Re-Activation of the Expression of Glyoxysomal Genes in Green Plant Tissue
R alf Birkhan and Helmut Kindi
B iochem ie, F ach bereich C h em ie, U n iversität M arburg, H ans-M eerw ein-Straße,
D -3 5 5 0 M arburg, B undesrepublik D eu tsch lan d
Z. N atu rforsch.
45c,
1 0 7 - 111 (1990); received Septem ber 12, 1989
Isocitrate L yase c D N A , M alate Syn thase c D N A , G ly o x y so m e, L eaf P eroxisom e,
T ransition o f O rganelles
G ly o x y so m es are being replaced by leaf-typ e peroxisom es during the greening o f darkgrow n cucum ber co ty led o n s. L ight fu n ction s in this process as negative m od u lator o f the gene
expression o f glyo x y la te cycle enzym es but as p ositive regulator for the activation o f glycollate
oxid ase form ation . T he differential gene exp ression w as investigated at the level o f m R N A
am ou n ts using c D N A prob es hyb rid izin g w ith m alate synthase m R N A , isocitrate lyase
m R N A , and g lycollate o xid ase m R N A . H yb rid ization probes were obtained from a c D N A
library com p lem en tary to the germ in ation -sp ecific m R N A s o f cucum ber cotyled on s. The
process o f replacem ent o f g ly o x y so m a l protein s by lea f peroxisom al proteins w as reversed to a
certain extend w h en greened co ty le d o n s w ere brought back in the dark. H ybrid ization on
N orthern b lots provided evid en ces that in greened c o tyled on s the am oun t o f m alate synthase
m R N A and isocitrate lyase m R N A starts to increase up on dark treatm ent.
Introduction
Genes may be susceptible to activation by extra­
cellular factors. Horm ones produced within the
organism, metabolites provided from outside,
light or biotic signals bring about differential gene
expression in a particular cell. As an example, p ro ­
teins are synthesized preferentially and assembled
within a differentiated form o f an organelle be­
cause they take part in a metabolic process dom i­
nating in the cell at this stage [1-3].
Environmental and nutritional signals for gene
control are transduced by plants and may lead to
the form ation of intracellular structures which are
needed for special m etabolic tasks. In cells o f cu­
cumber cotyledons, two such extreme m etabolic
situations are known. In the dark, the seedling
grows heterotrophically and the organism depends
on the extensive fat mobilization in the storage tis­
sue which is in the cotyledon in this particular case
[4, 5]. In the light, the cotyledons become green,
and photoautotrophic growth based mainly on the
role o f chloroplasts is established [6, 7], The gene
activation required for the expression o f the form ­
er function seems to be horm one regulated [8, 9]
while the changes in gene activity preceding the as-
R eprint requests to H. K indi.
Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen
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sembly of intracellular structures necessary for the
later function are light-triggered processes [10].
Parallel to the change in metabolic function of
cotyledon cells, the forms o f microbodies in these
cells are changed. According to their function, we
distinguish between glyoxysomes [4, 5, 11] housed
in dark-grown cotyledons and leaf peroxisomes
which are characteristic o f the fully greened cotyle­
dons [11, 12].
M icrobody proteins are encoded by nuclear
genes and synthesized in the cytoplasm on free
polysomes [11, 13]. Then, a presently unknown
process follows by which the cytoplasmically made
precursors are transferred into the organelle and
assembled to mature enzymes by acquiring of co­
factors and oligomerization. Experiments on gene
expression at the level o f enzyme activity and
m R N A activity indicated that the control of
m icrobody formation may primarily be at the
transcriptional level [14]. We found that the com ­
position of microbodies concerning their function­
al enzymes is virtually identical with the com posi­
tion of the cytoplasmic pools of precursors des­
tined for the im port into the microbody [11, 15].
During the transition of glyoxysomes into leaf
peroxisomes, which takes place during the first
50 h when dark-grown seedling were brought into
light, the gene expression of glyoxysomal proteins
is reduced [15, 16] while the one of leaf peroxi­
somal proteins is increased [17]. In this paper, we
dem onstrate this change in gene expression at the
level o f the amounts of individual m RNAs. In ad-
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108
R. Birkhan and H. Kindl ■ Re-Aetivation o f G lyoxysom al Functions
dition, it is shown that this process can be reversed
if light is eliminated and fat reserves are still pres­
ent in the cotyledons.
M aterials and Methods
Plant material, RNA isolation and translation
in vitro
Cucum ber seeds ( Cucumis sativus L. Chinesische
Schlangengurken) were germinated at 26 C on
sterilized vermiculite for 4 d. Seedlings were then
placed under different light/dark regimes. White
light of 10,000 lux was provided by fluorescent
lamps. Cotyledons were used as the source of RNA
as described previously [18, 19]. Poly(A) + RNA
was isolated by chrom atography on oligo(dT)-cellulose. Subfractionation of poly(A)+ RNA was
performed by electrophoresis on agarose gels con­
taining methylmercuric hydroxide [18, 19].
Poly(A)+ R N A was subfractionated, extracted
from the gel, purified by phenolization and precip­
itation, and used for the preparation of cDNA.
Total R N A or poly(A)+ R NA was translated
in vitro according to [18].
Preparation o f a cucumber cDNA library
in pB R 322 and characterization o f clones
Double-stranded cD NA was prepared from
poly(A)+ RNA, highly enriched in the size of
1.4-2.7 kb, according to [18, 19]. The cDNA was
tailed with dC and annealed to dG-tailed pBR 322
at the PstI site. E. coli DH 1 was transformed ac­
cording to [19]. Tetracycline-resistant and ampicillin-sensitive colonies were grown on nitrocellulose.
They were prepared for hybridization by the meth­
od of G runstein and Hogness [20].
The cD N A bank was screened with various sin­
gle-stranded cDNAs prepared from poly(A) +
R N A from cotyledons of etiolated seedlings or
greening seedlings, from poly(A)+ RNA of green
leaves, or from several poly(A)+ RNA subfrac­
tions obtained after electrophoretic isolation. Dif­
ferential screening by colony hybridization yielded
a num ber o f clones coding for RNAs characteristic
of the fat degrading stage of the cotyledons. In ad­
dition, the cD N A used for screening was enriched
in sequences which encode proteins in the M r
range of 50,000-80,000. Plasmid DNA was isolat­
ed by the method of Birnboim and Doly [21] and
further purified by centrifugation in CsCl density
gradients.
Final identification of clones was done by hy­
brid selection [22] o f m R N A , in vitro translation,
and im m unoprecipitation of the respective pro­
teins [18]. D N A sequence determ ination was car­
ried out by the m ethods of M axam and Gilbert
[23] on various fragments of cD N A which were la­
belled at the 3'-end by T4-polym erase (3'-overhangs) or Klenow fragment of E. coli polymerase I
(5'-overhangs). Alternatively, sequences were de­
termined by the dideoxyribonucleotide method
[24] after recloning in M 13.
For probing glycollate oxidase m R N A , clone
E 56 of a lentil cD N A library [18] was used.
Northern blot analysis
RNA was separated by electrophoresis on 1%
agarose gels containing formaldehyde as described
by Lehrach et al. [25], Transfer to nitrocellulose
membranes followed standard procedures [26].
The conditions described by [22] were used for hy­
bridization with D N A labelled with [a-32P]dCTP
by using a multiprim e kit. D ot blots were done
according to [22].
Results
Preparation and screening o f a
cucumber cD N A library
A cD N A library enriched in sequences coding
for proteins in the M Trange of 50,000-80,000 was
constructed from poly(A)+ R N A prepared from
cotyledons of etiolated cucumber seedlings. Lipid
metabolism in cotyledons at this stage is generally
associated with elevated levels of the m R N A activ­
ity for enzymes of the glyoxylate cycle and fatty
acid ß-oxidation (referred to as glyoxysomal en­
zymes). Thus, cotyledons of 4 d old seedlings are
rich in m R N A coding for m alate synthase and iso­
citrate lyase. Taking into account this enrichment,
the initial screening o f cD N A clones for germination-inducible genes was based on differential hy­
bridization. cD N A from m R N A o f cotyledons
with fully expressed glyoxylate cycle, cD NA from
m RNA o f hypocotyls lacking the enzymes of
glyoxylate cycle, cD N A from m R N A enriched by
size separation, and cD N A derived from m RNA
of green leaves were utilized for colony hybridiza-
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R. Birkhan and H. Kindl • Re-Activation o f G lyoxysom al Functions
109
Gene expression at the level o f mRNA
1
2
3
4
5
6
7
1 2 3 4 5
Fig. 1. A n a ly sis o f translation w ith hybride-selected
R N A s. c D N A s prepared from clon es p 7 -5 6 and p 6 -7 4
were used to isolate specific m R N A s. Their activity, a n a ­
lyzed by tran slation in vitro in a reticulocyte lysate, yield ­
ed rad ioactive peptides (A; lane 1) w hich were character­
ized by their size by SD S gel electrophoresis. A , lane 2
and B, lane 1 have radioactive glyoxysom al proteins as
internal stan dards. A , lane 3 and 4 are for isocitrate lyase
m R N A selected w ith the p 6 -7 4 clon e, prior to im m u n oprecipitation (lane 3) and afterwards (lane 4). B, lane 2
and 3 are for m alate synthase m R N A selected with the
p 7 -5 6 clo n e, prior to im m unop recip itation (lane 2) and
afterw ards (lane 3). C on tam in ation w ith a 50,000 D a
translation artefact (A , lane 5 and B, lane 4) w as n ot
present after im m unop recip itation. A , lane 6 and B,
lane 5 are c o n tro ls with preim m une serum. A , lane 7
con tain s p rotein s not precipitable w ith a mixture o f a n ti­
serum against isocitrate lyase and m alate synthase.
tion. Twenty clones were selected which appeared
to hybridize with cDNA characteristic of fat-de­
grading tissue and to encode proteins in the M r
range o f approxim ately 60,000. Translation of hybrid-selected m RNA, followed by im munoprecipi­
tation and electrophoresis (Fig. 1) revealed that
two clones, p 1-55 and p6-74, encode sequences of
isocitrate lyase, and that one clone (p7-56) had se­
quences com plementary to malate synthase
m RNA. Clone p7-56 and p6-74 were further char­
acterized by sequencing. A 200 bp sequence of
clone p7-56 was almost identical with the
C-terminal sequence for malate synthase pub­
lished by Smith and Leaver [27], The 1200 bp in­
sert o f p6-74 showed 75% homology with the iso­
citrate lyase cD N A prepared from castor bean
m R N A [28]. The cDNA inserts of p7-56 and
p6-74 were used as probes for determining the
m alate synthase m RNA and isocitrate lyase
m R N A levels in cotyledons at different develop­
m ental stages.
The change in gene expression was analyzed by
determining the pools o f individual mRNAs.
N orthern blots o f RNAs, prepared after exposing
the seedlings the light for various times, were hy­
bridized with the radioactively labelled inserts of
p7-56 and p6-74. M alate synthase m RNA was
characterized by a size o f approximately 2.05 kb
while the m RNA encoding isocitrate lyase was
with 2.2 kb somewhat larger. We compared the
levels o f m RNAs coding for isocitrate lyase and
malate synthase during the light-transition period.
During the beginning o f the greening phase, the
levels o f these m RN A s were not significantly
altered. Fig. 2 dem onstrates that the level of iso­
citrate lyase m R N A starts to decline only after
18 h following the onset of light. After 36 h of
light, however, the m R N A levels fell down to Zs-'A
of the values obtained with cotyledons left in the
dark. Thus, the illumination time required for the
reduction of stationary m R N A levels was proba­
bly close to 18 h, which is slightly shorter than the
time-period characteristic for the decrease in syn­
thesis of malate synthase and isocitrate lyase
in vivo (unpublished results).
Com parison of the glyoxysomal enzyme
m RNAs with the increase of glycollate oxidase
m RNA reveals that the m R N A coding for a per­
oxisomal enzyme was present long before any re­
duction in the pools of glyoxysomal m RNAs was
detectable. As dem onstrated in Fig. 2, exposure to
light for 36 h is sufficient to reduce the level of
*
•
- 2.05 kb
M a la te synthase mRNA
Is o c itra te lya s e mRNA
0 12 18 36 h
Fig. 2. D ecrease in the level o f m alate synthase m R N A
and isocitrate lyase m R N A in c o ty led o n s during the illu­
m ination o f 4 d old dark-grow n seedling. T he a u to ­
radiography o f respective parts o f N orthern blots sh ow s
signals at the p osition o f m R N A visualized by hybridiza­
tion w ith labelled c D N A probes. T he four lanes contain
the R N A s prepared from c o ty led o n s w hich were illum i­
nated for the tim es indicated.
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R. Birkhan and H. Kindl • Re-Activation o f Glyoxysomal Functions
1 1 0
gene expression for glyoxysomal function to a low
level. At that time, the cotyledons were greened
(100 (ig chlorophyll per cotyledon) but still con­
tained some lipid reserves (1.3 mg triglyceride per
cotyledon). Transfer of these plants into the dark
and analysis of m RNAs after various times in the
dark dem onstrated that darkness in the presence
of lipid effects the reactivation of the genes encod­
ing malate synthase and isocitrate lyase (Fig. 3).
This is in contrast to other green tissues, e.g.
leaves, where darkness does not bring about the
expression o f glyoxysomal functions.
the assumption that mainly transcriptional con­
trol, but not import or other posttranslational
processes, are relevant for the protein com position
of the various microbody species. Therefore, an in­
crease in the levels o f m RNAs coding for glyoxy­
somal proteins are taken as first signs of glyoxy­
somal functions. This is a sensitive means to detect
the time point where the alteration in the functions
of peroxisomes begins.
On this basis, we compare in the light regime the
expression o f glycollate oxidase which takes less
than 3 h [18] and the reduction of glyoxysomal
gene activity which starts after 18 h (Fig. 2). In the
dark, the re-activation o f genes encoding glyoxy­
somal enzymes needs less than 30 h (Fig. 3) which
is a shorter period of time than the long lag period
of glyoxysome formation observed during the ear­
ly stage o f germination in the dark. We have
argued [18] that the velocity of this kind of gene ex­
pression may be somehow controlled by the space
of microbodies present in the cell.
The increase in isocitrate m RNA level and
m alate synthase m RNA level in a photosynthetic
tissue has not been observed till now. However,
low levels of enzyme activity of isocitrate lyase or
malate synthase have been detected in leaves under
senescent conditions [29, 30]. As for the suscepti­
bility to activation o f glyoxysomal genes, it is
probable that not auxins make the difference be­
tween greened cucumber cotyledons and leaves.
R ather the nutritional control as it is also observed
in yeasts growing on oleate [31] may be the decisive
signal.
Discussion
A cknowledgemen ts
An increasing am ount o f data support the hypo­
thesis that in cells at different developmental
stages, the microbody proteins are assembled ac­
cording to the set of genes who are activated at
that time [11, 13]. The results are consistent with
The authors like to acknowledge the excellent
technical assistance o f Simona Ahlborn. We are
grateful for the support by the Deutsche F o r­
schungsgemeinschaft (Projekt Ki 186/8-1) and the
Fonds der Chemischen Industrie.
- Malate synthase mRNA
" Isocitrate tyase mRNA
0
6 18 30 h
U l
•
•
•
/
•
— Glycol la te oxidase mRNA
F ig. 3. Increase in the am ou n t o f m alate synthase
m R N A and isocitrate lyase m R N A variou s tim es after
the transfer o f greened c o ty led o n s into the dark. T he in­
tensities o f the signals are p rop ortion al to the am ou n t o f
m R N A present in the respective total R N A preparation.
O nly the parts o f the N orth ern b lots are sh ow n w hich en ­
com p ass the p o sitio n s o f the respective m R N A s. The
tim es indicated at the four lanes represent the length o f
dark period. Besides labelled m alate syn thase c D N A
and isocitrate lyase c D N A , also g lycollate oxidase
c D N A w as used as hyb rid ization probe.
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R. B irkhan an d H. K indl • R e-A ctivation o f G lyoxysom al F u n ctio n s
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