J. Sci. Fd Agric. 1974,25, 67-71

Amylase Isozymes of Oats (Avena sativa L.)

James B. Smith and Michael D. Bennett

Plant Breeding Institute, Maris Lane, Trumpington, Cambridge, England (Manuscript received 9 July 1973 and accepted 1I September 1973)

Amylase isozymes were studied in six varieties of Avena sativa L. using agar gel electrophoresis. Three distinct zymograms were detected in aqueous extracts of endosperm of 4 to 5-day old seedlings. No intravarietal variation was observed. These results indicate that amylase zymograms may be useful markers for helping in the identification of oat varieties. Using the varieties Maris Tabard and Maris Titan it was found that all the isozymes in 4 to 5-day old seedlings, except one, had the properties of a-amylase. The zymograms of extracts from ungerminated grains ofthese twovarieties, although apparently identical to thoseof4 to 5-day old seedlings, differed in that all the a-isozymes in the former were found to be heat labile whereas in the latter they were heat stable.

1. Introduction

Amylase isozymes have been studied in a number of different cereals and those of wheat and barley have received considerable With the exception of Nummi et aL7, who included a variety (Tammi) of Avena sativa in their comparative study of j-amylases of several different cereal species, oat amylases have been very little studied.

Agar gel electrophoresis offers a simple method for the rapid and effective separation of amylase isozymes. In the present work it has been used to investigate the amylase isozymes in the germinated grain of several varieties of Avena sativa. The original purpose was to investigate the possibility of using amylase isozymes to distinguish between two varieties, Maris Titan and Maris Tabard, bred at the Plant Breeding Institute. Later the work was extended to include some other varieties and to investigate the properties of oat amylase isozymes.

2. Experimental

The six oat varieties Maris Tabard, Maris Titan, Condor, Manod, Rodney and CC/4146/4 used in this study were obtained from the Cereals Department of the Plant Breeding Institute. Both Maris Tabard and Maris Titan were derived from the cross CC/4146/4 x Condor, whereas Rodney and Manod were unrelated to the other varieties and had only the variety Victoria in common in their immediate pedigrees.

The dehusked grains were germinated at 23 "C on moist filter paper in plastic Petri dishes. Seedlings were routinely used after 4 to 5 days of germination either fresh or after

3* 67

68 J. B. Smith and M. D. Bennett

storage at -20 "C. Cold storage did not affect the amylase isozyme pattern. After removal of the shoot and roots a single grain was macerated in a small volume (0.4 ml) of distilled water. After centrifugation the supernatant was used immediately for gel electrophoresis.

Ungerminated grain was ground in a pestle and mortar with a small amount of sand. Samples of the meal were extracted overnight at 10 "C with 0.1 M-phosphate buffer plus 2.5 %, NaC17 in the ratio of 1 ml of buffer for every 1 g of meal. After centrifugation at 38 000 g for 15 min the supernatant was dialysed against 0.01 M-phosphate buffer, pH 7, for 24 h.

Isozymes were separated using 1 % Ionagar (Oxoid) in phosphate buffer, pH 7.7, 0.02 ionic strength.' The same buffer was used in the electrophoresis tank. Sufficient hot agar to give a layer approximately 1-mm thick was pipetted onto either 12 x 8 cm or 7.5 x 5 cm glass plates. After cooling, 1 -cm wide slots were formed in the gel, towards the cathode end, by pressing a double thickness of Whatman 3MM chromatography paper into the gel.

During the course of the electrophoretic separation the tank was kept in a refrigerator at 10 "C. A constant current of 10 mA per small plate or 25 mA for the larger plate was applied for 50 min. After separation the plate was placed in contact with one of similar size coated with 1 soluble starch or fi-limit dextrin, the latter substrate being used for the more specific identification of a-amylase isozymes. The sandwich was incubated at 45 "C for 15 to 30 min and the zymograms developed by staining both gels with I,KI solution.

agar in distilled water plus either 0.5

3. Results

3.1. Zymograms from the germinated grain

Figure I(b) and (c) show typical amylase zymograms of Maris Tabard and Maris Titan, respectively. after 4 to 5 days of germination. Under the conditions of electro- phoresis employed all the isozymes migrated towards the anode.

L

n

.

1 GroupA - +

Figure 1. Amylase zymograms of aqueous extracts of endosperm of 4 to 5-day old seedlings of A. sativa. (a) Variety CC/4146/4 (b) variety Maris Tabard (c) variety Maris Titan. Bands showing high amylase activity are blacked in.

Amylase isoenzymes of oats 69

For convenience of description the isozymes have been divided into three groups A, B and C (see Figure 1). In both varieties group A contained three bands and these were generally relatively weak in activity. A consistent difference between the two varieties was found in group B. Here, although there were three bands present in both, all those of Maris Tabard were always of high activity whereas in Maris Titan only two were highly active, the third always being much less so. Furthermore, the most anodic band in group B was always nearel to the anode in Maris Titan than in Maris Tabard [Figure I(b) and (c)]. In group C there were two bands of similar mobility in both varieties but the most anodic of the pair was not always detected. Several hundred coded grains each of Maris Tabard and Maris Titan were individually analysed and it was always possible, using the group B pattern, to distinguish between the two varieties.

Of the four other varieties included in this study, Manod had a pattern identical to that of Maris Tabard whereas Rodney and Condor gave zymograms identical to that of Maris Titan. The variety CC/4146/4, however, gave a zymogram that was distinct from the others [Figure l(a)]. There were three bands in group A which apparently gave similar mobilities to those of the Maris Tabard and Maris Titan types. Group B contained four bands all of approximately equal activity. The mobility of the most anodal band was similar to that of the most anodal of this group in Maris Titan. Only weak diffuse activity was found in the group C region of CC/4146/4. This variety was derived from a cross between A . sativa and another hexaploid species A . sterilis.

3.2. Characterisation of the isozymes The varieties Maris Tabard and Maris Titan were used in this part of the study to characterise the isozymes. When P-limit dextrin was used as the substrate all bands were present except the most anodal of the group C pair. This band also disappeared when a few drops of lop4 M-mercuric chloride solution were included between the plates during incubation. If an aliquot of the sample was heated at 70 "C for 15 min prior to electro- phoresis the most anodal group C band did not appear. This heat treatment only reduced the intensity of the other bands very slightly and it often resulted in the strong bands of the group B region having sharper outlines. The inclusion of a few drops of 20% sodium hexametaphosphate in the sandwich during incubation to remove the Ca2+ greatly reduced the activity of all bands except the most anodal of group C.

The ability to digest P-limit dextrin, the dependence of activity on the presence of Ca2+, and resistance to heavy metal ions indicate that all the bands were u-amylases,l except for the most anodal of group C. This latter was heat labile, did not attack P-limit dextrin and was inhibited by heavy metal ions. This suggests that it represented a P-amylase is0zyme.l

3.3. Amylases in the ungerminated grain Although the bands were for the most part less distinct and intense, the zymogram of the extract from ungerminated grains of Maris Titan was apparently identical to that found in the grain of a 4 to 5-day old seedling. The activity of the isozymes toward /3-limit dextrin was also identical. When the extract from the ungerminated grain was heated at 70 "C for 15 min, however, all the amylase activity was destroyed. A similar

70 J. B. Smith and M. D. Bennett

result was obtained using Maris Tabard although in this case the original extract contained much less amylase activity.

3.4. Changes during germination

Grains of Maris Titan were sampled after 24 h of germination and aqueous extract of single grains were made (0.1 mllgrain). An aliquot of each extract was heated at 70 "C for 15 min. The zymograms obtained using either soluble starch or P-limit dextrin as the substrate are shown in Figure 2. Only a trace of activity could be detected in the group A region probably due to the low total activity of the extract. The one band in group C was not present in the heated extract. Of the three bands in region B only the one of intermediate mobility was found to be heat stable [Figure 2(b)] although some- times the band of least mobility was represented by a trace of activity in the heated extract.

Origin

U - ( a 1 ( b )

+

Group A

Group B

Group C

Figure 2. Amylase zymograms of aqueous extracts of endosperm of Maris Titan after germination for 24 h. (a) Untreated extract (b) extract after heating at 70 "C for 15 min.

4. Discussion

Three different amylase zymograms were found amongst the six oat varieties studied. Although using amylase zymograms it was possible to distinguish with certainty between the two closely related varieties Maris Tabard and Maris Titan, it was not possible to distinguish between Rodney and Condor nor between Manod and Maris Tabard. Many more varieties would have to be examined to determine the full range of amylase isozyme variation amongst oat varieties and whether such variation might be useful in their classification or identification.

No intravarietal variation in amylase zymograms was found although Williamson, Kleese and Snyder9 found such variation in the esterase zymograms in the three oat varieties that they examined.

The finding of a-amylase in the ungerminated grain may have been due to the fact that some oat varieties have a high level of amylase activity in the grain at maturity or that the grain was not completely ripe. In the latter instance Olered and Jonsson5 found that although the a-amylase found in developing wheat grain is continuously inactivated during the ripening process it could be regenerated again by raising the moisture content of the grain. They also found that the a-amylase in the developing

Amylase isoenzymes of oats 71

grain became more heat sensitive as it became fully ripe and was more heat sensitive than those of germinated wheat. Using polyacrylamide gel electrophoresis Kruger3 found two types of a-amylases of different mobilities in malted wheat. One set was identical with those found in the unripe grain and the other set was produced de nouo during germination.

Perhaps there are two sets of a-amylase in A . sativa, one heat labile set formed during development of the grain and a second heat stable set of similar electrophoretic mobility formed de nouo during germination of the grain. It has been well established that a- amylase production occurs de nouo during the germination of cereal grains under the control of gibberellins.lO* The one heat stable band in group B of the zymogram of Maris Titan after germination for 24 h [Figure 2(b)] may represent the first of the heat stable a-amylases formed during germination, while the other heat labile a-amylases may represent those found in the ungerminated grain. A further study of the amylase isozymes formed during the course of the growth and maturation of the grain is being undertaken to investigate this possibility.

References 1. Frydenberg, 0.; Nielsen, G. Hereditas 1965, 54, 123. 2. Kruger, J. E. Cereal Chem. 1972,49,379. 3. Kruger, J. E. Cereal Chem. 1972,49, 391. 4. Van Onckelen, H. A.; Verbeck, R. Planta 1969, 88,255. 5. Olered, R.; Jonsson, G . J. Sci. Fd Agric. 1970,21, 385. 6. Stoddart, J. L. Planta 1971, 97, 70. 7. Nummi, M.; Daussant, J.; Niku-Paavola, M.-L.; Kalsta, H.; Enari, T.-M. J. Sci. Fd Agric.

1970,21, 258. 8. Cooper, A. H.; Pollock, J. R. A. J. Znst. Brew. 1957, 63, 24. 9. Williamson, J. A,; Kleese, R. A.; Snyder, J. R. Nature, Lond. 1968,220,1134.

10. Tanaka, Y . ; Ito, T.; Akazawa, T. PI. Physiol. Lancaster 1970, 46, 650. 11. Varner, J. E.; Chandra, G. R. Proc. Nut. Acad. Sci. U.S.A. 1964,52, 100.