14-3-3 Proteins in Cell Regulation
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Received I I March 2002
Transgenic 14-3-3 isoforms in plants: the metabolite profiling of repressed 14-3-3
protein synthesis in transgenic potato plants
J. Szopa'
Institute of Biochemistry and Molecular Biology, University of Wroctaw, Przybyszewskiego 63/77,
5 I - I48 Wroctaw, Poland
Abstract
tuber number and an increase in tuber size; an
increase in the fresh weight of the transgenic
tubers was also detected. T h e increased catecholamine level was accompanied by an increased
ratio of soluble sugars to starch in overexpressed
transformant. T h e opposite effect was detected in
14-3-3-repressed transgenic plants. All the repressed plants showed significant increases in
nitrate reductase (NR) activity, suggesting that
the regulation of NR occurs in vivo, and is not
isoform-dependent. T h e increase in NR activity
resulted in a significant decrease in nitrate level.
T h e level of sucrose phosphate synthase activity
was also significantly increased in all 14-3-3underexpressed transgenes, and remarkably the
increase in enzyme activity was accompanied by
respective changes in sucrose levels in the tubers.
T h e most intriguing finding was the significant
(2-3-fold) increase in ethylene content in all the
14-3-3-repressed transgenic lines, which probably
resulted from a methionine level increase. T h e
substantial increase of ethylene level in the
repressed forms might explain the significant
shortening of the vegetation period of the analysed
transgenic plants.
14-3-3 proteins are abundant eukaryotic proteins
that interact with many other proteins, thereby
modulating their function and thus cell metabolism. T h e data from mRNA analysis confirm
the developmental regulation of 14-3-3 isoform
expression in potato plants. In order to test
whether or not 14-3-3 protein expression affects
plant phenotype and metabolism, transgenic potato plants either overexpressing Cucurbita pep0
14-3-3 or underexpressing endogenous 14-3-3
isoforms were analysed. An increase in tuber
number and a decrease in tuber size in the overexpressed transformant was observed ; the transgenic plants contain more chlorophyll than the
control and they lose it more slowly than the control when transferred to the dark. T h e 14-3-3repressed transgenic plants showed a decrease in
Key words: metabolite profile, Solonurn tuberosurn, transgenic
plant.
Abbreviations used: NR, nitrate reductase; SPS, sucrose phosphate synthase.
'e-rnail [email protected]
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0 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 4
Introduction
T h e 14-3-3s are a family of highly homologous
proteins, first discovered in brain tissue and now
thought to be present in all eukaryotic cells [1,2].
14-3-3 proteins are a family of acidic 30 kDa
molecules which form homo- and hetero-dimers
and have been found in cytosol, nuclei and nuclear
matrix [3]. They are modulators of enzyme activities [4]. This suggestion comes from two lines
of evidence; first, that 14-3-3 is immunoprecipitated with enzyme proteins, and second, that
recombinant 14-3-3 inhibits enzyme activities.
Many recent reports pointed out the great importance of 14-3-3 proteins in plant metabolic
pathways. It is suggested that these proteins
regulate nitrogen assimilation by binding to nitrate
reductase (NR) and sucrose synthesis by binding
to sucrose phosphate synthase (SPS). T h e binding
of 14-3-3s to their partners is phosphorylationdependent [ S ] , and in the case of NR is Mg2+stimulated [6]. T o date, many proteins known to
be targets for 14-3-3s have been identified, and the
list of partner proteins is still growing.
While there has been substantial progress in
the identification of the diverse partners of 14-3-3
in recent years, at least two important questions
remain to be answered: is there any specificity
within the 14-3-3 isoforms in the binding of
diverse partners, and does this binding affect plant
metabolism or physiology in vivo? T o answer
these questions, investigations were done on the
number of 14-3-3 isoforms, the tissue specificity
of their expression and their age-related synthesis
in potato plants. Also, transgenic potato plants
were created with two isoforms of 14-3-3 repressed, and the phenotype and carbohydrate
metabolism of the transgenic plants were analysed.
Potato 14-3-3 isoforms
I n potato plants, by means of three different
cDNA library screenings, six cDNAs encoding
14-3-3 isoforms were recognized. T h e sequence
analysis of these cDNAs revealed their high
homology, ranging from 65 to 9 5 O b nucleotide
identity. Northern analysis revealed that all the
isoforms are present in each analysed tissue and
that there is no clear correlation between the
isoforms and the organ where they are expressed.
However, the ratio between the isoforms seems
to be characteristic for the tissues examined
(Figure 1).
T h e data obtained strongly suggest nonuniform expression of 14-3-3 isoforms in leaves
along the plant stem. T h e total amount of 14-3-3
mRNA decreases with leaf age, a fact which was
reported previously, based on Western analysis
[71.
I 4-3-3 overexpression
T h e first transgenic plants with overexpressed
14-3-3 protein were potato plants transformed
with cDNA derived from the Cucurbita pep0
Figure I
The organ distribution (Northern blot) of isoforms 16r, 20r, 29g, 30g, 34g and 35g
FI, Db, I 1-14, R, St and T represent flower, dormant bud, internodes I4 (from the top of the plant down), root, stolon and tuber
respectively; L I -L I0 are the first and subsequent leaves from the top of the plant down
IF1
DbI1 12 I3 I4 R St
0 2002 Biochemical Society
L1 L2 L3 L4 L5 M L7 L8 L9 L10
T
406
14-3-3 Proteins in Cell Regulation
cDNA library. An increase in tuber number and
a decrease in tuber size was observed in the transformants [3]. T h e transgenic plants contained
more chlorophyll than the controls, and they lost
it more slowly than the controls when transferred
to the dark; they also showed changes in lipid content and composition. T h e transgenic potato
tubers contained 69 Ol0 more total fat than the wildtype plants [8]. In another study, the overexpression of 14-3-3s derived from Vicia faba caused an
enhanced K + conductance of the plasmalemma in
tobacco mesophyll cells, but the plants displayed a
normal phenotype [9]. T h e intriguing thing is that
in both cases the transgenic potato plant tissues
displayed an additional m R N A band encoding
14-3-3 in Northern analysis, whereas the tobacco
plant tissues showed a 14-3-3 protein band detected by immunostaining. It was suggested that
gene co-activation occurs in the case of transformed potato plants, and covalent modification of
the exogenous isoform in the case of tobacco. It is
thus speculated that a dynamic reaction occurs as
a result of 14-3-3 overexpression in these different
plant species.
Interesting results were obtained from analysis of bacterial cells with overexpressed N terminally deleted 14-3-3 from C . pep0 cDNA,
103 amino acids deleted from the N-terminus.
T h e expression of truncated 14-3-3 resulted in
dramatic changes in the genomic D N A structure
and cell growth. T h e occurence of 50 kb D N A
fragments and growth retardation were the
characteristic features of these cells [lo]. However, these did not appear when cells deficient
in nuclease ( x t k A gene, coding for endonuclease 11; BW9109) were transformed and analysed
(A. Korobczak and J. Szopa, unpublished work),
which might suggest nuclease up-regulation by
cleavage products of 14-3-3 and provide a signal
for apoptotic cell death.
Phenotype of transgenic potato
In order to analyse the function of these proteins
more precisely, three transgenes were created, all
Figure 2
Potato tubers from 14-3-3-modified plants
RE, OV and W,repressed, overexpressed and wild-type, respectively: tuben from t w o plants
are shown in each case
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0 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 4
with 14-3-3repression. Earlier-created transgenic
plants where the 20r isoform was repressed
showed the involvement of 14-3-3in the regulation of the plant vegetation period; such plants
lose chlorophyll faster during their growth than
the control plants. A 2-3 week shorter vegetation
time was a characteristic feature of potato plants
with repressed 20r mRNA when grown under
greenhouse conditions [7]. It was of interest
whether the change in the vegetation period of
these potato plants was due to the 20r isoform (the
transgenic lines referred to as J4),or rather was a
result of a decrease in total 14-3-3.T o test this,
two other transgenic lines were generated, in
which the 29g (the transgenic lines referred to as
JS) and simultaneously both the 29g and 20r
isoforms (the transgenic lines referred to as G1)
were repressed. T h e transgenic lines were visually
indistinguishable from the control plants when
grown in tissue culture. However, under greenhouse conditions the plants with reduced 14-3-3
content had shortened vegetation periods. There
was no differences in the vegetation period between the single and double transformants. However, a significant change in the phenotype of the
tubers formed was observed (Figure 2).
T h e transgenic plants showed a decrease in
tuber number and an increase in tuber size; an
increase in the fresh weight of transgenic tubers
was also detected. Again there was no visible
difference between the single and double transformed lines [l13.
Catecholamines
T h e first reported function ascribed to 14-3-3s
was the activation of neuronal tyrosine and tryptophan hydroxylases dependent on their prior
phosphorylation [12].Recently, it was reported
[13]that transgenic potato plants overexpressing
14-3-3contain increased quantities of dopamine,
noradrenaline (' norepinephrine ') and normetanephrine (the last is a novel catecholamine derivative) [14].T h e increased catecholamine level
was accompanied by an increased ratio of soluble
sugars to starch. T h e opposite effect was detected
in 14-3-3-repressed transgenic plants.
Nitrogen-fixation enzymes
It was reported several times that 14-3-3proteins
might be involved in nitrogen fixation by modulating NR activity [15].This suggestion is based
on two lines of evidence; first, that 14-3-3 is
0 2002 Biochemical Society
408
immunoprecipitated with NR, and second, that
recombinant 14-3-3inhibits enzyme activity. It
should be pointed out that in in vitro experiments
there was only a slight non-significant difference
between the isoforms in NR inhibition. T h e
results of enzyme incubation with potato recombinant proteins derived from 20r cDNA or 29g
cDNA or with 14-3-3 from C. pep0 showed
that they are similarly effective. However, to
date there is no evidence that the same may
occur in vivo. So, transgenic plants grown in
tissue culture were analysed for enzyme activity.
T h e nitrate contents and NR activity were measured for leaves from plants with repression of
a single isoform, 20r (54plants) or 29g (JSplants),
and with both 20r and 29g repressed (G1 plants).
All the plants showed significant increases in
NR activity, suggesting that the regulation of
NR occurs in vivo and is not isoform-dependent.
T h e increase in NR activity resulted in a significant decrease in nitrate level. Two other
enzymes involved in nitrogen fixation, nitrite
reductase and glutamine synthetase, were also
analysed but their activity was only very slightly
changed in the transgenic plants [l11.
Metabolites of nitrogen assimilation
T h e generated transgenic plants with repressed
14-3-3synthesis were analysed for the content of
metabolites which appear in nitrogen fixation.
As a consequence of an NR increase and a
decrease in nitrate content, an increase in glutamine and asparagine levels was expected. T h e
single transformants 54 and JS showed a significant
increase in asparagine content, while glutamine
content was decreased in all the transformants. It
is interesting that the level of glutamic acid was
significantly increased in the single transformant
54 and double transformant G1, which might
suggest that carbon-skeleton synthesis might be
affected by 14-3-3in these plants [ll].
Carbohydrate metabolism
In in vitro experiments, a strong interaction of the
14-3-3 protein with SPS has been reported.
However, there was no preferential interaction of
either of the analysed isoforms, 20r and 29g, with
SPS [ll].So, analyses were done for the crucial
plant metabolites in the created transgenic plants.
Measurements were conducted on two plant
organs, leaves and tubers.
14-3-3 Proteins in Cell Regulation
cal pathway between glycolysis and the tricarboxylic acid cycle.
Leaves
T h e level of SPS activity was significantly increased in all the transgenic lines, a fact which
confirms the in vitro data. However, the measured
sucrose level did not correspond to respective
enzyme activation. Thus, although there is a clear
influence of 14-3-3 on enzyme activity, its consequences for leaf metabolism are rather negligible.
Since carbohydrates provide the carbon skeleton
for the synthesis of many metabolites, it can be
speculated that the excess of sucrose is immediately used in cellular synthesis. A substantial
increase was observed in the a-ketoglutarate level
and subsequently in the glutamic/pyroglutamic
acid level in the leaves of 14-3-3-repressed potato
plants. A significant increase was found in the
content of glucose 6-phosphate, pyruvate and
citrate, suggesting the activation of the biochemi-
Tubers
T h e level of SPS activity was significantly increased in all the transgenic lines [ll] and, remarkably, the increase in enzyme activity was
accompanied by respective changes in sucrose
levels. Thus, the in vitro finding was again confirmed in in vivo experiments. T h e glucose and
starch content is increased in the J4 and G 1 lines.
T h e content of phosphorylated glucose and fructose was not affected by the repression of either
single 14-3-3 isoform, but these decreased in the
double antisense G1 potato tubers.
In summary, the consequences of N R and
SPS activation via 14-3-3 protein repression are
somewhat different for leaf and tuber metabolism.
Figure 3
The content of the selected metabolites in the potato tubers
____________
The shlkimate
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Transgenic lines
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Transgenic lines
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Transgenic lines
~ _ _ _ _ _
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0 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 4
T h e precise identification of the molecular background of these differences needs further investigation.
significant shortening of the vegetation period of
the analysed transgenic plants. Of the other important metabolites, significant decreases in flavonoid and steroidal glycoalkaloid contents were
detected.
Field-trialled potato analysis
Transgenic potato plants with underexpression of
the 14-3-3 protein grown in the field (1997-2001)
were analysed. T h e transgenic plants showed a
significant increase in NR and SPS activities and
a decrease in nitrate level. T h e reduction in
14-3-3 protein level consistently resulted in a
catecholamine level decrease concomitantly with
starch content increase, and in an increase in the
ratio of soluble sugars to starch in the tubers. By
contradiction, potato plants with overexpressed
14-3-3 showed an increase in dopamine and
noradrenaline levels, a decrease in starch content
and a decrease in the ratio of soluble sugars to
starch. T h e preliminary data on transgenic potato
plant analysis where the human dopamine receptor
had been expressed showed a significant increase
in catecholamine content concomitant with a
decrease in starch level (A. Swiedrych and
J. Szopa, unpublished work). It is thus speculated
that, similarly to the situation in human and
animal cells, catecholamines may also control the
ratio of soluble sugars to starch in plant cells. An
increase in starch content was also recently reported [ 161 for Arabidopsis plants with repressed
14-3-3 protein synthesis. I t was suggested that this
resulted from the direct interaction of 14-3-3 with
starch synthase [16]. Whatever the mechanism,
14-3-3 down-regulates starch content in potato
and Arabidopsis plants.
T h e determination of amino acid composition
in the tubers showed a significant increase in
methionine, proline and arginine contents and a
slight but consistent increase in hydrophobic
amino acid and lysine contents in the transgenic
potato plants. T h e most intriguing finding is the
significant (2-3-fold) increase in ethylene content
(Figure 3) in all the transgenic lines (J. Kepczynski
and J. Szopa, unpublished work), which probably
resulted from methionine increase.
T h e substantial increase in ethylene level in
the repressed potato plants might explain the
0 2002 Biochemical Society
I thank Professor J. Biemat and Professor J. Kepczynski for helpful
discussions and PhD. students I. Matysiak-Kata, A. Prescha, A.
Swiqdrych, M. Wrobel and M. Zuk for providing data. This
research was supported by grant nos. 6P04A 02018. 5P06A
023 I9 and PBZ/029/PO6/2000 from National Scientific Committee and Projet Concerte de Cooperation Scientifique entre
la France et la Pologne "Polonium".
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