Download Investigating the role of plant SNFI

Transgenic Plants and Plant Biochemistry
~~
Investigating the role of plant SNF I -related protein kinases
Nigel G. Halford*$, Angela L. Man*, Jacqueline H. A. Barker*, Wendy Monger*, Peter R. Shewry*,
Alison Smitht and Patrick C. Purcell*
*Department of Agricultural Sciences, University of Bristol, Institute of Arable Crops Research, Long Ashton
Research Station, Bristol BSI 8 9AF, U.K. and tJohn lnnes Institute, Institute of Plant Science Research, Colney Lane,
Norwich NR4 7UH, U.K.
Introduction
W e have isolated plant homologues of the SNFI
(sucrose non-fermenting l)/AMP-activated protein
kinase gene family. This gene family regulates
carbon catabolite repression/derepression in yeast
[ 1 I. and plays a major role in the control of lipid
metabolism in mammals 12-4 I. Their involvement
in these fundamental processes of metabolic regulation makes these genes particularly interesting and
we aim to determine their function in plants using
transgenic techniques, focusing particularly on their
efrects on storage product accumulation in harvested organs.
The SNFl protein kinase in budding
yeast
In budding yeast (Saccharomyces cerevisiae), the
availability of sufficient levels of glucose in the environment leads to the repression of numerous
genes in a process termed carbon catabolite repression [ 1 I. The derepression of these glucose-repressible genes when the yeast is deprived of glucose
requires the function of a complex signal-transduction pathway, not all of which has been elucidated.
Genes encoding several components of the pathway
have been isolated by screening for mutants defective in the regulation of the invertase gene SUC2
[ 5-7). One of these, SNFI (sucrose non-fermenting
I), encodes a protein with a sequence characteristic
of protein serinelthreonine kinases [XI and it has
been shown that the SNFl protein has protein
kinase activity and that this activity is essential for
its function in vivo 191. snfl mutants are unable to
utilize sucrose or raffinose (both substrates of invertase), galactose, maltose or non-fermentable carbon
sources such as ethanol and glycerol. SNFl can
therefore justifiably be termed a global regulator of
carbon metabolism in yeast.
Abbreviations used: CaMV, cauliflower mosaic virus;
HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HKK,
HMGCoA reductase kinase.
$To whom correspondence should be addressed.
Plant SNF I-related protein kinase
genes
The first plant SNFI-related gene to be characterized was cRKIN1, a cDNA isolated from a rye
endosperm cDNA library [ 101. The cDNA encodes
a protein of 502 amino acid residues with an M,of
57710. It contains all the invariant residues and conserved domains typical of eukaryotic protein
kinases, including the sequence Asp-Leu-Lys-ProGlu-Asn, which is indicative of serinelthreonine
specificity [ 111. It is significantly more similar to the
SNFl protein kinase than to any other; the GAP
program [ 121 calculates a sequence identity of
66.9% between the two proteins, a strikingly high
figure given the evolutionary divergence of rye and
yeast. The protein kinase catalytic domain comprises 220 amino acid residues in the N-terminal
half of the two proteins, which is more highly conserved than the C-terminal half.
A functional relationship between RKlNl and
SNFl was tested by expressing the RKINl protein
in a yeast snfl mutant [lo]. Transformants expressing the W I N 1 protein were able to grow on
glycerol minimal medium, showing that SNFl function had been restored.
W e have now isolated SNFl-related genomic
clones, cDNAs and PCR products from Arabidopsis
[ 1.31 and barley [ 141, and partial sequences from
sugar beet and potato. A tobacco homologue has
also been isolated and characterized [ 1.51. Arabidopsis contains a single SNFI-related gene which is
expressed in all tissues, whereas barley and potato
contain multigene families with 10-20 and 5-10
copies per haploid genome respectively. The fulllength sequences all encode proteins with M,s of
58000. The barley gene family appears to contain
two classes, typified by clones RKIN2 and HKIN12.
HKIN2-type transcripts are detectable in all tissues,
whereas the HKIN12-type is only present in the
seed. The HKIN12 type is more similar to RKIN1,
whereas the HKIN2 type is more similar to the
Arabidopsis, potato and sugar beet homologues, suggesting that the cereal seed-specific form has
diverged further from the ancestral sequence.
The isolation of these genes invites the intriguing question of whether a carbon catabolite rep-
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ression and derepression system, similar to that of
yeast, exists in plants. There is evidence for the
regulation of gene expression by carbon metabolites
in plants. For example, the repression of transcription of photosynthetic genes by sugars, including
glucose, has been demonstrated in maize mesophyll
protoplasts [ 161 and Chenopodium cell-suspension
cultures [ 171, and the expression of a-amylase
genes in cultured rice cells is repressed by available
carbohydrate nutrients and induced by carbohydrate nutrient starvation [ 181. As yet there is no
evidence as to how the signals to activate and inactivate these genes are created or transduced.
The mammalian AMP-activated
protein kinase
The mammalian AMP-activated protein kinase is
activated allosterically by Y-AMP [ 191. The enzyme
plays an important role in the regulation of lipid
metabolism [Z-41, inactivating by phosphorylation
both acetyl-CoA carboxylase [20-23], the enzyme
which catalyses the first step in fatty aid synthesis,
and 3-hydroxy-3-methylglutaryl-CoA reductase
(HMG-CoA reductase) 1241, a regulatory enzyme in
isoprenoid biosynthesis. A synthetic peptide (the
SAMS peptide, with the sequence HMRSAMSG1,€II,VKRR) derived from the rat acetyl-CoA carboxylase phosphorylation site can act as a substrate
and phosphorylation of this peptide in a convenient
I
-;&!
l*Rcrr,)
assay for activity of the kinase [25]. A full-length
cDNA encoding rat AMP-activated protein kinase
has been sequenced and found to be very similar to
SNFl from yeast and its homologues in plants (261.
An alignment of catalytic regions of the yeast, rat
and barley proteins is shown in Figure 1. The similarity is high enough ( 46% sequence identity) to
strongly suggest that the SNFl kinase, AMP-activated protein kinase and the kinases encoded by the
plant SNFI-related genes, which we have isolated,
all belong to the same family and that they will have
some substrates and functions in common. There is
already some biochemical evidence supporting this
hypothesis; for example, both the SNFl protein
kinase [27] and AMP-activated protein kinase [28]
will phosphorylate and inactivate yeast acetyl-CoA
carboxy lase.
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Purification of the plant SNF I-related
protein kinase
A protein kinase with similar biochemical properties to the AMP-activated protein kinase and the
SNFl protein kinase has been purified from a
number of plant extracts [29]. and has been called
IIMG-CoA reductase kinase (€IRK)-A. Like the
SNFl protein kinase, HRK-A is not activated by
AMP. It will phosphorylate and inactivate IIMGCoA reductase from animals and plants. We have
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PILEUP [ 121 alignment of the catalytic regions of the barley BKIN12 [ 141, yeast SNFl [8] and rat AMP-activated
Barley
Rat
Yeast
Barley
Rat
Yeast
Barley
Rat
Yeast
Barley
Rat
Yeast
Barley
Rat
Yeast
Barley
Rat
Yeast
Volume 22
Transgenic Plants and Plant Biochemistry
A series of constructs have been made containing a 503 bp potato SNFI-related PCR fragment
inserted in the antisense orientation downstream of
a twin cauliflower mosaic virus (CaMV) 35s gene
promoter [33], a patatin gene promoter [34], and a
potato ST-ISl gene promoter 1351 (Figure 2).
These promoters drive constitutive, tuber-specific,
and photosynthetic tissue-specific expression
respectively. The constructs have been introduced
into the potato genome by Agrobucterium-mediated
transformation of leaf discs. Despite several independent attempts, only a single plant has been
generated containing the CaMV 3%-based construct. This may indicate that constitutive expression of the antisense SNFI-related sequence is
lethal, but other causes cannot be ruled out. In
contrast, several lines of transgenic plants have been
generated with the other two constructs and grown
to maturity under standardized greenhouse conditions. No morphological differences in the aerial
parts of the plant were observed, and the number
and size of the tubers was normal, except in one line
from each construct where Southern blot analysis
revealed that gross rearrangement of the genome
had occurred. However, a screen of the tubers of
the plants containing the patatin gene promoter-
used the SAMS peptide phosphorylation assay to
monitor purification of a related protein kinase from
barley endosperm extracts. This protein kinase is
recognized by an antibody raised against a peptide
with a sequence present in the deduced amino acid
sequences of the plant SNFI-related protein
kinases. Its M, of 58000 is also consistent with the
size predicted for the SNF 1-related protein kinases.
This is further evidence that the protein kinases
encoded by the SNFl -related genes and cDNAs
which we have isolated are the equivalent of HRKA.
Antisense expression of SNF I-related
sequences in transgenic potato
Clearly, SNF 1-related protein kinases could have a
number of functions in plants, as they do in yeast
and animals. We aim to focus on their role in the
harvested organs of crop plants, particularly their
effects on storage product accumulation, and we
have chosen potato as a model system in which to
use transgenic techniques towards this end. Potato
is readily amenable to Agrobucten'um-mediated
transformation, and antisense expression of transgenes in potato has already been extensively used in
the investigation of carbon metabolism [ 30-321.
Design of pBlN 19-based vectors [ 371 for the introduction and expression
of an antisense SNFf-related sequence in potato
Kan, kanomycin-resistance gene; LB. left border; NOS, nopaline synthase; NPT, neomycin
phosphotransferase;OCS. octopic synthase.
Eco RI
Born HllBgl II
ECO RI
Born HI
Promoter
I. 2 x CaMV 355
2. Patatin
3. ST-LSI
Hind 111
Fusion
-503 bp Potato
kinase
fragment
ocs 3'
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Protein kinase activity in wild-type (WT) and transgenic (PAT) potato plants using the SAMS peptide as
a substrate
The transgenic plants contained an antisense SNF 1 -related
sequence under the control of a patatin gene promoter
3 0.03
However, we have already shown that we can
reduce SNF1-related protein kinase activity in
potato tubers using antisense techniques, and that
the SAMS peptide phosphorylation assay is a convenient and rapid means of screening transformants. We believe that the transgenic plants which
we are generating will provide excellent experimental material for dissecting the complex systems
in which these protein kinases are operating.
L
1 Gancedo. J. M. (1992) Bur. J. Hiochem. 206.297-31 3
2 Iiardie, I). G. and MacKintosh. H. W. (1002) I h -
Plant
based construct for protein kinase activity against
the SAMS peptide has revealed that this activity has
been significantly reduced in 8 out of 11 plants, and
in one plant was only 10% of the level in untransformed plants (Figure 3). These plants should provide excellent material for biochemical and
molecular analyses to determine the effects of
reducing SNF 1-related protein kinase activity in the
potato tuber.
As an alternative to the use of constitutive and
tissue- or development-regulated gene promoters to
express the antisense sequence, we are using the
tetracycline-inducible system described by Gatz et
al. [ 361. Primary transformants expressing the
T n I 0-encoded tet repressor-operator protein have
been successfully generated and the highest expressors are being transformed again with the antisense
SNFI-related sequence under the control of a
modified CaMV 35s promoter which contains three
tet regulatory elements. Expression of the antisense
sequence should be switched off until it is induced
by the application of tetracycline, allowing for relatively fine control of its expression in tissue culture.
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Conclusions
It is becoming apparent that SNFl-related protein
kinases are ubiquitous among eukaryotes. and that
they regulate several fundamental metabolic pathways. This makes them extremely interesting to
study, but also very difficult to study, as effects in
one pathway may be coupled to effects in others.
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