Investigating the role of plant SNFI-related protein kinases

Transgenic Plants and Plant Biochemistry

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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

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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 'r i+i;;dj

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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

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Promoter I. 2 x CaMV 355 2. Patatin 3. ST-LSI

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Fusion

-503 bp Potato kinase fragment

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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

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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.

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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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