Thermostabilities of grain ß-amylase and ß-glucanase in Finnish landrace barleys and their putative past adaptedness

Hereditas 132: 1 1 1 - 1 18 (2000) Thermostabilities of grain p-amylase and p-glucanase in Finnish landrace barleys and their putative past adaptedness HANNU AHOKAS and MARJA-LEENA MANNINEN Crops and Soil, Agricultural Research Centre, Jokioinen, Finland Ahokas, H. and Manninen, M.-L. 2000. Thermostabilities of grain (3-amylase and (3-glucanase in Finnish landrace barleys and their putative past adaptednes-Hereditas 132: 11 1118. Lund, Sweden. ISSN 0018-0661. Received November 19, 1999. Accepted March 6, 2000 Thermostability of (3-amylase activity was a general feature in a sample of 32 Finnish barley landraces. One of two Finnish landraces probably contributed the thermostability to cv. 'Pirkka' in crosses performed about 70 years ago. The stability is less evolved in P-glucanase activity although the most tolerant types appeared in landraces and in Pirkka with a Finnish landrace background. Selection'pressure for thermostability in grains may have been a feature of traditional crop management practices among Finns in the past: drying grain crops, including premature barley, above an oven in a special drying house at temperatures exceeding 55"C, and germination in black, sunlit slash-and-burn soils, with a measured surface temperature of 63°C. A positive, though small correlation between the thermotolerance ratios of the two enzymes may be a remnant of their common long selection pressure ending tens of generations prior to collection in the 1960s and 1970s. Hunnu Ahokus, Crops and Soil, Agricultural Research Centre, Myllytie 10, FIN-31600 Jokioinen, Finland. E-mail: [email protected] Samples of Finnish landrace cereals studied previously proved highly variable in several traits, evidently containing a rich variety of genes and combinations of genes. The landrace populations have been mixtures, some approaching mixtures of unique genotypes in the past (AHOKAS 1998; AHOKAS and POUKKULA 1999). The reasons for this variation are evidently historical, environmental and selective (AHOKAS and MANNINEN 2000). Methods of crop and field management prior to about 1930 in Finland may have generated environments which unconsciously selected thermostability of various vital characteristics at germination and harvest. The thermostability of enzymes of malting barley (Hordeum uulgare L.) is generally a desired characteristic, and preferred malting barleys apparently have significant P-amylase thermostability (KIHARA et al. 1998). They also show pedigrees with simple inheritance of thermostability (KIHARA et al. 1998). Since high thermostability of P-amylase exceeding 65 YO remaining activity seems to be a rare but an inherited characteristic (KIHARA et al. 1998, 1999), there is reason to study thermostability in the sample, probably managed with slash-and-burn culture (see HEIKINHEIMO 191 5) and riihi-heated drying (see GROTENFELT 1899; TALVE 1961) still some tens of generations earlier. MATERIAL AND METHODS Plant material The material analyzed in this study, which has been described earlier (AHOKAS and POUKKULA 1999), was from the 1996 harvest and of good quality. Most of the Finnish landrace selections have been accessioned by the US Department of Agriculture, Beltsville, MD: PI 349678-PI 349681, PI 415017-PI 415019, PI 467622-PI 467627 and PI 467629-PI 467653. Sound grains were weighed and hulled partly by hand and further with a 50% H,SO, wash followed by water rinses, and then germinated aseptically on washed, sterile quartz sand in groups of 15 in the dark at 15.5 f 0.5"C for 120 hours. Thereafter, the germinants were homogenised aseptically in a buffer of pH 4.6 containing 40 mM sodium acetate, 40 mM sodium phosphate and 0.001 % sodium azide as previously described (AHOKAS and POUKKULA 1999). The extract supernatants were stored at -70°C or temporarily at -20°C until used. The assays were replicated and replicates which differed by more than four percentage points were analyzed for a third time. All the results are given as means of the determinations. 112 H. Ahokas and M.-L. Manninen Hereditas 132 (2000) Assay of @-amylase Aliquots (40 pl) of the extract were mixed with 3960 pl of a cold buffer of 50 mM MOPS, pH 7.0 with 1 % BSA (Sigma A-751 1). A sample of 200 p1 was kept on ice and another heated for 30 min at 56.7"C in a thermostatic circulator (LKB 2219 Multitemp I1 Thermostatic Circulator using 20 YO Shell Antifreeze 402 coolant in the water bath). The actual temperature ranged from 56.3 to 57.0"C during the incubation as measured by the instrument and an external thermocouple (Pt 1000, Knick). Samples of 25 pl of the heated and +0"C control samples were further diluted with 225 p1 of buffer B (100 mM maleic acid, 1 mM EDTA, 0.1% w/v of BSA, NaOH until pH 6.2 was reached and 0.02% sodium azide) and assayed with a p -nitrophenyl maltopentaoside substrate containing a-glucosidase purchased from Megazyme. Table 1. p-Amylase activity in extracts of germinated grains after 30 min incubation at 56.7"C Landrace or reference Remaining activity (ratio) Activity without incubation at 56.7"C (arbitrary units for grain mass) Landrace selections HA 22 HA 44 HA 52 HA 9 HA 20 HA 10 HA 38 HA 31 HA 42 HA 53 HA 17 HA 29 HA 19 HA 49 HA 18 HA 40 HA 9-63-4 HA 70-3 HA 6-33-02 HA 9-63-8 HA 70-2 HA 5 HA 48 HA 11 HA 12 HA 33 HA 9-63-2 HA 9-63-1 HA 3 HA 14 HA 45 Global barleys Haruna Nijo Pirkka Noire 2R Montpellier PI 391421 Adorra F(6, lines a HA 52 x Adorra HA 52 x Adorra HA 52 x Adorra HA 52xAdorra HA 52 x Adorra HA 146-04-1 0.90 0.82 0.77 0.77 0.76 0.76 0.75 0.75 0.75 0.74 0.74 0.74 0.74 0.73 0.73 0.73 0.73 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.71 0.71 0.71 0.70 0.41 0.39 0.87 0.75 0.40 0.38 0.38 0.65 0.44 0.41 0.40 0.40 0.62 0.74 0.69 1.63 0.86 0.49 1.05 1.13 0.78 1.01 0.54 0.47 0.51 0.98 0.68 1.12 0.77 1.03 0.64 0.65 0.48 0.89 0.52 0.98 0.81 1.08 0.81 0.61 0.89 0.76 0.24 0.40 0.66 0.68 0.36 0.36 0.57 0.34 0.36 0.30 0.88 0.61 a Grains from several F, plants. Hereditas 132 (2000) Enzyme thermostability in barley landraces 1 13 Table 2. Spearman coefjcients of rank correlution between ratios of p-glucanase thermostability and other independent measurements in the 32 barley landrace samples Second variable rs Significance Activity of P-glucanase without 0.140 NS (P = 0.56) heat treatment (for grain mass) heat treatment (for extract volume) Activity of b-glucanase without 0.058 NS (P = 0.75) Thermostability of P-amylase 0.277 P = 0.12 Thermostability of P-amylase 0.351 P = 0.057 excluding two extreme variants (Fig. 2) ' Without heat treatment, activity of P-glucanase for grain mass vs extract volume, rs = 0.944, P < 0.001. The assay was conducted according to the supplier's instructions and took 10 niin at 40°C. The dilutions of the extracts were 1000-fold for the assay, diluting putative endogenous thermoprotecting molecules, e.g. maltose (TAKAHATA et al. 1994), and enzyme inhibitors to insignificant levels. Assay of B -glucanase Melted and well-mixed extracts were diluted 3.76-fold with Na-acetate buffer (25 mM, 0.02Y0 wjv of Naazide, final pH 4.43) and 1 YO w/v of BSA (Sigma A-751 1). Samples of 550 pl were either heat-treated for 15 min at + 45.0"C as described or kept on ice. Both the samples were left to stand for 30 min at room temperature, with subsequent assaying of 500 p1 at +30°C for 15 rnin with a Beta-Glucazyme tablet (Megazyme) based on Azurine-crosslinked barley Pglucan. The reaction was terminated with 6 ml of 1 YO w/v Trizma base in water, vortexed twice at 5 min intervals, filtered (Whatman 1, 0 9 cm) and absorbances were measured at 590 nm as instructed by the supplier (Megazyme). The final assay pH was 4.8 at 30"C, and was maintained during the heat treatment. In Na-acetate buffer the maximal activity has been observed at pH 5 (KOTAKE et al. 1997). Absorbances were determined in arbitrary units based on the extract volume or the original grain mass. RESULTS p-amylase The activity remaining after heating is presented in Table 1 as the ratio for 32 landrace seIections, five global barleys and five lines of the cross HA 52 x 'Adorra'. Among the global barleys, 'Haruna Nijo', known to have thermostable P-amylase based on extracts of ungerminated grains (KIHARA et al. 1998), appeared to have thermostable P-amylase in this study of germinated samples (Table 1). 'Pirkka' (also studied as a4459), known to have highly active P-amylase (SIMBERG 1950; ALLISON and SWANSTON 1974), proved to have highly thermostable P-amylase in this study, while the other global barleys have the lowest ratios, with levels ranging from 0.38 to 0.40. Fifty per cent of the parentage of Pirkka is from two Finnish landraces (SIMBERG 1950; KIVI 1969), the Specific parental lines of landraces crossed about 70 years ago not being maintained. The ratio distribution of the landrace samples varies from 0.38 to 0.90 with a mean SEM of 0.72 f 0.09, their total distribution deviating highly significantly from normality (x' = 511, P << 0.001). The central fraction, landraces with the two highest and two lowest ratios removed, ranges from 0.70 to 0.77 with a mean SEM of 0.73 f 0.003, and fits a normal distribution (x2 = 2.154, P > 0.80). This suggests that the range of 0.70 to 0.77 is produced by a single allele or several allele types having the same effect. There seem to be other alleles involved, putatively one causing 0.39 to 0.41 ratios, and two others, one giving a ratio of 0.81 and the other giving 0.90 (Table 1). There is no correlation between the ratio of thermostability and the total unheated P-amylase activity in a given volume of the sample (rs = 0.196, P > 0.30), or between the ratio and the activity per unit of grain mass in the sample (rs = 0.176, P > 0.40). p -Glucanuse The activity of P-glucanase is in general less thermostable than that of P-amylase. The activity of the non-heat-treated extracts varied from 0.51 to 1.29 arbitrary units per ml in the samples of the 32 landraces and from 0.75 to 1.21 arbitrary units in the samples of the five global barleys. Relative to grain mass, the variations in activity were 0.80 to 2.24 arbitrary units in the 32 landrace samples and 1.05 to 1.86 arbitrary units in the five global barley samples. The correlation of these activity determinations of the 32 landraces was rs =0.944 (P < O.OOl), and that of the five global barleys rs = 0.60 (NS) (Table 2). The ratios of the activity of the remaining heattreated (15 rnin at 45°C) P-glucanase to the original activity varied from 0.52 to 0.81 with a mean of 0.67 in the 32 landraces, from 0.40 to 0.82 with a mean of 0.59 in the five global barleys. Among the global barleys, Pirkka, with a 50% Finnish landrace background, has the highest remaining activity ratio of 0.82, the other ratios being 0.71 (Haruna Nijo), 0.59 (Adorra), 0.45 (Noire 2R Montpellier) and 0.40 (PI 391421). 114 H. Ahokus and M.-L. Munninen Hereditas 132 (2000) The distribution of thermostability in the landrace sample (Fig. 1) deviates from normality due to flatness and is bimodal with central modes of 0.61 and 0.73. Various correlations are presented in Table 2: there is a small positive correlation between P-amylase and P-glucanase thermostabilities, rs = 0.277, P = 0.12, and if two extreme variants are exluded, rs = 0.351, P = 0.057 (Fig. 2). DISCUSSION -amylase High thermostability of P-amylase in barley cultivars appeared rare and displayed inheritance in the known pedigrees (KIHARA et al. 1998). In these landraces, the total P-amylase activity for grain mass or soluble protein is highly variable (AHOKAS and POUKKULA 1999). The lack of correlation indicates that activity level and thermostability are separate phenomena and probably have a different genetic basis. The ratios of the five F(6) lines of the cross HA 52 x Adorra, parents with 0.77 and 0.38 ratios, respectively (Table l), indicate that the thermostability ratio has a simple inheritance as shown by other material (KIHARA et al. 1998). The line with a 0.65 ratio may still have a heterogeneous minority of grains with the low-ratio allele. Allelic differences in the final amino acyl sequence P-amylases have been detected in barley (KREIS et al. 1987; ERKKILA et al. 1998) or induced in cloned barley sequencies (OKADA et al. 1995). Amino acyl residue changes have been found to confer thermostability (OKADA et al. 1995; EGLINTON et al. 1998; MIKAMI et al. P-Amylases as proteins appear to be multifunctional in various plant species and their different tissues (PAN et al. 1988; AHOKAS and NASKALI 1990; GANA et al. 1998), the enzyme activity not necessarily being the objective of natural selection e.g. under thermostress. 1999). REMAINING ACTIVITY (%I 50 55 60 65 70 75 80 4 % ic 33 a Y 0 12 = E 1 0 g 0.25 i? 6 0.50 3: 0.75 t 2 1.00 1.25 1.50 v) c IF U U -I - u_ ~- I 1 I I I I II t I' Fig. 1. The bimodal distribution of the remaining activity of P-glucanase percentages among the 32 landrace lines (upper plot), and the original untreated activities (lower plot). Hereditas 132 (2000) Enzyme thermostabilitv in barley landraces 115 em Ip ** ** ** ** *.* I 1 1 I I I 40 50 60 70 80 90 B-AMYLASE, REMAIIUING ACTIVITY (%) Fig. 2. The distributions of B-amylase and /3-glucanase thermostabilities in the 32 landrace lines suggesting an influence of past coadaptation. Spearman coefficients of rank correlation, rs = 0.277, P = 0.12 for the whole sample; if the two extreme variants to the left are excluded, rs = 0.351, P = 0.057. - Glucanase The activity of unheated P-glucanase showed less variation than beta-amylase or a-amylase (AHOKAS and POUKKULA 1999). This holds true for a wild barley sample of 257 H. spontaneum entries (AHOKAS and POUKKULA 1999). Since P-glucanase loosens cellular walls, increasing permeation (H~J and FINCHER 1995), its excessive activity results in the danger of leakages from the germinating grain may also be a disadvantage. While 1 + 3-P-glucanase has pathogenesis-related effects against fungi, increasing their cellwall permeability (see e.g. GRENIER et al. 1999), the 1 -+ 3,l -+ 4-P-glucanase makes the cell walls of the wetted and germinating grain tissue more susceptible to invading organisms. Up to 5 QTLs for finished malt glucanase and 3 QTLs for green malt glucanase were detected (HAN et al. 1995) with two structural genes for isoenzymes of (1 + 3,1+ 4)-P-glucanase (LITTS et al. 1990; WOLF 1992). One of these, EII, is restricted to the aleurone layer of germinated grain, while EI is also transcribed in scutalla on young leaves and roots at germination in addition to aleurone (SLAKESKI et al. 1990; SLAKESKI and FINCHER 1992). The P-glucanase isoenzyme I1 was found to be glycosylated with 3.6% carbohydrate (WOODWARD and FINCHER 1982). Glycosylation may be the source of thermostability in bacterial P-glucanase (OLSEN and THOMSEN 199 1) and many other types of proteins (e.g. Gu et al. 1989; NAKAMURA et al. 1998; YANEz et al. 1998). The level of glycosylation is possibly subject to multigenic variation. The observed bimodality suggests two alleles, perhaps two types of glycosylation EII gene product, and may also mean a more complicated dependence with two levels of EI activity masking the EII activity levels. The unheated and heated activity did not show any correlation (Table 2), suggesting that thermostability is independent of activity in this landrace sample. The high thermostability found in Pirkka, with a 50% landrace parentage (SIMBERG 1950; KIVI 1969), probably has its origins in Finnish landraces. This small sample does not necessarily reveal either the extreme activities or the thermostability of the past variation in the Finnish landraces. A genetically modified bacterial P-glucanase has high thermal sta- 116 H. Ahokas and M.-L. Manninen Hereditas 132 (2000) bility (JENSEN et al. 1996, 1998). Transgenic barley expressing bacterial P-glucanase has shown stability of the gene over a few generations (JENSEN et al. 1998) and may hence serve as an artificial alternative to the endogeneous resource in barley, although landraces have not yet been throughly screened. General discussion Selection of stable protein forms by repeated external heat may have occurred in the landraces. There are two stages during which Finnish landrace cereals were often subjected to heat in the past. The harvested mature and premature straws were frequently dried over a special oven called a kiuas, giving off perfusive smoke in a special building called a riihi (TALVE 1961). During such drying, crops were commonly subjected to initial temperatures of 55-60°C; the temperatures were later raised, and excessive heating sometimes occurred (GROTENFELT 1899, 1922). Viable grain tissues were sometimes subjected to denaturating heat. The other stage at which grains may have been subjected to extra heating occurred at germination in black slash-and-burn soils. Different variants of burning as a mode of cultivation (e.g. burn over peatland) did not end in Finland until the 1940s (AHOKAS and MANNINEN 2000) and burning was the prevailing method of field management in the past (HEIKINHEIMO 1915). A dark soil surface, such as that of a burned area, absorbs more solar radiation and thus becomes relatively hot. During different summers in Finland at N latitudes of 61'40' and 61 '52', respectively, the maximum temperatures measured in the surface layer of burned black soil has been 52.8"C (LIPAS and MAKI-PErAYs 1961; VIRO 1974), and up to 63°C on the soil surface (VAARTAJA 1949). In the past, barley commonly germinated in early June, and hence the burned soils served as a heat-selective agent due to their darkness. Soil temperature maxima exceeding 50°C would be exceptional during the germination season even in the subtropical desert habitats of wild barley, but are reached for barley at the seasonal end or post-seasonally (GUTTERMAN 1997). Wild barley has also been a source of thermostability in grain P-amylase (EGLINTON et al. 1998; AHOKAS and NASKALI, unpublished). Due to the apparent multifunction of barley P-amylase, other reasons for the enzyme thermostability cannot be excluded. The barley enzyme P-glucanase is induced at germination (e.g. BRUNSWICK et al. 1987; SLAKESKI and FINCHER 1992). Therefore, slash-and-burn management putatively provided a thermoselective environment for barley. The 1 + 3,l +4+-glucanase isoenzymes EI and EII are the principle activities expected to appear in samples germinated for five days (BRUNSWICK et al. 1987; LOI et al. 1987; MCFADDEN et a]. 1988), with substrate specificity towards mixed-linked 1 -+ 3,l -+ 4-P-glucans (HBJ and FINCHER 1995). Malted barley (1 -3,l +4)-P-glucanases were found to be thermolabile (BRUNSWICK et al. 1987). The significance for brewing is indicated by the fact that the activity of P-glucanase during malting is positively correlated with malt extract (STUART et al. 1988). The fate of cereal landraces It has turned out to be a substantial loss for local plant breeders that the Finnish landraces, themselves a part of the national heritage, have not been adequately maintained. 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