Enzymes of carbohydrate metabolism in developing Hordeum distichum grain

Phytochcmatry,1973, Vol 12, PP 1923 to 1928

Per~amonPress. Prmtedm England.

ENZYMES OF CARBOHYDRATE METABOLISM IN DEVELOPING HORDEUM DISTICHUM GRAIN E DENISE BAXTER* and CAROL M. DUFFUS Department

of Agrtcultural Bmchemtstry, School of Agrrculture, Umversrty of Edmburgh, West Mams Road, Edmburgh 9. Scotland (Received 19 December 1972

Key Word Index-Hordeum rsm , GIP synthesis

drstlchum, Grammeae,

Accepted 1 March 1973)

barley, endosperm,

amyloplast, carbohydrate

metabol-

Abstract-Vartattons m acttvrty of several enzymes assoctated wrth carbohydrate metabohsm were recorded durmg the development of barley endosperm The enzymes mvestrgated were sucrose_UDP (ADP) glucosyl transferase , mvertase, UDPG (ADPG) pyrophosphorylase , hexokmase , glucosed-phosphate ketotsomerase , phosphoglucomutase, and nucleostdedrphosphokmase INTRODUCTION INITIATION of short-cham glucan primers may be carried out by phosphorylase (E.C 2 4 1, l), with glucose-l-phosphate (GIP) serving as the glucosyl donor le2 The nucleotide sugars, UDPG (UDP-glucose) and ADPG (ADP-glucose) which, with these primers, are the substrates for starch synthesIs3*4 may be synthesized from sucrose by sucrose-UDP (ADP) glucosyl transferase (E C 2 4 1 13),5 or from GIP by ADPG (UDPG) pyrophosphorylase (E C 2 7 7 9) 6 GIP, itself a product of sucrose metabohsm, 1sthus a key intermediate m the conversion of sucrose to starch, and variations m its concentration may provide a regulatory mechamsm m starch biosynthesis Enzyme systems concerned m the biosynthesis and metabolism of GIP and the nucleotlde sugars were therefore investigated m barley endosperm, m order to determine changes m activity durmg maturation RESULTS

Major Blochemrcal Constrtuents The relative contrlbutlons of starch, reducmg sugars, and protein to the dry weight of the gram, and of water to the fresh weight, are shown m Fig 1 The proportion of reducing sugars fell as starch accumulated m the endosperm Protein levels increased during the first 20 days after anthesa, and then remained fairly constant during maturation Water content was high m the young gram, reaching a maximum around 14 days after anthesis, then falling steadily as the gram ripened The levels of blochemlcal constituents described here were approximately the same as those m the gram used for enzyme assays (see below) * Present address The Brewing Industry Research Foundatron,

Nutfield, Redhdl. Surrey RHl 4HY.

1 SLABNIK, E and FRYDMAN,R (1969) Blochem Blophys Res Commun 38,709 2 BAXTER, E D and DUFFUS, C M (1973) Phytochemrstry 12, m press. 3 DE FEKIZT~,M A R and LELO~R,L F (1960) Nature 187,918 4 JENNER,C F (1968) PIant Physlol 43,41 s DELMAR, D P and ALBERSHEIN,P (1970) Plant Physrof 45,788. 6 TURNER, J F , TURNER, D H and LEE, J B (1957) Austruluzn J El01 Scz 10,407. 1923

E D BAXTER andC

1924

M DUFFUS

Sucrose- UDP (A DP) Glucosyltransferase

Actlvlty with UDP, which was conslderably greater than with ADP m soluble endosperm extracts, could be detected at 5-7 days, and increased rapldly until 25 days (Fig 2) Actlvlty wtth ADP was not apparent until 8 days, and rose gradually to a maxlmum around 34 days No actlvlty with eltl& UDP or ADP was detected in amyloplast fractions

08

. UDP o ADP A

lnvertase

actwty

e c 06 2* ti 0 \2 0 c-4

g 2 z : 02

0

IO

20

Days

30 40 after anthesis

50

60

FIG 1. STARCH, SOLUBLE REDUCING SUGARS AND TOTAL SOLUBLE PROTEIN AS A 'A OF dry wt. AND WATER AS A % OF fr wt OF WHOLE ENDOSPERM DURING MATURATION

0

30 IO 20 Days after onthesls

4(

FIG 2 SUCROSE-UDP (ADP) GLUCOSYL TRANSPERASE ACTIVITY WITH mP AND ADP, AND INVERTASE ACTIVITY IN SOLUBLE ENDOSPERM EXTRACTSOFDEVELOPINGBNDOSPERM

Inverrase (E C 3 2 1 26)

This was measured both at pH 4 8 and 7 2, and activity was low m each case The vanatlon m activity at pH 7 2 throughout maturation m the soluble fraction of endosperm IS shown m Fig 2 Acttvlty was detected around 10 days after anthesls and remained fairly constant during maturation, de&rung after 30-34 days By 21 days after anthesls a sign& cant proportion of the total gram actlvlty was located m the soluble fraction of the embryo UDPG (ADPG) Pyrophosphorylase

The UDPG enzyme could be detected m the soluble endosperm fraction by 7 days after anthesls Actlvlty increased rapldly at first, levelhng off around 16 days, then rising again to a peak at 24 days (Fig 3) The ADPG enzyme appeared somewhat later-around 12 days, and a maximum actlvlty, slightly lower than that of the UDPG enzyme, was reached by 21 days Inorganrc Pyrophosphatase (E C 3 6 1.1)

Figure 4 shows that actlvlty m the early stages of maturation closely resembled the pattern of ADPG pyrophosphorylase Maximum actlvlty, however, was attained around the same

Enzymes of carbohydrate metabohsm m developmg Hordeum dutlchum

grain

1925

period as UDPG pyrophosphorylase Activity was chtefly located m the soluble fraction, but some could be detected III the amyloplasts from 12 days after anthesrs

. UDPG q

ADPG

Fro 3 UDPG

AND

Days after anttmio

Days after

ADPG

PYROPHOSPHORYLASE ACTIVITYINSoLUBLBBNDoSPERMEXTRACTS.

anthescs

FIG 4

INORGANIC PYROPHOSPHATASE ACTMN IN SOLUBLE ENDOSPBRM EXTRACTS AND AMYWPLASTS

Hexokznase (E C 2 7 1 1)

Thts enzyme was present m both soluble and amyloplast fracttons from 7 days after anthesrs and Increased steaddy throughout the nntral stages of maturatron, levelhng off around 21 days Stmtlar results were obtained with glucose and fructose as substrates The results expressed m Fig. 5 were obtamed usmg glucose. Amyloplast actlvny was approximately half that of the soluble fractton 6-

.A*

25-

E 206 &

o Endosperm

sG6P

extracts

. Amyloplasts

2-

q

ketolsomerase

,/

Phosphoglubnutase!

.6 -

4-

0

5

IO

Days after

FIG. 5.

I5

20

25

0

anthew

H&IXOKUUSS ACTIVITY IN SOLUBLB sPBRMBXTRACTsANDAKYL.oPLASl3.

.A 6

I6

Doys after ENDo-

24

32

40

anthesls

FIQ. 6 G6P KETOLWbfERASE AC’RvlTv IN fun01 F6P/mm/10 @WUs AND PHOSPHOClLUCOMUTASE m pm01 P,/mm/lO grams (vfiues x 2) IN sounca ENDoSPBRMBXTRAcr3

1926

E D BAXTER and C M DUFFUS

Phosphoglucomutase (E C 2 7 5 I) and Glucose-6-phosphate ketozsomerase (E C 5 3 I 9) Both enzymes were present m the soluble endosperm extract very early m development, and exhIbIted slmllar patterns of actlvlty (Fig 6) There was no appreciable change until I8 days, when actlvlty increased sharply, between 20 and 22 days Glucose-6-phosphate (G6P) ketolsomerase actlvlty began to level off around 25 days, when phosphoglucomutase was already declmmg Nucleosrde drphosphokmase (E C 2 7 4 6) Sucrose-UDP glucosyl transferase and ADPG pyrophosphorylase exhlblt their greatest Increase m actlvlty around 15-20 days after anthesls Nucleoslde dlphosphokmase was therefore assayed m soluble endosperm extracts of this age to determme whether mterconverslon of the nucleotldes formed m these reactlons was possible The presence of the enzyme was confirmed, and It was found to be more active with UTP (7 5 nmoI/mm/gram) than wrth GTP (4 5 nmol/mm/gram) DISCUSSION

Patterns of changes m reducmg sugars closely resembled those recorded m hheat’ and peas,8 and are consistent with a mechanism by which starch 1s syntheslsed from a precursor pool of soluble reducing sugars That IS, the pool IS mltlally depleted during the onset of rapid starch synthesis but 1s subsequently mamtamed at a constant (but lower) level, presumably by the contmumg supply of sucrose to the gram 7 The Increase m protern levels which lmmedlately precedes the period of rapld starch accumulation may reflect synthesis of enzyme protein, since an increase m the actlvlty of most enzymes mvestlgated was observed during this stage of development However, maintenance of protein concentrations during later maturation may be attributed to the accumulation of protem bodies m the starchy endosperm g The mltlal stages of starch synthesis, namely the formatlon of GIP and nucleotlde sugars from translocated sucrose, appear to be locahzed m the soluble fraction of the endosperm, rather than m the amyloplasts The relatively high actlvlty of sucrose-UDP glucosyl transferase m young endosperm suggests that most of the sucrose entering the endosperm IS converted to nucleotlde sugars, predommantly UDPG This contrasts with the sltuatton m maize, where sucrose-UDP glucosyl transferase cannot be detected until I2 days after anthesls lo In this case sucrose IS thought to be metabolized to glucose and fructose via Invertase, at least m the early stages of development ll Fructose 1s also a product of the sucrose-UDP glucosyl transferase reaction, and may be converted to GIP via hexokmase, G6P ketolsomerase and phosphoglucomutase actlvlty,12** 3 smce these enzymes are all present m barley endosperm soon after anthesls In the young endosperm GIP synthesized m this way may be lmmedlately polymerized by phosphorylase activity, formmg short-chain a-l ,Cglucans which could serve as primers for transglucosylase activity ‘s2*14 ’ JENNINGS,A C and MORTON, R K (1963) Australzan J BIOI Scr 16, 318 8 TURNER, J F (1969) Australian J Bzol SCI 22, 1145 9 BUWROSE, M S (1960) J Ultrast Res 4, 231 lo TSAI, C Y , SALAMMI,F and NELSON, 0 E (1970) Plant Physzol 46, 299 I1 SHANNON,J C (1972) Plant Physzol 49, 198,203 z2 DE FEKETE, M A R and CARDINI, C E (1964) Arch Bzochem Brophys 104, 173 I3 DE FEKETE, M A R (1969) PIanta 87, 311 z* BIRD, I F (1969) Ph D Them, University of London

Enzymes of carbohydrate

metabohsm

m developmg Hordeum dntrchum gram

1927

Some of the UDPG formed by sucrose-UDP glucosyl transferase actlvrty may be converted to starch by UDPG-lmked starch synthetase. However, durmg the period from 10 to 15 days after anthesls, increasing UDPG pyrophosphatase actlvlty, m conJunctlon with low levels of morgamc pyrophosphatase would favour pyrophosphorylysls of UDPG, forming UTP and GIP Thus sucrose could be converted to GIP as proposed by Turner et al ,6 and De Fekete and Cardml I2 The subsequent increase m ADPG-pyrophosphorylase and morgamc pyrophosphatase would faclhtate transfer of glucose from GIP to ADPG and thence to starch via ADPG-lmked starch synthetase Detectlon of nucleoslde dlphosphokmase m endosperm extracts mdlcates that UTPformed from UDPG (via UDPGpyrophosphorylase) may be utlhzed to reform the ATP-required for ADPG synthesis 2

--,UTP

/ ,’ I

+ ADP

GIP :

, 4

ScHEhiE 1 PROPOSED SCHEMEOF SYNTHESISOFSTARCH

1 2 3 4

FROM SUCROSE IN Hordeum drstrchum ENDOSPERM

Sucrose-UDP glucosyl transferase UDPG pyrophosphorylase UDPG-starch synthetase Nucleoslde dlphosphokmase

5 6 7 8

Starch phosphorylase ADPG pyrophosphorylase Inorgamc pyrophosphatase ADPG-starch synthetase

In the reaction scheme outlined m Scheme 1, it is suggested that starch synthesis m very young endosperm 1s mltlated by phosphorylase activity (usmg GIP as glucosyl donor), together with UDPG starch synthetase Couplmg of the UDPG and ADPG pyrophosphorylase reactions would allow rapld conversion of sucrose to starch, via ADPG starch synthetase This would agree with previous results m which UDPG starch synthetase appeared earher m development than the ADPG enzyme l5 EXPERIMENTAL Plant materral The 2-row barley Hordeum drstzchum (L) Lam C V Mans Baldrlc was used throughout. Condltlons of growth and methods used to determme the date of anthesls were as described by Merritt and Walker I6 Preparatron of soluble endosperm extracts and amyloplast fractions The husk and testa-pencarp were removed by hand and the remammg endosperm (mcludmg the aleurone layer) together with the embryo constituted the ‘whole gram’ subsequently referred to This must be dlstmgulshed from the ‘whole gram’ of several other workers (for example, Jennmgs and Morton’) which Includes the testa perlcarp To prepare soluble endosperm extracts, the ‘whole gram’ was suspended m the appropriate buffer at 4” and homogemzed by hand m an all-glass homogemzer The homogenate was filtered through muslm to remove cell debris and centrifuged at 4” for 10 mm at 10 000 g The supernatant solution formed the soluble endosperm extract The pelleted material was washed once with buffer, re-centnfuged and re-suspended m buffer This fraction was composed mamly of amyloplasts and 1s referred to as the amyloplast fraction It was routmely exammed by hght microscopy and the age of the gram correlated with amyloplast diameter The number of grams used for extraction was varied from 60 (at 2-3 days) to 10 (18 days onwards) per 2 ml buffer The buffer required m the subsequent assay system was used as the extraction medmm m each case I5 BAXTER, E D and DUFFUS, C M (1971) Phytochemzstry 10, 2641 16 MERRIIT, N R and WALKER, J T (1969) J Inst Brewmg 75, 156

E D BAXTERand C M DUFFUS

1928 Chemxal

analyses

Reducmg

sugars,‘7*18 carbohydrate,”

and soluble

protem“’ were measured

by

standard methods Enzyme aways Sucrose-UDP glucosyl transferasezl and mvertase,“’ were assayed by Increase m reducmg sugars ADPG (UDPG) pyrophosphorylase,* hexokmase,l” and nucleoslde dlphosphokmase” were estlmated spectrophotometrlcally by couphng a product of the reactlon to the G6P dehydrogenase reactlon s Inorgamc pyrophosphatase,23 phosphoglucomutase” and G6P ketolsomerase,‘” were estimated by standard colorlmetrlc methods

Acknowled~emenrs-The authors wish to thank the Scottish Plant Breedmg Statlon, Pentlandfield, East Lothlan, for supplymg the plant material The mlcroscope, a Vlckers M15C, was purchased with a Royal Society grant The work was supported by the Agricultural Research Council ” SOMmYI, M (1945) J Bd Chem 160, 69 I* NELSON,N (1944) J Blol Chem 153, 375 jr) MORRIS, D L (1948) Screrzce 107, 254 2o LOWRY,0 H , ROSERROUCH,N J, FARR, A L and RANDALL,R J (1951) J BIOI Chem 193, 265 *’ MILNER, Y and AVIGAD, G (1964) Israel J Chem 2, 316 *’ DICKINSON,D B and DAVIES,M D (1971) Plant Celi Physrol 12, 157 ” HFPPEL9 L A (1955) m Methods m Enzymology (COLOWICK,S P and KAPLAN, N 0, eds ), Vol 11, p 570, Academic Press, New York