsis
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:6, s. e11342-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Calreticulin (CRT) is a ubiquitous ER protein involved in multiple cellular processes in animals, such as protein folding and calcium homeostasis. Like in animals, plants have evolved divergent CRTs, but their physiological functions are less understood. Arabidopsis contains three CRT proteins, where the two CRTs AtCRT1a and CRT1b represent one subgroup, and AtCRT3 a divergent member. Methodology/Principal Findings: Through expression of single Arabidopsis family members in CRT-deficient mouse fibroblasts we show that both subgroups have retained basic CRT functions, including ER Ca2+-holding potential and putative chaperone capabilities. However, other more general cellular defects due to the absence of CRT in the fibroblasts, such as cell adhesion deficiencies, were not fully restored. Furthermore, in planta expression, protein localization and mutant analyses revealed that the three Arabidopsis CRTs have acquired specialized functions. The AtCRT1a and CRT1b family members appear to be components of a general ER chaperone network. In contrast, and as recently shown, AtCRT3 is associated with immune responses, and is essential for responsiveness to the bacterial Pathogen-Associated Molecular Pattern (PAMP) elf18, derived from elongation factor (EF)-Tu. Whereas constitutively expressed AtCRT1a fully complemented Atcrt1b mutants, AtCRT3 did not. Conclusions/Significance: We conclude that the physiological functions of the two CRT subgroups in Arabidopsis have diverged, resulting in a role for AtCRT3 in PAMP associated responses, and possibly more general chaperone functions for AtCRT1a and CRT1b.
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| 5. |
- Christensen, Anna, et al.
(författare)
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Overexpression of the Ca2+-binding protein calreticulin in the endoplasmic reticulum improves growth of tobacco cell suspensions (Nicotiana tabacum) in high-Ca2+ medium
- 2005
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 123:1, s. 92-99
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Tidskriftsartikel (refereegranskat)abstract
- Calreticulin (CRT) is a eukaryotic, highly conserved, Ca2+-binding protein predominantly located in the endoplasmic reticulum (ER) lumen. In addition to being involved in the regulation of cellular Ca2+, calreticulin is a key quality control element during protein folding in the ER lumen. Tobacco (Nicotiana tabacum L.) suspension cells overexpressing a maize CRT (CRT1a) were used here to examine the properties of CRT in growing plant cells with respect to stress exposure. The endogenous CRT gene was induced rapidly after subculturing of the cells to new medium. In accordance, the CRT protein levels increased, peaking at days 3-4. At day 5, when the CRT transcript levels had levelled off, a further increase in endogenous CRT expression was obtained when the cells were treated with excess Ca2+ or the N-linked glycosylation inhibitor tunicamycin. Whereas the response to Ca2+ occurred within 30 min, the induction by tunicamycin took several hours to be established. Transforming tobacco cells with maize CRT1a, under a constitutive mannopine synthase promoter, resulted in a stable level of expressed CRT1a during the growth cycle compared with endogenous CRT. The CRTs showed differences in attached glycans, but both contained the high mannose-rich-type glycans characteristic of ER proteins. In agreement with an ER location, both tobacco CRT and the transgene product CRT1a codistributed with the ER marker NADH cytochrome c reductase after density gradient centrifugation of microsomal fractions from tobacco cells. Increased production of CRT, as was obtained in the transgenic tobacco cell lines, made cells more tolerant than wild-type cells to high Ca2+ during growth. These data suggest that overexpression of CRT1a in plant cells results in a more efficient calcium buffering capacity in the ER.
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| 6. |
- Gomez, Federico, et al.
(författare)
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Biochemical aspects of carrot processing
- 2003
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Ingår i: International Symposium on Future Technologies for Food Production and Future Food Scientists, Proceedings. - 0280-9737. ; :162, s. 87-87
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Konferensbidrag (refereegranskat)
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| 7. |
- Gomez, Federico, et al.
(författare)
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Changes in the carrot (Daucus carota L. cv. Nerac) cell wall during storage
- 2004
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Ingår i: Food Research International. - : Elsevier BV. - 0963-9969. ; 37:3, s. 225-232
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to examine biochemical changes in cell wall carbohydrates and extensin proteins during long-term storage of carrots (Daucus carota L. cv. Nerac). During the storage period of 6 months, cell wall fractions were isolated from the carrot at various times for carbohydrate and protein analysis. Signs of extensin cross-linking and its concomitant insolubilisation in the cell wall were found after 7 and 12 weeks of storage. During the same period the concentration of galactose and arabinose decreased, while other carbohydrate components as well as the degree of methylesterification remained virtually unchanged. After the 12th week of storage no changes in the extensin or carbohydrates were detected. Oxidative cross-linking between extensin molecules in the cell wall has been implicated in cell wall strengthening and may be part of the mechanism behind the storage-induced firmness of carrots. (C) 2003 Elsevier Ltd. All rights reserved.
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| 8. |
- Gomez, Federico, et al.
(författare)
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Cold acclimation of carrots during storage mechanical properties and antifreezing protein.
- 2003
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Ingår i: Acta horticulturae : technical communications of ISHS. - 0567-7572. ; 599, s. 699-703
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Konferensbidrag (refereegranskat)abstract
- Changes in the composition of carrot cell wall proteins were investigated, associating metabolic changes during long-term storage with changes in mechanical properties. Harvested carrots accumulate an antifreezing protein in their cell walls reaching a maximum level after 12 weeks of storage at 0°C, followed by a gradual decrease. During the same period of time, there is a decrease in the slicing force during the first 7 weeks of storage followed by an increase until the 12th week. The appearance and accumulation of the antifreezing protein suggest that structural changes leading to changes in mechanical properties during the first 12 weeks of storage might be associated with a cold acclimation process.
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| 9. |
- Gomez, Federico, et al.
(författare)
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Influence of cold acclimation on the mechanical strength of carrot (Daucus carota L.) tissue
- 2004
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Ingår i: European Journal of Horticultural Science. - 1611-4434. ; 69:6, s. 229-234
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the influence of cold acclimation on the mechanical strength of carrot (Daucus carota L.) taproots. Changes in the mechanical strength were monitored when cold acclimation was induced in carrot plants cultivated in a growth chamber under strict climate control and in taproots harvested from field cultivation, where the plants had been exposed to the natural variations in climate. The appearance and accumulation of an antifreeze protein in the cell wall isolated from cold-stored taproots showed that a cold acclimation process is in progress in the harvested taproot derived from carrot plants grown in the field. The force needed to slice the taproots significantly increased during the first 12 weeks of storage, where the higher concentration of the antifreeze protein indicated the highest development of cold acclimation during that period of time. The increase in tissue rigidity during cold acclimation was also shown by the increase of the Young's modulus in taproot tissue from carrot plants acclimated 11 weeks under controlled temperature conditions. After 24 weeks of storage there was a significant increase in slicing force that was accompanied by signs of cell membrane deterioration, as measured by relative electrolyte leakage. Thus, the later increase in tissue strength might be related with a senescence process.
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| 10. |
- Gomez, Federico, et al.
(författare)
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On the induction of cold acclimation in carrots (Daucus carota L.) and its influence on storage performance
- 2005
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Ingår i: Food Research International. - : Elsevier BV. - 0963-9969. ; 38:1, s. 29-36
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Tidskriftsartikel (refereegranskat)abstract
- We investigated the role of cold acclimation in carrot plants with respect to its influence on the storage performance of the harvested taproots. The induction of cold acclimation was followed in plants cultivated in a growth chamber under strict climate control and in taproots harvested from two separate field cultivations where the plants had been exposed to the natural variations in climate. Under controlled growth conditions, levels of antifreeze protein (AFP) mRNA were used as a marker for cold acclimation in carrot taproot tissue. Expression of this gene was induced by cold in discs excised from harvested taproots and this induction was clearly affected by the growth temperature of the plants from which the taproots were taken. These in vitro data were consistent with those from field-grown plants. In the cell wall of taproots harvested in year 2000, where the intact plants had frequently been exposed to temperatures below 6degreesC, a 36 kDa AFP accumulated to higher levels during storage than in the taproots harvested from plants grown in year 2001, where cultivation temperatures had rarely dropped below 6degreesC. The taproots from 2001 exhibited poor storage performance as shown by an earlier increase in relative electrolyte leakage and decrease in dry matter compared to taproots harvested in 2000. The capacity of the AFP to accumulate during storage was consistent with a high storage performance.
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