PlantCell Reports
Plant Cell Reports (1986) 5:430-434
© Springer-Verlag 1986
Protein synthesis in a maize callus exposed to NaCI and mannitol Subbanaidu R a m a g o p a l * U.S, Department of Agriculture, Agricultural Research Service, Western Regional Research Centre, Albany, CA 94710, USA Received April 10, 1986 / Revised version received July 18, 1986 - Communicated by I. K. Vasil
Abstract A maize (Zea mays, L) callus was exposed to media containing different levels of NaCI (0 to 3%) and mannitol (0 to 18.2%) for a period of /tweeks, and the changes in growth and protein synthesis were determined. Cells are able to tolerate and grow in NaCI up to I% (0.17 M) or mannitol up to 9.1% (0.5 M), but the relative overall growth rates are about I/6 and I/8 of the control, respectively. Protein synthesis, as assessed by pulselabeling of the cells with 35S-methionine after exposure to the stress reagents at various times of incubation, suggests that the relative rates of amino acid uptake and its incorporation into proteins are inhibited as early as /4 h after exposure, and the extent of inhibition does not increase appreciably until after I week. Severe inhibition of uptake and protein synthesis results from prolonged exposures at growth-inhibitory concentrations of NaCI and mannitol. In general, the overall mean inhibition of cellular uptake and protein synthesis in the first 2-week period are approximately 50% and 35% for the NaCI (1%) and mannitol (7.3 %) treatments, respectively. No detectable differences are apparent in the abundant, steady state protein population as revealed by SDS-PAGE and on staining with Coomassie blue or silver, but random losses of individual proteins occur after 2 weeks at 2% and 3% NaCI and at 18.2% mannitol. Of the newly-synthesized proteins, discernible changes are found in 7 and zt polypeptides in NaCI and mannitol treatmerits, respectively. Apparently three new proteins (74 kd, 28.5 kd, and 26.2 kd) are induced de novo under both treatments. Other proteins (39.5 kd, 39.0 kd, 30 kd, and 16.5 kd) show an increased or decreased level of synthesis. NaCI levels above 0.5% or mannitol levels above 3.6% da not alter the pattern of newly-synthesized proteins. This altered expression of newly-made proteins in the maize callus occurs only after a week of exposure to salinity or osmotic stress and coincides with the cell growth phase. INTRODUCTION The decline in plant productivity due to salinity and drought in saline and arid lands necessitates the development of stress-tolerant crops. Of the various approaches, cell culture, selection for mutants has received increasing attention in recent years (Maliga, 198b,; Meredith, 198/4; Rains e t a l , 1980). Salt-tolerant cell lines have been isolated in a number of plant species (Maliga, 198b,; Flick, 1983), and the tolerant trait has also been claimed to be maintained in regenerated plants of Nicotiana (Nabors et al., 1980) and Datura (Tyagi et al., 19817. Studies so far suggest that both genetic and epigenetic factors may be involved in the development of stresstolerance in cultured cells (Maliga, 1984; Meredith, 1984). * P r e s e n t address:
However, which of these factors is more important and what adaptive mechanisms actually operate in the tolerant cells are not understood. A knowledge of the molecular responses such as gene expression during cellular adaptation to stress would be helpful and might lead to the identification of the altered putative genes. Recently, this approach was applied in a tobacco suspension culture system, and the investigators showed the appearance of specific proteins in saltadapted cells (Ericson and Alfinit% 1984; Singh et al., 1985). Here, 1 report our studies on protein synthesis in a maize cell line treated with NaCI and mannitol to induce salinity and water stress. MATERIALS AND METHODS Cell line. Callus cultures of Zea mays var. Black Mexican Sweet obtained from Z. R. Sung (The University of California, Berkeley) were maintained in the dark at 27*C on Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with I mg per liter of 2, Zt-D and I% agar. NaCI and mannitol were incorporated in the medium. Four to S callus pieces of 25 to 30 mg FW each were placed on a single plate. Three to five plates were used for each treatment. Measurement of in vivo protein synthesis. At the end of treatment period as specified, calli (100 to 200 mg FW) were transferred to I ml of a labeling medium. This medium had the same composition as the corresponding treatment medium without agar and included 100 ~Ci of 3SS-methionine (I,098Ci/mmole, New England Nuclear). The cells were labeled by agitating the tubes for 2 h at IS0 rpm at 25*C, harvested on 2 layers of Miracloth by gentle suction, rinsed thoroughly with cold water, and frozen immediately in liquid nitrogen. Samples for labeling were collected by pooling 5 to 8 independent callus pieces from each treatment. The frozen cells were ground in a mortar under liquid nitrogen and transferred to 200 to It00 ]JI of a buffer consisting of S0 mM Tris HCI (pH 7.5), 10 mM Na2EDTA (pH 7.It), 5% 2-mercaptoethanol, 2% sodium dodecylsulfate (SDS), and 10% glycerol. The samples were mixed in a vortex mixer, boiled for 10minutes, centrifuged for 5minutes in an Eppendorf microcentrifuge (Brinkman Model 5414), end the supernatants saved. Uptake and incorporation were determined as described (Ramagopal et al., 1977). Five to 10 1JI of this supernatant was spotted directly on a glass fiber (Whatman GF/C) disc, dried, and counted in a liquid scintillation counter to determine the total cellular uptake of 35S-methionine. The incorporation of 35S-methionine int 9
USDA/ARS, Experiment Station, HSPA, P.O. Box 1057, Aiea, HI 96701, USA
431 cellular proteins was measured by precipitating 5 to 10 ~1 aliquots of the supernatants with 9% (final concentration) cold trichloroacetic acid (TCA) for 30 to 45 minutes on ice and collecting the precipitated proteins on glass fiber discs. The precipitates were further washed with ice-cold TCA (3 x S ml) and 5 ml of 95% ethanol, dried, and counted. Two to three determinations were made an each point and the means are presented in Tables I and 2. The value of each determination was within 5% of the reported means. Electropharesis of proteins. The proteins extracted as described above were resolved on a 12.5% SDS-polyacrylamide gel (Laemmli, 1970) along with molecular weight standards (Bia Rad Laboratories). The gels were stained with Coomassie blue (Ramagopal, 1976) or silver (Merrill et al., 1981). Radioactive gels, after fixation of proteins, were treated with En3hance (New England Nuclear), dried, and exposed to Kodak X-AR5 film with an intensifying screen (Du Pont).
RESULTS Callus Growth The effects of NaCI and mannitol on the growth of maize cultures were examined at a range of concentrations. Cells were grown on media containing up to 3% (0.SIM) NaCI or up to 18.2% (IM) mannitol for a period of 4 weeks (Fig. I). Control cultures began ta grow in a week (Fig. I A); fresh weight increases were about 2, 7, and 30 fold in I, 2, and It weeks, respectively. Cultures treated with NaCI and mannitol, on the other hand, grew slowly and showed changes in morphology.
Protein Synthesis The relative capacity for protein synthesis was measured soon after the cells were transferred to NaCI or mannitol media and during their subsequent incubation period. The cells were pulse-labeled with 3SS-methionine, and its uptake and incorporation into proteins were determined. The ability of cells to take up the labeled amino acid varied with the stress agent and duration of exposure (Table I). The uptake was reduced by about It0 to 50% after 4 h of NaCI treatment; the reductions were abaut 60 to 80% at later periods. The concentration of NaCI apparently did not have any bearing on the inhibition until I week, but after 2 weeks, NaCI levels above 2% reduced the uptake by greater than 90%. The effects of mannitol on uptake were less severe compared to NaCI. At 3.6%, mannitol had no effect and sometimes stimulated the uptake up to a week, but at 2 week, it caused a reduction of 70%. Higher mannitol concentrations inhibited the uptake up to a week by 20 to 40% and by 70% after 2 weeks. The proportion of cellular uptake which was utilized in protein synthesis was apparently independent of stress duration but indicated marked variation dependent on the stress agent. Approximately 58% of the label taken up was utilized in pratein synthesis in untreated control cultures (Table I). NaCI treatment reduced the protein fraction below control levels by about 42%, 77%, and 87% at I%, 2%, and 3% levels, respectively; mannitol at 3.6% had no effect but caused a reduction by about 25% and 70% respectively, at 7.3% and 14.6%.
200 6O0-
I
[] I Day
m
Table I. Effects of NaCI and mannital on relative rates of 35S-methionine uptake and incorporation into proteins.
B
~10o
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'
1NIC,{%) ' 2
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3
i-lmll FI~•,
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' 14.6 j
[7nn 0
, O NaCl(%) 51 2
3
l/
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3691182 Mannltol(%)
Fig. I. Effects of NaCI and mannitol on the growth of maize callus. Each FW value is a mean of 25 to 45 separate pieces of the inoculated call[. Panels A and B represent independent experiments. Panel A. Growth during the first 2 week period. Panel B. Callus growth at it weeks. All NaCI-treated cells appeared healthy until l day but those an high salts turned brown at 7 day. Browning of callus was very slight up to I% NaCI, but above 2% NaCI it was extensive. At 0.5% and I% NaCI levels, fresh weight increases were real and delayed somewhat compared to control. Fresh weight increases at 0.5% NaCI were about 3 and 8 fold a t 2 and 4 weeks, respectively. Calli on I% NaCI showed a 2 fold increase in fresh weight in 2 weeks and an additianal 2 fold at the end of 4 weeks. Cells failed to grow at 2 and 3% NaCl. Higher concentrations of mannitol were inhibitory to callus growth; calli started to turn brown in a week at 7.3% or above. There was a limited growth in a week at 3.6% mannitol wh!ch improved later on; fresh weight increases were about S fold in 2 weeks and an additional 3 fold between 2 and 4 weeks. At 7.3% mannitol, a 50% increase in fresh weight was observed after 2 weeks of culture, and it improved to an overall increase of 2 fold at 4 weeks. In other experiments, no significant differences were observed in callus growth at 7.3% or 9.1% mannitol levels.
Treatment Control
It h 100
I day
(58)* 100
(57)
I week 100
2 week
(58) 100
(56)
NaCI(%)
I
60
(34)
29
(29)
73
(50)
42
(37)
2 3
52 56
(13) (8)
22 25
(9) (0.6)
21 18
(19) (12)
II 8
(17) (13)
3.6 7.3
ll3 88
(62) (42)
85
74
(56) (83)
103 64
(76) (52)
31 35
(56) (44)
14.6
61
(19)
46
(24)
70
(13)
36
(16)
Mannitol(%)
The maximal control values (100) for rates of 35S-methionine uptake (expressed as cpm/100 m~ FW cells/2h) were 8.9xl06, 7.2x106, 3.3x106, and 5.2xl0 b for It h, 1 day, I week and 2 week cultures, respectively. * Values in parentheses represent the percentages of the total 35S-methionine counts which were incorporated into the TeA-insoluble protein fraction. The relative rate of amino acid incorporation into proteins as measured by TCA-insoluble radioactivity was significantly altered in treated cultures. Because of the observed differences in label uptake (Table I), the protein synthesis data are compared before and after correction for uptake differences (Table 2), both of which lead to similar conclusions. Increased levels of NaCI inhibited protein synthesis as early as 4 h. The extent of inhibition did not seem to vary with the duration of stress as it varied with the concentration of NaCI employed. Protein synthesis reductions were in excess of 80% above 2% NaCI. Protein synthesis was nat altered at 3.6% mannitol until about 2 weeks. Higher mannitol concentrations reduced the rates but to a lesser extent than the NaCI treatments.
432 Table 2. Effects of NaCI and mannitol on relative rates of protein synthesis.
Steady State and De Novo Proteins
Treatment
4h
I day
I week
2 weeks
Control
I00
IO0
I00
I00
NaCl(%)
1
35
(48) 15
(24)
63
2
12
(17)
4
(7)
7
(12)
3
(7)
3
8
(12)
0.2
(0.5)
4
(7)
2
(3)
(121) 31 (79) 28 (21) I0
(52) (45) (17)
Mannitol(%) 3.6 121 7.3 63 14.6 19
(I06) 83 (71) 56 (27) 20
(95) (71) (29)
137 58 16
(79) 28
(45)
The maximal control values (100) for rates of protein synthesis (expressed as cpm/100mg FW cells/2h) were 5.2x10% 4.1x106, 1.9x106, and 2.9xl06 for 4h, I day, I week, and 2 week cultures, respectively. Values in parentheses represent data after correction for the uptake of label.
Changes in protein synthesis were also assessed after 4 weeks of treatment which indicated the relative effects were quite similar to those observed at 2 week. In addition, no major differences were found between mannitol levels, 7.3% and 9.1% or 14.6% and 18.2% (data not shown).
Fig. 2.
The NaCI and mannitol treatments on individual cellular proteins were assessed by resolving them on SDSp olyacrylamide gels. The cultures were sampled beginning at 4 h of treatment and continued until 4 weeks of incubation. Changes in the steady state population of abundant proteins are compared at 2 and 4 weeks after staining the gels with Coomassie blue (Fig. 2). The protein patterns were all similar for the entire culture period, but cells treated with higher concentrations of NaCI and mannitol started to indicate massive losses of proteins at about 2 weeks (Fig. 2A, lanes 5 and 8; 2B, lanes 4, 5, and 8). Protein lass was evident in 2 weeks at 3% NaCI, but at 4 weeks, it was found at 2% NaCI as well. Mannitol at 18.2% caused protein loss at 2 weeks (Fig. 2A), and the loss was greater on further incubation of cultures (Fig. 2B). Larger amounts of proteins were applied to the gels at treatment levels where protein loss was observed to ascertain whether it was specific. The prateins lost belonged to both low and high molecular weight groups (Fig. 2A and B, arrows). Relatively greater loss occured in NaCI stress than in the mannitol stress. Not all proteins were lost, however. A few protein bands appeared to show intensified staining on stress (Fig. 2A and B, closed circles). Of the proteins that were retained, most were similar in both NaCI and mannitol treatments. No additional differences could be detected in the steady state protein population if stained with a more sensitive silver method (Merrill et al., 1981) (data not shown).
Steady state protein patterns of maize callus exposed to NaCI and mannitol. The cultures were exposed for 2 (A) or 4 (B) weeks, proteins extracted, electrophoresed, and stained with Coomassie blue. Treatments and mg FW of cells giving rise to the protein extract applied on the gel are indicated for each lane: Panel A. (1)Control, 8; (2)0.5% NaCI, 4; (3)1% NaCI, 4; (4)2°,6 NaCI, 20; (5)3% NaCI, 40; (6)3.6% mannitol, 8; (7) 9.1% mannitol, 12; (8) 18.2% mannitol, 40. Panel B. The lane designations are the same as in Panel A except for the amount of protein applied for electrophoresis which is indicated: lane (3) 10; (4) 50; (5) 50; (6) 3; (7) 10; (8) 25. Arrows indicate some of the prominent protein bands which are undetectable after treatments with NaCI or mannitol. Closed circles indicate protein bands which show intensified staining.
433 An analysis of the newly-synthesized proteins revealed changes depending on treatment. As above for steady state proteins, patterns of the newly-made proteins were examined far the entire incubation period beginning 4 h of treatment. Majority of the proteins were similar, and no differences were apparent until I week; however, distinct expressional changes in at least 7 polypeptides were evident after 2 weeks (Fig. 3). Apparently, three new proteins (74 kd, 28.5 kd, and 26.2 kd) were induced de nova in both NaCI and mannitol treated cultures. The 74 kd protein was expressed at about the same level in both treatments, but the two low mw proteins were expressed more strongly in NaCI cultures. The expression of one protein of 30 kd increased with increased levels of NaCI (0.5 and I%) and mannitol (3.6 and 9.1%) but seemed to be depressed at higher levels. Two proteins, 39.5 kd and 39 kd, were not at all made at NaCI levels 2% and above but were continued to be made at a detectable level in all mannitol treatments tested. A newly-made 16.5 kd protein was lacking from all NaCI treatments but was present in all mannitol treatments. No additional changes in protein pattern were apparent in cultures treated far 4 weeks.
Kd
74,0~1b
-66.2
-45,0
-31.0 26.2~
-21.5
16.5-* -14A
Fig. 3.
Patterns of newly-synthesized proteins of maize callus exposed to NaCI and mannital. Cells were treated with NaCI (lanes 1-4) or mannitol lanes 5-7) for 2 weeks and labeled with 5S-methionine and processed as described in materials and methods. Equal amount of TCAprecipitable radioactivity (2 x 105 cpm except I x 105 cpm for lane 7) was applied on the gels far electrophoresis.
Lane (I)control, (2) 0.5% NaCI, (3) I% NaCI, (4) 2% NaCI, (5)3.6% mannitol, (6)9.1% mannitol, (7) 18.2% mannitol.
DISCUSSION Plant cells may react to stress in various ways. Early on, they may be shocked by the stress-treatment and show transient changes which will be followed by a period of physiological and metabolic adjustments leading to survival of adapted cells. Protein synthesis is an essential metabolic component of cell survival and growth. The results of this study demonstrate that the relative rates of protein synthesis and the stability and expression of specific proteins are altered when cultured maize cells are exposed to conditions of salinity and water stress. In the initial phase, up to a week, characterized by shock effects of sudden exposure and adjustment to different stress environments, a primary effect appears to be inhibition of amino acid uptake and globular protein synthesis. Synthesis of proteins is hindered more strongly with severe stress than amino acid uptake. In the later phase, after about a week, protein synthesis continues, although at a declining rate, and allows cultures exposed to NaCI in the range of I% and mannitol to about 9.1% to show increases in fresh weight. The abundant steady state protein population is apparently unchanged throughout the test period except that there i~ a random loss of individual proteins after 2 weeks at the highest stress levels studied (Fig. 2). Cells in the later phase regulate the specific expression of certain de nova proteins (Fig. 3). We found only a small fraction of the newly made proteins, about 7 discernible polypeptides, are regulated during stress in maize culture. Three are induced de nova and 4 others indicate a reduced or enhanced level o f ~ sis. While our studies were in progress, the results of a related study in tobacco culture were reported (Singh et al., 1985). These authors compared the protein patterns in cultures with and without exposure to 1% NaCI and describe the synthesis of a 26 kd protein which may be analogous to the 26.2 kd protein we have observed in maize. However, in contrast to maize, they were able to detect synthesis of the tobacco protein in both cultures (control and NaCl-treated) if the cells were labeled with 35S but only in NaCI stressed cells if stained with Coomassie blue. Therefore, the regulation of the 26 kd protein expression seems to differ somewhat in maize and tobacco. Both osmotic and ionic stresses arise from NaCI treatment but only the osmotic stress is expected with mannitol. We find that 3 new proteins of similar molecular weights are induced under both treatments. Increasing the NaCI concentration greater than 0.5% or mannitol greater than 3.6% apparently does not cause any additional changes in protein patterns. Two similar proteins were also found in NaCI and PEG treated tobacco cells (Singh et al., 1985). However, whether these common proteins are encoded by identical genes is nat known. Nevertheless, the findings in maize and tobacco so far suggest that salinity and osmotic stress may generate a common signal which in turn controls the expression of these proteins. The responses elicited by salinity and water stress in cultured plant cells are apparently distinct from those of the widely investigated temperature stress. In tobacco and ather cultures, an increase in temperature causes the synthesis of new proteins within 30 to 60 minutes (Kanabus et al., 1984; Lopata and Gleba, 1985). Therefore, temperature stress is related to a shock effect on cells and leads to a transient, altered gene expression. The role of the heat shock proteins in developing tolerance to temperature stress is not defined. Some indication that the newly made proteins during salinity and water stress may be important in the development of tolerance emerges by an analysis of the timing of the cellular response towards these signals. Unlike the temperature stress, the altered protein pattern seen during salinity and water stress does not occur immediately on exposure. In maize cultures, this is evident after
434 a long exposure time and occurs only in cells that are growing or about to grow. Similar results were also obtained in tobacco where the 26 kd protein was expressed after 12 days of exposure to NaCI and coincided with the growth phase (Singh et al., 1985). A detailed study of the altered proteins would lead ta an understanding of their role(s) in cellular adaptation to salinity and water stress.
Maliga P (1984) Ann IRev Plant Physiol 35:519-542 Meredith CP (1984) In: Gustafson JP (ed) Gene Manipulation in Plant Improvement, 16th Stadler Genetics Symposium Plenum Press, New Yark, pp 503-520 Merrill CR, Goldman D, Sedman SA, Ebert MH (1981) Science 21 I : 11437-1438
ACKNOWLEDGEMENT Murashige T, Skoog F (1962) Physiol Plant 15:473-497 I thank Roger Thorn and Patricia Bryant for technical assistance.
Nabors MW, Gibbs SE, Bernstein CS, Meis ME (1980) Z. Pflanzenphysiol 97:13-17
REFERENCES Ericson MC, Alfinito SH (1984) Plant Physiol 74:506-509
IRains DW, Croughan TP, Stavarek SJ (1980) In: Rains DW, Valentine IRC, Hollander A (eds) Genetic Engineering of Osmoregulation, Plenum Press, New York, pp 279-292
Flick CE (1983) In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of Cell Culture, vol I, MacMillan, New York, pp 393-441
IRamagopal S (1976) Eur J Biochem 69:289-297
Kanabus J, Pikaard CS, Cherry JH 0984) Plant Physiol 75: 639-644 Laemmli UK (1970) Nature 227:680-685 Lopato SV, Gleba YY (1985) Plant Cell Reports 4:19-22
IRamagopal S, Huang B, Marcus A (1977) J Cell Physiol 93:319-330 Singh NK, Handa AK, Hasegawa PM, Bressan IRA (1985) Plant Physiol 79:126-137 Tyagi AK, IRashid A, Maheshwari, SC (1981) Protoplasma 105:327-332
Plant Cell Reports (1986) 5:430-434
© Springer-Verlag 1986
Protein synthesis in a maize callus exposed to NaCI and mannitol Subbanaidu R a m a g o p a l * U.S, Department of Agriculture, Agricultural Research Service, Western Regional Research Centre, Albany, CA 94710, USA Received April 10, 1986 / Revised version received July 18, 1986 - Communicated by I. K. Vasil
Abstract A maize (Zea mays, L) callus was exposed to media containing different levels of NaCI (0 to 3%) and mannitol (0 to 18.2%) for a period of /tweeks, and the changes in growth and protein synthesis were determined. Cells are able to tolerate and grow in NaCI up to I% (0.17 M) or mannitol up to 9.1% (0.5 M), but the relative overall growth rates are about I/6 and I/8 of the control, respectively. Protein synthesis, as assessed by pulselabeling of the cells with 35S-methionine after exposure to the stress reagents at various times of incubation, suggests that the relative rates of amino acid uptake and its incorporation into proteins are inhibited as early as /4 h after exposure, and the extent of inhibition does not increase appreciably until after I week. Severe inhibition of uptake and protein synthesis results from prolonged exposures at growth-inhibitory concentrations of NaCI and mannitol. In general, the overall mean inhibition of cellular uptake and protein synthesis in the first 2-week period are approximately 50% and 35% for the NaCI (1%) and mannitol (7.3 %) treatments, respectively. No detectable differences are apparent in the abundant, steady state protein population as revealed by SDS-PAGE and on staining with Coomassie blue or silver, but random losses of individual proteins occur after 2 weeks at 2% and 3% NaCI and at 18.2% mannitol. Of the newly-synthesized proteins, discernible changes are found in 7 and zt polypeptides in NaCI and mannitol treatmerits, respectively. Apparently three new proteins (74 kd, 28.5 kd, and 26.2 kd) are induced de novo under both treatments. Other proteins (39.5 kd, 39.0 kd, 30 kd, and 16.5 kd) show an increased or decreased level of synthesis. NaCI levels above 0.5% or mannitol levels above 3.6% da not alter the pattern of newly-synthesized proteins. This altered expression of newly-made proteins in the maize callus occurs only after a week of exposure to salinity or osmotic stress and coincides with the cell growth phase. INTRODUCTION The decline in plant productivity due to salinity and drought in saline and arid lands necessitates the development of stress-tolerant crops. Of the various approaches, cell culture, selection for mutants has received increasing attention in recent years (Maliga, 198b,; Meredith, 198/4; Rains e t a l , 1980). Salt-tolerant cell lines have been isolated in a number of plant species (Maliga, 198b,; Flick, 1983), and the tolerant trait has also been claimed to be maintained in regenerated plants of Nicotiana (Nabors et al., 1980) and Datura (Tyagi et al., 19817. Studies so far suggest that both genetic and epigenetic factors may be involved in the development of stresstolerance in cultured cells (Maliga, 1984; Meredith, 1984). * P r e s e n t address:
However, which of these factors is more important and what adaptive mechanisms actually operate in the tolerant cells are not understood. A knowledge of the molecular responses such as gene expression during cellular adaptation to stress would be helpful and might lead to the identification of the altered putative genes. Recently, this approach was applied in a tobacco suspension culture system, and the investigators showed the appearance of specific proteins in saltadapted cells (Ericson and Alfinit% 1984; Singh et al., 1985). Here, 1 report our studies on protein synthesis in a maize cell line treated with NaCI and mannitol to induce salinity and water stress. MATERIALS AND METHODS Cell line. Callus cultures of Zea mays var. Black Mexican Sweet obtained from Z. R. Sung (The University of California, Berkeley) were maintained in the dark at 27*C on Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with I mg per liter of 2, Zt-D and I% agar. NaCI and mannitol were incorporated in the medium. Four to S callus pieces of 25 to 30 mg FW each were placed on a single plate. Three to five plates were used for each treatment. Measurement of in vivo protein synthesis. At the end of treatment period as specified, calli (100 to 200 mg FW) were transferred to I ml of a labeling medium. This medium had the same composition as the corresponding treatment medium without agar and included 100 ~Ci of 3SS-methionine (I,098Ci/mmole, New England Nuclear). The cells were labeled by agitating the tubes for 2 h at IS0 rpm at 25*C, harvested on 2 layers of Miracloth by gentle suction, rinsed thoroughly with cold water, and frozen immediately in liquid nitrogen. Samples for labeling were collected by pooling 5 to 8 independent callus pieces from each treatment. The frozen cells were ground in a mortar under liquid nitrogen and transferred to 200 to It00 ]JI of a buffer consisting of S0 mM Tris HCI (pH 7.5), 10 mM Na2EDTA (pH 7.It), 5% 2-mercaptoethanol, 2% sodium dodecylsulfate (SDS), and 10% glycerol. The samples were mixed in a vortex mixer, boiled for 10minutes, centrifuged for 5minutes in an Eppendorf microcentrifuge (Brinkman Model 5414), end the supernatants saved. Uptake and incorporation were determined as described (Ramagopal et al., 1977). Five to 10 1JI of this supernatant was spotted directly on a glass fiber (Whatman GF/C) disc, dried, and counted in a liquid scintillation counter to determine the total cellular uptake of 35S-methionine. The incorporation of 35S-methionine int 9
USDA/ARS, Experiment Station, HSPA, P.O. Box 1057, Aiea, HI 96701, USA
431 cellular proteins was measured by precipitating 5 to 10 ~1 aliquots of the supernatants with 9% (final concentration) cold trichloroacetic acid (TCA) for 30 to 45 minutes on ice and collecting the precipitated proteins on glass fiber discs. The precipitates were further washed with ice-cold TCA (3 x S ml) and 5 ml of 95% ethanol, dried, and counted. Two to three determinations were made an each point and the means are presented in Tables I and 2. The value of each determination was within 5% of the reported means. Electropharesis of proteins. The proteins extracted as described above were resolved on a 12.5% SDS-polyacrylamide gel (Laemmli, 1970) along with molecular weight standards (Bia Rad Laboratories). The gels were stained with Coomassie blue (Ramagopal, 1976) or silver (Merrill et al., 1981). Radioactive gels, after fixation of proteins, were treated with En3hance (New England Nuclear), dried, and exposed to Kodak X-AR5 film with an intensifying screen (Du Pont).
RESULTS Callus Growth The effects of NaCI and mannitol on the growth of maize cultures were examined at a range of concentrations. Cells were grown on media containing up to 3% (0.SIM) NaCI or up to 18.2% (IM) mannitol for a period of 4 weeks (Fig. I). Control cultures began ta grow in a week (Fig. I A); fresh weight increases were about 2, 7, and 30 fold in I, 2, and It weeks, respectively. Cultures treated with NaCI and mannitol, on the other hand, grew slowly and showed changes in morphology.
Protein Synthesis The relative capacity for protein synthesis was measured soon after the cells were transferred to NaCI or mannitol media and during their subsequent incubation period. The cells were pulse-labeled with 3SS-methionine, and its uptake and incorporation into proteins were determined. The ability of cells to take up the labeled amino acid varied with the stress agent and duration of exposure (Table I). The uptake was reduced by about It0 to 50% after 4 h of NaCI treatment; the reductions were abaut 60 to 80% at later periods. The concentration of NaCI apparently did not have any bearing on the inhibition until I week, but after 2 weeks, NaCI levels above 2% reduced the uptake by greater than 90%. The effects of mannitol on uptake were less severe compared to NaCI. At 3.6%, mannitol had no effect and sometimes stimulated the uptake up to a week, but at 2 week, it caused a reduction of 70%. Higher mannitol concentrations inhibited the uptake up to a week by 20 to 40% and by 70% after 2 weeks. The proportion of cellular uptake which was utilized in protein synthesis was apparently independent of stress duration but indicated marked variation dependent on the stress agent. Approximately 58% of the label taken up was utilized in pratein synthesis in untreated control cultures (Table I). NaCI treatment reduced the protein fraction below control levels by about 42%, 77%, and 87% at I%, 2%, and 3% levels, respectively; mannitol at 3.6% had no effect but caused a reduction by about 25% and 70% respectively, at 7.3% and 14.6%.
200 6O0-
I
[] I Day
m
Table I. Effects of NaCI and mannital on relative rates of 35S-methionine uptake and incorporation into proteins.
B
~10o
• 0
[-1WI 71P, O
'
1NIC,{%) ' 2
"
3
i-lmll FI~•,
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[7nn 0
, O NaCl(%) 51 2
3
l/
E]~
3691182 Mannltol(%)
Fig. I. Effects of NaCI and mannitol on the growth of maize callus. Each FW value is a mean of 25 to 45 separate pieces of the inoculated call[. Panels A and B represent independent experiments. Panel A. Growth during the first 2 week period. Panel B. Callus growth at it weeks. All NaCI-treated cells appeared healthy until l day but those an high salts turned brown at 7 day. Browning of callus was very slight up to I% NaCI, but above 2% NaCI it was extensive. At 0.5% and I% NaCI levels, fresh weight increases were real and delayed somewhat compared to control. Fresh weight increases at 0.5% NaCI were about 3 and 8 fold a t 2 and 4 weeks, respectively. Calli on I% NaCI showed a 2 fold increase in fresh weight in 2 weeks and an additianal 2 fold at the end of 4 weeks. Cells failed to grow at 2 and 3% NaCl. Higher concentrations of mannitol were inhibitory to callus growth; calli started to turn brown in a week at 7.3% or above. There was a limited growth in a week at 3.6% mannitol wh!ch improved later on; fresh weight increases were about S fold in 2 weeks and an additional 3 fold between 2 and 4 weeks. At 7.3% mannitol, a 50% increase in fresh weight was observed after 2 weeks of culture, and it improved to an overall increase of 2 fold at 4 weeks. In other experiments, no significant differences were observed in callus growth at 7.3% or 9.1% mannitol levels.
Treatment Control
It h 100
I day
(58)* 100
(57)
I week 100
2 week
(58) 100
(56)
NaCI(%)
I
60
(34)
29
(29)
73
(50)
42
(37)
2 3
52 56
(13) (8)
22 25
(9) (0.6)
21 18
(19) (12)
II 8
(17) (13)
3.6 7.3
ll3 88
(62) (42)
85
74
(56) (83)
103 64
(76) (52)
31 35
(56) (44)
14.6
61
(19)
46
(24)
70
(13)
36
(16)
Mannitol(%)
The maximal control values (100) for rates of 35S-methionine uptake (expressed as cpm/100 m~ FW cells/2h) were 8.9xl06, 7.2x106, 3.3x106, and 5.2xl0 b for It h, 1 day, I week and 2 week cultures, respectively. * Values in parentheses represent the percentages of the total 35S-methionine counts which were incorporated into the TeA-insoluble protein fraction. The relative rate of amino acid incorporation into proteins as measured by TCA-insoluble radioactivity was significantly altered in treated cultures. Because of the observed differences in label uptake (Table I), the protein synthesis data are compared before and after correction for uptake differences (Table 2), both of which lead to similar conclusions. Increased levels of NaCI inhibited protein synthesis as early as 4 h. The extent of inhibition did not seem to vary with the duration of stress as it varied with the concentration of NaCI employed. Protein synthesis reductions were in excess of 80% above 2% NaCI. Protein synthesis was nat altered at 3.6% mannitol until about 2 weeks. Higher mannitol concentrations reduced the rates but to a lesser extent than the NaCI treatments.
432 Table 2. Effects of NaCI and mannitol on relative rates of protein synthesis.
Steady State and De Novo Proteins
Treatment
4h
I day
I week
2 weeks
Control
I00
IO0
I00
I00
NaCl(%)
1
35
(48) 15
(24)
63
2
12
(17)
4
(7)
7
(12)
3
(7)
3
8
(12)
0.2
(0.5)
4
(7)
2
(3)
(121) 31 (79) 28 (21) I0
(52) (45) (17)
Mannitol(%) 3.6 121 7.3 63 14.6 19
(I06) 83 (71) 56 (27) 20
(95) (71) (29)
137 58 16
(79) 28
(45)
The maximal control values (100) for rates of protein synthesis (expressed as cpm/100mg FW cells/2h) were 5.2x10% 4.1x106, 1.9x106, and 2.9xl06 for 4h, I day, I week, and 2 week cultures, respectively. Values in parentheses represent data after correction for the uptake of label.
Changes in protein synthesis were also assessed after 4 weeks of treatment which indicated the relative effects were quite similar to those observed at 2 week. In addition, no major differences were found between mannitol levels, 7.3% and 9.1% or 14.6% and 18.2% (data not shown).
Fig. 2.
The NaCI and mannitol treatments on individual cellular proteins were assessed by resolving them on SDSp olyacrylamide gels. The cultures were sampled beginning at 4 h of treatment and continued until 4 weeks of incubation. Changes in the steady state population of abundant proteins are compared at 2 and 4 weeks after staining the gels with Coomassie blue (Fig. 2). The protein patterns were all similar for the entire culture period, but cells treated with higher concentrations of NaCI and mannitol started to indicate massive losses of proteins at about 2 weeks (Fig. 2A, lanes 5 and 8; 2B, lanes 4, 5, and 8). Protein lass was evident in 2 weeks at 3% NaCI, but at 4 weeks, it was found at 2% NaCI as well. Mannitol at 18.2% caused protein loss at 2 weeks (Fig. 2A), and the loss was greater on further incubation of cultures (Fig. 2B). Larger amounts of proteins were applied to the gels at treatment levels where protein loss was observed to ascertain whether it was specific. The prateins lost belonged to both low and high molecular weight groups (Fig. 2A and B, arrows). Relatively greater loss occured in NaCI stress than in the mannitol stress. Not all proteins were lost, however. A few protein bands appeared to show intensified staining on stress (Fig. 2A and B, closed circles). Of the proteins that were retained, most were similar in both NaCI and mannitol treatments. No additional differences could be detected in the steady state protein population if stained with a more sensitive silver method (Merrill et al., 1981) (data not shown).
Steady state protein patterns of maize callus exposed to NaCI and mannitol. The cultures were exposed for 2 (A) or 4 (B) weeks, proteins extracted, electrophoresed, and stained with Coomassie blue. Treatments and mg FW of cells giving rise to the protein extract applied on the gel are indicated for each lane: Panel A. (1)Control, 8; (2)0.5% NaCI, 4; (3)1% NaCI, 4; (4)2°,6 NaCI, 20; (5)3% NaCI, 40; (6)3.6% mannitol, 8; (7) 9.1% mannitol, 12; (8) 18.2% mannitol, 40. Panel B. The lane designations are the same as in Panel A except for the amount of protein applied for electrophoresis which is indicated: lane (3) 10; (4) 50; (5) 50; (6) 3; (7) 10; (8) 25. Arrows indicate some of the prominent protein bands which are undetectable after treatments with NaCI or mannitol. Closed circles indicate protein bands which show intensified staining.
433 An analysis of the newly-synthesized proteins revealed changes depending on treatment. As above for steady state proteins, patterns of the newly-made proteins were examined far the entire incubation period beginning 4 h of treatment. Majority of the proteins were similar, and no differences were apparent until I week; however, distinct expressional changes in at least 7 polypeptides were evident after 2 weeks (Fig. 3). Apparently, three new proteins (74 kd, 28.5 kd, and 26.2 kd) were induced de nova in both NaCI and mannitol treated cultures. The 74 kd protein was expressed at about the same level in both treatments, but the two low mw proteins were expressed more strongly in NaCI cultures. The expression of one protein of 30 kd increased with increased levels of NaCI (0.5 and I%) and mannitol (3.6 and 9.1%) but seemed to be depressed at higher levels. Two proteins, 39.5 kd and 39 kd, were not at all made at NaCI levels 2% and above but were continued to be made at a detectable level in all mannitol treatments tested. A newly-made 16.5 kd protein was lacking from all NaCI treatments but was present in all mannitol treatments. No additional changes in protein pattern were apparent in cultures treated far 4 weeks.
Kd
74,0~1b
-66.2
-45,0
-31.0 26.2~
-21.5
16.5-* -14A
Fig. 3.
Patterns of newly-synthesized proteins of maize callus exposed to NaCI and mannital. Cells were treated with NaCI (lanes 1-4) or mannitol lanes 5-7) for 2 weeks and labeled with 5S-methionine and processed as described in materials and methods. Equal amount of TCAprecipitable radioactivity (2 x 105 cpm except I x 105 cpm for lane 7) was applied on the gels far electrophoresis.
Lane (I)control, (2) 0.5% NaCI, (3) I% NaCI, (4) 2% NaCI, (5)3.6% mannitol, (6)9.1% mannitol, (7) 18.2% mannitol.
DISCUSSION Plant cells may react to stress in various ways. Early on, they may be shocked by the stress-treatment and show transient changes which will be followed by a period of physiological and metabolic adjustments leading to survival of adapted cells. Protein synthesis is an essential metabolic component of cell survival and growth. The results of this study demonstrate that the relative rates of protein synthesis and the stability and expression of specific proteins are altered when cultured maize cells are exposed to conditions of salinity and water stress. In the initial phase, up to a week, characterized by shock effects of sudden exposure and adjustment to different stress environments, a primary effect appears to be inhibition of amino acid uptake and globular protein synthesis. Synthesis of proteins is hindered more strongly with severe stress than amino acid uptake. In the later phase, after about a week, protein synthesis continues, although at a declining rate, and allows cultures exposed to NaCI in the range of I% and mannitol to about 9.1% to show increases in fresh weight. The abundant steady state protein population is apparently unchanged throughout the test period except that there i~ a random loss of individual proteins after 2 weeks at the highest stress levels studied (Fig. 2). Cells in the later phase regulate the specific expression of certain de nova proteins (Fig. 3). We found only a small fraction of the newly made proteins, about 7 discernible polypeptides, are regulated during stress in maize culture. Three are induced de nova and 4 others indicate a reduced or enhanced level o f ~ sis. While our studies were in progress, the results of a related study in tobacco culture were reported (Singh et al., 1985). These authors compared the protein patterns in cultures with and without exposure to 1% NaCI and describe the synthesis of a 26 kd protein which may be analogous to the 26.2 kd protein we have observed in maize. However, in contrast to maize, they were able to detect synthesis of the tobacco protein in both cultures (control and NaCl-treated) if the cells were labeled with 35S but only in NaCI stressed cells if stained with Coomassie blue. Therefore, the regulation of the 26 kd protein expression seems to differ somewhat in maize and tobacco. Both osmotic and ionic stresses arise from NaCI treatment but only the osmotic stress is expected with mannitol. We find that 3 new proteins of similar molecular weights are induced under both treatments. Increasing the NaCI concentration greater than 0.5% or mannitol greater than 3.6% apparently does not cause any additional changes in protein patterns. Two similar proteins were also found in NaCI and PEG treated tobacco cells (Singh et al., 1985). However, whether these common proteins are encoded by identical genes is nat known. Nevertheless, the findings in maize and tobacco so far suggest that salinity and osmotic stress may generate a common signal which in turn controls the expression of these proteins. The responses elicited by salinity and water stress in cultured plant cells are apparently distinct from those of the widely investigated temperature stress. In tobacco and ather cultures, an increase in temperature causes the synthesis of new proteins within 30 to 60 minutes (Kanabus et al., 1984; Lopata and Gleba, 1985). Therefore, temperature stress is related to a shock effect on cells and leads to a transient, altered gene expression. The role of the heat shock proteins in developing tolerance to temperature stress is not defined. Some indication that the newly made proteins during salinity and water stress may be important in the development of tolerance emerges by an analysis of the timing of the cellular response towards these signals. Unlike the temperature stress, the altered protein pattern seen during salinity and water stress does not occur immediately on exposure. In maize cultures, this is evident after
434 a long exposure time and occurs only in cells that are growing or about to grow. Similar results were also obtained in tobacco where the 26 kd protein was expressed after 12 days of exposure to NaCI and coincided with the growth phase (Singh et al., 1985). A detailed study of the altered proteins would lead ta an understanding of their role(s) in cellular adaptation to salinity and water stress.
Maliga P (1984) Ann IRev Plant Physiol 35:519-542 Meredith CP (1984) In: Gustafson JP (ed) Gene Manipulation in Plant Improvement, 16th Stadler Genetics Symposium Plenum Press, New Yark, pp 503-520 Merrill CR, Goldman D, Sedman SA, Ebert MH (1981) Science 21 I : 11437-1438
ACKNOWLEDGEMENT Murashige T, Skoog F (1962) Physiol Plant 15:473-497 I thank Roger Thorn and Patricia Bryant for technical assistance.
Nabors MW, Gibbs SE, Bernstein CS, Meis ME (1980) Z. Pflanzenphysiol 97:13-17
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