The Plant Journal (2011) 66, 863–876
doi: 10.1111/j.1365-313X.2011.04547.x
Interplay of SLIM1 and miR395 in the regulation
of sulfate assimilation in Arabidopsis
Cintia G. Kawashima1,†, Colette A. Matthewman2,†, Siqi Huang1, Bok-Rye Lee2,‡, Naoko Yoshimoto3,4, Anna Koprivova2,
Ignacio Rubio-Somoza5, Marco Todesco5, Tina Rathjen1, Kazuki Saito3,4, Hideki Takahashi4,‡, Tamas Dalmay1,* and
Stanislav Kopriva2,*
1
School of Biological Sciences, University of East Anglia, Norwich, UK,
2
Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK,
3
Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University,
Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan,
4
RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan, and
5
Department of Molecular Biology, Max Planck Institute for Developmental Biology, (VI) Spemannstr. 37-39 D-72076,
Tübingen, Germany
Received 7 January 2011; revised 18 February 2011; accepted 21 February 2011; published online 4 April 2011.
*
For correspondence (fax +44 1603 592250; e-mail T.Dalmay@uea.ac.uk or +44 1603 450014; Stanislav.Kopriva@bbsrc.ac.uk).
†
These authors contributed equally to this work.
‡
Present address: Department of Biochemistry and Molecular Biology, Michigan State University, 209 Biochemistry Building, East Lansing, MI 48824-1319, USA.
SUMMARY
MicroRNAs play a key role in the control of plant development and response to adverse environmental
conditions. For example, microRNA395 (miR395), which targets three out of four isoforms of ATP sulfurylase,
the first enzyme of sulfate assimilation, as well as a low-affinity sulfate transporter, SULTR2;1, is strongly
induced by sulfate deficiency. However, other components of sulfate assimilation are induced by sulfate
starvation, so that the role of miR395 is counterintuitive. Here, we describe the regulation of miR395 and its
targets by sulfate starvation. We show that miR395 is important for the increased translocation of sulfate
to the shoots during sulfate starvation. MiR395 together with the SULFUR LIMITATION 1 transcription factor
maintain optimal levels of ATP sulfurylase transcripts to enable increased flux through the sulfate assimilation
pathway in sulfate-deficient plants. Reduced expression of ATP sulfurylase (ATPS) alone affects both sulfate
translocation and flux, but SULTR2;1 is important for the full rate of sulfate translocation to the shoots. Thus,
miR395 is an integral part of the regulatory circuit controlling plant sulfate assimilation with a complex
mechanism of action.
Keywords: microRNA, sulfate deficiency, sulfate assimilation, ATP sulfurylase, Arabidopsis.
INTRODUCTION
Sulfur is an essential macronutrient found in plants in the
amino acids cysteine and methionine, which are indispensable components of proteins and peptides, in co-enzymes
and prosthetic groups, and in a range of secondary metabolites. Plants take up inorganic sulfate from the soil, reduce it
into sulfide and incorporate it into the amino acid cysteine
(Kopriva, 2006; Takahashi et al., 2011). In the sulfate assimilation pathway, sulfate is first activated by ATP sulfurylase
(ATPS) to adenosine-5¢-phosphosulfate (APS). APS is
reduced by APS reductase (APR) to sulfite, which is subsequently reduced to sulfide by sulfite reductase. Sulfide
reacts with O-acetylserine (OAS) to form cysteine in a reaction catalyzed by OAS-(thiol)lyase. Sulfate assimilation is
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd
highly regulated in a demand-driven manner, and is
coordinated with the metabolism of nitrogen and carbon
(Lappartient and Touraine, 1996; Kopriva and Rennenberg,
2004; Davidian and Kopriva, 2010).
Particular attention has been given to understanding the
regulation of plant responses to sulfate starvation, including
transcriptomics and metabolomics approaches (Hirai et al.,
2003, 2005; Maruyama-Nakashita et al., 2003, 2005, 2006;
Nikiforova et al., 2003; Wawrzyńska et al., 2005). In response
to sulfate deficiency, plants induce sulfate uptake and the
rate of sulfate reduction, by the coordinated induction of
mRNA levels for sulfate transporters and APR, the key
enzyme of sulfate assimilation (Vauclare et al., 2002; Hirai
863
864 Cintia G. Kawashima et al.
et al., 2003; Maruyama-Nakashita et al., 2003; Nikiforova
et al., 2003). Whereas the physiological responses are well
understood, the molecular basis of this regulation remains
largely unknown. The exception is the identification of a
SULFUR LIMITATION 1 (SLIM1) transcription factor, which
is responsible for increasing the expression of sulfate
transporters and other genes induced by sulfate deficiency
(Maruyama-Nakashita et al., 2006). However, SLIM1 cannot
be the only factor involved in the regulation of the pathway
by sulfate starvation, because, for example, induction of APR
mRNA is not compromised in slim1 mutants (MaruyamaNakashita et al., 2006).
Another player in the sulfate starvation regulatory circuit is
microRNA395 (miR395). Computational analysis of the Arabidopsis genome revealed that two components of sulfate
assimilation, the low-affinity sulfate transporter SULTR2;1
and ATPS, are targeted by miR395 (Jones-Rhoades and
Bartel, 2004), and that the mRNAs encoding isoforms ATPS1
and ATPS4 of ATPS, and SULTR2;1, are cleaved by miR395
(Jones-Rhoades and Bartel, 2004; Allen et al., 2005; Kawashima et al., 2009). The accumulation of miR395 is strongly
induced by sulfate starvation. Higher levels of miR395 were
also found in phloem of Brassica napus plants after sulfate
starvation (Buhtz et al., 2008). In these respects miR395
resembles miR399, which is induced by phosphate deficiency and acts as a long-distance signal in the regulation of
phosphate homeostasis (Chiou et al., 2006; Pant et al., 2008).
Recently, a systematic analysis of all six miR395 loci and their
targets revealed that ATPS1, ATPS4 and SULTR2;1 are
targets of the miRNA in both leaves and roots, whereas
ATPS3 was cleaved in about 50% of cases in the leaves
(Kawashima et al., 2009). Two loci (miR395d and miR395f)
are expressed very weakly in both high and low sulfate
conditions, but the other four loci are strongly induced by
sulfate deficiency. Whereas miR395a and miR395b are
expressed most strongly in roots in the absence of sulfate,
miR395c and miR395e were strongly upregulated in both
roots and leaves grown on sulfate-free media (Kawashima
et al., 2009). The increased accumulation of miR395 following sulfate starvation is dependent on SLIM1 (Kawashima
et al., 2009). However, levels of SULTR2;1 transcript actually
increase in roots of S-starved plants, despite the accumulation of miR395. Comparison of the tissue-specific expression
of miR395 and SULTR2;1 revealed that the role of miR395
might be to limit the expression of SULTR2;1 to xylem cells
(Kawashima et al., 2009).
Very recently Liang et al. (2010) showed that miR395 is
responsible for the accumulation of sulfate in leaves, and its
translocation from old to young leaves, and showed that
SULTR2;1, ATPS1 and ATPS4 play a role in this process.
However, very little is known about the effects that miR395
regulation has on ATPS, on its enzyme activity and downstream effects on the whole sulfate assimilation, particularly
the fluxes through the pathway. This is important as ATPS
is localized in both plastids and the cytosol, although the
identity of the cytosolic isoform is not yet known (Kopriva
et al., 2009). We also addressed the interplay between SLIM1
and miR395 in the regulation of the sulfate-starvation
response, and the contribution of ATPS and SULTR2;1 to
the observed regulation. We show that despite strong
downregulation of mRNA levels of the miR395-targeted
ATPS4, total ATPS activity is only moderately affected by
sulfate starvation. By comparing plants overexpressing
miR395, plants in which miR395 activity was reduced by a
target mimic (Franco-Zorrilla et al., 2007; Todesco et al.,
2010) and slim1-1 mutants, we have been able to dissect the
contributions of these different regulatory components to
the control of responses to sulfate starvation in Arabidopsis.
RESULTS
Regulation of ATPS during sulfate starvation
We exposed Arabidopsis seedlings to 4 days of sulfate
starvation, which led to the induction of miR395 levels in
leaves and roots (Figure 1a). mRNA levels for the APR isoform APR2, as a marker for sulfate limitation-responsive gene
expression, and the miR395-target SULTR2;1 (Kawashima
et al., 2009), were increased in the roots of sulfate-starved
plants (Figure 1b). However, the two other confirmed targets
of miR395 responded differently to the treatment. Levels of
ATPS4 mRNA were reduced strongly by sulfate starvation,
but ATPS1 transcript levels were unaffected (Figure 1b). The
mRNA levels of the non-targeted ATPS2 were also unaffected
by the treatment, whereas ATPS3 mRNA levels were
increased in the roots of sulfate-starved plants. In agreement
with the transcript data, APR activity was increased in sulfatedeficient roots. Total ATPS activity was slightly reduced
(Figure 1c). Sulfate starvation resulted in slightly but not
significantly decreased levels of glutathione (GSH) in roots,
whereas cysteine levels were unaffected (Figure 1d).
As the effect of sulfate starvation on ATPS1 mRNA
accumulation was only marginal, despite the strong induction of miR395, we suspected that the action of miR395
might be counterbalanced by increased transcription of the
ATPS1 gene. We analyzed the effect of sulfate starvation on
plants expressing GFP either under the control of the ATPS1
promoter or fused C-terminally to ATPS1 and expressed
from the same ATPS1 promoter. The GFP fluorescence in
the ATPS1PRO::GFP lines followed the transcriptional regulation of the promoter. The transcript of ATPS1:GFP fusion is
targeted by the miR395, and any alteration in fluorescence of
this fusion protein therefore represents the combined effects
of both transcriptional and miR395 regulation. Sulfate
starvation increased GFP fluorescence in the roots of two
independent transgenic ATPS1PRO::GFP lines, but the GFP
fluorescence in lines expressing the ATPS1PRO::ATPS1:GFP
construct was not affected by sulfate deficiency (Figure 2).
qPCR analysis of GFP mRNA in these lines confirmed that
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
miR395 in the control of S assimilation 865
(a)
(b)
levels were compared with TIP41 or UBC (APR2), and the levels in plants
grown on GM were set to 1. (c) ATPS and APS reductase (APR) enzyme
activities were determined in root extracts. (d) Cysteine and glutathione levels
in root tissues were determined by HPLC. Results are presented as
means SDs from three independent biological replicates. *Significant
difference between S0 and control plants (Student’s t-test; P £ 0.05).
there were significantly higher steady-state levels in sulfatestarved ATPS1PRO::GFP lines, but not in ATPS1PRO::ATPS1:
GFP lines (Figure 2k). Therefore, the ATPS1 promoter
appears to respond to sulfate starvation in a similar way to
the sulfate starvation-inducible promoters of the SULTR1;1,
SULTR1;2 and APR genes (Maruyama-Nakashita et al., 2005,
2006). As the total ATPS1 mRNA levels were unaffected by
the treatment (Figure 1b), it seems that the induced levels
of miR395 prevent a high accumulation of ATPS1 mRNA in
sulfate-deficient plants.
Contribution of miR395 and SLIM1 to the sulfate-starvation
response
(c)
(d)
Figure 1. Regulation of miR395 and its target ATP sulfurylase (ATPS) in roots
by sulfate deficiency.
Arabidopsis (Col-0) plants were grown on growth media containing 1500 lM
sulfate (GM) for 2 weeks, and were then transferred to either GM or media
lacking a sulfur source (S0) and grown for a further 4 days. (a) Northern blot
analysis of miR395; the membrane was exposed for 1 day. For a control, the
membranes were stripped and re-probed with a U6-specific probe. (b) RNA
was isolated from the roots and subjected to quantitative RT-PCR with
primers specific to the four ATPS genes, SULTR2;1 and APR2. The mRNA
Both miR395 and SLIM1 are components of the regulatory
network controlling the response to sulfate starvation.
However, as miR395 is under the control of SLIM1 (Kawashima et al., 2009), we tested their individual contributions
to the sulfate-starvation response. To increase the levels of
miR395 in a SLIM1-independent manner, we constitutively
overexpressed miR395c and miR395e, which represent the
two classes of miR395, differing in one nucleotide. To block
the effect of miR395 during sulfate deficiency we used the
target mimic described by Todesco et al. (2010). In addition,
we analysed the response of ATPS to sulfate starvation in
the slim1-1 mutant (Maruyama-Nakashita et al., 2006). Twoweek-old seedlings were used for these experiments, and at
this stage the plants were phenotypically indistinguishable,
in contrast to the findings of Liang et al. (2010), who showed
that miR395-overexpressing plants are smaller at later
developmental stages. As expected, whereas in Col-0
miR395 was highly induced by sulfate starvation in both
roots and leaves; in slim1-1 plants no increase in miR395
levels was observed (Figure 3). Overexpression of both
variants of miR395 resulted in the strong accumulation of
the miRNA in both roots and shoots, irrespective of sulfate
status. No increase of miR395 accumulation by sulfate limitation was observed for the two lines overexpressing
miR395, probably because of saturation of the northern blot.
In the target mimic line, MIM395, miR395 was highly abundant under sulfate-deficient conditions in both organs (Figure 3). However, in vivo most of this miRNA would be
bound to the target mimic, and therefore inactive. Interestingly, low levels of miR395 were detected in MIM395 and
slim1-1 roots at normal sulfate supply, probably because of
low sulfate contents in the leaves of these plants (see below).
The manipulation of miR395 expression did not alter transcript levels of SLIM1 under either control or sulfatedeficient conditions (Figure S1).
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
866 Cintia G. Kawashima et al.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
Figure 2. Sulfate deficiency upregulates GFP
expression driven by the ATPS1 promoter.
Fluorescence microscopy images of seedlings
grown on GM media containing 1500 lM sulfate
(GM) for 2 weeks, and were then transferred to
either GM (a–e) or S0 media lacking sulfate (f–j)
and grown for a further 4 days: (a, f) Col-0; (b, g)
ATPS1PRO::GFP line 1p1; (c, h) ATPS1PRO::GFP
line 1p2; (d, i) ATPS1PRO::ATPS1:GFP line 1g4;
(e, j) ATPS1PRO::ATPS1:GFP line 1g10. (k) RNA
was isolated from the transgenic seedlings and
subjected to quantitative RT-PCR with primers
specific to GFP. Transcript levels were compared
with TIP41, and the levels in plants grown on GM
was set to 1. No GFP was detected in Col-0.
Results are presented as means SDs from
three independent biological replicates. *Significant difference between S0 and control plants
(Student’s t-test; P £ 0.05).
(k)
Figure 3. Expression of miR395.
Seedlings of wild-type Col-0, two lines overexpressing miR395, 35S::miR395c
and 35S::miR395e, target mimic line MIM395 and slim1-1 were grown on
growth media (GM) for 2 weeks, before transfer to either GM media or GM
lacking a sulfur source (S0), for a further 4 days. RNA isolated from shoots
and roots was subjected to northern blot analysis of miR395, the membrane
was exposed for 1 day. The membranes were stripped and re-probed with a
U6-specific probe as a control.
The regulation of ATPS gene expression by sulfate
starvation was compared in the roots of these five genotypes (Figure 4). As observed previously (Figure 1), the
ATPS1 mRNA level was not affected by sulfate deficiency
in Col-0. Similarly, no regulation of ATPS1 by sulfate
starvation was detected in the miR395-overexpressing lines.
The overexpression of both forms of miR395, however,
resulted in significantly reduced steady-state levels of
ATPS1 in both conditions. In the MIM395 line the ATPS1
transcript was highly increased by sulfate starvation (Figure 4a). In contrast, ATPS1 was suppressed by sulfate
starvation in slim1-1 (Figure 4a). Transcript levels of ATPS2,
which is not targeted by miR395, were reduced by sulfate
deficiency in Col-0, despite being not regulated in a previous
experiment, but remained unchanged in the miR395-overexpressing plants, and slim1-1 and MIM395 lines (Figure 4b). ATPS3 mRNA levels were induced by sulfate
limitation in Col-0 and MIM395 plants, but not in slim1-1 or
any of the 35S::miR395 lines (Figure 4c). Overexpression of
miR395 led to only a small reduction in ATPS3 steady-state
levels compared with Col-0, which agrees with ATPS3 not
being a target for miR395-directed cleavage in the roots
(Kawashima et al., 2009). The steady-state level of ATPS4
mRNA was strongly reduced by sulfate starvation in Col-0,
but mutation of SLIM1 or changes in miR395 levels resulted
in the loss of this regulation (Figure 4d). Overexpression of
miR395 resulted in much lower levels of ATPS4 transcript in
both conditions, whereas in the MIM395 roots ATPS4 mRNA
was more abundant than in Col-0. In leaves of Col-0, ATPS2,
ATPS3 and ATPS4 were regulated by sulfate starvation in
the same way as in the roots, whereas ATPS1 was slightly
but not significantly reduced (Figure S2). Overexpression of
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
miR395 in the control of S assimilation 867
(a)
(b)
(c)
(d)
(e)
(f)
Figure 4. Effect of modulation of miR395 accumulation on regulation of ATPS transcript levels in roots by sulfate deficiency.
Arabidopsis Col-0, two lines overexpressing miR395, 35S::miR395c and 35S::miR395e, plants with reduced accumulation of miR395 by target mimicry (MIM395) and
slim1-1 mutant plants were grown on growth media containing 1500 lM sulfate (GM) for 2 weeks, and were then transferred to either GM or media lacking a sulfur
source (S0) and grown for a further 4 days. RNA was isolated from the roots and subjected to quantitative RT-PCR with primers specific to (a) ATPS1, (b) ATPS2, (c)
ATPS3 and (d) ATPS4, and to (e) SULTR2;1 and (f) APR2 as controls. The mRNA levels were compared with TIP41 or UBC (APR2); the levels in Col-0 plants grown on
GM are set to 1. Results are presented as means SDs from three independent biological replicates analysed in technical triplicates. *Significant difference
between S0 and control plants (Student’s t-test; P £ 0.05). Values marked with different letters are significantly different between genotypes grown under the same
conditions (P £ 0.05).
miR395 led to a decrease of ATPS1 and ATPS4 transcripts in
the leaves, similar to the roots, but in addition the ATPS3
mRNA level was also diminished (Figure S2).
Despite the large changes in transcript levels for ATPS
isoforms, little variation of ATPS activity was observed.
Sulfate deficiency slightly reduced the enzyme activity
in roots of Col-0, but the activity was unaffected by
the treatment in MIM395, whereas in 35S::miR395c,
35S::miR395e and slim1-1 roots the small observed decrease
was not statistically significant (Figure 5a). In 35S::miR395c,
but not 35S::miR395e, the enzyme activity was slightly
decreased at normal sulfate nutrition. In the leaves, ATPS
activity was reduced by sulfate starvation in Col-0 and slim11, and, compared with Col-0, by overexpression of both
forms of miR395 under both sulfate conditions (Figure S3).
Taken together the expression data show clearly that
miR395 has an important role in controlling ATPS1 and
ATPS4 mRNA accumulation, but has only a small impact on
the ATPS enzyme activity. SLIM1 has a dual function in the
regulation of ATPS, directly affecting transcription of the
ATPS1 and ATPS4 genes and indirectly regulating the mRNA
levels through miR395 expression.
The transcript levels of another target of miR395,
SULTR2;1, were elevated by sulfate starvation in Col-0 and
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
868 Cintia G. Kawashima et al.
(a)
(b)
(c)
(d)
(e)
(f)
Figure 5. Effect of modulation of miR395 accumulation on the regulation of sulfate assimilation in the roots by sulfate deficiency.
Arabidopsis Col-0, two lines overexpressing miR395, 35S::miR395c and 35S::miR395e, plants with reduced accumulation of miR395 by target mimicry (MIM395) and
slim1-1 mutant plants were grown on growth media containing 1500 lM sulfate (GM) for 2 weeks, and were then transferred to either GM or media lacking a sulfur
source (S0) and grown for a further 4 days. Roots were collected and (a) ATP sulfurylase (ATPS) and (b) APS reductase (APR) activity was determined. (c) Cysteine,
(d) glutathione and (e) sulfate content was measured by HPLC. (f) The seedlings were incubated for 4 h with their roots submerged in nutrient solution adjusted to
sulfate concentration of 0.2 mM and supplemented with 6.7 lCi [35S]sulfate. Shoot and root material was harvested separately, and the flux was determined as
incorporation of 35S from [35S]sulfate to thiols and proteins. Results are presented as means SDs from three independent biological replicates. *Significant
difference between S0 and control plants (Student’s t-test; P £ 0.05). Values marked with different letters are significantly different between genotypes grown under
the same conditions (P £ 0.05).
slim1-1 roots, as described previously (Kawashima et al.,
2009), and also in MIM395. No upregulation of SULTR2;1
was observed in the miR395 overexpressing lines: the
mRNA levels were lower than in Col-0 roots under both
conditions (Figure 4e). By contrast, SULTR2;1 transcript
levels were reduced by sulfate starvation in the leaves in
all lines but slim1-1 (Figure S2). APR2 mRNA was induced by
sulfate starvation in the roots of all genotypes, although the
induction was less pronounced in MIM395 and slightly more
intensive in slim1-1 (Figure 4f). Regulation of APR was thus
not disturbed in any genotype and, correspondingly, the
enzyme activity was higher in sulfate-starved roots than in
control roots (Figure 5b).
The concentration of cysteine and glutathione were not
affected by sulfate starvation in the roots of any plant line
except 35S::miR395c, where they were unexpectedly higher.
Glutathione content was lower in slim1-1 than in Col-0 under
both conditions (Figure 5c,d). Similarly, no differences in the
reduction of root sulfate contents by sulfate limitation were
observed between the five genotypes. However, slim1-1
plants possessed lower sulfate levels in both control
and sulfate-deficient conditions (Figure 5e). Substantial
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
miR395 in the control of S assimilation 869
differences were determined in the sulfate contents of the
leaves. Again, for all five lines lower sulfate contents were
measured in sulfate-limited plants than in controls
(Figure 6a). However, in the miR395-overexpressing lines
sulfate levels were substantially higher than in Col-0 under
both conditions. Under normal sulfate supply, the sulfate
content of slim1-1 and MIM395 leaves was lower than in
Col-0 (Figure 6a). Contents of glutathione were decreased by
sulfate deficiency in leaves of all five genotypes, with
MIM395 and slim1-1 possessing lower levels under both
conditions (Figure S3). Sulfate starvation reduced cysteine
levels in the leaves of Col-0, 35S::miR395c and MIM395.
Thus, miR395 plays an important role in the control of
sulfate content and distribution between leaves and roots. It
must be noted, however, that the changes in thiols and
sulfate levels correspond to only moderate sulfate starvation, capable of triggering all the known physiological
responses, but without full depletion of sulfur-containing
metabolites.
miR395 controls sulfate translocation to the shoots
We determined the effect of sulfate starvation on flux
through the sulfate assimilation pathway using [35S]sulfate.
In Col-0 and MIM395 plants, sulfate starvation increased the
rate of translocation of sulfate to the shoots (Figure 6b).
Plants overexpressing miR395c and slim1-1 reacted to these
conditions in the opposite way, and the translocation rate to
the shoots was lower, whereas the decrease was not significant in 35S::miR395e plants (Figure 6b). Compared with
Col-0, overexpression of miR395 increased the sulfate
translocation rate to shoots under normal conditions, but
not under sulfate deficiency. Thus, under normal sulfate
supply, 40% of total 35S taken up by the roots was translocated to the shoots of Col-0 (Figure 6c). During sulfate starvation, in accordance with the increased translocation rate,
about 70% of the label was transported to the leaves. Overexpression of miR395 increased the portion of sulfate
translocated to shoots under normal sulfate conditions, as
was the case for slim1-1 mutants (Figure 6c). On the other
hand, the increase in sulfate transported to the shoots of
MIM395 plants during sulfate starvation was less prominent
than in the other lines. The flux through the pathway,
determined as the incorporation of 35S into proteins and
thiols, was increased in the shoots of Col-0 and MIM395
by sulfate limitation, but not in the other lines (Figure 6d).
Under normal conditions, no differences in the flux were
observed in shoots of the transgenic plants or slim1-1
compared with Col-0. Sulfate starvation caused increases in
Figure 6. Effect of modulation of miR395 accumulation on sulfate translocation and assimilation in leaves.
Arabidopsis Col-0, two lines overexpressing miR395, 35S::miR395c and 35S::miR395e, plants with reduced accumulation of miR395 by target mimicry (MIM395) and
slim1-1 mutant plants were grown on growth media containing 1500 lM sulfate (GM) for 2 weeks, and were then transferred to either GM or media lacking a sulfur
source (S0) and grown for a further 4 days. The seedlings were incubated for 4 h with their roots submerged in nutrient solution adjusted to sulfate concentration of
0.2 mM and supplemented with 6.7 lCi [35S]sulfate. Shoot and root material was harvested separately, and the flux was determined as incorporation of 35S from
[35S]sulfate to thiols and proteins. (a) Sulfate content in the leaves. (b) Translocation rate of sulfate from roots to shoots. (c) Percentage of 35S transported to leaves
from the [35S]sulfate taken up. (d) Flux through the pathway in the leaves. Results are presented as means SDs from three independent biological replicates.
*Significant difference between S0 and control plants (Student’s t-test; P £ 0.05). Values marked with different indices are significantly different between genotypes
grown under the same conditions (P £ 0.05).
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
870 Cintia G. Kawashima et al.
the flux through sulfate assimilation in the roots of Col-0,
35S::miR395c and slim1-1 plants, whereas there was no
change in 35S::miR395e or MIM395 plants (Figure 5f). These
results show that SLIM1, both through the action of miR395
and independently, is responsible for the increased translocation of sulfate from roots to shoots during sulfate starvation, and also for the increased rate of sulfate reduction.
As miR395 targets two steps in sulfate metabolism, we
analysed the effects of sulfate starvation on mutants in
ATPS1 and SULTR2;1, and in plants with a reduced expression of ATPS4 by RNAi (Liang et al., 2010). Under normal
sulfate supply little variation in the contents of sulfate and
thiols was detected, except for higher sulfate levels in leaves
of atps1 and ATPS4-RNAi plants (Figure 7a), and lower
sulfate in the roots of ATPS4-RNAi (Figure 7b). Sulfate
starvation resulted in reduced sulfate and glutathione levels
in leaves of all genotypes, and lower cysteine content of Col0, sultr2;1 and ATPS4-RNAi (Figures 7 and S4). In roots,
glutathione levels were not altered by sulfate starvation,
whereas sulfate was reduced in all genotypes but atps1.
However, feeding with [35S]sulfate revealed significant
effects of the mutations on the rates of sulfate translocation
and reduction. Compared with Col-0, the sulfate translocation rate from roots to shoots (Figure 7c) and the portion
of sulfate transported to leaves (Figure 7d) were lower in
sultr2;1 under normal sulfate supply, confirming the role
Figure 7. Effect of modulation of expression of miR395 targets on sulfate translocation and assimilation.
Arabidopsis Col-0, sultr2;1 and atps1 mutants, and ATPS4-RNAi plants were grown on growth media containing 1500 lM sulfate (GM) for 2 weeks, and were then
transferred to either GM or media lacking a sulfur source (S0) and grown for a further 4 days. The seedlings were incubated for 4 h with their roots submerged in
nutrient solution adjusted to a sulfate concentration of 0.2 mM and supplemented with 6.7 lCi [35S]sulfate. Shoot and root material was harvested separately, and
the flux was determined as the incorporation of 35S from [35S]sulfate to thiols and proteins. (a) Total sulfate content in the leaves. (b) Total sulfate content in the
roots. (c) Translocation rate of sulfate from roots to shoots. (d) Percentage of 35S transported to leaves from the [35S]sulfate taken up. (e) Flux through the sulfate
assimilation in the leaves. (f) Flux through the sulfate assimilation in the roots. Results are presented as means SDs from three independent biological replicates.
*Significant difference between S0 and control plants (Student’s t-test; P £ 0.05). Values marked with different indices are significantly (P £ 0.05) different between
genotypes grown under the same conditions.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
miR395 in the control of S assimilation 871
of this low affinity transporter in root-to-shoot translocation of sulfate. On contrary, reduction of ATPS1 or ATPS4
expression resulted in increased sulfate translocation to the
shoots (Figure 7c,d). Sulfate starvation led to increase in the
translocation rate and to increased portion of 35S transported to the shoots in all four genotypes. Mutations
in individual miR395 targets did not alter the induction of
flux through sulfate assimilation by sulfate deficiency in
the shoots (Figure 7e). In the roots the flux was induced in
sultr2;1 and to much lower extent in ATPS4-RNAi, while no
increase could be measured for atps1 roots (Figure 7f).
Mutation of ATPS1 resulted in reduction of flux in both
organs, particularly during sulfate starvation. Reduction in
ATPS4 expression led to similar effects on the flux, however,
under normal sulfate supply, the reduction of flux in leaves
was not statistically significant, which agrees with predominant expression of this isoform in the roots (Noji et al.,
2006). Thus, both SULTR2;1 and ATPS contribute to the
increased translocation of sulfate to the roots caused by
miR395, and both ATPS1 and ATPS4 are important for the
full rate of sulfate reduction.
DISCUSSION
Regulation of miR395 and its targets by sulfur starvation
Our study revealed that miR395 has a complex and noncanonical role in the regulation of sulfate assimilation.
MiR395 levels are induced by sulfate starvation (JonesRhoades and Bartel, 2004; Kawashima et al., 2009); however,
the levels of mRNA of its targets are not regulated in the
expected ways. The transcript levels of three miR395targeted genes show different responses to sulfate starvation in roots: SULTR2;1 mRNA levels increase, ATPS4 levels
decrease and ATPS1 levels are unaffected (Figure 1). Similar
results were recently obtained by Liang et al. (2010), who
showed a decrease in ATPS4 and a small increase in ATPS1
and ATPS3 transcripts in response to sulfate limitation.
It seems, therefore, that miR395-mediated mRNA cleavage
regulates the functions of its targets by three different
mechanisms. The mRNA levels of SULTR2;1 increase in
roots during sulfate starvation, and its expression can be
spatially limited to xylem parenchyma cells (Kawashima
et al., 2009). ATPS4 undergoes a canonical regulation by
miR395, because its mRNA levels decrease following
induction of miR395. ATPS1 levels were not affected by
sulfate deficiency in our study, in contrast to that of JonesRhoades and Bartel (2004), who observed a decrease in
ATPS1 mRNA. However, our experiments differed from that
undertaken by Jones-Rhoades and Bartel (2004): we investigated short-term effects of sulfate deficiency on roots,
whereas whole plants grown on sulfate-limited nutrient
media for 2 weeks were analysed in the other study. However, a small decrease in ATPS1 transcript was observed in
leaves (Figure S2a). Microarray analyses of gene expression
in plants subjected to sulfate starvation consistently show
a reduction of ATPS4 transcript levels, but not of ATPS1
transcript levels (Hirai et al., 2003; Maruyama-Nakashita
et al., 2003, 2006; Nikiforova et al., 2003), which supports
our observations. ATPS1 transcript is cleaved by miR395
(Kawashima et al., 2009), thus the stable steady-state levels
of ATPS1 during sulfate starvation are probably maintained
by increased rates of transcription. Indeed, analysis of
ATPS1PRO::GFP lines revealed that the ATPS1 promoter is
activated during sulfate deficiency (Figure 2). Correspondingly, ATPS1 mRNA levels were increased by sulfate deficiency, when the activity of miR395 was inhibited in MIM395.
The increased expression of the ATPS1 gene is SLIM1
dependent (Figure 4), which is similar to the responses of
many other genes of sulfate assimilation (MaruyamaNakashita et al., 2006). Thus, the miR395-induced cleavage
of ATPS1 counteracts the increased transcription of its gene
so that the steady-state levels of ATPS1 mRNA are not
altered during sulfate deficiency.
The reduction in ATPS activity by sulfate limitation is
counter-intuitive in the context of plant responses to sulfate
starvation. Plants react to sulfate deficiency by increasing
their capacity for sulfate uptake and reduction by inducing
the mRNA levels for high-affinity sulfate transporters and
APS reductase (Reuveny et al., 1980; Smith, 1980; Takahashi
et al., 1997; Hirai et al., 2003; Maruyama-Nakashita et al.,
2003; Nikiforova et al., 2003). However, a reduction in ATPS
mRNA levels and enzyme activity should lead to a lower
entry of sulfate into the assimilation pathway, counteracting
the increased activity of APR during sulfate starvation.
Indeed, the flux through sulfate assimilation was reduced
in plants deficient in either ATPS1 or ATPS4 (Figure 7),
whereas usually there is a good correlation between high
APR activity and increased flux (Vauclare et al., 2002;
Scheerer et al., 2010; Mugford et al., 2011). However, the
flux was increased in sulfate-limited plants, thereby confirming that APR exerts more control over the pathway, as
observed previously (Vauclare et al., 2002), and its increased
activity can compensate for reductions in ATPS activity
caused by miR395. Therefore, it seems that the miR395driven reduction of ATPS contributes to the plant response
to sulfate limitation rather subtly, and not by bulk changes in
enzyme activity. It is possible that cell-specific differences
in the expression of miR395 and ATPS genes contribute to
tissue-specific increases in ATPS activity, which are masked
in the measurements from whole organs. Such localized
increases would be in line with the increase in APR activity
and the flux through sulfate assimilation.
SLIM1 and miR395 have distinct functions in the
sulfate-starvation response
The analysis of plants overexpressing miR395, or those in
which its function was diminished either by a target mimic
(Todesco et al., 2010) or by a mutation in SLIM1 (Kawashima
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
872 Cintia G. Kawashima et al.
et al., 2009), allowed us to dissect the direct effect of SLIM1
(without miR395) and miR395 alone. Our results show
clearly that the SLIM1-dependent induction of miR395 is
important for the increased translocation of sulfate to the
shoots during sulfate deficiency, so as to enable more efficient sulfate assimilation in the leaves. The function of
SLIM1 as a regulator of the sulfate starvation response is
firmly established (Maruyama-Nakashita et al., 2006). Surprisingly, however, although sulfate uptake and assimilation
are both increased by sulfate deficiency, the mechanisms of
the regulation seem to be different. Sulfate uptake and the
mRNA levels of high-affinity sulfate transporters SULTR1;1
and SULTR1;2 are increased by S starvation in a SLIM1dependent manner (Maruyama-Nakashita et al., 2006). This
is not the case for APR, which is clearly upregulated by
sulfate deficiency, independently of SLIM1 (Figure 5b;
Maruyama-Nakashita et al., 2006). How the function of SLIM1
itself is connected with sulfate deficiency is not known, as
its mRNA levels are not affected by sulfate deficiency
(Figure S1; Maruyama-Nakashita et al., 2006), nor by the
manipulation of miR395 expression. It should also be noted
that as judged from metabolite concentrations, the extent of
sulfate deficiency in this study is not as severe as in previous
studies (Hirai et al., 2003; Maruyama-Nakashita et al., 2003,
2006), but still the response of the key genes is identical.
The analysis of ATPS mRNA accumulation in both
35S::miR395 lines confirmed that in roots ATPS1 and ATPS4
are the targets of the miRNA, whereas ATPS3 can also be
cleaved under sulfate-limiting conditions. Although the
reduction of ATPS1 and ATPS4 agrees with the results of
Liang et al. (2010), we could not confirm a decrease in ATPS3
levels in roots during normal sulfate availability. However,
in leaves all three ATPS isoforms potentially targeted by
miR395 are cleaved, which agrees with the results of
Kawashima et al. (2009) and expression analyses of Liang
et al. (2010). Similar to ATPS1 (Figures 2 and 4a), ATPS4
levels are also controlled by both SLIM1 and miR395. SLIM1
is necessary for the full rate of ATPS4 gene expression,
whereas the miR395 seems to be important in controlling
ATPS4 levels, even under normal sulfate supply (Figure 4d).
However, despite significant variation in ATPS transcript
levels in the different genotypes, very little difference in total
root ATPS activities has been observed. The reduction in
ATPS activity by sulfate limitation was small but consistent
over several experiments (Figures 1 and 5a), and therefore
probably represents a significant part of the response to
sulfate starvation. However, this reduction was limited to
Col-0. ATPS enzyme activity was only marginally affected
in roots of miR395-overexpressing plants, despite the high
reduction in ATPS1 and ATPS4 mRNA levels, or in MIM395
plants where the transcript levels were significantly higher.
In addition, no compensatory changes in ATPS2 or ATPS3
transcript levels were observed. In leaves this effect is even
more prominent. In miR395-overexpressing lines the tran-
script levels of ATPS1, ATPS3 and ATPS4 are reduced to 10–
30% of wild-type (WT) levels, and even ATPS2 mRNA level is
slightly reduced. Nevertheless, the total foliar ATPS activity
in these lines is only approximately 20% lower than in Col-0
(Figures S2 and S3). This indicates a robust post-transcriptional mechanism capable of maintaining ATPS enzyme
activity, despite large variation in the mRNA levels. This is
similar to the previously observed uncoupling of regulation
of APR mRNA levels and activity by salt stress in numerous
mutants disrupted in hormone signalling (Koprivova et al.,
2008).
The manipulation of miR395 had a strong effect on leaf
sulfate content, as observed previously (Liang et al., 2010).
Steady-state levels of sulfate were higher in leaves of the
35S::miR395 lines than in Col-0 during both normal and
sulfate-deficient conditions (Figure 6a), but the mechanisms
leading to this accumulation differed. Whereas under normal sulfate supply the accumulation of miR395 results in the
increased translocation of sulfate from the root to the shoot
(Figure 6b,c), this is not the case during sulfate limitation.
Instead, sulfate limitation increases flux through sulfate
assimilation in leaves of Col-0, but not in miR395-overexpressing lines (Figure 6d), thus leading to sulfate accumulation in the latter. This is the same mechanism leading to
sulfate accumulation in mutants of the APR2 isoform of APR
(Loudet et al., 2007). Correspondingly, eliminating the regulatory effect of miR395 in MIM395 plants resulted in a lower
sulfate content under normal sulfate supply, which can be
related to a lower translocation of sulfate to the shoots
(Figure 6). Remarkably, on several occasions the results
from overexpression of miR395c were different to those with
miR395e, e.g. the accumulation of thiols or the regulation of
flux by sulfate deficiency (Figure 5c,d,f). This agrees with
different effects of stress on germination of seeds of
Arabidopsis overexpressing these two miR395 species
(Kim et al., 2010). However, although these authors
observed changes in cleavage pattern of the target ATPS
transcripts under stress, this was not the case in this study.
The mechanisms of the different responses to the overexpression of miR395c and miR395e thus remain to be
elucidated.
Also, mutants deficient in ATPS isoforms were shown to
accumulate sulfate (Liang et al., 2010). Interestingly, in our
experimental system the mutants in ATPS behaved exactly
as plants overexpressing miR395: under normal sulfate
nutrition the sulfate translocation to shoots was greater than
in Col-0, whereas the flux through sulfate assimilation was
reduced, and therefore they accumulated sulfate in the
leaves (Figure 7). During sulfate deficiency, however, the
sulfate content was higher in ATPS4-RNAi only, corresponding to reduced flux but no changes in translocation. It seems
thus that the effect of miR395 overexpression can be
explained solely by a reduction in ATPS. However, the
contribution of SULTR2;1 cannot be excluded, as analysis of
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
miR395 in the control of S assimilation 873
the sultr2;1 mutant revealed that this transporter is important for the full rate of sulfate translocation from roots to
shoots. Thus changes in SULTR2;1 tissue-specific expression caused by miR395 most probably contribute to the high
increase in sulfate translocation to the shoots during conditions of sulfate deficiency.
The measurements of flux through sulfate assimilation
revealed that in wild-type plants sulfate starvation results
not only in higher sulfate uptake and activity of APR, but also
in a higher sulfate reduction rate. However, the increase in
APR activity by sulfate starvation alone is not enough to
cause the higher reduction rate, as the flux remained
unchanged in 35S::miR395e and MIM395 lines (Figure 5b,f).
The increased flux in roots of miR395-overexpressing plants
under normal conditions occurs despite minimal alterations
in total root ATPS or APR activity. This indicates that
variation in the ratio of different ATPS isoforms, as a
consequence of cleavage of ATPS1 and ATPS4, has a
significant effect on the performance of plant sulfur metabolism. This could be because of differences in catalytic
properties or (sub)cellular distribution.
MicroRNAs in control of plant nutrition
Our finding that miR395 is an integral component of the
sulfate assimilation regulatory network, and plays a critical
role in the control of sulfate partitioning between shoots and
roots, fits well with the plethora of functions microRNAs
have been reported to play in plants and animals. The first
targets identified for plant miRNAs were transcription factors involved in development (Llave et al., 2002; Rhoades
et al., 2002). However, soon targets involved in other aspects
of plant life were identified, in abiotic and biotic stress
responses and in nutrient uptake and assimilation (JonesRhoades and Bartel, 2004; Sunkar and Zhu, 2004; Fujii et al.,
2005; Ruiz-Ferrer and Voinnet, 2009). MiR395 was amongst
the first miRNAs identified that did not target developmental
processes (Jones-Rhoades and Bartel, 2004). Other miRNAs
have been linked to nutrient deficiencies, most importantly
miR399, which is induced by phosphate starvation, and
miR398, which responds to copper deficiency (Fujii et al.,
2005; Yamasaki et al., 2007). In a systematic survey, 20
miRNAs were shown to be differentially expressed under
conditions of deficiency in nitrogen or phosphate (Pant
et al., 2009). Many of these nutrient-responsive miRNAs
were identified in phloem of B. napus, indicating their role in
the long-distance communication of nutrient status (Buhtz
et al., 2008).
The best characterized from the nutrient-responsive miRNAs is miR399 (Fujii et al., 2005; Chiou et al., 2006; FrancoZorrilla et al., 2007; Pant et al., 2008). MiR399 targets the
PHO2 gene encoding a ubiquitin-conjugating E2 enzyme
24 (Aung et al., 2006). The pho2 mutants over-accumulate
phosphate in the shoots as a result of the increased uptake
and translocation from the roots. Phosphate deficiency
induces miR399 and thus causes downregulation of PHO2,
and improves the maintenance of phosphate homeostasis
during these conditions (Figure 8; Chiou et al., 2006). Thus
the transport of miR399 from shoots to roots is important for
its function (Buhtz et al., 2008). Similar to miR399, miR395 is
induced by sulfate starvation in a SLIM1-dependent manner
(Figure 8). However, in contrast to miR399 the upregulation
occurs both in roots and leaves (Kawashima et al., 2009).
Figure 8. Comparison of function of miR395 and miR399.
(a) Under normal phosphate (Pi) supply PHO2 inhibits phosphate uptake. Phosphate deficiency in shoots induces miR399 expression, the miRNA is transported to
roots where it inhibits PHO2. This relieves the inhibition of phosphate uptake and phosphate is translocated to the shoots. (b) Under normal sulfate (SO24 ) supply,
SLIM1 and miR395 maintain levels of ATPS1 and ATPS4 transcripts for the optimal assimilation of sulfate into organic sulfur compounds (Sred). Sulfate limitation
activates SLIM1, which directly induces root sulfate transporters to increase sulfate uptake. SULTR2;1 is induced by sulfate limitation. SLIM1 induces accumulation
of miR395, which limits expression of SULTR2;1 to xylem parenchyma, thus enhancing sulfate translocation to the shoots and inhibiting shoot-to-root transport in
phloem. miR395 inhibits ATPS4 transcript levels and together with SLIM1 regulates ATPS to allow increased flux through the sulfate assimilation pathway achieved
by the SLIM1-independent induction of APR. The phloem transport of miR395 is indicated by an arrow, and its unknown function is indicated by a question mark.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
874 Cintia G. Kawashima et al.
Under normal sulfate supply, miR395 and SLIM1 are
involved in the fine-tuning of ATPS transcript levels and
activity to match the needs of plants for reduced sulfur.
During sulfate limitation, SLIM1 directly triggers increased
sulfate uptake and an unknown mechanism induces APR
(Maruyama-Nakashita et al., 2006), leading to increased flux
through the sulfate assimilation pathway in both roots and
shoots. SLIM1 also induces miR395, which through the
combination of limiting SULTR2;1 expression to xylem
parenchyma cells and reducing ATPS expression, and
consequently the flux through sulfate assimilation, causes
increased translocation of sulfate to the shoots. In addition,
ATPS transcript levels are maintained by SLIM1 and miR395
to optimal levels for meeting the increased sulfate reduction
rate. MiR395 is also transported in phloem (Buhtz et al.,
2008), and can accumulate in the roots of hen1-1 plants
when grafted with Col-0 scions (Buhtz et al., 2010). Because
miR395 promoters are active in both roots and shoots the
functional significance of this transport is still not known.
However, as the low levels of miR395 detected in roots of
MIM395 and slim1-1 correlated with low sulfate levels in the
leaves of these lines, a role as a systemic signal of foliar
sulfate status cannot be excluded. The uncertainty about the
significance of phloem transport of miR395 is especially
apparent when compared with the function of miR399,
which is dependent on shoot-to-root translocation (Pant
et al., 2008). Accordingly, miR399 is found in vascular plants
only (http://www.mirbase.org), whereas miR395 is present
in the moss, Physcomitrella patens (Fattash et al., 2007).
Therefore, the original function of miR395 was most probably associated with the regulation of sulfate assimilation,
independent from the regulation of long-distance transport,
which may have been a secondary function acquired during
evolution.
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
In this study, wild-type Arabidopsis thaliana (ecotype Col-0), slim1-1
mutant plants (Maruyama-Nakashita et al., 2006), and atps1, sultr2;1,
and ATPS4-RNAi line 5 (Liang et al., 2010) were used. Unless
otherwise stated, plants were grown for 2 weeks on vertical plates
with GM-agarose media (Valvekens et al., 1988) at 20C under 16-h
light/8-h dark cycles, before being transferred to fresh GM plates
(control) or treatment plates for a further 4 days. Sulfur-deficient
medium (S0) was made in accordance with Kawashima et al. (2009)
by exchanging all sulfate salts for chlorides. Three independent
pools of roots from at least 40 plants and shoots of 20 plants were
homogenized and used for all subsequent measurements.
RNA isolation and expression analysis
For the detection of miRNAs, total RNA was extracted from Arabidopsis shoot and root tissues using the TRIZOL reagent (Invitrogen, http://www.invitrogen.com). Total RNA (10 lg) was loaded
onto each lane and resolved on a 15% denaturing polyacrylamide
gel, and carbodiimide-mediated cross-linking of RNA to Hybond-NX
was performed according to the method described by Pall et al.
(2007). The membranes were labeled with DNA oligonucleotides
complementary to the miR395a sequence that was pre-labeled with
[c-32P]ATP using T4 polynucleotide kinase (Invitrogen). Blots were
hybridized overnight at 37C in ULTRAhyb-Oligo hybridization
buffer (Ambion, http://www.ambion.com). They were then washed
two times with 0.2 · SSC and 0.1 · SDS for 30 min at 37C. The
membranes were then exposed to the phosphoimager.
For expression analysis of the miR395 targets and GFP, total RNA
was isolated from roots by phenol:chloroform:isoamylalcohol
(25:24:1) extraction and LiCl precipitation (Sambrook et al., 1989),
and repurified using an RNeasy Plant Mini kit (Qiagen, http://
www.qiagen.com), including the DNAse treatment. Expression of
ATPS1-4, SULTR2;1, APR2 and sGFP genes was analysed by realtime quantitative RT-PCR (qPCR), using the fluorescent intercalating
dye SYBR Green (Applied Biosystems, http://www.appliedbiosystems.com) in a DNA engine OPTICON2 continuous fluorescence
detector (Bio-Rad, http://www.bio-rad.com). The extracted RNA was
reverse-transcribed into cDNA, using SuperScriptII Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions.
Subsequently, the cDNA was used as a template for qPCR with
gene-specific primers (for primer sequences see Table S1). The
Arabidopsis TIP41 gene was used as a standard for ATPS and
SULTR2;1 measurements, TIP41-A for sGFP, and the UBC gene was
used for the determination of APR2. Relative quantification of
expression levels was performed using the comparative cycle
threshold (Ct) method (following the manufacturer’s instructions,
bulletin 2; Applied Biosystems). At least three independent RNA
preparations from independently grown plants were analyzed with
three technical replicates for the qPCR.
Enzyme assays
The roots were homogenized 1 : 20 (w/v) in 50 mM Na/K phosphate
buffer pH 8 supplemented with 30 mM Na2SO3, 0.5 mM 5¢-AMP and
10 mM dithioerythritol, and the extract was centrifuged for 30 sec at
1500 g to remove cell debris. APR activity was measured in the
supernatants as the production of [35S]sulfite, assayed as acid volatile radioactivity formed from [35S]APS and DTE (Koprivova et al.,
2008). ATPS activity was determined by a coupled assay based on
APS and pyrophosphate-dependent formation of ATP (Cumming
et al., 2007). NADH was measured at a wavelength of 340 nm using
a SpectraMax 340PC384 plate reader (Molecular Devices, http://
www.moleculardevices.com).
The protein concentration in the extracts was determined by BioRad protein assay with bovine serum albumin as a standard.
Measurements of sulfur-containing compounds
Sulfate contents were determined by the ion-exchange HPLC
method, as described in Scheerer et al. (2010). Cysteine and glutathione (GSH) were analysed by HPLC as described by Koprivova
et al. (2008) from 20–30 mg of plant material.
Creation of GFP constructs and transgenic plants
The chimeric gene construct of the ATPS1 promoter and sGFP for
plant transformation was created as follows: oligonucleotide
primers, ATPS1-prom-FXh and ATPS1-prom-RBm (Table S2) were
used to amplify genomic DNA fragments from the 5¢ promoter
region, 3020 bp upstream of the translational initiation site, and
terminated just after the translational initiation site of ATPS1. PCR
was performed on genomic DNA prepared from Arabidopsis Col-0
ecotype using KOD plus DNA polymerase (Toyobo, http://
www.toyobo.co.jp/e). The resultant PCR-amplified fragment was
cloned into pCR-Blunt II-TOPO (Invitrogen) and fully sequenced.
The XhoI–BamHI fragment of the ATPS1 promoter was inserted
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 863–876
miR395 in the control of S assimilation 875
between the SalI and BamHI sites of the promoter-less GFP binary
plasmid, pBI-GFP (Mugford et al., 2009).
For the creation of the ATPS1::ATPS1:sGFP fusion gene construct,
oligonucleotide primers ATPS1-prom-FBm and ATPS1-CDSnostopRNco (Table S2) were used to amplify DNA fragments starting from
the 5¢ promoter region, 3020 bp upstream of the translational
initiation site, and terminating just before the ATPS1 translational
stop site. PCR was performed on Col-0 genomic DNA using KOD
plus DNA polymerase (Toyobo), and the PCR-amplified fragment
was cloned into the pCR-Blunt II-TOPO vector (Invitrogen) and fully
sequenced. The BamHI–NcoI fragment of the ATPS1 gene and the
NcoI–EcoRI fragment of pTH2 (Chiu et al., 1996), containing the
fusion gene cassette of the sGFP(S65T) and the nopaline synthase
terminator (NOSter), were ligated. The resultant BamHI–EcoRI
fragment of the ATPS1::ATPS1:GFP:NOSter fusion cassette was
placed into the position of b-glucuronidase gene and the NOSter in
the binary plasmid, pBI101 (Clontech, http://www.clontech.com).
The chimeric gene constructs 35S::miR395c and 35S::miR395e for
the overexpression of miR395 were created as follows: oligonucleotide primers OEmiR395e_Forward-F-BamHI/OEmiR395e_Forward-R-ScaI (Table S2) and OEmiR395c-Forward-F-BamHI/
OEmiR395c-Forward-R-ScaI, respectively, were used to amplify
genomic DNA fragments. PCR was performed on genomic DNA
prepared from Arabidopsis Col-0 ecotype using KOD plus DNA
polymerase (Toyobo). The resultant PCR-amplified fragments were
cloned into pCR-Blunt II-TOPO (Invitrogen) and fully sequenced. The
BamHI–ScaI fragments of pre-miR395 were inserted between the
BamHI and ScaI sites of the CaMV35S promoter plasmid, pBI-121
(Kawashima et al., 2009).
The resulting binary plasmids were transformed into Agrobacterium tumefaciens GV3101 (pMP90) (Koncz and Schell, 1986) by the
freeze–thaw method (Höfgen and Willmitzer, 1988). Col-0 plants
were transformed according to the floral-dip method (Clough and
Bent, 1998). Transgenic plants were selected on GM agar medium
(Valvekens et al., 1988) containing 50 mg L)1 kanamycin sulfate.
Seeds were collected from kanamycin-resistant T2 progenies and
used to grow plants for the analysis.
MIM395 was developed as other MIM constructs (Todesco et al.,
2010). The 23-nucleotide, miR399-complementary motif in IPS1
(Franco-Zorrilla et al., 2007) has been exchanged for a sequence
complementary to miR395, and the construct was expressed in
Arabidopsis under the control of the 35S promoter.
Microscopy and imaging of GFP
Fluorescence of ATPS1:GFP or GFP under the influence of the
ATPS1 promoter was recorded using a Leica MZFLIII stereomicroscope coupled to a Nikon Coolpix 990 camera (Nikon, http://
www.europe-nikon.com/en_GB/).
Determination of flux through sulfate assimilation
The flux through sulfate assimilation was measured as the incorporation of 35S from [35S]sulfate to thiols and proteins, essentially as
described in Kopriva et al. (1999) and Vauclare et al. (2002). Plants
were grown on vertical GM plates for 14 days before transfer to
either GM plates (control) or S0 plates for a further 4 days. Subsequently the plants were transferred into 24-well plates, with the
roots submerged in 1 ml of nutrient solution adjusted to a sulfate
concentration of 0.2 mM, and supplemented with 6.7 lCi [35S]sulfate (Hartmann Analytic, http://www.hartmann-analytic.de) to a
specific activity of 1860 kBq nmol sulfate)1, and incubated then in
light for 4 h. After incubation, seedlings were washed three times
with 1 ml of cold non-radioactive nutrient solution, and then carefully blotted with paper tissue. Roots and shoots were separated,
weighed, transferred into 1.5-ml tubes and frozen in liquid nitrogen.
The quantification of 35S in plant material and in different S-containing compounds was performed exactly as described by
Mugford et al. (2011).
ACKNOWLEDGEMENTS
SK’s work is supported by the British Biotechnology and Biological
Sciences Research Council (BBSRC). CAM was supported by BBSRC
CASE studentship with Plant Bioscience Ltd. This work was also
supported by the European Commission (FP6 Integrated Project
SIROCCO LSHG-CT-2006-037900) to TD and the Weigel lab at the
MPI for Developmental Biology, by a Special Postdoctoral Fellowship from RIKEN (to NY), and by Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan (to HT, YN and KS). We would like to thank
Dr Diqiu Yu, Key Laboratory of Tropical Forest Ecology Kunming,
China, for the kind donation of seeds of sultr2;1 and ATPS4-RNAi
lines.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Expression of SLIM1.
Figure S2. Effect of the modulation of miR395 accumulation on
regulation of ATPS transcript levels in leaves by sulfate deficiency.
Figure S3. Effect of modulation of miR395 accumulation on the
regulation of sulfate assimilation in leaves by sulfate deficiency.
Figure S4. Effect of modulation of expression of miR395 targets on
regulation of thiol contents by sulfate deficiency.
Table S1. Primer sequences for expression analysis by qPCR.
Table S2. Primer sequences for the creation of transformation
constructs.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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