- Email: [email protected]
Changes in ATP Level in Crowns of Winter Wheat Seedlings during Hardening to Frost
J. PlantP/rysiol. Vol. 136. pp. 635-637(1990)
Short COlnnlunication
Changes in ATP Level in Crowns of Winter Wheat Seedlings during Hardening to Frost ZBIGNIEW RYBKA
Department of Plant Biochemistry and Physiology, Institute of Plant Breeding and Acclimatization, Radzik6w, P.O. Box 1019, 00-950 Warszawa, Poland Received February 21,1990 . Accepted March 7,1990
Summary During 8 days of frost hardening of winter wheat seedlings, the ATP level, as well as AEC and phosphorylation potential, increased in the crowns of frost resistant winter wheat cultivars, whereas these values remained unchanged or decreased in the susceptible cultivars.
Key words: Triticum aestivum L.,frost resistance, hardening, ATP. Abbreviation: AEC
=
adenylate energy charge.
Introduction A significant increase in the ATP level was observed during hardening of winter rape plants (Sobczyk and KacperskaPalacz 1978) and winter wheat (Perras and Sarhan 1984). It seems that respiration is a main energy source at low temperature during hardening (above O°C) since hardiness can be evoked in darkness (Roberts 1985). However, our previous experiments (Rybka 1989) showed that respiration of crowns of winter wheat cultivars adaptable to frost but differing in frost resistance was similar and decreased gradually Crowns are the object during 8 days of hardening at +2 of our special interest since roots and leaves are regenerated from crowns after frost damage. It seemed interesting to compare ATP levels in crowns of winter wheat cultivars differing in frost resistance.
0c.
Material and Methods Four winter wheat cultivars of drastically different frost resistances were used: Mironovska 808 (Tk 50 = - 22 0q, San Pastore (Tk SO = -13 0q (Rybka 1989), Odesska 16 (frost resistance comparable to Mironovska 808 on the basis of field experiments) and Super X (comparable to San Pastore). The seedlings were grown in climatic chambers (type KTLK 1250, GDR). The day/night temperature regime was 18/14 °C and relative humidity 70/80 %, respectively, with a 16h photoperiod, light intensity 120Wm- 2• During © 1990 by Gustav Fischer Verlag, 5tultgan
hardening the regime was changed to + 2°C, 24 h photoperiod, light intensity 40 Wm - 2 and relative humidity 80 %. Crowns of 10-day-old seedlings were extracted before and during 8 days of hardening. Excised crowns (100 mg) were frozen in liquid nitrogen and adenine nucleotide was extracted with cold perchloric acid. After neutralization the final extracts were stored at - 40°C. Concentration of adenine nucleotides was determined luminometrically using the luciferin-luciferase complex according to Hampp (1985). Adenylate energy charge was calculated according to Atkinson (1977). Inorganic phosphate was determined according to Fiske and SubbaRow (1925) and the acid phosphatase activity was assayed according to Moss (1984). The phosphorylation potential ([ATP]/ [ADP]x[Pi]) was calculated according to Kraayenhof (1969).
Results and Discussion ATP content in crowns of frost resistant winter wheat cultivars increased during 8 days of hardening by 50 % and 120 % in Odesska 16 and Mironovska 808 cultivars, respectively (Fig. 1 A). In the frost sensitive cultivars a negligible (cv. Super X) or even substantial (cv. San Pastore) increase in ATP content was observed on the 4th day of hardening, but it dropped below the control level after 8 days of cold treatment (Fig. 1 B). The difference found probably should not be ascribed to ATPase since this enzyme, being a component of plant membranes, rather responds only to membrane injury
636
ZBIGNIEW RYBKA
B
A
..... "5,1200 CIJ ~
>.
c..
....."0
1000
101 VI
CIJ
"0 E
c:
800
Q. ~
ct
600
c
8
4
C
4
B
days of hardening
Table 1: Adenylate energy charge (AEC) in crowns of frost resistant (Mironovska 808 and Odesska 16) and frost sensitive (San Pastore and Super X) cultivars during 8 days of hardening. Adenylate energy charge cultivar
control
hardened
Mironovska 808 Odesska 16 San Pastore Super X
0.85 0.85 0.92 0.98
0.98 0.99 0.92 0.94
4
8
c
days of hardening
Fig. 2: The inorganic phosphate content in crowns of four winter wheat cultivars (symbols are the same as in Fig. 1).
Fig. 1: ATP content in crowns of frost resistant (A) and frost sensitive (B) winter wheat cultivars. Mironovska 808: ,( ¢ ), Odesska 16: (0), San Pastore: (0), Super X: (1:.). Bars indicate ± SD.
(Iswari and Palta 1989), which does not take place under our experimental conditions. The AEC ratio in control plants was high (0.85-0.98) and increased slightly during hardening of the resistant cultivars, whereas in the frost sensitive ones it remained at the same or a slightly lower level than before hardening (Table 1). Concentration of Pi varied in control plants but beginning from the 4th day of hardening the Pi level was significantly higher in crowns of the frost sensitive cultivars (Fig. 2). The activity of non-specific acid phosphatases of resistant (Mironovska 808) cultivar was slightly lower than that of susceptible San Pastore cultivar but the pattern of changes was similar and was neither correlated with inorganic phosphate level nor with frost tolerance (data not shown). It should be emphasized that the phosphorylation potential (Table 2) was up to 5-10 times higher in the frost resistant cultivars as a result of hardening. Phosphorylation potential also proved to be a more sensitive indicator of changes during hardening of wheat plants to water stress compared to the AEC value (Zagdanska, unpub!. data). It is of considerable interest that despite similar patterns of changes in apparent photosynthesis in the green part of seedlings (data unpublished) and similar patterns of changes in respiration both in crowns and leaves (Rybka 1989) and soluble sugar content (data not shown), ATP content increases exclusively during hardening in frost resistant cultivars. It seems that a higher ATP level and higher phosphorylation potential in frost resistant cultivars might explain their higher capability for frost survival and/or regrowth after stress.
Table 2: Phosphorylation potential (in mM- I) of crowns of winter wheat cultivars during 8 days of hardening. Phosphorylation potential (mM-I) cultivar
control
hardened
Mironovska 808 Odesska 16 San Pastore Super X
0.32 0.47 0.38 1.29
2.45 2.06 0.28 0.42
Acknowledgements
I would like to thank Professor Konstancja Raczynska-Bojanowska for valuable comments and critical reading of the manuscript. This work was partially supported by a grant No. 05.02.03. from the Polish Academy of Science.
ATP and frost resistance
References ATKINSON, D. E.: Cellular energy metabolism and its regulation. Academic Press, New York (1977). FISKE, CH. and V. SUBBARow: J. BioI. Chern. 66, 375-400 (1925). HAMPP, R.: Adenosine 5' -diphosphate and adenosine 5' -monophosphate: Luminometric method. In: Methods of Enzymatic Analysis. III ed, vol. VII, 370-379 (1985). BERGMAYER, H. U. (ed.): Verlag Chemie Weinheim, Deerfield Beach, Florida, Basel.
637
ISWARI, S. and J. P. PALTA: Plant Physiol. 90, 1088-1095 (1989). KRAAYENHOF, R.: Biochem. Biophys. Acta 180, 213-215 (1969). Moss, D. W.: «Acid phosphatases.» In: Methods in Enzymatic Analysis, vol. IV, 92-106. BERGMAYER, H. U. ed.: Verlag Chemie Weinheim, Deerfield Beach, Florida, Basel (1984). PERRAS, M. and F. SARHAN: Physiol. Plant. 60, 129-132 (1984). ROBERTS, D. W. A.: Can. J. Plant Sci. 65, 893 -900 (1985). RYBKA, Z.: J. Plant Physiol. 134, 17 -19 (1989). SOBCZYK, E. A. and A. KACPERSKA-PALACZ: Plant Physiol. 62, 875 - 878 (1978).
Short COlnnlunication
Changes in ATP Level in Crowns of Winter Wheat Seedlings during Hardening to Frost ZBIGNIEW RYBKA
Department of Plant Biochemistry and Physiology, Institute of Plant Breeding and Acclimatization, Radzik6w, P.O. Box 1019, 00-950 Warszawa, Poland Received February 21,1990 . Accepted March 7,1990
Summary During 8 days of frost hardening of winter wheat seedlings, the ATP level, as well as AEC and phosphorylation potential, increased in the crowns of frost resistant winter wheat cultivars, whereas these values remained unchanged or decreased in the susceptible cultivars.
Key words: Triticum aestivum L.,frost resistance, hardening, ATP. Abbreviation: AEC
=
adenylate energy charge.
Introduction A significant increase in the ATP level was observed during hardening of winter rape plants (Sobczyk and KacperskaPalacz 1978) and winter wheat (Perras and Sarhan 1984). It seems that respiration is a main energy source at low temperature during hardening (above O°C) since hardiness can be evoked in darkness (Roberts 1985). However, our previous experiments (Rybka 1989) showed that respiration of crowns of winter wheat cultivars adaptable to frost but differing in frost resistance was similar and decreased gradually Crowns are the object during 8 days of hardening at +2 of our special interest since roots and leaves are regenerated from crowns after frost damage. It seemed interesting to compare ATP levels in crowns of winter wheat cultivars differing in frost resistance.
0c.
Material and Methods Four winter wheat cultivars of drastically different frost resistances were used: Mironovska 808 (Tk 50 = - 22 0q, San Pastore (Tk SO = -13 0q (Rybka 1989), Odesska 16 (frost resistance comparable to Mironovska 808 on the basis of field experiments) and Super X (comparable to San Pastore). The seedlings were grown in climatic chambers (type KTLK 1250, GDR). The day/night temperature regime was 18/14 °C and relative humidity 70/80 %, respectively, with a 16h photoperiod, light intensity 120Wm- 2• During © 1990 by Gustav Fischer Verlag, 5tultgan
hardening the regime was changed to + 2°C, 24 h photoperiod, light intensity 40 Wm - 2 and relative humidity 80 %. Crowns of 10-day-old seedlings were extracted before and during 8 days of hardening. Excised crowns (100 mg) were frozen in liquid nitrogen and adenine nucleotide was extracted with cold perchloric acid. After neutralization the final extracts were stored at - 40°C. Concentration of adenine nucleotides was determined luminometrically using the luciferin-luciferase complex according to Hampp (1985). Adenylate energy charge was calculated according to Atkinson (1977). Inorganic phosphate was determined according to Fiske and SubbaRow (1925) and the acid phosphatase activity was assayed according to Moss (1984). The phosphorylation potential ([ATP]/ [ADP]x[Pi]) was calculated according to Kraayenhof (1969).
Results and Discussion ATP content in crowns of frost resistant winter wheat cultivars increased during 8 days of hardening by 50 % and 120 % in Odesska 16 and Mironovska 808 cultivars, respectively (Fig. 1 A). In the frost sensitive cultivars a negligible (cv. Super X) or even substantial (cv. San Pastore) increase in ATP content was observed on the 4th day of hardening, but it dropped below the control level after 8 days of cold treatment (Fig. 1 B). The difference found probably should not be ascribed to ATPase since this enzyme, being a component of plant membranes, rather responds only to membrane injury
636
ZBIGNIEW RYBKA
B
A
..... "5,1200 CIJ ~
>.
c..
....."0
1000
101 VI
CIJ
"0 E
c:
800
Q. ~
ct
600
c
8
4
C
4
B
days of hardening
Table 1: Adenylate energy charge (AEC) in crowns of frost resistant (Mironovska 808 and Odesska 16) and frost sensitive (San Pastore and Super X) cultivars during 8 days of hardening. Adenylate energy charge cultivar
control
hardened
Mironovska 808 Odesska 16 San Pastore Super X
0.85 0.85 0.92 0.98
0.98 0.99 0.92 0.94
4
8
c
days of hardening
Fig. 2: The inorganic phosphate content in crowns of four winter wheat cultivars (symbols are the same as in Fig. 1).
Fig. 1: ATP content in crowns of frost resistant (A) and frost sensitive (B) winter wheat cultivars. Mironovska 808: ,( ¢ ), Odesska 16: (0), San Pastore: (0), Super X: (1:.). Bars indicate ± SD.
(Iswari and Palta 1989), which does not take place under our experimental conditions. The AEC ratio in control plants was high (0.85-0.98) and increased slightly during hardening of the resistant cultivars, whereas in the frost sensitive ones it remained at the same or a slightly lower level than before hardening (Table 1). Concentration of Pi varied in control plants but beginning from the 4th day of hardening the Pi level was significantly higher in crowns of the frost sensitive cultivars (Fig. 2). The activity of non-specific acid phosphatases of resistant (Mironovska 808) cultivar was slightly lower than that of susceptible San Pastore cultivar but the pattern of changes was similar and was neither correlated with inorganic phosphate level nor with frost tolerance (data not shown). It should be emphasized that the phosphorylation potential (Table 2) was up to 5-10 times higher in the frost resistant cultivars as a result of hardening. Phosphorylation potential also proved to be a more sensitive indicator of changes during hardening of wheat plants to water stress compared to the AEC value (Zagdanska, unpub!. data). It is of considerable interest that despite similar patterns of changes in apparent photosynthesis in the green part of seedlings (data unpublished) and similar patterns of changes in respiration both in crowns and leaves (Rybka 1989) and soluble sugar content (data not shown), ATP content increases exclusively during hardening in frost resistant cultivars. It seems that a higher ATP level and higher phosphorylation potential in frost resistant cultivars might explain their higher capability for frost survival and/or regrowth after stress.
Table 2: Phosphorylation potential (in mM- I) of crowns of winter wheat cultivars during 8 days of hardening. Phosphorylation potential (mM-I) cultivar
control
hardened
Mironovska 808 Odesska 16 San Pastore Super X
0.32 0.47 0.38 1.29
2.45 2.06 0.28 0.42
Acknowledgements
I would like to thank Professor Konstancja Raczynska-Bojanowska for valuable comments and critical reading of the manuscript. This work was partially supported by a grant No. 05.02.03. from the Polish Academy of Science.
ATP and frost resistance
References ATKINSON, D. E.: Cellular energy metabolism and its regulation. Academic Press, New York (1977). FISKE, CH. and V. SUBBARow: J. BioI. Chern. 66, 375-400 (1925). HAMPP, R.: Adenosine 5' -diphosphate and adenosine 5' -monophosphate: Luminometric method. In: Methods of Enzymatic Analysis. III ed, vol. VII, 370-379 (1985). BERGMAYER, H. U. (ed.): Verlag Chemie Weinheim, Deerfield Beach, Florida, Basel.
637
ISWARI, S. and J. P. PALTA: Plant Physiol. 90, 1088-1095 (1989). KRAAYENHOF, R.: Biochem. Biophys. Acta 180, 213-215 (1969). Moss, D. W.: «Acid phosphatases.» In: Methods in Enzymatic Analysis, vol. IV, 92-106. BERGMAYER, H. U. ed.: Verlag Chemie Weinheim, Deerfield Beach, Florida, Basel (1984). PERRAS, M. and F. SARHAN: Physiol. Plant. 60, 129-132 (1984). ROBERTS, D. W. A.: Can. J. Plant Sci. 65, 893 -900 (1985). RYBKA, Z.: J. Plant Physiol. 134, 17 -19 (1989). SOBCZYK, E. A. and A. KACPERSKA-PALACZ: Plant Physiol. 62, 875 - 878 (1978).