Proteins Associated with Adaptation of Cultured Tobacco Cells ... - NCBI

Plant Physiol. (1985) 79, 126-137

0032-0889/85/78/0126/1 2/$0 1.00/0

Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl1 Received for publication December 18, 1984 and in revised form May 1, 1985

NARENDRA K. SINGH, AVTAR K. HANDA, PAUL M. HASEGAWA, AND RAY A. BRESSAN* Department of Horticulture, Purdue University, West Lafayette, Indiana 47907 ABSTRACT Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 1 1-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands. Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate 3sS into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

Plants typically exhibit significant morphological and metabolic changes in response to perturbation to their normal environment. Many of these changes are believed to be adaptive responses which make the organism more fit in the altered environment. In many such instances, the phenotypic changes are complex and difficult to evaluate, thus making it difficult to establish a very quantitative relationship between the environmental parameter and phenotypic response. In addition, precise control over environmental parameters and uniformity of response ofplant tissues can be difficult to achieve. Such difficulties can be greatly reduced by the use of cultured cells of plants which have been adapted to different levels of stress (23). It is often assumed that the altered phenotype of cells with enhanced ability to survive and grow in the presence of high levels of NaCl is largely the result of altered gene expression. Unfortunately, there is no a priori reason to believe that the

altered expression of any particular gene after adaptation to NaCl necessarily provides increased survival or growth. The first step in und-erstanding such expressional changes would seem to be the establishment of a correlation between the level of different gene products, i.e. mRNA and/or protein and the degree of adaptation to NaCl. There seem to be two basic approaches which can be taken if identification and quantitation of proteins is used to provide a correlation between the altered expression of specific genes and changes in the environment. One can look for changes in the expression of genes which code for proteins which would logically be involved in adaptation based on known metabolic processes, such as proteins involved in ion transport. A second approach is to characterize the new and abundant proteins of the cell that occur during adaptation and after the cells have become adapted-to the stress. Although it is not possible to assign immediately a function to such proteins, they can be used to study the regulation of gene expression during exposure to increased levels of NaCl stress. Several new proteins which are synthesized in response to an altered environment have been reported as 'stress proteins' or shock proteins in plants (1, 2, 11, 14, 16, 17, 24, 25, 28, 30, 32, 33, 36-38). However, only a few of these proteins have been found to be involved in known physiological or metabolic processes (19, 20, 33). Most of these proteins appear as an immediate response by the organism to an altered environment (temperature, anoxia, osmotic stress, and wounding), and it is questionable whether many of them are associated with increased growth and survival of the plants in the new environment. Osmotic shock also leads to the synthesis of new proteins and the extracellular release of several proteins (1, 17, 32). However, it is not clear how osmotic stress causes changes in the synthesis of specific proteins (6). Some of these shock proteins are considered to be involved in possible transport functions (1, 32). We have been interested in understanding the molecular basis for NaCl adaptation by cultured cells of tobacco. We examined the levels of abundant proteins in the cells adapted to increasing levels of NaCl and a preliminary result has been presented (34). Our results indicate that there may be an involvement of a 26kD polypeptide in the adaptation of cells to NaCl, and a close association between the synthesis of certain abundant proteins and the degree of adaptation to NaCl stress. MATERIALS AND METHODS Cell Culture. Cell suspension cultures of Nicotiana tabacum L. var Wisconsin 38 are maintained in our laboratory as described (5, 22). NaCl adapted and unadapted cells are maintained

' Supported by Purdue University Agricultural Experiment Station Program Improvement Fund and by the United States Department of Energy grant DE 13109. Journal Paper 10,148, Purdue University Agricultural Experiment Station.

with their respective concentrations of NaCl in the medium. Unadapted cells are designated S-0 and cells adapted to growth in 10, 16, 20, and 25 g NaCl L' of culture medium are designated 126

PROTEINS ASSOCIATED WITH NaCl ADAPTATION

127

92.5

66.2 45.0

31.0 21.5 1-7 7%, 4g*

14.0 ., .,. 'i,."... '1., -

1

2

3

4

5

6

7

8

FIG. 1. SDS-PAGE on 10 to 16% acrylamide gradient gel of equal amounts of total cellular protein at stationary phase of culture growth from cells adapted for growth at 25, 20, 16, 10, and 0 g NaCI/L medium (lanes 1-5, respectively). Lanes 6 and 7 represent protein extracted from cells adapted to 25 and 30% polyethylene glycol. Lane 8 contains mol wt standards.

S-10, S-16, S-20, and S-25, respectively. Cells adapted to 25 and 30 g PEG (100 ml)-' medium were isolated as previously described for cultured cells of tomato (9) and are designated P-25 and P-30, respectively. All adapted cell lines were grown in media containing the designated level of NaCl or PEG for at least 100 generations before use in these experiments. In Vivo Labeling of Protein. Cells from late log phase of culture growth were used to inoculate fresh medium at a density of 8 g fresh weight L` for S-0 and S-10 cells and 20 g fresh weight L' for S-25 cells. Cultures were grown in 1-L Erlenmeyer flasks containing 200 ml medium on a gyratory shaker as described (22). Twenty-five ml of culture were withdrawn every 2 or 3 d after inoculation and transferred into a 1 25-ml Erlenmeyer flask with 100 oCi H2350S4 (from New England Nuclear) and incubated for 24 h on a gyratory shaker at 26° under 16-h illumination. There was no detectable change in the pH of the medium as a result of addition of H235S04. The 35S-fed cells were harvested by filtration and fresh weight was recorded. To determine the amount of 35S incorporation into protein, a known fresh weight of cells was suspended in 10% TCA containing 2 mM Na2SO4 and placed in boiling water bath for 10 min. After cooling on ice, the cells were filtered on glass fiber filter discs (Whatman No. 934-4H) which were washed with 10 ml cold 5% TCA containing 2 mm Na2SO4 followed by 10 ml cold ethanol wash. The 35S incorporation was determined, using Bray's scintillation cocktail (8), in a Beckman LS 6800 scintillation spectrometer.

Extraction of Cellular Protein. Cells were harvested by filtration and were washed twice in cold acetone (-20°C). Acetone was removed by filtration followed by air drying. The acetone

powdered cells were stored at -20°C for subsequent protein extraction. Protein for SDS-PAGE was extracted in modified Laemmli's buffer (26) containing 65 mm Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 5% ,3-mercaptoethanol, 2 mm EDTA, and 1 mm phenylmethylsulfonylfluoride. The cell samples in protein extraction buffer were placed in a boiling water bath for 3 min. Clear supematants obtained after centrifugation in an Eppendorf centrifuge for 2 min were used for electrophoresis. For isoelectrofocusing, protein was extracted in O'Farrell's buffer (29) containing 9 M urea, 2% (v/v) Nonidet NP-40, 5% ,B-mercaptoethanol, and 2% (v/v) Ampholyte (pH 3-10). Samples were sonicated for 10 s and the clear supernatants were used for electrofocusing. Electrophoresis. One-dimensional electrophoresis was carried out using the procedure of Laemmli (26) with some modifications. Slab gels (16 cm x 18 cm x 0.75 mm) with a 10 to 16% polyacrylamide gradient in the running gel and a 4% polyacrylamide stacking gel were used for electrophoresis at a constant current of 12 mamp/gel slab. Either equal amounts of protein as determined by the Bradford method (7) or equal amounts of TCA-precipitated 3IS cpm were applied in each lane. After electrophoresis, gels were stained with 0.05% Coomassie brillant blue R-250 in 40% methanol: 10% acetic acid. Gels were destained in 10% methanol:7% acetic acid and preserved in 7% acetic acid:5% glycerol. Stained gels were scanned with a Beckman DU-8 spectrophotometer at 550 nm. Two-dimensional gel electrophoresis of protein was performed according to the procedure of 0-Farrell (29). The isoelectrofocusing tube gels, 2 x 113 mm, containing the following: 9 m urea, 4% acrylamide (17.5:1 w/w acrylamide:bis-acrylamide),

Plant Physiol. Vol. 79, 1985

SINGH ET AL.

128

z




-v

-

.....

Alwil i.,,

5

!w

s

, ~

~

~

~

~

~

~

~

-

3

During preparation of this manuscript, a report appeared showing a comparison of protein differences between S-0 and S-10 cells which had been obtained from this laboratory. Three polypeptides, 32, 26, and 20 kD were shown to be associated with S10 cells (16). Altered gene expression during NaCl and PEG adaptation by the cultured cells is also suggested by the amino acid composition of total soluble protein. The dramatic reduction of hydroxyproline in the soluble protein suggests that adaptation to NaCl leads to inhibition of the synthesis of hydroxyproline containing protein, and/or perhaps more rapid turnover of hydroxyproline containing protein with a corresponding increase in the free pool of hydroxyproline. A similar situation does exist in Bouvardia ternifolia adapted to NaCl (15). The physiological significance of these changes is unclear, although a reduction in hydroxyproline rich arabinogalactan proteins or precursor wall proteins might explain this. Increased deposition of hydroxyproline rich precursor wall proteins could possibly explain decreased expansion of adapted cells with higher turgor (5). Synthesis of a major 26-kD polypeptide coincident with both the initiation of growth of unadapted cells in medium containing NaCl and the period of osmotic adjustment by the cells (5) also suggests a physiological role for the 26-kD polypeptide in adaptation. However, we do not know the function of this and other polypeptides in the process of adaptation, or the mechanism

regulating the synthesis of these proteins. It has been suggested that the regulation of protein synthesis under salinity may be at the level of translation and that the translational regulation is caused by the activation of RNAse in the cytoplasm due to increased concentration of NaCl leading to degradation of messages (35). On the other hand, translational regulation may take place due to dissociation of polyribosomes and formation of a set of new polyribosomes under the conditions of desiccation stress (4, 12, 13, 30). However, there is evidence which suggests that this is not always the case (3). It should be made clear that a correlation between the quantity of protein and the level of NaCl stress can arise purely by the fact that either the message for these polypeptides or the proteins themselves are protected or less inhibited by the increased concentration of cytoplasmic NaCl in the adapted cells. Since our results and results of others have shown that overall protein synthesis is repressed in response to salinity and water deficit (3, 4, 6, 12-15, 35), it is also likely that general protein synthesis is inhibited by high levels of NaCl. However, the synthesis of some of these polypeptides may not be inhibited or in fact may be stimulated by the presence of NaCl. Our present results do not provide evidence against or for these possibilities. However, the correlation of NaCl adaptation level with the increasing concentration of these polypeptides suggests quantitative changes in gene expression after adaptation to NaCl.

PROTEINS ASSOCIATED WITH NaCl ADAPTATION

1

2

FIG. 10. Polypeptide map of 26-kD polypeptide after partial proteolytic digestion. Lanes I and 2 represent peptide map of 35S labeled 26kD band from S-0 and S-25 cells on 16th and 12th d of culture growth, respectively.

>ws1-sR ¢t. .s

sr ts&tss *tKt2E :stRs sttt9 t.'oF.

1

2

FIG. 1 1. Immunoelectrophoresis of protein extracts from S-25 (1), S(2), and P-30 (3), cells at the stationary phase of culture growth. Amounts of total protein extract applied in different wells were 25, 160, and 30 ,gg in lanes 1, 2, and 3 respectively. O

135

It seems from our results that the 26-kD polypeptide which appears during NaCl adaptation is not a salt shock protein, primarily because its synthesis commences only around the period when the cells begin to grow in culture (i.e. during adaptation to NaCl) and does not start immediately in response to exposure to NaCl. A comparison of known shock proteins synthesized by sudden exposure of cells to altered external factors such as heat shock proteins (2, 11, 24, 25), osmotic shock proteins (1, 17), and anaerobic shock proteins (19, 20, 28, 33) with the polypeptides associated with NaCl tolerance in our system do not provide any basis of similarity. This is consistent with the fact that the adapted cells are no longer under shock and the proteins that they synthesize are associated with a physiological state of adaptation and not a physiological state of trauma. The adapted cells can undergo shock when transferred to levels of NaCl which are higher or lower than those to which they are adapted. Newly synthesized proteins appear in response to such shock exposure but none are the major polypeptides associated with adapted cells (data not shown). We have partially characterized the 26-kD polypeptide which constitutes about 10% of the total cellular protein in adapted cells. Our results demonstrate that at least two types of 26-kD polypeptides are synthesized in adapted cells, one type which appears early in the growth cycle, the major portion of which has a pI greater than 8.2, and another which appears later in the growth cycle and the major portion of which has a pl of 7.8. There seems to be only one type of major 26-kD polypeptide(s) with a pl of 7.8 which appears later in the growth cycle in unadapted cells. In unadapted cells, this polypeptide either has a high rate of turnover and degradation or the rate of synthesis is very low, and we can visualize it only immunologically or by 35S-fluorography apparently because of a relatively high S amino acid content (>8%) of the polypeptide (our unpublished result). The fact that the 26-kD polypeptide(s) which is unique to adapted cells and the 26-kD polypeptide appearing late during culture growth of unadapted cells show immunological cross-reactivity and have a similar partial proteolysis peptide map would suggest that these two polypeptides are the products of the same or similar genes. From two-dimensional electrophoresis, it seems that the main 26-kD polypeptide(s) from adapted cells is more basic and heterogeneous with a pl greater than 8.2, and the main 26-kD polypeptide from unadapted cells has a pl of 7.8 (Fig. 8, A and B). Differences in the isoelectric points of the immunologically related 26-kD polypeptides would suggest either a posttranslational modification of the polypeptide by glycosylation or phosphorylation of amino acid residues (which may perhaps protect it from degradation in adapted cells) or that the two types of polypeptides are the products of related genes which have undergone divergent evolution by amino acid substitution(s). To understand the possible functions of this 26-kD protein in adjustment of plant cells to NaCl, its localization in the cell would be helpful. However, more importantly, isolation of DNA sequences coding for the 26-kD protein should allow the construction of a plasmid vector to introduce these DNA sequences under constitutive or modulatable expression into unadapted cells to test the effect of altered expression of this protein on adaptability of the cells. Also a DNA probe for such a gene could be used to examine its presence, organization, and regulation of expression in organisms differing widely in their ability to adapt to and tolerate salinity. Such a probe could be used to identify species not containing this gene for use as recipients of the gene in transformation experiments. Acknowledgments-We are thankful to D. Rhodes, Zoecon Corporation, Palo Alto, CA, for assistance in the identification of hydroxyproline by mass spectrometry and to G. McClatchy, J. Clithero, and E. A. Frankenberger for excellent technical assistance.

136

Plant Physiol. Vol. 79, 1985

SINGH ET AL.

I

Table II. Amino Acid Composition of Soluble Protein from Cells at Stationary Phase of Culture Growth Amino Acid 5-10 S-25 S-0 P-30 S-20 ng amino acid/ug protein 67 54 64 Ala 56 49 56 108 56 60 53 Gly 41 37 Val 32 26 29 44 44 Thr 38 36 43 64 37 52 49 62 Ser 64 64 Leu 66 50 53 Ile 33 26 15 36 23 Pro 64 42 42 45 51 92 92 105 93 92 Asp 32 Phe 31 36 43 45 114 125 95 84 Glu 108 44 47 59 58 53 Lys 27 28 Tyr 46 28 39 7 19 6 125 13 Hyp LITERATURE CITED 1. AMAR L, L REINHOLD 1973 Loss of membrane transport ability in leaf cells and release of protein as a result of osmotic shock. Plant Physiol 51: 620625 2. BARNETr T, M ALTSCHULER, CN McDANIEL, JP MASCARENHAS 1980 Heat shock induced proteins in plant cells. Dev Genet 1: 331-340 3. BEWLEY JD, KM LARSON 1980 Cessation of protein synthesis in water stressed pea roots and maize mesocotyls without loss of polyribosomes. Effects of lethal and non-lethal water stress. J Exp Bot 31: 1245-1256 4. BEWLEY ID, MJ OLIVER 1983 Responses to a changing environment at the molecular level: does desiccation modulate protein synthesis at the transcriptional or translational level in a tolerant plant? In Current Topics in Plant Biochemistry. Uhiversity of Missouri Press, Columbia, pp 145-164 5. BINZEL ML, PM HASEGAWA, AK HANDA, RA BRESSAN 1985 Adaptation of tobacco cells to NaCl. Plant Physiol 79: 118-125 6. BOYER JS 1976 Stress relationships in protein synthesis: water and temperature. In Genetic Improvement of Seed Proteins. National Academy of Science, Washington, pp 159-171 7. BRADFORD M 1976 A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 8. BRAY GA 1960 A simple efficient liquid scintillation for counting aqueous solution in a liquid scintillation counter. Anal Biochem 1: 279-285 9. BRESSAN RA, AK HANDA, S HANDA, PM HASEGAWA 1982 Growth and water relations of cultured tomato cells after adjustment to low external water potentials. Plant Physiol 70: 1303-1309 10. CLEVELAND DW, SG FISCHER, MW KIRSCHNER, UK LAEMMLI 1977 Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by

FIG. 12. Rocket immunoelectrophoresis of equal amounts of total protein extracted from S0, 5-10, 5-16, S-20, and S-25 cell lines at the stationary phase of culture growth in wells I to 5, respectively, showing increasing height of rocket in cell lines from 5-0 to S-25. Rockets migrated towards cathode.

gel electrophoresis. J Biol Chem 252: 1102-1106 11. COOPER P, THD Ho 1983 Heat shock proteins in maize. Plant Physiol 71: 215-222 12. DHINDSA RS, JD BEwLEY 1976 Plant desiccation: polyribosome loss not due to ribonuclease. Science 191: 181-182 13. DHINDSA RS, JD BEWLEY 1978 Messenger RNA is conserved during drying of drought tolerant Tortula ruralis. Proc Natl Acad Sci USA 75: 842-846 14. DIAZ DE LEON L, L ARcos, JL DIAZ DE LEON, H Soro 1980 Protein biosynthesis by Bouvardia ternifolia cell cultures adapted to NaCI. In Proceedings of Second International Workshop on Biosaline Research. LaPaz, Mexico, pp 455-460 15. DIAZ DE LEON JL, H SOTO, MT MERCHANT, L DIAZ DE LEON 1980 Biochemical and ultrastructure changes induced by NaCI in cell culture derived from Bouvardia ternifolia. In Proceedings of Second International Workshop on Biosaline Research. LaPaz, Mexico, pp 461-466 16. ERICSON ME, SH ALFINITO 1984 Protein produced during salt stress in tobacco cell cultures. Plant Physiol 74: 506-509 17. FLECK J, A DURR, C FRITSCH, T VERNET, L HIRTH 1982 Osmotic shock 'Stress Protein' in protoplast of Nicotiana sylvestris. Plant Sci Lett 26: 159-165 18. HANDA AK, RA BRESSAN, S HANDA, PM HASEGAWA 1983 Clonal variation for tolerance to polyethylene glycol-induced water stress in cultured tomato cells. Plant Physiol 72: 645-653 19. HANSON AD, JV JACOBSON 1984 Control of lactate dehydrogenase, lactate glycolysis and c-amylase by 02 deficit in barley aleurone layers. Plant Physiol 75: 566-572 20. HANSON AD, JV JACOBSON, JA ZWAR 1984 Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers. Plant Physiol 75: 573-581 21. HARBOE N, A INGILD 1973 Immunization, isolation of immunoglobulins, estimation of antibody titre. In Manual of quantitative immunoelectrophoresis: methods and applications. Scand J Immunol 2: Suppl 161164 22. HASEGAWA PM, RA BRESSAN, AK HANDA 1980 Growth characteristics of NaCI-selected and non-selected cells of Nicotiana tabacum L. Plant Cell Physiol 21: 1347-1355 23. HASEGAWA PM, RA BRESSAN, S HANDA, AK HANDA 1984 Cellularmechanism of tolerance to water stress. Proceedings of the symposium on somatic cell genetics: prospect for development of stress tolerance. Hortscience 19: 371377 24. KANABUS J, CS PIKAARD, JH CHERRY 1984 Heat shock proteins in tobacco cell suspension during growth cycle. Plant Physiol 75: 639-644 25. KEY JL, CY LIN, YM CHEN 1981 Heat shock proteins of higher plants. Proc Natl Acad Sci USA 78: 3526-3530 26. LAEMMLI UK 1970 Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680-685 27. LAURELL CB 1966 Quantitative estimation of proteins by electrophoresis in aarose containing antibodies. Anal Biochem 15: 45-52 28. MACQUOT B, C PRAT, C MOUCHES, A PRADET 1981 Effect of anoxia on energy chare and protein synthesis in rice embryo. Plant Physiol 66: 636-6 29. O'FARRELL PH 1975 High resolution two dimensional electrophoresis of proteins. J Biol Chem 250: 4007-4021 30. OLIVER MJ, JD BEWLEY 1984 Plant desiccation and protein synthesis VI. Changes in protein synthesis elicited by desiccation of the moss Tortula ruralis case affected at the translational level. Plant Physiol 74: 923-927

PROTEINS ASSOCIATED WITH NaCl ADAPTATION 31. RHODES D, AC MYERS, G JAMIESON 1981 Gas chromatography-mass spectrometry of N-heptafluorobutyryl isobutyl esters of amino acids in the analysis of kinetics of ['5N]H4' assimilation in Lemna minor L. Plant Physiol 68: 11971205 32. RUBENSTEIN, B 1982 Regulation of H+ excretion: role of protein released by osmotic shock. Plant Physiol 69: 945-949 33. SACHS MM, M FREELING, R OKIMOTO 1980 The anaerobic proteins of maize. Cell 20: 761-767 34. SINGH NK, AK HANDA, PM HASEGAWA, RA BRESSAN 1983 Electrophoretic protein patterns in cultured cells of tobacco adapted to NaCl. Plant Physiol 72: S-535

137

35. STROGNOV BP 1973 Structure and Function of Plant Cells in Saline Habitat. A Halsted Press Book translated from Russian by A. Mercado. John Wiley and Sons, New York 36. STUART DA, JE VARNER 1980 Purification and characterization of a saltextractable hydroxyproline-rich glycoprotein from aerated carrot discs. Plant Physiol 66: 787-792 37. TYMMS MJ, DF GAFF, ND HALLMAN 1982 Protein synthesis in desiccation tolerant angiosperm Xerophyta villosa during dehydration. J Exp Bot 33: 323-343 38. WEBSTER PL 1980 "Stress" protein synthesis in pea root meristem? Plant Sci Lett 20: 141-145