- Email: [email protected]
Accumulation of Dehydrin-like-proteins in the Mitochondria of Cold-treated Plants
• JOURNAL OF • PLANT PHYSIOLOGY
J Plant Physiol. Vol. 156. pp. 797-800 (2000) http://www.urbanfischer.de/journals/jpp
© 2000 URBAN & FISCHER Verlag
I
Short Communication
I
Accumulation of Dehydrin-like-proteins in the Mitochondria of Cold-treated Plants Genadii B. Borovskii 1 *, Irina V. Stupnikova 1, Anna I. Antipina 1, Craig A. Downs 2 and Victor K. Voinikov l 1
Institute of Plant Physiology and Biochemistry, Russian Academy of Sciences, Irkutsk-33, P.O. Box 1243, Irkutsk, Russia 664033
2
U.S. National Oceanic and Atmospheric Administration, Marine Biotechnology Division, 219 Ft. Johnson Rd., Charleston, SC 29412, USA
Received May 10, 1999· Accepted December 6, 1999
Summary
A number of proteins have been found to accumulate in plants during cold tolerance. One class of cold proteins that is associated with cold tolerance acclimation is the glycine-rich, hydrophilic, D-II LEA (late embryogenesis abundant) proteins known as dehydrins. Several members of the dehydrin-class of proteins are known to associate with the nucleus, in the cytosol, and with the plasma membrane. Dehydrins that localise with the mitochondria have not been found. We provide evidence that two dehydrin-like proteins (dips) accumulate in the mitochondria only after low temperature treatment. We also provide evidence for a positive correlation between the relative accumulation of these proteins in the mitochondria in response to cold stress and relative cold tolerance of several species of plants.
Key words: cold stress, cold tolerance, dehydrins, mitochondria, Secale cereale (L.), Triticum aestivum (L.), Zea mays (L.). Abbreviations: dip = dehydrin-like-protein; LEA = late embryogenesis abundant; SDS-PAGE dodecyl sulfate polyacrylamide gel electrophoresis. Introduction
Plants respond to cold and freezing temperatures through physiological, morphological, and metabolic processes. Nearfreezing and freezing temperatures can induce cellular dehydration (drought) by which water from within the cell migrates to the outside of the cell (Guy, 1990). A number of studies have demonstrated a role for osmoprotectants and changes of membrane composition in cold-stress tolerance (Galinski, 1993; Bohnert et ai., 1995; Hare et al., 1998), though the specific function for many of the cold- and drought-stress induced proteins remain unknown.
* Correspondence. E-mail: [email protected]
= sodium
One class of proteins that is induced by both cold and drought stress is the dehydrin family of proteins. Dehydrins, also referred to as Group II late embryogenesis abundant (LEA) proteins, are glycine-rich, hydrophilic, and thermostable. Dehydrins are evolutionarily conserved among photosynthetic organisms including angiosperms, gymnosperms, ferns, mosses, liverworts, algae and cyanobacteria, as well as in some non-photosynthetic organisms such as yeast (Close et al., 1993 a; Close, 1996; Campbell and Close, 1997; Mtwisha et al., 1998). A number of studies have established positive correlation between drought and cold stress tolerance and dehydrin accumulation in a number of different plant species and different genotypes of a species (Labhilili et al., 1995; Moons et al., 1995; Close, 1996; Pelah et al., 1997). Dehyd0176-1617100/156/797 $ 12.0010
798
Genadii B. Borovskii, Irina V. Stupnikova, Anna I. Antipina, Craig A. Downs and Victor K. Voinikov
rins have been hypothesised to function by stabilising largescale hydrophobic interactions such as membranes structures or hydrophobic patches of proteins (Dure, 1993; Close, 1996, 1997). Highly-conserved polar regions of dehydrins have been suggested to hydrogen bond with polar regions of macromolecules, acting essentially as a surfactant, to prevent coagulation during conditions of cellular dehydration or low temperatures. Recently, an acidic dehydrin has been determined to localise to close proximity to the plasma membrane during cold acclimation, supporting the role of cryoprotection of the plasma membrane during dehydration and freezing stress (Danyluk et al., 1998). To date, immuno-localisation and subcellular fractionation results have established that members of the dehydrin family localise in proximity to the nucleus, cytoplasm, and plasma membrane, but it is unknown whether other organelle or cell structures have dehydrins associated with them in response to cold or dehydration stress. Mitochondria are highly susceptible to changes in intracellular concentration of solutes induced by cellular dehydration of cold temperatures (DeSantis et al., 1999). To date, plant mitochondrial responses and adaptations to cellular dehydration and cold stress have been relatively unexplored. The objective of this study was to determine whether dehydrin-like proteins (dips) localise in proximity to plant mitochondria in response to cold stress.
Materials and Methods Three-day-old etiolated seedlings of Triticum aestivum (L.) (winter wheat) and Secale cereale (L.) (winter rye) were grown at 20·C. Zea mays (L.) (maize) was grown at 27·C. Unstressed plants were maintained under growth conditions for 1 day. Cold treatment was performed by subjecting seedlings to 4·C for wheat and rye and to 10·C for maize. Control and cold-treated seedlings were compared at similar growth stages. Crude mitochondria were isolated from control and cold-treated seedling by a method described elsewhere (Voinikov et al., 1991) with modifications (Voinikov et al., 1998). Further purification was performed on a discontinuous Percoll gradient (De Virville et al., 1994). Puriry and integrity of mitochondria were determined by measurement of cytochrome c oxidase activity (EC 1.9.3.1) (De Virville et al., 1994). Mitochondria were sonicated for disruption in 100 mmol/L TrisHCl (pH 7.6) containing 1 mmollL phenylmethylsulfonyl fluoride. Undisrupted mitochondria were precipitated by centrifugation at 17,000 gn for 5 min and discarded. The supernatant was divided into two fractions: (1) fraction 1 was boiled for 20 min to isolate thermostable proteins; and (2) fraction 2 was subjected immediately to protein precipitation. Proteins from fractions 1 and 2 were precipitated with 10 % trichloroacetic acid, washed with cold acetone, and dissolved in sample loading buffer for SDS-PAGE. Proteins were subjected to SDS-PAGE using a mini-Protean PAGE cell (Bio-Rad, USA) according to the manufacturer's instruction. Western blotting and immunodetection were carried out as described by Timmons and Dunbar (1990) using anti-dehydrin primary antibody (1: 1,000 dilution), kindly provided by Dr. T.]. Close (Close et aI., 1993 b). Western blot images were analysed by Sigma Scan Pro Software (Sigma Chemicals, USA). Protein concentrations of samples were determined according to Esen (1978).
Results and Discussion
Dehydrins accumulate in response to cold stress in the nucleus or cytoplasm, but it is unknown if they can accrue in mitochondria or chloroplasts. The accumulation of another family of proteins associated with freezing tolerance was demonstrated for several cereal species (Close, 1996; Sarhan et ai., 1997). In wheat and other cereals, the wcs120 gene family is coordinately regulated by low temperatures and accumulates to high levels in freezing tolerant members of Poaceae (Houde et al., 1995; Limin et ai., 1995). This family shares homology with the dehydrin family of proteins (Dure, 1993). Immunogold labelling indicated that the WCS 120 protein family is located in both the nucleus and cytoplasm of cold acclimated tissues. Mitochondria were found to contain few gold particles. However, cellular fractionation did not reveal the presence of any member of this protein family in the mitochondrial fraction (Houde et al., 1995). To determine whether dehydrin-like proteins (dip) accumulated in the mitochondria in response to cold stress, mitochondria from control and cold-stressed plants from wheat, rye, and maize plants were isolated and subjected to SDS-PAGE. Protein profiles as detected by Coomassie Blue staining indicated that there were no differences in both total (Fig. 1 A) and thermostable protein fractions (Fig. 1 B) between control and cold-acclimated plants in any of the speCles. Immunoblot analysis using the dehydrin antibody of total mitochondrial protein indicated the presence of 54 kDa and 60 kDa dehydrin-like proteins (dlp54 and dlp60). The analysis with primary antibodies blocked by dehydrin peptide confirmed that corresponding proteins are really dehydrins (data not shown). Both proteins were expressed in control and cold-stressed plants for all species examined (Fig. 1 C). Immuno-analysis of the mitochondrial thermostable-protein fraction of control and cold-stressed samples indicated that both proteins were thermostable (Fig. 1 D). Relative accumulation of dlp60 after cold acclimation was higher in rye than in wheat, and dlp60 accumulation in maize was barely detectable and below the linear range for densitometric analysis (Fig. 2 A). Densitometric analysis of the mitochondrial thermostable proteins indicated a small, but distinct accumulation of the relative abundance of dlp54 and dlp60 in rye, wheat, and maize (Fig. 2 B). The differences between plant species in the relative abundance of these two proteins were significantly less for total mitochondrial thermostable protein content than in total mitochondrial protein content. This difference between total mitochondrial protein content (Fig. 2 A) and mitochondrial thermostable protein content (Fig. 2 B) suggests that the accumulation of dlp54 and dlp60 after cold treatment occurred mostly due to the accumulation in mitochondria of all thermostable proteins rather than the dips alone; but nonetheless, this accumulation was determined in the thermostable fraction also. Mitochondria are sensitive to solute concentration in the medium. Therefore, it is very likely that water content in these organelles would be low during ice crystal formation. The concentration of proteins in the matrix of mitochondria is rather high even under normal conditions, over 50 % by dry-weight (Douce, 1985). Cellular dehydration is thought to arise con-
799
Dehydrin-like-proteins of the Plant Mitochondria 234
A
5
6
kD
2
C
-66
3
4
5
~~~~'M' J~_F"~t•.
•
kD
!"""'--"" -60 -54
-.
-45
6 _..,..,.;.
-24 -18
I
B
Fig. 1: SDS-PAGE protein profiles and Western blots of isolated plant mitochondria. The total (A & C) or thermostable (B & D) mitochondrial proteins of winter wheat (In. 1 & 2), winter rye (In. 3 & 4), and maize (In. 5 & 6) of control (in. 1, 3 & 5) and cold-treated seedlings (in. 2, 4 & 6) were visualised with Coomassie blue (A & B), or electro blotted onto nitrocellulose membranes, and ptobed with antibody against dehydrin (C & D). 151lg of protein was loaded per lane. Molecular masses of protein standards (A & B) or molecular masses of dips (C & D) are indicated on the right.
2
3
1000 c:
·f 2
.5
2500 2000
OJ
l
a
i; co
"
I
500
1500
.dlp54 (control) OdlpS4(acclim.)
Ea dlp60 (control) mdlp60 (acclim.)
1000 500
a wheat
rye
2
D
3
4
5
6
-18
1500
::J
6
-24
A
~
5
"'iiil~~nn~
2500 2000
4
maize
Plant species
Fig. 2: Relative abundance of the dehydrin-like proteins in the total (A) and thermostable (B) protein fractions of mitochondria. Protein separation and Western blotting were the same as in Fig. 1. Membranes were scanned and total intensities of the bands corresponding to dlp54 and dlp60 were measured. Means and standard errors of the means are shown.
comitantly with freezing temperatures. Dehydrins accumulate not just in response to cold-stress temperatures, but also in reponse to dehydration (Close, 1993 a). Our results concerning the accumulation of dehydrin-like proteins in mitochondria with the cold treatment indicated that mitochondrial dIps may
be involved in freezing- and dehydrative-tolerance mechamsms. The function of mitochondrial dips remains unknown. Recently, immunoelectron microscope analyses revealed that protein WCOR41O, an acidic dehydrin, accumulates in the vicinity of the plasma membrane. WCOR41O is a peripheral protein and not an integral protein. The authors proposed that this dehydrin may function by preventing the destabilisation of the plasma membrane that occurs during dehydrative conditions (Danyluk et al., 1998). The dIps found in the mitochondria could possibly function by preventing destabilisation of membranes and/or stabilising in a chaperonelike function the macromolecules in the matrix or in the intermembrane space. Precise localisation of these proteins in the organelles would give more information about the function of mitochondrial dIps. Besides preventing or repairing damage induced by cold stress and dehydration, mitochondrial dIps may also play a role in regulating mitochondrial function during cold or osmotic stress. It has been suggested that RAB 17, a dehydrin related protein, mediates the transport of specific nuclear targeted proteins during stress (Goday et al., 1994). Mitochondrial dIps may have an analogous role by regulating protein transport or mitochondrial respiration. Finally, it is worth noting from our data that the mitochondria of various monocot species accumulated dIps of similar molecular masses, suggesting a conserved response to dehydrative/freezing stress. Acknowledgements
This research was funded by a grant from the Russian Foundation of Basic Research (project 99-04-48121) and a grant from the
800
Genadii B. Borovskii, Irina V. Stupnikova, Anna I. Antipina, Craig A. Downs and Victor K. Voinikov
Young Scientist Project Program of the Siberian Division of the Russian Academy of Sciences (G. B.). We sincerely thank Dr. T. J. Close for his generous gift of the dehydrin antibody.
References Bohnert, H. J., D. E. Nelson and R. G. Jensen: Plant Cell 7, 10991111 (1995). Campbell, S. A., and T. J. Close: New Phytol. 137, 61-74 (1997). Close, T. J.: Physiol. Plant. 97, 795-803 (1996). - Physiol. Plant. 100,291-296 (1997). Close, T. J., and P. J. Lammers: Plant Physiol. 101, 773-779 (1993a). Close, T. J., R. D. Fenton and F. Moonan: Plant Mol. BioI. 23, 279-286 (1993 b). Danyluk, J., A. Perron, M. Houde, A. Limin, B. Fowler, N. Benhamou and F. Sarhan: Plant Cell 10, 623-638 (1998). DeSantis, A., P. Landi and G. Genchi: Plant Physiol. 119, 743-754 (1999). De Virville, J., I. Aaron, M. F. Alin and F. Moreau: Plant Physiol. Biochem.32, 159-166 (1994). Douce, R.: Mitochondria in higher plants: structure, function, and biogenesis. Academic Press, London (1985). Dure, L. III.: In: Close, T. J. and Bray, E. A. (eds.), Current Topics in Plant Physiology: Plant Responses to Cellular Dehydration during Environmental Stress, pp. 91-103. ASPP Rockville, Rockville (1993).
Esen, A. A.: Anal. Biochem. 89,264-273 (1978). Galinski, E. A.: Experientia 49, 487-495 (1993). Goday, A., A. B. Jensen, F. A. Culianez-Macia, M. M. Alba, M. Figueras, J. Serratosa, M. Torrent and M. Pages: Plant Cell. 6, 351360 (1994). Guy, C. L.: Annu. Rev. Plant Physiol. Plant Mol. BioI. 41, 187-223 (1990). Hare, P. D., W. A. Cress and J. Van Staden: Plant Cell Environ. 21, 535-553 (1998). Houde, M., C. Daniel, M. LaChapelle, F. Allard, S. Laliberte and F. Sarhan: PlantJ. 8, 583-593 (1995). Labhilili, M., P. Joudrier and M.-F. Gautier: Plant Sci. 112, 219230 (1995). Limin, A. E., J. Danyluk, L. P. Chauvin, D. B. Fowler and F. Sarhan: Genome 38, 1023-1031 (1995). Moons, A., G. Bauw, E. Prinsen, M. Van Montagu and D. V. D. Straeten: Plant Physiol. 107, 177-186 (1995). Mtwisha, L., W. Brandt, L. McCready and G. G. Lindsey: Plant Mol. BioI. 37, 513-521 (1998). Pelah, D., W. Wang, A. Altman, o. Shoseyov and D. Bartels: Physiol. Plant. 99, 153-159 (1997). Sarhan, F., F. Ouellet and A. Vazquez-Tello: Physiol. Plant. 101, 439-445 (1997). Timmons, T. M., and B. S. Dunbar: Methods Enzymol. 182, 679688 (1990). Voinikov, V. K., N. N. Varakina and T. P. Pobezhimova: Rus. J. Plant Physiol. 38, 530-537 (1991). Voinikov, V. K., T. P. Pobezhimova, A. V. Kolesnichenko, N. N. Varakina and G. B. Borovskii: J. Therm. BioI. 23, 1-4 (1998).
J Plant Physiol. Vol. 156. pp. 797-800 (2000) http://www.urbanfischer.de/journals/jpp
© 2000 URBAN & FISCHER Verlag
I
Short Communication
I
Accumulation of Dehydrin-like-proteins in the Mitochondria of Cold-treated Plants Genadii B. Borovskii 1 *, Irina V. Stupnikova 1, Anna I. Antipina 1, Craig A. Downs 2 and Victor K. Voinikov l 1
Institute of Plant Physiology and Biochemistry, Russian Academy of Sciences, Irkutsk-33, P.O. Box 1243, Irkutsk, Russia 664033
2
U.S. National Oceanic and Atmospheric Administration, Marine Biotechnology Division, 219 Ft. Johnson Rd., Charleston, SC 29412, USA
Received May 10, 1999· Accepted December 6, 1999
Summary
A number of proteins have been found to accumulate in plants during cold tolerance. One class of cold proteins that is associated with cold tolerance acclimation is the glycine-rich, hydrophilic, D-II LEA (late embryogenesis abundant) proteins known as dehydrins. Several members of the dehydrin-class of proteins are known to associate with the nucleus, in the cytosol, and with the plasma membrane. Dehydrins that localise with the mitochondria have not been found. We provide evidence that two dehydrin-like proteins (dips) accumulate in the mitochondria only after low temperature treatment. We also provide evidence for a positive correlation between the relative accumulation of these proteins in the mitochondria in response to cold stress and relative cold tolerance of several species of plants.
Key words: cold stress, cold tolerance, dehydrins, mitochondria, Secale cereale (L.), Triticum aestivum (L.), Zea mays (L.). Abbreviations: dip = dehydrin-like-protein; LEA = late embryogenesis abundant; SDS-PAGE dodecyl sulfate polyacrylamide gel electrophoresis. Introduction
Plants respond to cold and freezing temperatures through physiological, morphological, and metabolic processes. Nearfreezing and freezing temperatures can induce cellular dehydration (drought) by which water from within the cell migrates to the outside of the cell (Guy, 1990). A number of studies have demonstrated a role for osmoprotectants and changes of membrane composition in cold-stress tolerance (Galinski, 1993; Bohnert et ai., 1995; Hare et al., 1998), though the specific function for many of the cold- and drought-stress induced proteins remain unknown.
* Correspondence. E-mail: [email protected]
= sodium
One class of proteins that is induced by both cold and drought stress is the dehydrin family of proteins. Dehydrins, also referred to as Group II late embryogenesis abundant (LEA) proteins, are glycine-rich, hydrophilic, and thermostable. Dehydrins are evolutionarily conserved among photosynthetic organisms including angiosperms, gymnosperms, ferns, mosses, liverworts, algae and cyanobacteria, as well as in some non-photosynthetic organisms such as yeast (Close et al., 1993 a; Close, 1996; Campbell and Close, 1997; Mtwisha et al., 1998). A number of studies have established positive correlation between drought and cold stress tolerance and dehydrin accumulation in a number of different plant species and different genotypes of a species (Labhilili et al., 1995; Moons et al., 1995; Close, 1996; Pelah et al., 1997). Dehyd0176-1617100/156/797 $ 12.0010
798
Genadii B. Borovskii, Irina V. Stupnikova, Anna I. Antipina, Craig A. Downs and Victor K. Voinikov
rins have been hypothesised to function by stabilising largescale hydrophobic interactions such as membranes structures or hydrophobic patches of proteins (Dure, 1993; Close, 1996, 1997). Highly-conserved polar regions of dehydrins have been suggested to hydrogen bond with polar regions of macromolecules, acting essentially as a surfactant, to prevent coagulation during conditions of cellular dehydration or low temperatures. Recently, an acidic dehydrin has been determined to localise to close proximity to the plasma membrane during cold acclimation, supporting the role of cryoprotection of the plasma membrane during dehydration and freezing stress (Danyluk et al., 1998). To date, immuno-localisation and subcellular fractionation results have established that members of the dehydrin family localise in proximity to the nucleus, cytoplasm, and plasma membrane, but it is unknown whether other organelle or cell structures have dehydrins associated with them in response to cold or dehydration stress. Mitochondria are highly susceptible to changes in intracellular concentration of solutes induced by cellular dehydration of cold temperatures (DeSantis et al., 1999). To date, plant mitochondrial responses and adaptations to cellular dehydration and cold stress have been relatively unexplored. The objective of this study was to determine whether dehydrin-like proteins (dips) localise in proximity to plant mitochondria in response to cold stress.
Materials and Methods Three-day-old etiolated seedlings of Triticum aestivum (L.) (winter wheat) and Secale cereale (L.) (winter rye) were grown at 20·C. Zea mays (L.) (maize) was grown at 27·C. Unstressed plants were maintained under growth conditions for 1 day. Cold treatment was performed by subjecting seedlings to 4·C for wheat and rye and to 10·C for maize. Control and cold-treated seedlings were compared at similar growth stages. Crude mitochondria were isolated from control and cold-treated seedling by a method described elsewhere (Voinikov et al., 1991) with modifications (Voinikov et al., 1998). Further purification was performed on a discontinuous Percoll gradient (De Virville et al., 1994). Puriry and integrity of mitochondria were determined by measurement of cytochrome c oxidase activity (EC 1.9.3.1) (De Virville et al., 1994). Mitochondria were sonicated for disruption in 100 mmol/L TrisHCl (pH 7.6) containing 1 mmollL phenylmethylsulfonyl fluoride. Undisrupted mitochondria were precipitated by centrifugation at 17,000 gn for 5 min and discarded. The supernatant was divided into two fractions: (1) fraction 1 was boiled for 20 min to isolate thermostable proteins; and (2) fraction 2 was subjected immediately to protein precipitation. Proteins from fractions 1 and 2 were precipitated with 10 % trichloroacetic acid, washed with cold acetone, and dissolved in sample loading buffer for SDS-PAGE. Proteins were subjected to SDS-PAGE using a mini-Protean PAGE cell (Bio-Rad, USA) according to the manufacturer's instruction. Western blotting and immunodetection were carried out as described by Timmons and Dunbar (1990) using anti-dehydrin primary antibody (1: 1,000 dilution), kindly provided by Dr. T.]. Close (Close et aI., 1993 b). Western blot images were analysed by Sigma Scan Pro Software (Sigma Chemicals, USA). Protein concentrations of samples were determined according to Esen (1978).
Results and Discussion
Dehydrins accumulate in response to cold stress in the nucleus or cytoplasm, but it is unknown if they can accrue in mitochondria or chloroplasts. The accumulation of another family of proteins associated with freezing tolerance was demonstrated for several cereal species (Close, 1996; Sarhan et ai., 1997). In wheat and other cereals, the wcs120 gene family is coordinately regulated by low temperatures and accumulates to high levels in freezing tolerant members of Poaceae (Houde et al., 1995; Limin et ai., 1995). This family shares homology with the dehydrin family of proteins (Dure, 1993). Immunogold labelling indicated that the WCS 120 protein family is located in both the nucleus and cytoplasm of cold acclimated tissues. Mitochondria were found to contain few gold particles. However, cellular fractionation did not reveal the presence of any member of this protein family in the mitochondrial fraction (Houde et al., 1995). To determine whether dehydrin-like proteins (dip) accumulated in the mitochondria in response to cold stress, mitochondria from control and cold-stressed plants from wheat, rye, and maize plants were isolated and subjected to SDS-PAGE. Protein profiles as detected by Coomassie Blue staining indicated that there were no differences in both total (Fig. 1 A) and thermostable protein fractions (Fig. 1 B) between control and cold-acclimated plants in any of the speCles. Immunoblot analysis using the dehydrin antibody of total mitochondrial protein indicated the presence of 54 kDa and 60 kDa dehydrin-like proteins (dlp54 and dlp60). The analysis with primary antibodies blocked by dehydrin peptide confirmed that corresponding proteins are really dehydrins (data not shown). Both proteins were expressed in control and cold-stressed plants for all species examined (Fig. 1 C). Immuno-analysis of the mitochondrial thermostable-protein fraction of control and cold-stressed samples indicated that both proteins were thermostable (Fig. 1 D). Relative accumulation of dlp60 after cold acclimation was higher in rye than in wheat, and dlp60 accumulation in maize was barely detectable and below the linear range for densitometric analysis (Fig. 2 A). Densitometric analysis of the mitochondrial thermostable proteins indicated a small, but distinct accumulation of the relative abundance of dlp54 and dlp60 in rye, wheat, and maize (Fig. 2 B). The differences between plant species in the relative abundance of these two proteins were significantly less for total mitochondrial thermostable protein content than in total mitochondrial protein content. This difference between total mitochondrial protein content (Fig. 2 A) and mitochondrial thermostable protein content (Fig. 2 B) suggests that the accumulation of dlp54 and dlp60 after cold treatment occurred mostly due to the accumulation in mitochondria of all thermostable proteins rather than the dips alone; but nonetheless, this accumulation was determined in the thermostable fraction also. Mitochondria are sensitive to solute concentration in the medium. Therefore, it is very likely that water content in these organelles would be low during ice crystal formation. The concentration of proteins in the matrix of mitochondria is rather high even under normal conditions, over 50 % by dry-weight (Douce, 1985). Cellular dehydration is thought to arise con-
799
Dehydrin-like-proteins of the Plant Mitochondria 234
A
5
6
kD
2
C
-66
3
4
5
~~~~'M' J~_F"~t•.
•
kD
!"""'--"" -60 -54
-.
-45
6 _..,..,.;.
-24 -18
I
B
Fig. 1: SDS-PAGE protein profiles and Western blots of isolated plant mitochondria. The total (A & C) or thermostable (B & D) mitochondrial proteins of winter wheat (In. 1 & 2), winter rye (In. 3 & 4), and maize (In. 5 & 6) of control (in. 1, 3 & 5) and cold-treated seedlings (in. 2, 4 & 6) were visualised with Coomassie blue (A & B), or electro blotted onto nitrocellulose membranes, and ptobed with antibody against dehydrin (C & D). 151lg of protein was loaded per lane. Molecular masses of protein standards (A & B) or molecular masses of dips (C & D) are indicated on the right.
2
3
1000 c:
·f 2
.5
2500 2000
OJ
l
a
i; co
"
I
500
1500
.dlp54 (control) OdlpS4(acclim.)
Ea dlp60 (control) mdlp60 (acclim.)
1000 500
a wheat
rye
2
D
3
4
5
6
-18
1500
::J
6
-24
A
~
5
"'iiil~~nn~
2500 2000
4
maize
Plant species
Fig. 2: Relative abundance of the dehydrin-like proteins in the total (A) and thermostable (B) protein fractions of mitochondria. Protein separation and Western blotting were the same as in Fig. 1. Membranes were scanned and total intensities of the bands corresponding to dlp54 and dlp60 were measured. Means and standard errors of the means are shown.
comitantly with freezing temperatures. Dehydrins accumulate not just in response to cold-stress temperatures, but also in reponse to dehydration (Close, 1993 a). Our results concerning the accumulation of dehydrin-like proteins in mitochondria with the cold treatment indicated that mitochondrial dIps may
be involved in freezing- and dehydrative-tolerance mechamsms. The function of mitochondrial dips remains unknown. Recently, immunoelectron microscope analyses revealed that protein WCOR41O, an acidic dehydrin, accumulates in the vicinity of the plasma membrane. WCOR41O is a peripheral protein and not an integral protein. The authors proposed that this dehydrin may function by preventing the destabilisation of the plasma membrane that occurs during dehydrative conditions (Danyluk et al., 1998). The dIps found in the mitochondria could possibly function by preventing destabilisation of membranes and/or stabilising in a chaperonelike function the macromolecules in the matrix or in the intermembrane space. Precise localisation of these proteins in the organelles would give more information about the function of mitochondrial dIps. Besides preventing or repairing damage induced by cold stress and dehydration, mitochondrial dIps may also play a role in regulating mitochondrial function during cold or osmotic stress. It has been suggested that RAB 17, a dehydrin related protein, mediates the transport of specific nuclear targeted proteins during stress (Goday et al., 1994). Mitochondrial dIps may have an analogous role by regulating protein transport or mitochondrial respiration. Finally, it is worth noting from our data that the mitochondria of various monocot species accumulated dIps of similar molecular masses, suggesting a conserved response to dehydrative/freezing stress. Acknowledgements
This research was funded by a grant from the Russian Foundation of Basic Research (project 99-04-48121) and a grant from the
800
Genadii B. Borovskii, Irina V. Stupnikova, Anna I. Antipina, Craig A. Downs and Victor K. Voinikov
Young Scientist Project Program of the Siberian Division of the Russian Academy of Sciences (G. B.). We sincerely thank Dr. T. J. Close for his generous gift of the dehydrin antibody.
References Bohnert, H. J., D. E. Nelson and R. G. Jensen: Plant Cell 7, 10991111 (1995). Campbell, S. A., and T. J. Close: New Phytol. 137, 61-74 (1997). Close, T. J.: Physiol. Plant. 97, 795-803 (1996). - Physiol. Plant. 100,291-296 (1997). Close, T. J., and P. J. Lammers: Plant Physiol. 101, 773-779 (1993a). Close, T. J., R. D. Fenton and F. Moonan: Plant Mol. BioI. 23, 279-286 (1993 b). Danyluk, J., A. Perron, M. Houde, A. Limin, B. Fowler, N. Benhamou and F. Sarhan: Plant Cell 10, 623-638 (1998). DeSantis, A., P. Landi and G. Genchi: Plant Physiol. 119, 743-754 (1999). De Virville, J., I. Aaron, M. F. Alin and F. Moreau: Plant Physiol. Biochem.32, 159-166 (1994). Douce, R.: Mitochondria in higher plants: structure, function, and biogenesis. Academic Press, London (1985). Dure, L. III.: In: Close, T. J. and Bray, E. A. (eds.), Current Topics in Plant Physiology: Plant Responses to Cellular Dehydration during Environmental Stress, pp. 91-103. ASPP Rockville, Rockville (1993).
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