Download SnRK1 Isoforms AKIN10 and AKIN11 Are

SnRK1 Isoforms AKIN10 and AKIN11 Are
Differentially Regulated in Arabidopsis Plants
under Phosphate Starvation1[C][OA]
Selene Fragoso, Laura Espı́ndola, Julio Páez-Valencia, Alicia Gamboa, Yolanda Camacho,
Eleazar Martı́nez-Barajas, and Patricia Coello*
Departamento de Bioquı́mica, Facultad de Quı́mica (S.F., L.E., E.M.-B., P.C.), Departamento Ecologı́a
Funcional, Instituto de Ecologı́a (J.P.-V., A.G.), and Departamento de Bioquı́mica, Instituto de Fisiologı́a
Celular (Y.C.), Universidad Nacional Autónoma de México, Mexico, Distrito Federal 04510
During phosphate starvation, Snf1-related kinase 1 (SnRK1) activity significantly decreases compared with plants growing
under normal nutritional conditions. An analysis of the expression of the genes encoding for the catalytic subunits of SnRK1
showed that these subunits were not affected by phosphate starvation. Transgenic Arabidopsis (Arabidopsis thaliana) plants
overexpressing the AKIN10 and AKIN11 catalytic subunits fused with green fluorescent protein (GFP) were produced, and
their localizations were mainly chloroplastic with low but detectable signals in the cytoplasm. These data were corroborated
with an immunocytochemistry analysis using leaf and root sections with an anti-AKIN10/AKIN11 antibody. The SnRK1
activity in transgenic plants overexpressing AKIN11-GFP was reduced by 35% to 40% in phosphate starvation, in contrast with
the results observed in plants overexpressing AKIN10-GFP, which increased the activity by 100%. No differences in activity
were observed in plants growing in phosphate-sufficient conditions. Biochemical analysis of the proteins indicated that
AKIN11 is specifically degraded under these limited conditions and that the increase in AKIN10-GFP activity was not due to
the phosphorylation of threonine-175. These results are consistent with an important role of AKIN10 in signaling during
phosphate starvation. Moreover, akin10 mutant plants were deficient in starch mobilization at night during inorganic
phosphate starvation, and under this condition several genes were up-regulated and down-regulated, indicating their
important roles in the control of general transcription. This finding reveals novel roles for the different catalytic subunits
during phosphate starvation.
Phosphorus (P) is an essential element required for
plant growth and development (Bieliski, 1973). The P
concentration in soil solutions is high but the assimilable form, inorganic phosphate (Pi), is always in a
limiting concentration ranging from 1 to 10 mM (Poirier
and Bucher, 2002); thus, plants are frequently growing
in Pi-limited conditions. Plants have evolved two
broad strategies to cope with limiting Pi: (1) responses
designed to increase Pi availability or acquisition, and
(2) metabolic adaptations to decrease Pi requirements
(Poirier and Bucher, 2002). Processes that lead to
enhanced uptake include increased production and
1
This work was supported by the DGAPA-UNAM (grant no.
IN202206), PAIP (grant no. 6290–13), and CONACyT (grant no.
52072). S.F. and L.E. received a scholarship from CONACyT, Mexico.
* Corresponding author; e-mail [email protected].
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:
Patricia Coello ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.133298
1906
secretion of phosphatases, exudation of organic acids,
and greater root growth along with modified root
architecture (Bates and Lynch, 1996; Ma et al., 2001;
López-Bucio et al., 2002). There are also processes that
conserve the use of Pi, such as the remobilization of
internal Pi, modifications in carbon metabolism that
bypass Pi-requiring steps, and alternative respiratory
pathways (Plaxton, 2004). During Pi-limited conditions, a reduction in cytoplasmic Pi, ATP, and ADP
directly affects glycolysis, since they are used as
cosubstrates of enzymes, such as ATP-dependent
phosphofructokinase, NAD-dependent glyceraldehyde3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. However, despite low
concentrations of cytosolic Pi and nucleoside phosphates, pyrophosphate (PPi) concentrations remain
high and PPi can serve as an energy donor under
certain conditions (Duff et al., 1989; Theodorou and
Plaxton, 1993). In this situation, in order to continue
with the production of carbon skeletons to conserve
the carbon flux, several PPi-dependent enzymes
are activated, such as UDP-Glc phosphorylase and
PPi-dependent phosphofructokinase (Duff et al.,
1989; Ciereszko et al., 2001). Another alternative glycolytic pathway known in plants is catalyzed by the
action of a nonphosphorylating NADP-dependent
Plant Physiology, April 2009, Vol. 149, pp. 1906–1916, www.plantphysiol.org Ó 2009 American Society of Plant Biologists
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Regulation of AKIN10 and AKIN11 during Phosphate Starvation
glyceraldehyde-3-phosphate dehydrogenase, which is
induced during Pi starvation and bypasses Pi-dependent
NAD-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (Duff et al.,
1989; Theodorou and Plaxton, 1993; Plaxton and Carswell,
1999).
Signal transduction mechanisms that produce adaptive changes during Pi deficiency are not clearly
known. Modifications in gene expression in response
to Pi deficiency can be grouped into the early genes
that respond rapidly and often nonspecifically to Pi
deficiency and the late genes that alter the morphology, physiology, or metabolism of plants upon prolonged stress. These late genes generally increase the
acquisition of Pi or promote the efficient use of Pi
within the plant (Vance et al., 2003). In bean (Phaseolus
vulgaris) plants, we identified early and late genes that
encode proteins that might be involved in signal
transduction (Camacho et al., 2008; P. Coello, A.
Ramı́rez, and E. Martı́nez-Barajas, unpublished data).
One of the late genes has high homology with the
a-subunit of the AMP-dependent protein kinase
(AMPK) complex in animal cells. The protein kinases
SNF1/AMPK/Snf1-related kinase 1 (SnRK1) are a
conserved family of kinases that play an important
role in energy balance control, as demonstrated by the
mammalian complex AMPK (Hardie and Sakamoto,
2006). In yeast, Snf1 (for Suc nonfermenting 1) is
known to be a key player in the diauxic shift from
fermentative to oxidative metabolism in response to
Glc deprivation (Rolland et al., 2006). In plants, SnRK1
seems to have important roles in controlling metabolic
homeostasis and stress signaling (Baena-González
et al., 2007; Baena-González and Sheen, 2008) and
during plant development (Zhang et al., 2001; Lu et al.,
2007). The SnRK1 kinases are heterotrimeric complexes formed by an a catalytic subunit and b and g
regulatory subunits. In Arabidopsis (Arabidopsis thaliana), each subunit is encoded by multiple genes,
giving rise to a large variety of possible heterotrimeric
combinations (Polge and Thomas, 2006). To explore
the possibility that SnRK1 complexes are involved in
Pi-starvation responses, we characterized the expression and the activity of two of the catalytic subunits
(AKIN10 and AKIN11) in Arabidopsis plants growing
in Pi-sufficient and Pi-starvation conditions. Our results indicate that both catalytic subunits are mainly
located in the chloroplast. They are differentially regulated during Pi starvation. Whereas complexes
formed with the AKIN10 catalytic subunit increase
in activity, those formed with AKIN11 are specifically
degraded. To explore the role of AKIN10 during Pi
starvation, we isolated a mutant with a T-DNA insertion in akin10. No differences were observed between
wild-type and mutant plants during their development and growth. However, at the biochemical level,
starch metabolism, especially starch degradation, was
impaired in the mutant during Pi starvation. The
transcriptional activation of genes coding for proteins
directly involved in carbon metabolism and chloro-
plast biogenesis was up-regulated in the akin10 plants,
and genes involved in light signaling, protein degradation, and GTP signaling were down-regulated. All
of these results suggest that the AKIN10 catalytic
subunit of the SnRK1 complex might be involved in
the regulation of some of the metabolic changes that
are observed during Pi starvation.
RESULTS
Expression and Activity of SnRK1 during
Phosphate Starvation
It has been very well documented that kinases of
the AMPK/SnRK1/SNF1 family are activated in lowenergy conditions and when Glc is absent from the
medium (Baena-González and Sheen, 2008). In plants,
Pi starvation is a condition that produces an important
reduction in the amount of ATP. To get information
about the regulation of SnRK1 during Pi deficiency, its
kinase activity was measured by the phosphorylation
of the SAMS peptide. The SnRK1 activity was lower in
leaf extracts from Pi-starved plants (Fig. 1). To evaluate
if the difference in activity was due to differential
expression of the catalytic subunits, we searched the
Genevestigator database and found that only two of
the three genes that encode the catalytic subunits
(AKIN10 and AKIN11) were expressed in rosette
leaves. No changes in gene expression were found in
plants grown under complete nutritional conditions
Figure 1. SnRK1 activity in plants grown for 10 d in Pi-sufficient (+Pi)
and Pi-starvation (2Pi) conditions. Activity was estimated using the
SAMS peptide kinase assay. Bars represent means 6 SD (n = 3).
Plant Physiol. Vol. 149, 2009
1907
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Fragoso et al.
(Fig. 2A). In addition, after 5 and 10 d of Pi starvation,
semiquantitative reverse transcription (RT)-PCR indicated that no visible changes occurred in the amount
of the AKIN10 and AKIN11 transcripts. Amplification
of actin was used as an internal control to indicate
equal loading. At4 is a Pi starvation-inducible gene
and was used as an indicator of the Pi status (Burleigh
and Harrison, 1999). In contrast with AKIN10 and
Figure 2. A, Average signal of gene expression for AKIN10, AKIN11,
and AKIN12 derived from the microarray data available at the
Genevestigator database (Zimmermann et al., 2005) and arranged
according to tissue. B, Effect of Pi starvation on the accumulation of
AKIN10 and AKIN11 transcripts. RT-PCR of total RNA from rosette
leaves from plants grown for 5 and 10 d under Pi-sufficient (+) and Pistarvation (2) conditions. At4 is a Pi-starvation control gene, and actin
was used as an internal control. [See online article for color version of
this figure.]
AKIN11, At4 induction was higher in Pi-starved plants
and was observed after 5 d of treatment (Fig. 2B).
Phosphate Starvation Induces Differential Changes in
AKIN10 and AKIN11 Catalytic Subunits
Having established that no changes in gene expression were observed in the catalytic subunits during Pi
starvation to explain the reduction in activity, we
analyzed the protein levels. Transgenic Arabidopsis
plants carrying AKIN10-GFP and AKIN11-GFP fusion
proteins were evaluated. Localization of GFP fusion
proteins by confocal microscopy showed that both
catalytic subunits colocalized with the chlorophyll,
suggesting a chloroplast localization (Fig. 3, A and D).
In the case of AKIN10, a low but detectable signal was
also observed in the cytoplasm (Fig. 3C). To confirm
the localization of the catalytic subunits, we raised
polyclonal antibodies using a polypeptide that recognized AKIN10 and AKIN11 to detect the subunits in
root and leaf sections. Immunolocalization analysis
indicated that the catalytic subunits are present in both
organs (Fig. 4, A and G). In leaf cells, the catalytic
subunits are localized in the chloroplast but the signal
was also detected in the cytoplasm (Fig. 4, A–C). In
roots, the catalytic subunits were present in all cells,
from the epidermis to the vascular tissue (Fig. 4, G–I).
To probe the chloroplast localization of the catalytic
subunits at the biochemical level, proteins obtained
from purified chloroplasts were separated with SDSPAGE and transferred to a nitrocellulose membrane.
Western-blot analysis identified a polypeptide band
around 58 kD, which is the expected molecular mass
for AKIN11 and AKIN10. The same blot was probed
with Rubisco antibodies (Fig. 5). Under Pi starvation
conditions, the detection of GFP fusion proteins
showed no important changes in AKIN10. Interestingly, the fluorescence corresponding to AKIN11-GFP
almost completely disappeared (Fig. 6). This reduction
in the fluorescence corresponding to one of the catalytic subunits was also observed in the leaf and root
sections of Pi-starved plants, visualized using the antia-antibody (Fig. 4, D and J). In leaf cells, the amount of
the catalytic subunits decreased notably, since the
chlorophyll signal was more intense than the Alexa
568 signal (Fig. 4, D–F). In roots, the catalytic subunits
were confined to the surrounding vascular tissue and
the signal in the cortex and epidermis disappeared
(Fig. 4, J–L). In addition, the activity of SnRK1 in
transgenic plants overexpressing AKIN10-GFP growing in Pi-starvation conditions was higher than in
plants growing in Pi-sufficient conditions, whereas in
AKIN11-GFP plants, Pi starvation reduced SnRK1
activity (Fig. 7A). The determination of AKIN10-GFP
and AKIN11-GFP levels in transgenic plants using
anti-GFP antibodies showed no changes in AKIN10
and a significant reduction in AKIN11 during Pi
starvation (Fig. 7, B and C). These results strongly
suggest a differential regulation of both catalytic sub-
1908
Plant Physiol. Vol. 149, 2009
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Regulation of AKIN10 and AKIN11 during Phosphate Starvation
Figure 3. Confocal fluorescence images of AKIN10 and AKIN11 fusion
proteins showing localization at the
chloroplast. The GFP signal and the
chlorophyll autofluorescence are indicated in green and red, respectively.
The merged images (Merge) of chlorophyll autofluorescence, GFP, and light
field are shown. Bars = 10 mm. [See
online article for color version of this
figure.]
units under this nutritional condition. Since no
changes in protein levels for AKIN10 were found,
the increase in its activity might be explained by a
posttranslational modification. Activation by phosphorylation of the Thr-175 present at the T-loop was
evaluated using an antibody that recognized the
mammalian phosphorylated Thr-172. The results
showed that plants growing in Pi starvation had the
same level of phosphorylated AKIN10 than plants
growing in Pi-sufficient conditions, indicating that
additional mechanisms are activating the AKIN10
complexes (Fig. 7B).
Figure 4. Immunolocalization of the catalytic subunits. Leaf and root sections of plants grown with or
without Pi were immunolocalized with anti-aantibodies. A to C, Leaf sections of plants grown under
+Pi conditions (603). D to F, Leaf sections of plants
grown under 2Pi conditions (603). G to I, Roots
grown under +Pi conditions (403). J to L, Roots grown
under 2Pi conditions (403). A, D, G, and J show goat
anti-rabbit Alexa 568 fluorochrome staining (green
signal). B and E show chlorophyll (red signal). H and K
show bright-field images of the transverse sections of
roots. C and F are overlaid images of Alexa 568
fluorochrome and chlorophyll for colocalization. I
and L are overlaid images of Alexa 568 and bright
field. Bars = 10 mm. [See online article for color
version of this figure.]
Plant Physiol. Vol. 149, 2009
1909
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Fragoso et al.
Figure 5. Western-blot detection of the a-catalytic subunit. A, Total
protein extracts (T) and chloroplast-extracted proteins (C) were separated by SDS-PAGE and transferred to nitrocellulose membranes.
Immunodetection of AKIN10 and AKIN11 was performed using the
enhanced chemiluminescence kit. B, The membrane in A was reblotted
using antibodies against Rubisco.
In plants, SnRK1 is directly involved in the regulation of carbohydrate metabolism (Halford et al., 2003;
Polge and Thomas, 2006). Antisense expression of
SnRK1 in barley (Hordeum vulgare) produces pollen
with no starch and arrests at the binucleate state of
development (Zhang et al., 2001). In Arabidopsis,
starch degradation was impaired in a transient
akin10, akin11 double mutant (Baena-González et al.,
2007). During Pi starvation, starch accumulation in
leaves is one of the most consistently observed biochemical responses (Ciereszko and Barbachowska,
2000; Bernal et al., 2005). To determine if AKIN10 has
a regulatory role in starch metabolism, the amount of
starch was measured in wild-type and mutant plants.
There was no difference in the starch levels between
plants growing in Pi-sufficient conditions. However,
starch accumulation was higher in the mutant than in
the wild-type plants during phosphate starvation (Fig.
9A). To determine if starch mobilization was affected,
plants were incubated with 14CO2 and the distribution
of the label was analyzed. The differences between the
labels associated with the starch granules at the end of
the day and at the end of the night are indications of
starch mobilization. The results showed that less mobilization occurred in akin10 during Pi starvation than
in the wild type (Fig. 9B).
It has been demonstrated that SnRK1 also regulates
gene expression (Baena-González et al., 2007). To
determine if akin10 mutants also had a transcriptional
effect during Pi starvation, we did a microarray analysis to identify genes that could be regulated by
SnRK1. We evaluated the transcript accumulation of
some genes that had changed levels of expression. The
results showed that some of these genes, which are
directly involved in carbohydrate metabolism, increased transcript levels in mutant plants grown under
Pi starvation. In contrast, other genes related to general
stress were down-regulated (Fig. 10).
Silencing of akin10 Affects Starch Metabolism during
Pi Starvation
The results presented so far suggest that SnRK1
complexes are formed with the AKIN10 catalytic subunit under Pi starvation. To better understand the role
of AKIN10 during Pi starvation, a T-DNA insertion
line in akin10 was identified and characterized. In
addition, RT-PCR, using gene-specific primers, indicated that no AKIN10 expression was found in the
mutant. actin and AKIN11 were expressed in both the
wild-type and mutant lines (Fig. 8A). When the presence of AKIN10 and AKIN11 was evaluated by western blot using two-dimensional SDS-PAGE, AKIN10
(pI = 8.36) was observed only in the wild type, whereas
AKIN11 (pI = 7.4) was identified in both wild-type and
mutant plants (Fig. 8B). No differences in SnRK1
activity were observed in the wild-type and mutant
plants growing in Pi-sufficient conditions. However,
SnRK1 activity decreased more in the mutant than in
the wild-type plants when they were grown in Pistarvation conditions (Fig. 8C).
DISCUSSION
We have identified genes that are up-regulated in Pistarvation conditions, and some of them encode proteins that are involved in signal transduction (Camacho
et al., 2008; Martinez et al., unpublished data). One of
these proteins has high homology with the catalytic
subunit of the AMPK from mammalian cells and with
the catalytic subunit of the Snf1 from yeast. In plants,
these kinases have been classified as SnRK1. AMPK
has been directly implicated in the regulation of the
cellular energetic balance, acting as a sensor (Hardie,
2007). Therefore, plant SnRK1 might be involved in
signaling changes during Pi starvation.
SnRK1 Activity during Pi Starvation
By analyzing the Genevestigator database, we observed that the expression of AKIN12 was very low
during Arabidopsis development. Therefore, we fo-
1910
Plant Physiol. Vol. 149, 2009
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Regulation of AKIN10 and AKIN11 during Phosphate Starvation
Figure 6. Confocal fluorescence images of AKIN10GFP and AKIN11-GFP. Plants were grown in Pisufficient (+Pi) or Pi-starved (2Pi) conditions. GFP
signal is indicated in green. Chlorophyll autofluorescence is indicated in red. Bars = 10 mm. [See
online article for color version of this figure.]
cused our study on AKIN10 and AKIN11. The activity
of SnRK1 decreases during Pi starvation, and this
difference is not due to changes in the expression of
the catalytic subunits during normal or Pi-starvation
conditions. Overexpressing both catalytic subunits as
GFP fusion proteins indicated that during Pi starvation, AKIN11-GFP complexes decreased in activity
whereas AKIN10-GFP complexes increased. In the
AKIN11 transgenic plants, this catalytic subunit is
degraded during Pi starvation, and its degradation
produces a reduction in activity. Interestingly, in yeast,
the catalytic subunit of Snf1 interacts with the Srb10/
Srb11 protein kinase. After phosphorylation of the
C-terminal region of RNA polymerase II, its activity is
inhibited and Srb11 is degraded in a proteasomedependent manner. Moreover, Snf1 has been directly
implicated in its degradation (Cooper et al., 1999). In
the same way, Arabidopsis AKIN10 and AKIN11
interact with the Skp1/ASK1 subunit of an E3 ubiquitin ligase and with the a4/PAD1 subunit of the 20S
proteasome (Farrás et al., 2001). It has been proposed
that SnRK1 is part of an SCF complex that phosphorylates its substrates. Recently, it was demonstrated
that AKIN10 interacts with PRL1, which is a receptor
substrate of the complex DDB1-CUL4 E3 ligase, leading to its degradation (Lee et al., 2008). We are currently investigating if AKIN11 is similarly degraded
and if Pi starvation is the only signal that triggers its
destruction. Furthermore, we evaluated if the activation of AKIN10 was due to the specific phosphorylation of the Thr-175, which is equivalent to the
mammalian Thr-172 that is phosphorylated at the
T-loop during the activation of the complex (Sugden
et al., 1999). In AKIN10, the increase in activity could
not be explained by the phosphorylation of the Thr175 present at the T-loop since it is phosphorylated all
the time, indicating that Pi starvation is not a stimulus
that activates the complex. It is possible that AKIN10 is
associated with other regulatory subunits during Pi
starvation and that those complexes have differences in
activity. In mammalian cells, g1, associated either with
a1 or a2, forms complexes with higher activity than
with the other two g-subunits (Cheung et al., 2000),
indicating that the subunit composition regulates their
activity. Altogether, these results suggest that Arabidopsis plants growing in Pi-sufficient conditions
mainly have complexes formed by the AKIN11 catalytic
subunit. When plants are transferred to Pi starvation,
Figure 7. Characterization of transgenic Arabidopsis plants overexpressing AKIN10-GFP and AKIN11-GFP fusion proteins. A, SnRK1
activity of transgenic plants measured by the phosphorylation of the
SAMS peptide. Plants expressing GFP were used as controls. Bars
represent means 6 SD (n = 3). B, Protein extracts from plants overexpressing AKIN10-GFP were separated by SDS-PAGE and transferred
to nitrocellulose membranes. Immunodetection was carried out using
anti-GFP antibodies. C, Protein extracts from plants overexpressing
AKIN11-GFP were separated and immunodetected as described above.
D, Protein extracts from transgenic plants overexpressing AKIN10-GFP
were separated and immunodetected using a specific P-Thr antibody.
Plant Physiol. Vol. 149, 2009
1911
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Fragoso et al.
Figure 8. Characterization of akin10 mutants. A, RTPCR of wild-type (wt) and akin10 plants detecting
AKIN10 and AKIN11 transcripts. actin was used as
a control. B, Protein extracts obtained from wildtype and mutant plants were separated by twodimensional SDS-PAGE. Proteins were transferred
to nitrocellulose membranes, and the catalytic subunits were identified using the anti-a-antibody. C,
SnRK1 activity was measured in protein extracts obtained from wild-type and mutant plants grown for
5 and 10 d in Pi starvation. Bars represent means 6
SD (n = 3).
AKIN11 is degraded, with a concomitant reduction in
activity. On the other hand, there are few complexes
formed by AKIN10, and their activation during Pi
starvation is not enough to produce an increase in the
total activity of SnRK1. However, when AKIN11 is
degraded, the complexes formed by AKIN10 might be
important in regulating some metabolic changes.
Localization of AKIN10 and AKIN11 Is
Mainly Chloroplastic
The subcellular localization of AKIN10 and AKIN11
indicated that both enzymes are chloroplastic and
cytoplasmic. Cytoplasmic localization was assumed,
since SnRK1 has been directly involved in the regulation and inactivation of enzymes such as Suc phosphate synthase, nitrate reductase, and HMG-CoA
reductase (Hey et al., 2005; Polge and Thomas, 2006;
Polge et al., 2008). However, the chloroplastic localization was unexpected. An analysis of the N-terminal
sequence in AKIN10 and AKIN11 using the Wolf PSort
subcellular localization predictor (Horton et al., 2007)
indicated cytoplasmic, nuclear, and chloroplastic localizations, although the last two had low scores. The
diverse roles of the SNF1/AMPK/SnRK1 family of
kinases raise the question of how such versatility is
achieved. Genetic studies in yeast suggest that isoforms of the b-subunits are important for functional
specificity. In fact, subcellular localization of Snf1
kinase is regulated by specific b-subunits (Vincent
et al., 2001). In the case of Arabidopsis, the regulatory
subunit bg has a recognizable chloroplast transit peptide, so interaction with that subunit could direct the
catalytic subunits to the chloroplast. On the other
hand, the subcellular localization prediction for b1 and
b2 regulatory subunits (Horton et al., 2007) indicated
that they are mainly nuclear and nuclear and chloroplast, respectively. We can hypothesize that the interaction of the catalytic subunits with those regulatory
subunits would localize the complex in different organelles. We are currently evaluating the subcellular
localization of the catalytic subunits by making deletions of the N-terminal sequence of AKIN10/AKIN11GFP fusion proteins and observing the final GFP
localization. In addition, we are evaluating if a direct
interaction with the bg-subunit directs the complex to
the chloroplast.
Although the chloroplastic localization has not been
completely explained, starch metabolism, especially at
night, is impaired in Arabidopsis plants with a transient reduction in AKIN10 and AKIN11 proteins
(Baena-González et al., 2007), opening the possibility
that SnRK1 directly or indirectly regulates enzymes
that are present in the chloroplast. This is, to our
knowledge, the first report indicating the chloroplastic
localization of SnRK1.
AKIN10 Is Involved in Starch Degradation and in Gene
Expression during Pi Starvation
In order to obtain more information about the role of
AKIN10 during Pi starvation, we identified and char-
1912
Plant Physiol. Vol. 149, 2009
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Regulation of AKIN10 and AKIN11 during Phosphate Starvation
tase has a glycogen-binding domain, which in plants is
recognized to bind starch. The model proposes that
SEX4 interacts with starch in vivo and is directly
necessary for its metabolism. Thus, SEX4 may directly
regulate the activity of starch-degrading enzymes or
may modulate the activity of a protein kinase, which in
turn may change the activity of enzyme(s) involved in
starch degradation (Niittylä et al., 2006). Additionally,
SnRK1 forms complexes with b-subunits that contain a
putative glycogen-binding domain (P. Coello, unpublished data), so we can hypothesize that SnRK1 may be
bound directly to starch through the b-subunits and
either be modulated directly by SEX4 or in an independent manner. We are currently investigating some
of these possibilities.
Research on plant SnRK1 has revealed its additional
role as a central regulator of the transcriptome under
multiple types of stress signals (Baena-González et al.,
2007; Baena-González and Sheen, 2008). Our results
with the akin10 mutant showed that transcription was
affected during Pi starvation. The induction of genes
potentially involved in Suc synthesis (SPS4 and SPS2),
sugar metabolism (GAL1 and HEX), and vesiclemediated formation of thylakoid membranes (THF1)
occurred under these conditions. In contrast, other
genes involved in the monosaccharide transportation
in tonoplast (TMT2), protein degradation (FBP and
SKP), GTP signaling (RAB), and light signal transduction (NPH) were down-regulated. Some of these
Figure 9. Starch metabolism in wild-type and akin10 plants. A, Starch
accumulation in wild-type (wt) and akin10 plants grown in Pi-sufficient
and Pi-starvation conditions. FW, Fresh weight. B, Starch mobilization
measured as the difference in the 14C label associated with the starch
granules at the end of the day and at the end of the night. Bars represent
means 6 SD (n = 5).
acterized a mutant plant lacking this subunit. The
evaluation of its role in starch metabolism indicated
that mutant plants mobilized less starch at night than
wild-type plants during Pi starvation. Starch accumulation during Pi deficiency has been directly associated
with less degradation at night (Bernal et al., 2005).
Enzymes involved in starch degradation are not fully
understood, and the importance of phosphorylation in
modulating their activities has not been investigated.
This is in contrast with the modulation of starch
synthesis, where isoforms of starch-branching enzyme
are activated by phosphorylation in the chloroplast
(Tetlow et al., 2004). Even more, the overexpression of
a SnRK1 potato (Solanum tuberosum) catalytic subunit
increased starch levels up to 30%, and there is no
evidence that changes in the activation state of the
ADP-Glc pyrophosphorylase exist in these transgenic
plants, indicating that some other mechanism, such as
direct phosphorylation of other enzymes, could be
involved (McKibbin et al., 2006). However, starch
degradation was reduced in a sex4 mutant, which
has an impaired protein phosphatase. This phospha-
Figure 10. AKIN10 modifies the transcription of different genes. RTPCR of total RNA from rosette leaves from wild-type (wt) and akin10
plants growing in Pi starvation. Genes involved in carbon metabolism,
such as SUCROSE PHOSPHATE SYNTHASE4 (SPS4), SUCROSE
PHOSPHATE SYNTHASE2 (SPS2), GALACTOKINASE1 (GAL1), and
HEXOKINASE (HEX ); genes involved in Glc sensing, such as THYLAKOID FORMATION1 (THF1); and genes involved in general stress
signaling, such as SKP1-INTERACTING PARTNER5 (SKP5 ), CYSTEINE
PROTEINASE (CP ), PHOTOTROPIC RESPONSIVE (NPH ), HEXOSE
TRANSPORTER2 (TMT2), F-BOX PROTEIN (FBP ), and RAB-RELATED
GTP-BINDING PROTEIN (RAB) were evaluated using specific primers.
actin was used as an internal control. The accession number of each
gene is indicated in “Materials and Methods.”
Plant Physiol. Vol. 149, 2009
1913
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Fragoso et al.
changes in gene expression might reflect multiple
types of stress convergence produced by energy deficiency. This is supported by the fact that some of the
Pi-starvation responses are mediated by sugar signaling (Liu et al., 2005; Karthikeyan et al., 2007). The
involvement of SnRK1 in Pi starvation signal transduction is in accordance with the idea that different
stresses are sensed as energy deficiencies, so future
work is needed to delimit these convergent transcriptional stress responses in order to be able to manipulate crop responses to Pi limitation.
MATERIALS AND METHODS
Growth of Plants and Isolation of Mutants
Arabidopsis (Arabidopsis thaliana) plants were grown in a growth chamber
at 21°C to 23°C under short-day conditions (8 h of light/16 h of dark) at a
light intensity of 120 mmol photons m22 s21 (fluorescent bulbs). akin10 mutant
was isolated from the SIGnAL T-DNA collection (line SALK_ 127939). Homozygous mutants were identified by PCR genotyping using the following
gene-specific primers: LP (5#-ACCACACGTTGGAAACTTTTG-3#) and RP
(5#-CAGATGGGTTCCTAACAGCAG-3#) in combination with the T-DNAspecific primer LBb1 (5#-GCGTGGACCGCTTGCTGCAACT-3#). For the
phosphate-starvation treatment, seedlings were transferred to Agrolite and
watered every third day with a Hoagland II solution (Jones, 1982), either
containing 500 mM ammonium phosphate (+Pi) as the only source of P or no
phosphate (2Pi), depending on the experiment.
RNA Isolation and RT-PCR
Total RNA was isolated using the Trizol reagent according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized using ImProm-II
Reverse Transcriptase according to the manufacturer’s instructions (Promega). The gene-specific primer pairs (forward and reverse) for detecting the
following genes were as follows: SPS4 (At4g10120), 5#-GCTAGCCTCAGGTTCAAGCT-3# and 5#-TGTGGAGGCCACCCAGTAAG-3#; SPS2 (At5g11110),
5#-TTCCGCTGGGGTGGAGAGAG-3# and 5#-AGAGCGACCTTAATGGCGTC-3#; HEX (At2g19860), 5#-TGGCGATATCGTCCCACCTA-3# and 5#-TGAGAGTGAGAAGCAGCAAG-3#; GAL1 (At3g06580), 5#-CCATACACGGCTGAGGAGAT-3# and 5#-AGAGCGACCTTAATGGCGTC-3#; THF1 (At2g20890),
5#-CAAGCGACCCATCCCGAGTA-3# and 5#-TGCTTCTTCTTCTCCCTCTC-3#;
TMT2 (At4g35300), 5#-TTGAGCAGGCGGGTGTCGGG-3# and 5#-GCAGCACGGGGAGACTGTAA-3#; FBP (At5g10770), 5#-CAGGGATCTTCGACGGCTCT-3# and 5#-CGTCCACACAAATTCAGGCG-3#; RAB (At3g09900), 5#-GATATGGGACACTGCTGGAC-3# and 5#-TGATCCCTTGGGGCTCGGCT-3#; SKP
(At3g54480), 5#-TTGCCTGGTCGGTGGAGGCG-3# and 5#-CGAACACGCTGCAGAACCAA-3#; CP (At3g19400), 5#-AACGCTGGATGTGATGGAGG-3#
and 5#-GGGTAAGAAGGCATCATTGC-3#; and NPH (At1g03010), 5#-AGCTTGTAGCCACGCAGCTC-3’ and 5#-GCCTTGCCACTCAAGGAAGG-3#. As
an internal control, the following forward and reverse primers of the ACTIN
gene were designed: 5#-TGGGGCAGAAGGATGCGTATG-3# and 5#-CAATACCAGTTGTACGGCCAC-3#. For a phosphate-starvation control, the At4
cDNA was amplified using the following primers: forward (5#-CCCCTAAAGAAACTGAAGCTCAAGAA-3#) and reverse (5#-GGAACATAAACATATAAGAGCTAT-3#). To analyze gene expression, equivalent amounts of total RNA
were input in cDNA synthesis and the same amount of cDNA was included in
PCR. The resultant PCR products were resolved on a 1% agarose gel for
comparison.
Transgenic Plant Construction
The coding regions of AKIN10 (At3g01090) and AKIN11 (At3g29160) were
amplified by PCR using the primers for AKIN10 (5#-CCCATGGATGGATCAGGCACA-3# and 5#-GAGGACTCGGAGCTGAGCAAG-3#) and AKIN11
(5#-CACCATGGATCATTCATCAAAT-3# and 5#-GATCACACGAAGCTCTGTAAG-3#). The fragment with the coding sequence was introduced into the
pENTR/D-TOPO vector (Invitrogen) and then recombined with an N-terminal
GFP fusion binary vector (pEarlyGate103) using recombinant Gateway
technology (Earley et al., 2006). Arabidopsis transformation using the Agrobacterium tumefaciens strain GV3101 was performed by the floral dip method
(Clough and Bent, 1998).
Kinase Activity Assay
Frozen leaves were homogenized in a chilled mortar with ice-cold extraction buffer containing 50 mM HEPES, pH 8.2, 50 mM NaF, 1 mM EDTA, 1 mM
EGTA, 1 mM Na3VO4, 50 mM NaCl, 10 mM b-glycerophosphate, 8% (v/v)
glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, and 50 mM leupeptin. The homogenate was centrifuged at
20,000g for 10 min at 4°C. Proteins obtained in the crude extracts were
precipitated by adding 11% (w/v) polyethylene glycol 10,000. The solution
was gently stirred for 20 min and centrifuged at 20,000g for 30 min. The pellet
was dissolved in 30 mL of extraction buffer, and the total protein was
evaluated according to the Bradford method (Bradford, 1976). One or 2 mg
of total protein was used in the assay. The SAMS peptide kinase activity was
determined as described by Davies et al. (1989), but with some modifications.
Peptide phosphorylation was assayed in a final volume of 25 mL containing
kinase buffer (40 mM HEPES, pH 7.0, 50 mM sodium fluoride, 25 mM
b-glycerophosphate, 50 mM NaCl, 8% [v/v] glycerol, 0.8 mM EDTA, 1 mM
dithiothreitol, 5 mM MgCl2,1 mM EGTA, and 1 mM Na3VO4), 200 mM SAMS
peptide (HMRSAMSGLHLVKRR), 200 mM AMP, and [g-32P]ATP (200 mM, 5
mCi mL21, 3,000 Ci mmol21). After incubation for 10 min at 30°C, 15-mL
aliquots were removed and spotted on 1-cm 3 1-cm squares of phosphocellulose paper (P81; Whatman), which were dropped into 500 mL of 1% (v/v)
H3PO4. The squares were washed three times for 20 min each in 1% H3PO4 and
then once with acetone, air dried, and counted by immersion in 5 mL of a
toluene-based scintillation fluid.
Chloroplast Extraction
Chloroplasts were isolated from Arabidopsis leaves and purified by
centrifugation through Percoll (Pharmacia) gradients as described by Weigel
and Glazebrook (2002).
Antibodies and Western Blots
A peptide corresponding to 10 conserved amino acids in the AKIN10 and
AKIN11 proteins was synthesized by Sigma. Peptide antigens were made by
linking synthetic peptide to keyhole limpet hemocyanin by a standard
protocol (Harlow and Lane, 1988). The hemocyanin-conjugated peptides
were used to raise antibodies in a female New Zealand White rabbit. For the
initial immunization, 200 mg of conjugate in Freund’s complete adjuvant was
delivered via several subcutaneous injections. The rabbit received two additional boosts, using 200 mg of hemocyanin-conjugated peptides in Freund’s
incomplete adjuvant at 2 and 6 weeks after initial injections. The final bleed
was performed by cardiac puncture 9 d after the final injection.
Antibodies that recognized the mammalian phosphorylated Thr at the
T-loop of the catalytic subunit were purchased from Santa Cruz Biotechnology. Antibodies that recognized the N terminus of the GFP were purchased
from Sigma.
Soluble or chloroplastic proteins were subjected to electrophoresis after
mixing with 23 Laemmli SDS-PAGE sample buffer and boiling for 5 min.
After electrophoresis, proteins were transferred to nitrocellulose membranes
using a mini gel apparatus (Bio-Rad). The blot was developed using an
enhanced chemiluminescence kit (GE Healthcare). Two-dimensional SDSPAGE was performed using the Multiphor II Electrophoresis Unit (Amersham) according to the manufacturer’s instructions.
Microscopy and Immunolabeling
Roots and leaves of Arabidopsis grown with or without phosphate were
harvested and fixed in 4% (v/v) formaldehyde in phosphate-buffered saline
(PBS), dehydrated in an ethanol series, and embedded in Paraplast Plus
(Polysciences). Sections of 6 to 7 mm were blocked with PBS plus 3% bovine
serum albumin, 0.01% sodium azide, and 0.1% Triton X-100 for 4 h at 4°C.
Sections were simultaneously incubated with the primary rabbit antia-subunit antibody (1:250 dilution) at 4°C overnight. Sections were then
rinsed with PBS and then incubated with secondary goat anti-rabbit Alexa
1914
Plant Physiol. Vol. 149, 2009
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Regulation of AKIN10 and AKIN11 during Phosphate Starvation
568-fluorochrome-conjugated antibody for 4 h at 4°C. Sections were examined
by confocal fluorescence
For the study of GFP fusion proteins, leaf sections were mounted on slides
and the GFP fluorescence was observed using a FluoView 1000 confocal
microscope (Olympus). Images were captured with a digital camera, and
photographs were processed for optimal presentation using Photoshop 8.0
14
CO2 Labeling, Starch Isolation, and Determination
Mature leaves were labeled with 14CO2 as described by Viola et al. (2001).
Five hours after the light period started, plants were incubated with the 14CO2
liberated from 50 mCi (2.18 GBq mmol21) of sodium [14C]bicarbonate by the
addition of 200 mL of 3 M lactic acid. Plants were exposed to 14CO2 for 1 h
before injecting an excess of 3 M KOH to neutralize the acid and to absorb any
remaining 14CO2. One group of plants was analyzed 4 h later, and another
group was incubated in the dark for 18 h. For starch isolation and quantification, leaf samples were homogenized in 2 to 3 mL of 80% ethanol and
extracted twice at 80°C for 30 min. The soluble fraction was discarded, and the
ethanol-insoluble fraction was resuspended in 1.5 mL of water and incubated
at 90°C for 4 h. After cooling, 220 units of amyloglucosidase (from Rhizopus;
EC 3.2.1.3) in 1.5 mL of 200 mM acetate buffer (pH 4.5) was added to each
sample and incubated at 37°C overnight. Glc and the label associated with the
starch were determined in the supernatant (Bernal et al., 2005).
ACKNOWLEDGMENTS
We thank Lorena Chávez González, Simón Guzmán León, José Luis
Santillán Torres, and Jorge Ramı́rez for assistance in the microarray determination; Gerardo Coello, Gustavo Corral, and Ana Patricia Gómez for
computer assistance; and Laurel Fabila and Carmen Parra for technical
assistance.
Received November 26, 2008; accepted February 5, 2009; published February
11, 2009.
LITERATURE CITED
Baena-González E, Rolland F, Thevelein JM, Sheen J (2007) A central
integrator of transcriptional network in plant stress and energy signalling. Nature 448: 938–943
Baena-González E, Sheen J (2008) Convergent energy and stress signaling.
Trends Plant Sci 13: 474–482
Bates TR, Lynch JP (1996) Stimulation of root hair elongation in Arabidopsis
thaliana by low phosphorus availability. Plant Cell Environ 19: 529–538
Bernal L, Coello P, Martı́nez-Barajas E (2005) Possible role of R1 protein on
starch accumulation in bean seedling (Phaseolus vulgaris L.) under
phosphate deficiency. J Plant Physiol 162: 970–976
Bieliski R (1973) Phosphate pools, phosphate transport and phosphate
availability. Annu Rev Plant Physiol 24: 225–252
Bradford MM (1976) A rapid and sensitive method for the quantitation of
protein utilizing the principle of protein-dye binding. Anal Biochem 72:
248–254
Burleigh SH, Harrison MJ (1999) The down-regulation of Mt4-like genes
by phosphate fertilization occurs systemically and involves phosphate
translocation to the shoots. Plant Physiol 119: 241–248
Camacho Y, Martınez-Castilla L, Fragoso S, Vázquez S, Martı́nez-Barajas
E, Coello P (2008) Characterization of a type A response regulator in the
common bean (Phaseolus vulgaris) in response to phosphate starvation.
Physiol Plant 132: 272–282
Cheung PCF, Salt IP, Davies SP, Hardie DG, Carling D (2000) Characterization of AMP-activated protein kinase c-subunit isoforms and their
role in AMP binding. Biochem J 346: 659–669
Ciereszko I, Barbachowska A (2000) Sucrose metabolism in leaves and
roots of bean (Phaseolus vulgaris L.) during phosphate deficiency. J Plant
Physiol 156: 640–644
Ciereszko I, Johansson H, Hurry V, Kleczkowski LA (2001) Phosphate
status affects the gene expression, protein content and enzymatic
activity of UDPglucose pyrophosphorylase in wild-type and pho mutants of Arabidopsis. Planta 212: 598–605
Clough FJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:
735–743
Cooper KF, Mallory MJ, Strich R (1999) Oxidative stress-induced destruction of the yeast C-type cyclin Ume3p requires phosphatidylinositolspecific phospholipase C and the 26S proteasome. Mol Cell Biol 19:
3338–3348
Davies SP, Carling D, Hardie DG (1989) Tissue distribution of the AMPactivated protein kinase, and lack of activation by cyclic-AMP-dependent
protein kinase, studies using a specific and sensitive peptide assay. Eur
J Biochem 186: 123–128
Duff SMG, Moorhead GBG, Lefebvre DD, Plaxton WC (1989) Phosphate
starvation inducible “bypasses” of adenylate and phosphate dependent
glycolytic enzymes in Brassica nigra suspension cells. Plant Physiol 90:
1275–1278
Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS
(2006) Gateway-compatible vectors for plant functional genomics and
proteomics. Plant J 45: 616–629
Farrás R, Ferrando A, Jaásik J, Kleinow T, Ocres L, Tiburcio A, Salchert K,
del Pozo C, Schell J, Koncz C (2001) SKP1-SnRK protein kinase
interactions mediate proteasomal binding of a plant SCF ubiquitin
ligase. EMBO J 20: 2742–2756
Halford NG, Hey S, Jhurreea D, Laurie S, McKibbin RS, Paul M, Zhang Y
(2003) Metabolic signalling and carbon partitioning: role of Snf-1 related
(SnRK1) protein kinase. J Exp Bot 54: 467–475
Hardie DG (2007) AMP activated/Snf1 protein kinases: conserved guardians of cellular energy. Nat Rev Mol Cell Biol 8: 774–785
Hardie DG, Sakamoto K (2006) AMPK: a key sensor of fuel and energy
status in skeletal muscle. Physiology (Bethesda) 21: 48–60
Harlow E, Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, pp 53–77
Hey SJ, Powers SJ, Beale MH, Hawkins ND, Ward JL, Halford NG (2005)
Enhanced seed phytosterol accumulation through expression of a modified HMG-CoA reductase. Plant Biotechnol J 4: 219–229
Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ,
Naka K (2007) WoLF PSORT: protein localization predictor. Nucleic
Acids Res 35: W585–W587
Jones JB (1982) Hydroponics: its history and use in plant nutrition studies.
J Plant Nutr 5: 1003–1030
Karthikeyan A, Varadarajan DK, Jain A, Held MA, Carpita NC, Raghothama
KG (2007) Phosphate starvation responses are mediated by sugar signaling
in Arabidopsis. Planta 225: 907–918
Lee JH, Terzaghi W, Gusmaroli G, Charron JB, Yoon HJ, Chen H, He YJ,
Xiong Y, Denga X (2008) Characterization of Arabidopsis and rice DWD
proteins and their roles as substrate receptors for CUL4-RING E3
ubiquitin ligases. Plant Cell 20: 152–167
Liu J, Samac DA, Bucciarelli B, Allan DL, Vance CP (2005) Signaling of
phosphorus deficiency-induced gene expression in white lupin requires
sugar and phloem transport. Plant J 41: 257–268
López-Bucio J, Hernandez-Abreu E, Sanchez-Calderon L, Nieto-Jacobo
MR, Simpson J, Herrera-Estrella L (2002) Phosphate availability alters
architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiol 129: 244–256
Lu CA, Lin CC, Lee KW, Chen JL, Huang LF, Ho SL, Liu HJ, Hsing YI, Yu
SM (2007) The SnRK1A protein kinase plays a key role in sugar
signaling during germination and seedling growth of rice. Plant Cell
19: 2484–2499
Ma Z, Bielenberg DG, Brown KM, Lynch JP (2001) Regulation of root hair
density by phosphorus availability in Arabidopsis thaliana. Plant Cell
Environ 24: 459–467
McKibbin RS, Muttucumaru N, Paul MJ, Powers SJ, Burrell MM, Coates
S, Purcell PC, Tiessen A, Geigenberger P, Halford NG (2006) Production of high-starch, low-glucose potatoes through over-expression of the
metabolic regulator SnRK1. Plant Biotechnol J 4: 409–418
Niittylä T, Comparot-Moss S, Lue WL, Messerli G, Trevisan M, Seymour
MDJ, Gatehouse JA, Villadsen D, Smith SM, Chen J, et al (2006)
Similar protein phosphatases control starch metabolism in plants and
glycogen metabolism in mammals. J Biol Chem 281: 11815–11818
Plaxton W (2004) Plant responses to stress: biochemical adaptations to
phosphate deficiency. In Encyclopedia of Plant and Crop Sciences.
Taylor and Francis, New York, pp 976–980
Plaxton WC, Carswell MC (1999) Metabolic aspects of the phosphate
starvation response in plants. In HR Lerner, ed, Plant Responses to
Environmental Stresses, from Phytohormones to Genome Reorganization. Marcel Dekker, New York, pp 350–372
Poirier Y, Bucher M (2002) Phosphate transport and homeostasis in
Plant Physiol. Vol. 149, 2009
1915
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Fragoso et al.
Arabidopsis. In CR Somerville, EM Meyerowitz, eds, The Arabidopsis
Book. American Society of Plant Biologists, Rockville, MD, doi/10.1199/
tab.0024, http://www.aspb.org/publications/arabidopsis/
Polge C, Jossier M, Crozet P, Gissot L, Thomas M (2008) b-Subunits of the
SnRK1 complexes share a common ancestral function together with expression and function specificities: physical interaction with nitrate reductase
specifically occurs via AKINb1-subunit. Plant Physiol 148: 1570–1582
Polge C, Thomas M (2006) SNF1/AMPK/SnRK1 kinases, global regulators
at the heart of energy control? Trends Plant Sci 12: 20–28
Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signaling
in plants: conserved and novel mechanisms. Annu Rev Plant Biol 57:
675–709
Sugden C, Donaghy PG, Halfors NG, Hardie DG (1999) Two SNF1 related
protein kinases from spinach leaf phosphorylate and inactivate
3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase
and sucrose phosphate synthase in vitro. Plant Physiol 120: 257–274
Tetlow IJ, Wait R, Lu Z, Akkasaeng R, Bowsher CG, Esposito S, KosarHashemi B, Morell MK, Emes MJ (2004) Protein phosphorylation in
amyloplast regulates starch branching enzyme activity and proteinprotein interaction. Plant Cell 16: 694–708
Theodorou ME, Plaxton WC (1993) Metabolic adaptation of plant respiration to nutritional phosphate deprivation. Plant Physiol 101: 339–344
Vance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use:
critical adaptations by plants for securing a nonrenewable resource.
New Phytol 157: 423–447
Vincent O, Townley R, Kuchin S, Carlson M (2001) Subcellular localization of the Snf1 kinase is regulated by specific b subunits and a novel
glucose signaling mechanism. Genes Dev 15: 1104–1114
Viola R, Roberts AG, Haupt S, Gazzani S, Handcock RD, Marmiroli N,
Machray GC, Oparka KJ (2001) Tuberization in potato involves a switch
from apoplastic to symplastic phloem unloading. Plant Cell 13: 385–398
Weigel D, Glazebrook J (2002) Arabidopsis: A Laboratory Manual. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 217–219
Zhang Y, Shewry PR, Jones H, Barcelo P, Lazzeri PA, Halford NG (2001)
Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley. Plant J
28: 431–441
Zimmermann P, Hennig L, Gruissem W (2005) Gene-expression analysis
and network discovery using Genevestigator. Trends Plant Sci 10:
407–409
1916
Plant Physiol. Vol. 149, 2009
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2009 American Society of Plant Biologists. All rights reserved.