β subunits of the SnRK1 complexes share a common ancestral function together with expression ...

39 Pages·2008·0.61 MB·English

Plant Physiology Preview. Published on September 3, 2008, as DOI:10.1104/pp.108.123026
β subunits of the SnRK1 complexes share a common ancestral function
together with expression and function specificities; physical interaction with
nitrate reductase specifically occurs via AKINβ1 subunit.
C. Polge1, 2, M. Jossier1, P. Crozet1, L. Gissot1, 3 and M. Thomas1*

1 Laboratoire Signalisation et Régulation Coordonnée du Métabolisme Carboné et Azoté, Institut
de Biotechnologie des Plantes (UMR8618), Université Paris-Sud, F-91405 Orsay Cedex, France.

This work was supported by the Ministère de l'Education Nationale et de la Recherche
(MNERT), France (to C.P., M.J., L.G. and P.C.).
2 Present address : Laboratoire de Bioénergétique Fondamentale et Appliquée, INSERM
U884, Université Joseph Fourier, F-38041 Grenoble, France.
3 Present address : Laboratoire de Biologie Cellulaire, Laboratoire Commun de Cytologie,
INRA Versailles, RD10, Route de Saint Cyr, F-78026 Versailles cedex, France.

* Corresponding author; e-mail [email protected]; fax [phone]
The author responsible for distribution of materials integral to the findings presented in this
article in accordance with the policy described in the Instructions for Authors
(www.plantphysiol.org) is: Martine Thomas ([email protected])

Running head: AKINβ subunits functional redundancy and diversity
Keywords: SnRK1, SNF1 kinase, nitrate reductase, SIP, AMPKβ.
Words: 9947
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Copyright 2008 by the American Society of Plant Biologists
Abstract

The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast,
mammals and plants in the control of energy balance. This heterotrimeric enzyme is
composed of one α-type catalytic subunit and two γ- and β-type regulatory subunits. In yeast
it has been proposed that the β-type subunits regulate both the localization of the kinase
complexes within the cell and the interaction of the kinase with its targets. In the present
work, we demonstrate that the three β-type subunits of Arabidopsis thaliana (AKINβ1, β2
and β3) restore the growth phenotype of the yeast sip1Δsip2Δgal83Δ triple mutant thus
suggesting the conservation of an ancestral function. Expression analyses, using AKINβ
promoter::GUS transgenic lines, reveal different and specific patterns of expression for each
subunit according to organs, developmental stages and environmental conditions. Finally our
results show that the β-type subunits are involved in the specificity of interaction of the kinase
with the cytosolic nitrate reductase (NR). Together with previous cell-free phosphorylation
data, they strongly support the proposal that NR is a real target of SnRK1 in the physiological
context. Altogether our data suggest the conservation of (an) ancestral basic function(s)
together with specialized functions for each β-type subunit in plants.

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Introduction
The SNF1/AMPK kinases family plays an important role in the control of the energy
balance. In yeast, SNF1 (Sucrose non fermenting 1) is as a key player in the shift from
fermentative to oxidative metabolism in response to glucose deprivation (diauxic shift)
(Hardie et al., 1998). Indeed, a quarter of the genome presents a SNF1-dependent change in
expression during this shift (Young et al., 2003). In mammals AMPK, the SNF1 homolog, has
been involved in sensing the cellular and whole-body energy levels. At the cellular level, this
kinase, once activated by an increase in AMP level, switches off ATP-consuming pathways
such as fatty acid, cholesterol and protein syntheses, and switches on ATP-producing
pathways such as fatty acid oxidation and glycolysis (Hardie, 2004). At the whole-body level,
AMPK is activated in response to muscle contraction, resulting in an increased fatty acid
oxidation and glucose uptake (Hardie and Carling, 1997; Merrill et al., 1997; Fryer et al.,
2002). This enzyme also plays an important role in glucose homeostasis since it participates in
the inhibition of insulin production and secretion by the islet β cells when glucose level in
blood is low (da Silva Xavier et al., 2000; da Silva Xavier et al., 2003) and to the inhibition of
glucose metabolism in liver (Foretz et al., 1998; Leclerc et al., 1998) (for review see (Rutter et
al., 2003)). AMPK also regulates the energy intake, its activation in hypothalamus leading to
a stimulation of food intake (Andersson et al., 2004; Minokoshi et al., 2004). In plants, several
reports have involved SnRK1 (SNF1 Related Protein Kinase 1) in the regulation of metabolic
pathways (for review see (Polge and Thomas, 2007)). Indeed, sucrose phosphate synthase
(SPS), nitrate reductase (NR) and trehalose phosphate synthase 5 (TPS5) have been identified
as in vitro phosphorylation targets of SnRK1 (Sugden et al., 1999; Harthill et al., 2006).
SnRK1 kinases also regulate the expression of several genes involved in carbohydrate
metabolism such as sucrose synthase and α-amylase (Purcell et al., 1998; Laurie et al., 2003)
and is involved in the regulation of starch synthesis (Zhang et al., 2001; Geigenberger, 2003;
Thelander et al., 2004). Recently a major contribution was brought by the work of Baena-
Gonzales and collaborators using transient expression experiments and transgenic plants.
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Their data implicate the two Arabidopsis SnRK1 kinases as central integrators of
transcriptional networks in response to stress and energy signalling (Baena-Gonzales et al.,
2007) thus reinforcing diverse previous reports (Nemeth et al., 1998; Farras et al., 2001;
Buitink et al., 2003; Bradford et al., 2003; Hao et al., 2003; Thelander et al., 2004; Gissot et
al., 2006; Radchuk et al., 2006).
In all eukaryotic kingdoms, SNF1/AMPK/SnRK1 kinases function as heterotrimeric
complexes composed of one catalytic subunit, the α-type subunit, and two regulatory
subunits, the γ- and β-type subunits (Davies et al., 1994; Mitchelhill et al., 1994; Jiang and
Carlson, 1997; Bouly et al., 1999). In mammals, the formation of the heterotrimer is necessary
for AMPKα activity (Dyck et al., 1996; Woods et al., 1996). In yeast, SNF4 (the γ-subunit)
deletion or simultaneous deletions of the three β-subunits totally inactivates the SNF1 activity
in vivo (Carlson et al., 1981; Schmidt and McCartney, 2000) indicating that they all play
important roles within the complex.
Concerning the β-type subunits, the conservation between the yeast, mammals, and
plants SIP/AMPKβ/SnRKβ proteins spread along three regions, the ASC, the KIS and the
GBD domains. In yeast, the ASC domain (“Association with Snf1 Complex”), located at the
C-terminus, allows the interaction of the β-subunits (SIP1, SIP2, GAL83) with SNF4 (Jiang
and Carlson, 1997). The internal KIS (“Kinase-Interacting Sequence”) region allows the
interaction of the β-subunits with SNF1 (Jiang and Carlson, 1997). More recently another
domain, overlapping the previously defined yeast KIS domain and presenting the
characteristics of an N-isoamylase domain, has been described (Hudson et al., 2003). Since in
some cases this region has been shown to bind to glycogen, it has been named GBD for
Glycogen Binding Domain (Polekhina et al., 2003).
Plant β-subunits can be grouped in two classes. One class of two plant β-subunits is
composed of proteins presenting the characteristics of the three yeast and the two mammalian
β-subunits in that they all have the three conserved domains previously described (Yang et al.,
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1992; Erickson and Johnston, 1993; Bouly et al., 1999; Kemp et al., 2003; Buitink et al.,
2003). In Arabidopsis thaliana, the two proteins of this class, AKINβ1 and AKINβ2, present
an overall identity of 49% and 55% within these regions (Bouly et al., 1999). The second
class is composed of shorter β-subunits, named AKINβ3 in A. thaliana, lacking the GBD and
the N-terminal region of the protein (Gissot et al., 2004).
Several studies have highlighted an emerging importance of the β-type subunits within
the complex, making them particularly attractive. For instance, in plants, a 90-95% decrease
in StubGAL83 expression, a potato β-type subunit, leads to an abnormal development of roots
and tubers (Lovas et al., 2003) suggesting an important role of this subunit in plant
development. In yeast, the β-subunits have been proposed to mediate the interaction of the
kinase with its targets (Vincent and Carlson, 1999). Moreover, the authors proposed that each
β-subunit would interact with specific sets of targets and that this specificity is dependent on
their variable N-terminal region. β-type subunits could also regulate the localization of the
kinase complexes within the cell, through their N-terminal and GBD regions (Vincent et al.,
2001; Wojtaszewski et al., 2002; Hedbacker et al., 2004). In plants, it has been shown recently
that A. thaliana AKINβ1 and AKINβ2 are N-Myristoylated and that their sub-cellular
localization may vary depending on this mechanism together with changes in kinase activity
(Pierre et al. 2007). These data suggest that β-subunits might play a major role in the
regulation and specialization of the SNF1 complexes. For instance, SNF1 complexes have
been involved in various aspects of the invasive filamentous yeast growth depending on the β-
subunit integrated in the complex (Vyas et al., 2003).
Yeast SNF1, mammalian AMPK and plant SnRK1 kinases are now considered as
global metabolic regulators. To gain more insights into the role of plant SnRK1 complexes,
we have focused our study on the three β-type subunits of A. thaliana, AKINβ1, AKINβ2 and
AKINβ3. We demonstrate that these three subunits restore the phenotype of the yeast
sip1Δsip2Δgal83Δ mutant suggesting the conservation of (an) ancestral basic function(s).
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However, each of these three subunits presents a specific pattern of gene expression during
development and in response to different environmental conditions. Finally, we report the first
evidence, to our knowledge, of a physical interaction between the major form of nitrate
reductase, NR2, and a SnRK1 complex containing AKINβ1. β-specific expression data
together with the specific interaction of AKINβ1 with NR2 strongly suggest specialized
functions for each β-type subunit in plants.

Results

AKINβ1, β2 and β3 share common ancestral function(s)
To get insights into the function of the plant β-subunits, we have performed yeast
functional complementation experiments using a yeast β-subunits triple mutant (Schmidt and
McCartney, 2000). The yeast strain presents complete deletions of the three yeast β-subunits
(sip1Δsip2Δgal83Δ) leading to a snf phenotype, thus is unable to grow on alternative carbon
sources such as ethanol and glycerol (Schmidt and McCartney, 2000). We have previously
shown that the plant specific subunit AKINβ3 can complement this mutant (Gissot et al.,
2004). In Figure 1, we show that the two other A. thaliana β-subunits, AKINβ1 and β2, can
also restore the growth ability of the mutant on a medium containing glycerol and ethanol as
carbon sources. This result suggests that plant β-type subunits can substitute for any of the
endogenous subunit to form a chimeric, active enzymatic complex, and share with yeast
subunits one or several common ancestral functions. However, the growth restoration appears
only partial with AKINβ2 despite its structural similarity with AKINβ1 and with the yeast β-
subunits.

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Analysis of AKINβ1, β2 and β3 genes reveals different potential regulatory elements
In order to determine whether the three plant β-type subunits could have redundant
functions, as suggested by yeast functional complementation experiments described above,
we have undertaken a detailed study of their promoter regions (Fig. 2).
The AKINβ1 promoter (772 bp from transcription start site) contains one 6 bp
consensus sequences known as “auxin response element” (Ulmasov et al., 1997) (Fig. 2).
However, digital northern analysis carried out with Genevestigator ((Zimmermann et al.,
2004); URL: https://www.genevestigator.ethz.ch) did not reveal any potential regulation by
auxin. One pollen box is present (Twell et al., 1991) as well as two dark induction or light
repression boxes (Dehesh et al., 1990; Inaba et al., 2000) (Fig. 2). Two antagonistic types of
boxes have also been found in this promoter, six sugar repressive (Lu et al., 1998; Morita et
al., 1998) and one sugar inducible boxes (Sun et al., 2003) (Fig. 2).
In the AKINβ2 promoter (998 bp from transcription start site), a domain similar to the
LAT56 pollen motif is present (Twell et al., 1991) (Fig. 2). However, this sequence differs
slightly from those previously identified (Twell et al., 1991; Weterings et al., 1995; Bate and
Twell, 1998) (TGTTGGTT instead of TGTGGTT). One sugar repressive box (Morita et al.,
1998) has also been found in this promoter (Fig. 2).
The AKINβ3 promoter (976 bp from transcription start site) contains 17 pollen boxes
(Fig. 2). Seven AAATGA boxes are on the major strand and three on the minus strand
(Wetering et al., 1995) as well as three LAT56-type pollen boxes (Twell et al., 1991). A dark
induction box (Dehesh et al., 1990) is present in the exonic region located between the two
leader introns (Fig. 2).

The detailed analysis of the promoters of the three AKINβ genes suggests a differential
regulation of gene expression. To confirm this assessment in vivo, we have studied the
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expression patterns of the three genes in different organs, at different developmental stages,
and in response to light conditions and to sugar availability.

AKINβ1, β2 and β3 present specific patterns of expression during the course of plant
development
To investigate expression patterns in different organs and at different developmental
stages we have produced AKINβ promoter::GUS transgenic lines. Translational fusions were
made between the UIDA reporter gene and fragments of AKINβ1, AKINβ2 and AKINβ3
promoters corresponding respectively to 772 bp, 998 bp and 976 bp upstream of the
transcription start, and containing the subset of regulation motifs in these regions described in
Figure 2. Staining data, shown in Figure 3, are representative of the results obtained for at
least three AKINβ1::GUS, AKINβ2::GUS and AKINβ3::GUS stable homozygous transgenic
lines.
GUS staining experiments indicate that there is at least one β-subunit expressed in all
the organs and tissues analyzed (Fig. 3D). Some organs and tissues express the three
AKIN::GUS constructs. This is found in the cotyledons soon after germination (data not
shown), and in roots from 72h-old seedlings (Fig. 3A, photographs b), in the primordia of
roots and reproductive organs (Fig. 3B), in the stamen filament and the pistil of young buds,
and in mature pollen (Fig. 3B, photographs f). These results suggest the existence of three
types of SnRK1 complexes in these tissues, αβ1γ, αβ2γ and αβ3γ (α representing α1 or α2
and γ being γ or βγ). Interestingly there are also tissues where the expression of only one β-
subunit is detected. AKINβ1 is mainly expressed in the sepals of young buds (Fig. 3B
photographs 1c, 1d) and in the ovary wall (Fig. 3B photographs 1e, 1f). AKINβ2 is the main
form in stigmata, in placenta with a strong coloration in septum (Fig. 3B, photographs e), in
floral pedicel and in the insertion area of the floral organs (Fig. 3B, photographs e and Fig.
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4A). AKINβ3 is the main form in the root tip of 48h-old seedlings (Fig. 3A photograph 3a), in
leaves primordia, in developing pollen, in ovules (Fig. 3C photographs 3e and 3f) and in
immature seeds (Fig. 3C photograph 3g).
Altogether, the staining data showed that at least one β-subunit is expressed in each
organ or tissue tested (Fig. 3D), suggesting a ubiquitous presence of the AKINβ-type proteins
in the whole plant. Thus these data also indirectly suggest the presence of AKIN kinase
complexes in all the organs of A. thaliana since we have previously shown that both the
catalytic AKINα subunit and AKINβγ, one AKINγ subunit, appear constitutively expressed
(Gissot et al., 2006). Another interesting result was the fact that in some organs the three β-
type subunits present specific patterns of expression which moreover vary during organ
development. It is noteworthy that, in opened flowers and siliques, there is nearly no overlap
between the patterns of expression of the three β-type subunits (Fig. 3C).

AKINβ1, β2 and β3 are up-regulated during the senescence
During the analysis of AKINβ2-GUS expression pattern, a strong staining was detected
in the pedicle during flower development (Fig. 4A). It is well known that in this region, after
flowering, cells enter senescence to allow the abscission of floral parts. The increase of
AKINβ2::GUS expression could be related to a program of cell death. The expression of
AKIN genes during the process of senescence was thus followed by northern blot experiments
at different leaf developmental stages (named a-i), throughout the development of the plant
(Fig. 4B and 4C). Our results show that expression of all three β-type subunits is up-regulated
at the advanced stages of senescence when most RNAs are degraded, as shown by
hybridization with EF1α and by the amount of intact 28S RNA (Fig. 4C, stages g, h and i).
Interestingly, our results also show that the mRNA levels of the other members of the AKIN
complex, AKINα1 and AKINβγ, are maintained at these stages, strongly suggesting the
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presence of different functional AKIN kinase complexes during senescence and thus
potentially several roles of the kinase in this process.

AKINβ genes are differently regulated by light
Regulatory sequences associated with dark induction have been identified in AKINβ1
and AKINβ3 promoters, reinforcing, for AKINβ1, previous data from our laboratory showing
differential light regulation (Bouly et al., 1999). Indeed, a rapid increase in the AKINβ1
transcripts level in the dark had been described, an effect that is reversed by light. No
information is available concerning AKINβ3 regulation. To determine whether the observed
variations in mRNA levels are due to a transcriptional regulation, as suggested for AKINβ1
and AKINβ3 by the presence of dark induction boxes, experiments were performed using the
AKINβ::GUS transgenic lines described earlier. β-glucuronidase activities were measured in
three-week-old plantlets after they were held for 2.5 d in the dark or kept for 2.5 d under a
16 hours photoperiod. Plants were harvested in the middle of the 16h light period. For each
construct, three independent transgenic lines were used and for each transgenic plant, two
independent experiments were performed with measurements made in triplicate (Fig. 5). The
results show an increase of GUS expression of around 1.5 - 2 when the plantlets are kept in
the dark for 2.5 d indicating a transcriptional regulation of AKINβ1 in these conditions
(Fig. 5A). They are reinforced by expression data on Genevestigator. In experiments 109 and
56 (experiment 109, AtGenExpress “response to different light treatment” and experiment 56
“Gene expression and carbohydrate metabolism through the diurnal cycle”, (Smith et al.,
2004)) the AKINβ1 transcripts levels increase 1.3- and 3.3-fold after 45- and 60-min in the
dark, respectively.
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