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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,
RESULTS AND DISCUSSION
which has at least four loci in the rice genome [1] plays
a vital role in carbohydrate metabolism and in the Mutant and control seedling proteins separated
production of triose phosphates and derivatives under non-denaturing conditions showed similar
important in signal transduction. banding patterns with subtle quantitative differences.
Mutants of rice, with enhanced lysine in endosperm However, 1-dimensional SDS-PAGE profiles of pro-
proteins [2] and some lines with enhanced seed pro- teins eluted from native gels showed the presence of
teins have a range of attributes which have been par- substantially greater Coomassie positive material at
tially characterized. We concluded from past experi- about 35 kD in the mutant than in the wild type.
ments that the mutant described here, PI564784, is Amino acid sequencing of the purified 35 kD band
constitutive for specific stress-related and possibly (Fig. 1) provides evidence that the peptide is an aldo-
membrane proteins significant in protein trans- lase. Figure 2 illustrates complete homology for 21
port/export, including fll,3-glucanases and chitinases amino acids with rice cDNA sequences derived from
[3]. Alterations in proteins, particularly membrane an expression library of rice seedlings. In addition to
and processing proteins, might be reflected in the the complete homology of proteins isolated from rice
activity of a ubiquitous enzyme such as aldolase. This there are many highly conserved regions which occur
work was based, in part, on the observations that across a wide range of species and tissue types includ-
electrophoretic profiles developed with Coomassie ing animal brain and liver extracts. Amino acids that
blue showed subtle quantitative differences in the are unique for plants and highly conserved include Y
banding patterns of mutants and controls separated (tyrosine) at position 3, K (lysine) at position 9, and
on both denaturing and non-denaturing acrylamide T (threonine) at position 30 and G (glycine) at position
gels. Proteins were eluted from the native gels of the 32. The N terminal end seems variable in plant systems
mutant and subsequently separated on SDS-PAGE 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
* Author to whom correspondence should be addressed. positions 6 and 7. Amino acids A (alanine) at 11, G
1335
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- cm deep vermiculite/potting soil mix in 10 cm clay
pots in chambers with 14 hr photoperiod from cool
O white fluorescent lamps at 90-100 #E m -2 s -1. Plants
were watered daily, cut at the soil level 10-14 days
Fig 1. SDS-PAGE electrophoretic profile of Coomassie blue after sowing and frozen in liquid N2. About 0.2 g
stained proteins eluted from non-denaturing gels of the frozen powder was transferred to a ground-glass tissue
mutant PI564784. Arrow marks the 35 kD band which was homogenizer and extracted at 0 ° with 4 to 4.5 ml of
microsequenced. extraction buffer consisting of 50 mM Bicine-NaOH,
pH 8.1, 5 mM MgC12, 1 mM EDTA, 10% glycerol,
at positions 18 and 20, and the triplet I (isoleucine), L 1% soluble PVP-40, 0.01% T r i t o n x 100 and 5 mM
(leucine) and A at positions 21, 22, 23, respectively, DTT. Homogenates were centrifuged at 27 000 g for
are highly conserved across Kingdoms as are L at 35, 15 rain at 2-4 °. Supernatants were quickly transferred
G at 39 and E and N (asparagine) at positions 41 to 1.5 ml micro centrifuge tubes and frozen in liquid
and 42 respectively. Finally, E at position 44 appears N2. The spectrophotometric assay for FBP-aldolase
highly conserved. These data suggest coding regions (EC 4.1.2.13) activity was performed essentially as
for sequence modifications which were likely to be described in ref [4]. Assay soln contained 50 mM Tris-
tolerated and regions in which changes would prob- HCI, ph 7.5, 1 mM EDTA, 1.6 mM fructose-l,6-
ably alter enzyme functions. bisphosphate, 0.2 mM N A D H , 10 units triose-phos-
Table 1 illustrates the aldolase activity in mutant phate isomerase, 1 unit alpha-glycerophosphate
and wild type extracts of rice seedlings. The aldolase dehydrogenase and 20/~1 of thawed leaf extract in a
activities in this experiment were generally lower in total vol. of 1 ml. Assays were performed at 25 ° and
the mutant than the controls when evaluated on the A changes measured at 340 nm. Soluble protein was
basis of crude extracts, leaf area, and fresh weight. measured according to ref [5] using BSA as standard
However, when expressed on a protein basis the spec- and following vendor's specifications.
ific enzyme activities (/~mol mg -1 protein) of the Preps of proteins for microsequencing: Seedlings
mutant and control were similar or higher in the ground in liquid N2 were extracted x 2 with 50 mM
mutant. An initial experiment designed to optimize Tris pH 6.8, 5% HOC2HsSH, 1 mM phe-
conditions produced similar enzyme patterns. The nylmethylsulphonylfluoride (PMSF) and insoluble
protein contents in crude extracts were also lower in PVP at 2-4 °. The proteins were pptd with 4 vol. of
the mutant than in the control. cold Me2CO and pelleted by centrifugation at 15 000
Our conclusions from this work are that aldolases g for 10 rain. The pellets were solubilized with 20 mM
isolated from mutant rice seedlings appear normal in NaOAc, pH 5.2, and 5% glycerol and layered onto
N-terminal amino acid sequences and have similar non-denaturing Tris/borate, pH 8.3, 5-16% poly-
enzymatic properties to the control enzymes. A further acrylamide gels. Margins of the gels were stained with
implication of this work is that the mutant phenotype 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 REFERENCES


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were blotted into PVDF membranes, visualized with Crop Science, 1994, 34, 1424.
amido black in 0.5 M HOAc and subjected to amino
3. Schaeffer, G. W. and Sharpe, F. T. Jr., Theoretical
acid microsequencing.
and Applied Genetics, 1990, 80, 841.
4. Kruger, I. and Schnarrenberger, C., European
Acknowled#ements--The authors express their
Journal of Biochemistry, 1983, 136, 101.
appreciation to T. Heutte for significant help in the
5. Bradford, M. M., Analytical Biochemistry, 1976,
microsequencing of the isolated proteins and to J. 72, 248.
Dudley for technical assistance.