Phytochemistry, Vol. 46, No. 8, pp.

1335-1338, 1997
~ Pergamon © 1997 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PH:S0031-9422(97)00470-6 0031-9422/97 $17.00+0.00

FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE ACTIVITY IN LEAVES

OF A RICE MUTANT SELECTED FOR ENHANCED LYSINE

GIDEON W. SCHAEFFER,*FRANK T. SHARPEand RICHARDC. SICHER

USDA, ARS, Plant Sciences Institute, BeltsviUe, Md. 20705, U.S.A.

(Receivedin revisedform 6 May 1997)

KeyWordIndex--Oryza sativa; Gramineae; aldolase; mutant; lysine.

Abstract--Unknown proteins isolated from mutant tissues of rice (Oryza sativa L.) recovered from inhibitor
selections were subsequently peptide microsequenced. Database searches putatively identified one peptide as
fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Tissues of mutant rice, PI564784, and wild type (cv Calrose
76) tissues were evaluated for aldolase activity. Total enzyme activities were slightly lower in the mutant than
the control but the differences were not significant. Although the mutant phenotype is for enhanced lysine and
protein, we ascribe the small aldolase differences to physiological adjustments, rather than to D N A modi-
fications of the aldolase gene(s). Homologies of rice peptides with aldolases from a range Of species, as well as
rice cell culture expressed sequence tags (ESTs) are presented. Some amino acids sequences are highly conserved.
The mutant phenotype expressing stress proteins is not likely to be defined by a change in rice aldolases.
© 1997 Elsevier Science Ltd

INTRODUCTION gels, blotted and sequenced. Microsequencing data

led to the identification of several proteins, including
Fructose-bisphosphate aldolase is a widely distributed
the aldolase reported here.
enzyme, and functions in both the chloroplasts and
cytoplasm of plants and in numerous animal tissue,
including the brain, muscle and liver. This enzyme,
which has at least four loci in the rice genome [1] plays
a vital role in carbohydrate metabolism and in the
production of triose phosphates and derivatives
important in signal transduction.
Mutants of rice, with enhanced lysine in endosperm
proteins [2] and some lines with enhanced seed pro-
teins have a range of attributes which have been par-
tially characterized. We concluded from past experi-
ments that the mutant described here, PI564784, is
constitutive for specific stress-related and possibly
membrane proteins significant in protein trans-
port/export, including fll,3-glucanases and chitinases
[3]. Alterations in proteins, particularly membrane
and processing proteins, might be reflected in the
activity of a ubiquitous enzyme such as aldolase. This
work was based, in part, on the observations that
electrophoretic profiles developed with Coomassie
blue showed subtle quantitative differences in the
banding patterns of mutants and controls separated
on both denaturing and non-denaturing acrylamide
gels. Proteins were eluted from the native gels of the
mutant and subsequently separated on SDS-PAGE
* Author to whom correspondence should be addressed.
1335
RESULTS AND DISCUSSION
Mutant and control seedling proteins separated
under non-denaturing conditions showed similar
banding patterns with subtle quantitative differences.
However, 1-dimensional SDS-PAGE profiles of pro-
teins eluted from native gels showed the presence of
substantially greater Coomassie positive material at
about 35 kD in the mutant than in the wild type.
Amino acid sequencing of the purified 35 kD band
(Fig. 1) provides evidence that the peptide is an aldo-
lase. Figure 2 illustrates complete homology for 21
amino acids with rice cDNA sequences derived from
an expression library of rice seedlings. In addition to
the complete homology of proteins isolated from rice
there are many highly conserved regions which occur
across a wide range of species and tissue types includ-
ing animal brain and liver extracts. Amino acids that
are unique for plants and highly conserved include Y
(tyrosine) at position 3, K (lysine) at position 9, and
T (threonine) at position 30 and G (glycine) at position
32. The N terminal end seems variable in plant systems
but more constant in animal systems. Amino acids
that are highly conserved across plant and animal
systems include E (glutamic acid) and L (leucine) at
positions 6 and 7. Amino acids A (alanine) at 11, G
1336 GIDEONW. SCHAEFFERet al.

which expresses altered stress enzyme activities com-

kD pared with the control is probably not conditioned by

12
J
a major change in rice aldolases. However, enzyme
activities do not provide information on protein
aggregation and post translational modifications such
43-- as phosphorylation and glycosylation which could
produce modified electrophoretic profiles.

-41 EXPERIMENTAL

Rice, (Oryza sativa L.) seeds were germinated in 1

30-
O white fluorescent lamps at 90-100 #E m -2 s -1. Plants
Fig 1. SDS-PAGE electrophoretic profile of Coomassie blue
stained proteins eluted from non-denaturing gels of the
mutant PI564784. Arrow marks the 35 kD band which was
microsequenced.
at positions 18 and 20, and the triplet I (isoleucine), L
(leucine) and A at positions 21, 22, 23, respectively,
are highly conserved across Kingdoms as are L at 35,
G at 39 and E and N (asparagine) at positions 41
and 42 respectively. Finally, E at position 44 appears
highly conserved. These data suggest coding regions
for sequence modifications which were likely to be
tolerated and regions in which changes would prob-
ably alter enzyme functions.
Table 1 illustrates the aldolase activity in mutant
and wild type extracts of rice seedlings. The aldolase
activities in this experiment were generally lower in
the mutant than the controls when evaluated on the
basis of crude extracts, leaf area, and fresh weight.
However, when expressed on a protein basis the spec-
ific enzyme activities (/~mol mg -1 protein) of the
mutant and control were similar or higher in the
mutant. An initial experiment designed to optimize
conditions produced similar enzyme patterns. The
protein contents in crude extracts were also lower in
the mutant than in the control.
Our conclusions from this work are that aldolases
isolated from mutant rice seedlings appear normal in
N-terminal amino acid sequences and have similar
enzymatic properties to the control enzymes. A further
implication of this work is that the mutant phenotype
cm deep vermiculite/potting soil mix in 10 cm clay
pots in chambers with 14 hr photoperiod from cool
were watered daily, cut at the soil level 10-14 days
after sowing and frozen in liquid N2. About 0.2 g
frozen powder was transferred to a ground-glass tissue
homogenizer and extracted at 0 ° with 4 to 4.5 ml of
extraction buffer consisting of 50 mM Bicine-NaOH,
pH 8.1, 5 mM MgC12, 1 mM EDTA, 10% glycerol,
1% soluble PVP-40, 0.01% T r i t o n x 100 and 5 mM
DTT. Homogenates were centrifuged at 27 000 g for
15 rain at 2-4 °. Supernatants were quickly transferred
to 1.5 ml micro centrifuge tubes and frozen in liquid
N2. The spectrophotometric assay for FBP-aldolase
(EC 4.1.2.13) activity was performed essentially as
described in ref [4]. Assay soln contained 50 mM Tris-
HCI, ph 7.5, 1 mM EDTA, 1.6 mM fructose-l,6-
bisphosphate, 0.2 mM N A D H , 10 units triose-phos-
phate isomerase, 1 unit alpha-glycerophosphate
dehydrogenase and 20/~1 of thawed leaf extract in a
total vol. of 1 ml. Assays were performed at 25 ° and
A changes measured at 340 nm. Soluble protein was
measured according to ref [5] using BSA as standard
and following vendor's specifications.
Preps of proteins for microsequencing: Seedlings
ground in liquid N2 were extracted x 2 with 50 mM
Tris pH 6.8, 5% HOC2HsSH, 1 mM phe-
nylmethylsulphonylfluoride (PMSF) and insoluble
PVP at 2-4 °. The proteins were pptd with 4 vol. of
cold Me2CO and pelleted by centrifugation at 15 000
g for 10 rain. The pellets were solubilized with 20 mM
NaOAc, pH 5.2, and 5% glycerol and layered onto
non-denaturing Tris/borate, pH 8.3, 5-16% poly-
acrylamide gels. Margins of the gels were stained with
Coomassie blue and 1-2 cm sections of the non stained

Table 1. Fructose-1,6 bisphosphate aldolase activity in crude extracts of seedlings and expanded (Epd) leaves of an in vitro-
derived mutant (PI564784) and Calrose 76 (Cal-76), the source cultivar

Leaf Area Rate Rate Rate

Protein mg Protein Protein (LA) Fr. wt. #molmin-~ #mol min-~ #molrain-
Sample (Fr. wt.)-I mg (cm-2) mg sample-~ cm2 g mg-' protein g-~ fr. wt. crn-2 LA

CaI-76 Epd Leaf 12.7 0.213 2.86 13.4 0.225 0.256 3.26 0.055
Mnt Epd Leaf 10.6 0.157 1.8 11.4 0.17 0.292 3.10 0.046

Cal-76 Seedling 32.3 0.447 8.73 18.3 0.27 0.389 12.54 0.185
Mut Seedling 26.1 0.383 5.4 15.0 0.22 0.394 10.28 0.151
 Fructose 1,6-bisphosphate aldolase in rice 1337

k
I 2 3 4 6 8 7 8 ¢ 10 I 11 12 I5 14 15 18 17 18 19 ~ I

Mutant

1 64
l (MS
$ O6
4 O4

I|
6 62
e 95
? 64
8 4 ,• -/ ~ .. .
. . . . . . . ~ . ~ /
6 95
10 M • / ~ o • v • • K ~ ' ~ /
11 66
12 4 S ~ A " / ~ . . . . . . ~ ' ~ /
1t 6 8 K E " I ~ * Y * " T " ~ * ~ 1
14 6 K K N I ~ A Y * O T " ~ K ~ I
16 47 ~ s A • / ~ - • v * • - ~ ' ~ /
16 6 S O K I I ~ L R * V A " ~ K ~ I
17 10 ~ E O K ! I I ~ L R " V T * ~ K ~ I
18 10 E Q K I /~H R * V A " ~ K ~ I
19 9 P E O K I I~Q R " V A S ~ K ~ I

20 21 22 23 24 25 23 27 28 29 30 / 31 32 33 34 35 36 37 38 39 40 I 41 42 43 44

Mutmt LI- ' ~ M I - 6 " ~ E ' - - 8 - 1 N A ~ / oL; "E-'-'N'I T

i
1 84
/
2 85
6 85
4 84
6 82
6 85 I
7 84
6 24

j
6 85
10 74

/il
11 86
1:l 24
I$ 26 T i N V I
14 26 N V /

mC:M
18 67

17 V G S I
T G S I
19 29 V G~I

1 Oryza eMiva 100%/21AA cONA, EST, Rice-10 Fl,TbiSPNd~ase"

2 " 100%/21 AA cDNA, EST, Rice-11 "
3 " 100%/21AA cDNA, EST, Rioe-12 "
4 " 100%/21 AA cONA, EST, Rioe-13 "
6 " 100%/21 AA ¢ONA, EST, R1¢e-14 "
6 " 95%/21/~ cONA, EST~Rk~-16 "
7 Amb~ thalllnl 86%/21 AA eDNA, EST, Arathallanl "
e Pleura mavum 95%/21AX ~kxol~ut, Jeer -
0 8pJmioJa ~ 81%/21 AA Chloroplast, precursor •
10 ONlilN(lammmm relnhardlJl 71%/21 AA Chlorofllmt
11 Orfm eatlvl 100% 144 AA cDNA, EST "
12 Plmm mllvum 95%/44 AA Chlor~ I~eOumor "
1:1 A n d l i d q z ~ thallmd 70% 144 AA Suboollulat, cytoplmmk; "
14 z~ m~m s~%/44 AA SuboenuW, cy~o~asm~ "
16 ~ ~ 91%/44 AA ChkxOl~t, precursor "
16 Homo ilaplen 50%n9 AA braln, human •
17 Idus mulct~8 50%/39 AA brain, mouse "
18 OryalolaOm ¢xmk:ulue 62%/39 AA muscle, rabbit "
19 Oallus gallus 48%/38 AA liver, chicken "

•"* (EC 4 . 1 . 2 . 1 3 ) Fructose-bisl~ aldolase

( D - F r . O m e - l , S - U ~ t e O-g~/oemUe~e-~.L.~a~/~*)
Fig 2. Homology of isolated/microsequenced proteins from mutant rice seedlings with Fructose-l,6-bisphosphate aldolase
from plant and animal sources retrieved from Genbank databases. Amino acids from the database corresponding with the
rice amino acids are designated by *. The highly conserved regions are indicated by * and shading.
1338 GIDEONW. SCHAEFFERet al.
portions representing the major protein bands were
eluted with H20 by shaking overnight. Eluted proteins
were re-precipitated with Me2CO and centrifuged. Pel-
lets were re-solubilized in SDS and sepd elee-
trophoretically in 12% SDS PAGE gels. Sepd proteins
were blotted into PVDF membranes, visualized with
amido black in 0.5 M HOAc and subjected to amino
acid microsequencing.
Acknowled#ements--The authors express their
appreciation to T. Heutte for significant help in the
microsequencing of the isolated proteins and to J.
Dudley for technical assistance.
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1. Nakamura, H., Satoh, W., Hidaka, S., Kagaya,
Y,, Ejriri, S. and' Tsutsumi, K., Plant Molecular
Biology, 1996, 30, 381.
2. Schaeffer, G. W., Sharpe, F. T. and Dudley, J. T.,
Crop Science, 1994, 34, 1424.
3. Schaeffer, G. W. and Sharpe, F. T. Jr., Theoretical
and Applied Genetics, 1990, 80, 841.
4. Kruger, I. and Schnarrenberger, C., European
Journal of Biochemistry, 1983, 136, 101.
5. Bradford, M. M., Analytical Biochemistry, 1976,
72, 248.