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

He edi as 132: 1 1 1 - 1 18 (2000) The mos abili ies o g ain p-amylase and p-glucanase in Finnish land ace ba leys and hei pu a i e pas adap edness HANNU AHOKAS and MARJA-LEENA MANNINEN C ops and Soil, Ag icul u al Resea ch Cen e, Jokioinen, Finland Ahokas, H. and Manninen, M.-L. 2000. The mos abili ies o g ain (3-amylase and (3-glucanase in Finnish land ace ba leys and hei pu a i e pas adap ednes-He edi as 132: 11 1- 118. Lund, Sweden. ISSN 0018-0661. Recei ed No embe 19, 1999. Accep ed Ma ch 6, 2000 The mos abili y o (3-amylase ac i i y was a gene al ea u e in a sample o 32 Finnish ba ley land aces. One o wo Finnish land aces p obably con ibu ed he he mos abili y o c . 'Pi kka' in c osses pe o med abou 70 yea s ago. The s abili y is less e ol ed in P-glucanase ac i i y al hough he mos ole an ypes appea ed in land aces and in Pi kka wi h a Finnish land ace backg ound. Selec ion'p essu e o he mos abili y in g ains may ha e been a ea u e o adi ional c op managemen p ac ices among Finns in he pas : d ying g ain c ops, including p ema u e ba ley, abo e an o en in a special d ying house a empe a u es exceeding 55"C, and ge mina ion in black, sunli slash-and-bu n soils, wi h a measu ed su ace empe a u e o 63°C. A posi i e, hough small co ela ion be ween he he mo ole ance a ios o he wo enzymes may be a emnan o hei common long selec ion p essu e ending ens o gene a ions p io o collec ion in he 1960s and 1970s. Hunnu Ahokus, C ops and Soil, Ag icul u al Resea ch Cen e, Mylly ie 10, FIN-31600 Jokioinen, Finland. E-mail: [email p o ec ed] Samples o Finnish land ace ce eals s udied p e i- ously p o ed highly a iable in se e al ai s, e i- den ly con aining a ich a ie y o genes and combina ions o genes. The land ace popula ions ha e been mix u es, some app oaching mix u es o unique geno ypes in he pas (AHOKAS 1998; AHOKAS and POUKKULA 1999). The easons o his a ia ion a e e iden ly his o ical, en i onmen al and selec i e (AHOKAS and MANNINEN 2000). Me hods o c op and ield managemen p io o abou 1930 in Finland may ha e gene a ed en i onmen s which un- consciously selec ed he mos abili y o a ious i al cha ac e is ics a ge mina ion and ha es . The he - mos abili y o enzymes o mal ing ba ley (Ho deum uulga e L.) is gene ally a desi ed cha ac e is ic, and p e e ed mal ing ba leys appa en ly ha e signi ican P-amylase he mos abili y (KIHARA e al. 1998). They also show pedig ees wi h simple inhe i ance o he - mos abili y (KIHARA e al. 1998). Since high he - mos abili y o P-amylase exceeding 65 YO emaining ac i i y seems o be a a e bu an inhe i ed cha ac e - is ic (KIHARA e al. 1998, 1999), he e is eason o s udy he mos abili y in he sample, p obably man- aged wi h slash-and-bu n cul u e (see HEIKINHEIMO 191 5) and iihi-hea ed d ying (see GROTENFELT 1899; TALVE 1961) s ill some ens o gene a ions ea lie . MATERIAL AND METHODS Plan ma e ial The ma e ial analyzed in his s udy, which has been desc ibed ea lie (AHOKAS and POUKKULA 1999), was om he 1996 ha es and o good quali y. Mos o he Finnish land ace selec ions ha e been acces- sioned by he US Depa men o Ag icul u e, Bel s ille, MD: PI 349678-PI 349681, PI 415017-PI 415019, PI 467622-PI 467627 and PI 467629-PI 467653. Sound g ains we e weighed and hulled pa ly by hand and u he wi h a 50% H,SO, wash ollowed by wa e inses, and hen ge mina ed asep ically on washed, s e ile qua z sand in g oups o 15 in he da k a 15.5 0.5"C o 120 hou s. The ea e , he ge minan s we e homogenised asep ically in a bu e o pH 4.6 con aining 40 mM sodium ace a e, 40 mM sodium phospha e and 0.001 % sodium azide as p e- iously desc ibed (AHOKAS and POUKKULA 1999). The ex ac supe na an s we e s o ed a -70°C o empo a ily a -20°C un il used. The assays we e eplica ed and eplica es which di e ed by mo e han ou pe cen age poin s we e analyzed o a hi d ime. All he esul s a e gi en as means o he de e mina ions. 112 H. Ahokas and M.-L. Manninen He edi as 132 (2000) Assay o @-amylase Aliquo s (40 pl) o he ex ac we e mixed wi h 3960 pl o a cold bu e o 50 mM MOPS, pH 7.0 wi h 1 % BSA (Sigma A-751 1). A sample o 200 p1 was kep on ice and ano he hea ed o 30 min a 56.7"C in a he mos a ic ci cula o (LKB 2219 Mul i emp I1 The mos a ic Ci cula o using 20 YO Shell An i eeze 402 coolan in he wa e ba h). The ac ual empe a- u e anged om 56.3 o 57.0"C du ing he incuba- ion as measu ed by he ins umen and an ex e nal he mocouple (P 1000, Knick). Samples o 25 pl o he hea ed and +0"C con ol samples we e u he dilu ed wi h 225 p1 o bu e B (100 mM maleic acid, 1 mM EDTA, 0.1% w/ o BSA, NaOH un il pH 6.2 was eached and 0.02% sodium azide) and assayed wi h a p -ni ophenyl mal open aoside subs a e con- aining a-glucosidase pu chased om Megazyme. Table 1. p-Amylase ac i i y in ex ac s o ge mina ed g ains a e 30 min incuba ion a 56.7"C Land ace o e e ence Remaining ac i i y ( a io) Ac i i y wi hou incuba ion a 56.7"C (a bi a y uni s o g ain mass) Land ace selec ions 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 ba leys Ha una Nijo Pi kka Noi e 2R Mon pellie PI 391421 Ado a F(6, lines a HA 52 x Ado a HA 52 x Ado a HA 52 x Ado a HA 52xAdo a HA 52 x Ado a 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 G ains om se e al F, plan s. He edi as 132 (2000) Enzyme he mos abili y in ba ley land aces 1 13 Table 2. Spea man coe jcien s o ank co elu ion be ween a ios o p-glucanase he mos abili y and o he independen measu emen s in he 32 ba ley lan- d ace samples Second a iable s Signi icance Ac i i y o P-glucanase wi hou 0.140 NS (P = 0.56) hea ea men ( o g ain mass) hea ea men ( o ex ac olume) Ac i i y o b-glucanase wi hou 0.058 NS (P = 0.75) The mos abili y o P-amylase 0.277 P = 0.12 The mos abili y o P-amylase 0.351 P = 0.057 excluding wo ex eme a i- an s (Fig. 2) ' Wi hou hea ea men , ac i i y o P-glucanase o g ain mass s ex ac olume, s = 0.944, P < 0.001. The assay was conduc ed acco ding o he supplie 's ins uc ions and ook 10 niin a 40°C. The dilu ions o he ex ac s we e 1000- old o he assay, dilu ing pu a i e endogenous he mop o ec ing molecules, e.g. mal ose (TAKAHATA e al. 1994), and enzyme in- hibi o s o insigni ican le els. Assay o B -glucanase Mel ed and well-mixed ex ac s we e dilu ed 3.76- old wi h Na-ace a e bu e (25 mM, 0.02Y0 wj o Na- azide, inal pH 4.43) and 1 YO w/ o BSA (Sigma A-751 1). Samples o 550 pl we e ei he hea - ea ed o 15 min a + 45.0"C as desc ibed o kep on ice. Bo h he samples we e le o s and o 30 min a oom empe a u e, wi h subsequen assaying o 500 p1 a +30°C o 15 nin wi h a Be a-Glucazyme able (Megazyme) based on Azu ine-c osslinked ba ley P- glucan. The eac ion was e mina ed wi h 6 ml o 1 YO w/ T izma base in wa e , o exed wice a 5 min in e als, il e ed (Wha man 1, 0 9 cm) and ab- so bances we e measu ed a 590 nm as ins uc ed by he supplie (Megazyme). The inal assay pH was 4.8 a 30"C, and was main ained du ing he hea ea men . In Na-ace a e bu e he maximal ac i i y has been obse ed a pH 5 (KOTAKE e al. 1997). Abso bances we e de e mined in a bi a y uni s based on he ex ac olume o he o iginal g ain mass. RESULTS p-amylase The ac i i y emaining a e hea ing is p esen ed in Table 1 as he a io o 32 land ace seIec ions, i e global ba leys and i e lines o he c oss HA 52 x 'Ado a'. Among he global ba leys, 'Ha una Nijo', known o ha e he mos able P-amylase based on ex ac s o unge mina ed g ains (KIHARA e al. 1998), appea ed o ha e he mos able P-amylase in his s udy o ge mina ed samples (Table 1). 'Pi kka' (also s udied as a4459), known o ha e highly ac i e P-amylase (SIMBERG 1950; ALLISON and SWANSTON 1974), p o ed o ha e highly he mos able P-amylase in his s udy, while he o he global ba leys ha e he lowes a ios, wi h le els anging om 0.38 o 0.40. Fi y pe cen o he pa en age o Pi kka is om wo Finnish land aces (SIMBERG 1950; KIVI 1969), he Speci ic pa en al lines o land aces c ossed abou 70 yea s ago no being main ained. The a io dis ibu ion o he land ace samples a ies om 0.38 o 0.90 wi h a mean SEM o 0.72 0.09, hei o al dis ibu ion de ia ing highly signi ican ly om no mali y (x' = 511, P << 0.001). The cen al ac ion, land aces wi h he wo highes and wo lowes a ios emo ed, anges om 0.70 o 0.77 wi h a mean SEM o 0.73 0.003, and i s a no mal dis ibu ion (x2 = 2.154, P > 0.80). This sugges s ha he ange o 0.70 o 0.77 is p oduced by a single allele o se e al allele ypes ha ing he same e ec . The e seem o be o he alleles in ol ed, pu a- i ely one causing 0.39 o 0.41 a ios, and wo o he s, one gi ing a a io o 0.81 and he o he gi ing 0.90 (Table 1). The e is no co ela ion be ween he a io o he - mos abili y and he o al unhea ed P-amylase ac i i y in a gi en olume o he sample ( s = 0.196, P > 0.30), o be ween he a io and he ac i i y pe uni o g ain mass in he sample ( s = 0.176, P > 0.40). p -Glucanuse The ac i i y o P-glucanase is in gene al less he - mos able han ha o P-amylase. The ac i i y o he non-hea - ea ed ex ac s a ied om 0.51 o 1.29 a bi a y uni s pe ml in he samples o he 32 land aces and om 0.75 o 1.21 a bi a y uni s in he samples o he i e global ba leys. Rela i e o g ain mass, he a ia ions in ac i i y we e 0.80 o 2.24 a bi a y uni s in he 32 land ace samples and 1.05 o 1.86 a bi a y uni s in he i e global ba ley samples. The co ela ion o hese ac i i y de e mina ions o he 32 land aces was s =0.944 (P < O.OOl), and ha o he i e global ba leys s = 0.60 (NS) (Table 2). The a ios o he ac i i y o he emaining hea - ea ed (15 nin a 45°C) P-glucanase o he o iginal ac i i y a ied om 0.52 o 0.81 wi h a mean o 0.67 in he 32 land aces, om 0.40 o 0.82 wi h a mean o 0.59 in he i e global ba leys. Among he global ba leys, Pi kka, wi h a 50% Finnish land ace back- g ound, has he highes emaining ac i i y a io o 0.82, he o he a ios being 0.71 (Ha una Nijo), 0.59 (Ado a), 0.45 (Noi e 2R Mon pellie ) and 0.40 (PI 391421). 114 H. Ahokus and M.-L. Munninen He edi as 132 (2000) The dis ibu ion o he mos abili y in he land ace sample (Fig. 1) de ia es om no mali y due o la ness and is bimodal wi h cen al modes o 0.61 and 0.73. Va ious co ela ions a e p esen ed in Table 2: he e is a small posi i e co ela ion be ween P-amylase and P-glucanase he mos abili ies, s = 0.277, P = 0.12, and i wo ex eme a ian s a e exluded, s = 0.351, P = 0.057 (Fig. 2). DISCUSSION -amylase High he mos abili y o P-amylase in ba ley cul i a s appea ed a e and displayed inhe i ance in he known pedig ees (KIHARA e al. 1998). In hese land aces, he o al P-amylase ac i i y o g ain mass o soluble p o ein is highly a iable (AHOKAS and POUKKULA 1999). The lack o co ela ion indica es ha ac i i y le el and he mos abili y a e sepa a e phenomena and p obably ha e a di e en gene ic basis. The a ios o he i e F(6) lines o he c oss HA 52 x Ado a, pa en s wi h 0.77 and 0.38 a ios, e- spec i ely (Table l), indica e ha he he mos abili y a io has a simple inhe i ance as shown by o he ma e ial (KIHARA e al. 1998). The line wi h a 0.65 a io may s ill ha e a he e ogeneous mino i y o g ains wi h he low- a io allele. Allelic di e ences in he inal amino acyl sequence P-amylases ha e been de ec ed in ba ley (KREIS e al. 1987; ERKKILA e al. 1998) o induced in cloned ba ley sequencies (OKADA e al. 1995). Amino acyl esidue changes ha e been ound o con e he mos abili y (OKADA e al. 1995; EGLINTON e al. 1998; MIKAMI e al. P-Amylases as p o eins appea o be mul i unc- ional in a ious plan species and hei di e en issues (PAN e al. 1988; AHOKAS and NASKALI 1990; GANA e al. 1998), he enzyme ac i i y no necessa ily being he objec i e o na u al selec ion e.g. unde he mos ess. 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 2 1.00 1.25 1.50 ) c I- F U U -I - u_ ~- I 1 I I I I II I' Fig. 1. The bimodal dis ibu ion o he emaining ac i i y o P-glucanase pe cen - ages among he 32 land ace lines (uppe plo ), and he o iginal un ea ed ac i i ies (lowe plo ). He edi as 132 (2000) Enzyme he mos abili in ba ley land aces 115 em Ip ** ** ** ** *.* I 1 1 I I I 40 50 60 70 80 90 B-AMYLASE, REMAIIUING ACTIVITY (%) Fig. 2. The dis ibu ions o B-amylase and /3-glucanase he mos abili ies in he 32 land ace lines sugges ing an in luence o pas coadap a ion. Spea man coe icien s o ank co ela ion, s = 0.277, P = 0.12 o he whole sample; i he wo ex eme a ian s o he le a e excluded, s = 0.351, P = 0.057. - Glucanase The ac i i y o unhea ed P-glucanase showed less a ia ion han be a-amylase o a-amylase (AHOKAS and POUKKULA 1999). This holds ue o a wild ba ley sample o 257 H. spon aneum en ies (AHOKAS and POUKKULA 1999). Since P-glucanase loosens cel- lula walls, inc easing pe mea ion (H~J and FINCHER 1995), i s excessi e ac i i y esul s in he dange o leakages om he ge mina ing g ain may also be a disad an age. While 1 + 3-P-glucanase has pa hogen- esis- ela ed e ec s agains ungi, inc easing hei cell- wall pe meabili y (see e.g. GRENIER e al. 1999), he 1 -+ 3,l -+ 4-P-glucanase makes he cell walls o he we ed and ge mina ing g ain issue mo e suscep ible o in ading o ganisms. Up o 5 QTLs o inished mal glucanase and 3 QTLs o g een mal glucanase we e de ec ed (HAN e al. 1995) wi h wo s uc u al genes o isoenzymes o (1 + 3,1+ 4)-P-glucanase (LITTS e al. 1990; WOLF 1992). One o hese, EII, is es ic ed o he aleu one laye o ge mina ed g ain, while EI is also ansc ibed in scu alla on young lea es and oo s a ge mina ion in addi ion o aleu one (SLAKESKI e al. 1990; SLAKESKI and FINCHER 1992). The P-glucanase isoenzyme I1 was ound o be glycosyla ed wi h 3.6% ca bohyd a e (WOODWARD and FINCHER 1982). Glycosyla ion may be he sou ce o he mos abili y in bac e ial P-glucanase (OLSEN and THOMSEN 199 1) and many o he ypes o p o eins (e.g. Gu e al. 1989; NAKAMURA e al. 1998; YANEz e al. 1998). The le el o glycosyla ion is possibly subjec o mul igenic a ia ion. The obse ed bimodali y sugges s wo alleles, pe - haps wo ypes o glycosyla ion EII gene p oduc , and may also mean a mo e complica ed dependence wi h wo le els o EI ac i i y masking he EII ac i i y le els. The unhea ed and hea ed ac i i y did no show any co ela ion (Table 2), sugges ing ha he mos a- bili y is independen o ac i i y in his land ace sam- ple. The high he mos abili y ound in Pi kka, wi h a 50% land ace pa en age (SIMBERG 1950; KIVI 1969), p obably has i s o igins in Finnish land aces. This small sample does no necessa ily e eal ei he he ex eme ac i i ies o he he mos abili y o he pas a ia ion in he Finnish land aces. A gene ically modi ied bac e ial P-glucanase has high he mal s a- 116 H. Ahokas and M.-L. Manninen He edi as 132 (2000) bili y (JENSEN e al. 1996, 1998). T ansgenic ba ley exp essing bac e ial P-glucanase has shown s abili y o he gene o e a ew gene a ions (JENSEN e al. 1998) and may hence se e as an a i icial al e na i e o he endogeneous esou ce in ba ley, al hough lan- d aces ha e no ye been h oughly sc eened. Gene al discussion Selec ion o s able p o ein o ms by epea ed ex e nal hea may ha e occu ed in he land aces. The e a e wo s ages du ing which Finnish land ace ce eals we e o en subjec ed o hea in he pas . The ha - es ed ma u e and p ema u e s aws we e equen ly d ied o e a special o en called a kiuas, gi ing o pe usi e smoke in a special building called a iihi (TALVE 1961). Du ing such d ying, c ops we e com- monly subjec ed o ini ial empe a u es o 55-60°C; he empe a u es we e la e aised, and excessi e hea ing some imes occu ed (GROTENFELT 1899, 1922). Viable g ain issues we e some imes subjec ed o dena u a ing hea . The o he s age a which g ains may ha e been subjec ed o ex a hea ing occu ed a ge mina ion in black slash-and-bu n soils. Di e en a ian s o bu ning as a mode o cul i a ion (e.g. bu n o e pea land) did no end in Finland un il he 1940s (AHOKAS and MANNINEN 2000) and bu ning was he p e ailing me hod o ield managemen in he pas (HEIKINHEIMO 1915). A da k soil su ace, such as ha o a bu ned a ea, abso bs mo e sola adia ion and hus becomes ela i ely ho . Du ing di e en summe s in Finland a N la i udes o 61'40' and 61 '52', espec i ely, he maximum empe a u es mea- su ed in he su ace laye o bu ned black soil has been 52.8"C (LIPAS and MAKI-PE AYs 1961; VIRO 1974), and up o 63°C on he soil su ace (VAARTAJA 1949). In he pas , ba ley commonly ge mina ed in ea ly June, and hence he bu ned soils se ed as a hea -selec i e agen due o hei da kness. Soil em- pe a u e maxima exceeding 50°C would be excep- ional du ing he ge mina ion season e en in he sub opical dese habi a s o wild ba ley, bu a e eached o ba ley a he seasonal end o pos -season- ally (GUTTERMAN 1997). Wild ba ley has also been a sou ce o he mos abili y in g ain P-amylase (EGLIN- TON e al. 1998; AHOKAS and NASKALI, unpub- lished). Due o he appa en mul i unc ion o ba ley P-amylase, o he easons o he enzyme he mos a- bili y canno be excluded. The ba ley enzyme P-glucanase is induced a ge mi- na ion (e.g. BRUNSWICK e al. 1987; SLAKESKI and FINCHER 1992). The e o e, slash-and-bu n manage- men pu a i ely p o ided a he moselec i e en i on- men o ba ley. The 1 + 3,l +4+-glucanase isoenzymes EI and EII a e he p inciple ac i i ies expec ed o appea in samples ge mina ed o i e days (BRUNSWICK e al. 1987; LOI e al. 1987; MCFADDEN e a]. 1988), wi h subs a e speci ici y owa ds mixed-linked 1 -+ 3,l -+ 4-P-glucans (HBJ and FINCHER 1995). Mal ed ba ley (1 -3,l +4)-P-glucanases we e ound o be he mo- labile (BRUNSWICK e al. 1987). The signi icance o b ewing is indica ed by he ac ha he ac i i y o P-glucanase du ing mal ing is posi i ely co ela ed wi h mal ex ac (STUART e al. 1988). The a e o ce eal land aces I has u ned ou o be a subs an ial loss o local plan b eede s ha he Finnish land aces, hemsel es a pa o he na ional he i age, ha e no been ade- qua ely main ained. The gene ical mix u es o lan- d aces o sel -pollina ed ce eals we e los in abou 50 yea s p io o 1955 (AHOKAS 2000). The endange ed s a e o he na ional land aces was poin ed ou by PESOLA (1951) a e he opic was discussed a he 8 h In e na ional Gene ic Cong ess in S ockholm (KIRK 1949), bu he u gen collec ing and main- ainance p oposed by PESOLA (1951) emained un ealized. REFERENCES Ahokas H, (1998). Mega educ ion o gene ic a ia ion in Finnish ce eals by b eeding in esponse o demands o echnology and mode niza ion ideology, 1905-1 955. 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