the plant journal 77, 604–615 doi: 10.1111/tpj.12407 ... · to the response of plants towards...
TRANSCRIPT
Sulfate availability affects ABA levels and germinationresponse to ABA and salt stress in Arabidopsis thaliana
Min-Jie Cao1,†, Zhen Wang1,2,†, Qing Zhao1, Jie-Li Mao1, Anna Speiser3,4, Markus Wirtz3, R€udiger Hell3, Jian-Kang Zhu5,6 and
Cheng-Bin Xiang1,*1School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province 230027, China,2Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei,
Anhui Province 230027, China,3Centre for Organismal Studies Heidelberg, Heidelberg University, Im Neuenheimer Feldman360, D-69120 Heidelberg,
Germany,4Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, Im Neuenheimer Feldman360,
D-69120Heidelberg, Germany,5Shanghai Center for Plant Stress Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences,
Shanghai 201602, China, and6Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA
Received 1 April 2013; revised 18 November 2013; accepted 3 December 2013; published online 13 December 2013.
*For correspondence (e-mail [email protected] or [email protected]).†These authors contributed equally.
SUMMARY
Sulfur-containing compounds play a critical role in the response of plants to abiotic stress factors including
drought. The phytohormone abscisic acid (ABA) is the key regulator of responses to drought and high-salt
stress. However, our knowledge about interaction of S-metabolism and ABA biosynthesis is scarce. Here we
report that sulfate supply affects synthesis and steady-state levels of ABA in Arabidopsis wild-type seedlings.
By using different mutants of the sulfate uptake and reduction pathway, we confirmed the impact of sulfate
supply on steady-state ABA content in Arabidopsis and demonstrated that this impact was due to cysteine
availability. Loss of the chloroplast sulfate transporter3;1 function (sultr3;1) resulted in significantly decreased
aldehyde oxidase (AO) activity and ABA levels in seedlings and seeds. These mutant phenotypes could be
reverted by exogenous application of cysteine or ectopic expression of SULTR3;1. In addition the sultr3;1
mutant showed a decrease of xanthine dehydrogenase activity, but not of nitrate reductase, strongly indicat-
ing that in seedlings cysteine availability limits activity of the molybdenum co-factor sulfurase, ABA3, which
requires cysteine as the S-donor for sulfuration. Transcription of ABA3 and NCED3, encoding another key
enzyme of the ABA biosynthesis pathway, was regulated by S-supply in wild-type seedlings. In contrast, ABA
up-regulated the transcript level of SULTR3;1 and other S-metabolism-related genes. Our results provide evi-
dence for a significant co-regulation of S-metabolism and ABA biosynthesis that operates to ensure sufficient
cysteine for AO maturation and highlights the importance of sulfur for stress tolerance of plants.
Keywords: SULTR3;1, cysteine, abscisic acid, Arabidopsis thaliana, moco factor.
INTRODUCTION
Sulfur is essential for plants because it participates in
many biological processes including the biosynthesis of
the two sulfur-containing amino acids, cysteine (Cys) and
methionine (Met), the resistance against diseases, and the
detoxification of reactive oxygen species (ROS), xenobiot-
ics, and heavy metals (Xiang et al., 2001; Takahashi et al.,
2011; Noctor et al., 2012). Sulfur metabolism is connected
directly via the Met salvage cycle to ethylene- and poly-
amine-related responses to abiotic stresses (Sauter et al.,
2013) and by formation of 3′-phosphoadenosine
5′-phosphate (PAP), a side product of sulfation reactions,
to the response of plants towards drought stress (Estavillo
et al., 2011). The polyamines are especially important for
seedling growth, development of vasculature and response
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd
604
The Plant Journal (2014) 77, 604–615 doi: 10.1111/tpj.12407
to drought stress by stabilizing negatively charged mole-
cules such as DNA, RNA and proteins (Waduwara-
Jayabahu et al., 2012; Chan et al., 2013).
Incorporation of sulfur into plant metabolism requires
uptake from the soil and coordinated transport of sulfate
from the root to the shoot by sulfate transporters (Sultr) of
group 1 and 2 (Takahashi et al., 2012). Mainly in the shoot,
but also in roots, sulfate is reduced by 50-adenylylsulfate(APS) reductase (EC 1.8.4.9, APR) and sulfite reductase (EC
1.8.7.1, SiR) to sulfide, which is then fixed by O-acetylserine
(thiol)lyase (EC 2.5.1.47, OAS-TL) into Cys. The synthesis of
the carbon- and nitrogen-containing acceptor of sulfide,
O-acetylserine, is catalyzed by serine acetyltransferase (EC
2.3.1.30, SAT). SAT and OAS-TL are encoded by small gene
families and their activities are present in the plastids, the
mitochondria and the cytosol (Hell and Wirtz, 2011). The
analysis of single OAS-TL loss-of-function mutants revealed
a significant exchange of Cys and sulfide between these
sub-cellular compartments (Heeg et al., 2008). The reduc-
tion of sulfate to sulfide is highly regulated at the level of
APR (Takahashi et al., 2011), which limits Cys synthesis and
takes place exclusively in plastids, as demonstrated by loss-
of-function mutants for SiR. Recently, we identified
SULTR3;1 (AT3G51895) as one of the transporters responsi-
ble for the uptake of sulfate into chloroplasts (Cao et al.,
2013). In Arabidopsis, 3′-phosphoadenosine-5′-phosphosul-
fate (PAPS) is the substrate of cytosolic sulfotransferases
(EC 2.8.2) that donate sulfate to target metabolites and
release PAP. PAPS is synthesized mainly in plastids, but to
a lower extent also in the cytosol, by activity of APS kinase
(EC 2.7.1.25), which competes in plastids with APR on their
common substrate adenosine-5′-phosphosulfate (APS;
reviewed in Chan et al., 2013).
The phytohormone ABA regulates many essential pro-
cesses including embryo maturation, seed dormancy, root
development, and responses to abiotic stresses. In the bio-
synthesis of ABA, the last reaction converting abscisic alde-
hyde to ABA is catalyzed by abscisic aldehyde oxidase (AO)
3 (EC 1.2.3.1, AAO3), which requires the molybdenum co-
factor (Moco). Moco must be sulfurated to act as the
co-factor of AAO3 (Mendel and Hansch, 2002; Schwarz and
Mendel, 2006). This sulfuration is catalyzed by Moco sulfur-
ase (EC 2.8.1.9, ABA3) that requires Cys as the sulfur donor
(Bittner et al., 2001; Xiong et al., 2001). As demonstrated by
the mutation in ABA3 (AT1G16540) leading to ABA defi-
ciency, the sulfuration of Moco is essential for ABA biosyn-
thesis (Xiong et al., 2001). Two additional sulfur atoms are
required in the cytosol for biosynthesis of metal-containing
pterin (MPT), the direct precursor of Moco. MPT synthase,
which consists of the two enzymes CNX6 and CNX7, pro-
duces MPT by transfer of two sulfur atoms and one copper
atom to cyclic pyranopterin monophosphate. As CNX7 car-
ries a single sulfur-binding site, a two-step reaction mecha-
nism for MPT synthesis is proposed. MPT synthase is
loaded with S and re-sulfurated by the rhodanese-like
domain of CNX5, which accepts S from a so-far unknown S-
donor in plants (Bittner and Mendel, 2009). Interestingly,
only two of the four Moco-containing plant enzymes (AAO3
and xanthine dehydrogenase, EC 1.17.1.4, XDH) specifically
accept a sulfurated Moco (S-Moco) from ABA3 and result in
a specific loss of AAO3 and XDH function in aba3 mutants.
In contrast, the remaining Moco-containing enzymes, sulfite
oxidase (EC 1.8.3.1) and nitrate reductase (EC 1.6.6.1, NR)
accept Moco directly or from Moco-binding proteins and
are not affected in aba3 (Schwarz and Mendel, 2006; Sch-
warz et al., 2009). The ABA3 catalyzed sulfuration step is
supposed to provide an efficient way to regulate the
amount of active AAO3 and XDH enzymes during different
physiological stresses (Bittner and Mendel, 2009). In fact,
transcription of ABA3 is rapidly induced by drought and salt
stress as well as upon treatment with ABA (Bittner et al.,
2001; Xiong et al., 2001). Therefore, AAO3 activity might
link ABA biosynthesis via ABA3 to sulfur metabolism.
Recent studies by numerous research groups have revealed
that sulfur-related metabolites are highly regulated by
drought stress (Chan et al., 2013). However, little informa-
tion is known about how sulfur metabolism and ABA func-
tion is coordinated in plants.
In this study we demonstrate that sulfate supply and
knock-out of SULTR3;1 function strongly affects ABA lev-
els. The results indicate that the essential nutrient sulfate
affects ABA biosynthesis in higher plants via the availabil-
ity of Cys and suggest an efficient mechanism by which
plants use sulfur to combat environmental stresses.
RESULTS
Sulfate availability affects the ABA content
The effect of exogenous sulfate availability on ABA content
was examined in wild-type seedlings that had been grown
on half-strength Murashige and Skoog (MS) medium for
2 weeks and subsequently treated with different concentra-
tions of sulfate (0–1500 lM) for 24 h prior to determination
of ABA contents. The steady-state ABA levels of wild-type
seedlings grown at non-limiting sulfate supply conditions
(150 and 1500 lM sulfate) were highest and indistinguish-
able from each other. After transfer of seedlings to limiting
sulfate supply conditions (0 and 15 lM), steady-state ABA
levels decreased significantly (Figure 1a). These results
demonstrate that the steady-state ABA level of wild-type
seedlings is positively correlated with exogenous supply of
sulfate.
Loss of SULTR3 transporters causes significant decrease
of ABA contents
In higher plants the formation of PAPS, the precursor of
PAP, a known regulator of the drought stress response
(Estavillo et al., 2011), is catalyzed in the cytosol and in
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
Sulfate availability affects ABA levels in Arabidopsis 605
plastids. In contrast, sulfate reduction takes place exclu-
sively in plastids. In order to test if the impact of sulfate
availability on ABA synthesis is caused by the cytosolic or
the plastid sulfate pool, we analyzed ABA contents in 2-
week-old seedlings of two SULTR3;1 loss-of-function
mutants (sultr3;1-2 and sultr3;1-4), which have decreased
rates of uptake of sulfate into chloroplasts (Cao et al.,
2013). The level of ABA in both mutants under non-
stressed conditions was about half that in the wild-type
(Figure 1b). The decreased ABA level in the knock-out
mutants could be reverted to the wild-type level by ectopic
expression of a cDNA that encoded SULTR3;1 under con-
trol of the 35S promoter (35S-SULTR3;1; Figure 1b). These
results demonstrate that disruption of SULTR3;1 function
leads to decreased ABA levels. However, when both
sultr3;1 mutants were stressed by high-salt treatment, ABA
contents were still induced. After 6 h of treatment with
200 mM NaCl, ABA levels in SULTR3;1 knock-out mutants
increased to similar levels as in the wild-type and 35S-
SULTR3;1 complementation line but were still about 50%
of the level in stressed wild-type plants (Figure 1b).
Recently, we have shown that isolated chloroplasts of
sultr3;2-2, sultr3;3-5, and sultr3;4 mutants also displayed
decreased sulfate uptake capacity (Cao et al., 2013). In
order to test if reduced capacity of sulfate uptake into chlo-
roplasts is always accompanied by decreased steady-state
levels of ABA, we tested the ABA level in 1-week-old seed-
lings of these mutants. Disruption of other genes of the
SULTR3 subfamily also resulted in significantly decreased
ABA levels (Figure 1c). The approximately 10-fold higher
steady-state ABA levels of 2-week-old wild-type seed-
lings (Figure 1b) in comparison with 1-week-old wild-type
seedlings (Figure 1c) indicated a significant ABA production
during early seedling growth. For that reason, all future
analyses regarding the effect of sulfur metabolism on ABA
biosynthesis were performed in the 2-week developmental
stage plants. The existence of five SULTR3 transporters in
Arabidopsis prompted us to test if growth and metabolite
profiles in later stages of vegetative development are signif-
icantly affected in the single mutants when grown under
normal and limited sulfur supply. All single sultr3 mutants
displayed wild-type-like growth and adaptation of sulfur-
related metabolites upon sulfate limitation in leaves of
6-week-old hydroponically grown plants (Figure S1), and
indicated at least partial redundancy in the function of the
SULTR3 transporters. The unchanged Cys levels in sultr3
mutants found here are not in disagreement with the pub-
lished lower Cys level in young leaves reported in Cao et al.
(2013), as here the total rosette was analyzed. In addition,
the ABA contents in freshly harvested seeds of sultr3;1
mutants were tested and found to be 25–50% of those in the
wild-type; this finding demonstrated that SULTR3;1 affects
ABA biosynthesis not only during early vegetative growth
but also during seed filling. The complementation line had
an even higher ABA content compared with the wild-type
seeds, possibly due to the 35S promoter-driven constitutive
expression of SULTR3;1 (Figure 1d).
These results show that disruption of SULTR3;1 affects
non-stressed induced ABA biosynthesis in both seeds and
seedlings, although growth and sulfur metabolites of the
sultr3;1 mutant are not affected significantly in the vegeta-
tive stage. Besides the plastid-localized SULTR3 subfamily,
disruption of SULTR3;5, a root-vasculature localized sulfate
transporter involved in root-to-shoot sulfate transport, also
**
**(a) (b)
(c) (d) ***
*
*
****
**
*
Figure 1. Decreased sulfate reduction results in
lower abscisic acid (ABA) steady-state levels of
seedlings.
(a) Sulfate supply affects ABA levels of wild-
type seedlings. Wild-type seedlings grown on
normal-sulfur medium for 2 weeks were trans-
ferred to medium that contained different levels
of sulfate (0-1500 lM sulfate as indicated) for
another day before ABA content was deter-
mined (n = 3).
(b) ABA contents in 2-week-old seedlings of the
SULTR3;1 loss-of-function mutants (sultr3;1-2
and sultr3;1-4), the wild-type (Col-0), and the
complemented sultr3;1-2 line (35S-SULTR3;1) in
absence or presence of 200 mM NaCl (n = 6).
(c) Loss of each SULTR3 subfamily member
results in a significant decrease of ABA content
in 1-week-old seedlings (n = 3).
(d) ABA contents in freshly harvested seeds of
the SULTR3;1 loss-of-function mutants
(sultr3;1-2 and sultr3;1-4), the wild-type (Col-0),
and the complemented sultr3;1-2 line (n = 3).
Statistically significant difference is determined
according to Student’s t-test and indicated by
***P < 0.001, **P < 0.01 or *P < 0.05. Values
represent means � standard deviation (SD).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
606 Min-Jie Cao et al.
resulted in decreased ABA content (Figure 1c). We con-
clude from this result that decreased ABA biosynthesis in
sulfur-deprived wild-type seedlings is caused by limitation
of the cellular sulfate pool, especially in plastids.
The decreased ABA levels in the sultr3;1 mutants can be
rescued by exogenous Cys
In order to dissect if limitation of plastid sulfate pool
affects ABA biosynthesis due to decreased sulfate reduc-
tion or decreased formation of PAPS in plastids, we tested
if exogenous application of Cys is able to rescue the phe-
notype of lowered ABA content in the sultr3;1 mutant.
Indeed, the ABA levels of the sultr3;1 knock-out seedlings
reverted to wild-type levels within 6 h by feeding with Cys
(Figure 2a), demonstrating that Cys availability limits ABA
synthesis in the sultr3;1 mutant. The ABA level in the wild-
type plants did not change significantly in response to
application of Cys (Figure 2a), suggesting that the Cys level
in the wild-type was sufficient.
To further characterize the impact of Cys availability on
ABA steady-state levels, we tested ABA contents in the
sir1-1 mutant. The sir1-1 mutant is a knock-down mutant
with a lower capability to reduce sulfate to sulfide in plast-
ids. However, sir1-1 shows a strong retardation of growth
and elevated Cys steady-state levels (Khan et al., 2010) that
has not been observed in sultr3;1 (Cao et al., 2013). Inter-
estingly, ABA levels in sir1-1 seedling were significantly
higher than in wild-type seedling (Figure 2b), these
findings supported the hypothesis that Cys availability
affects ABA synthesis. Another sulfur assimilation mutant,
apr2-2, showed lower ABA content (Figure 2c) and
decreased cysteine (Figure 2d) and glutathione content
(Figure 2e) when compared with the Col-0 wild-type. The
thiols and ABA level in the Col-0 wild-type and apr2-2
mutant decreased synchronously under sulfur-limited con-
dition (Figure 2c,d). These results further confirmed the
positive correlation of endogenous Cys content and ABA
synthesis.
Taken together, analyses of mutants for three crucial
steps in the sulfate assimilation pathway in combination
with Cys supplementation experiments suggested that Cys
availability affected ABA steady-state levels in seedlings,
and provides an explanation for the sulfate-dependent
alteration of ABA steady-state levels in wild-type seedlings
(Figures 1a and 2d).
S-Moco-dependent AO and XDH activities are decreased in
the sultr3;1 mutants
The most obvious link between Cys availability and ABA
synthesis is activity of abscisic aldehyde oxidase 3 (AAO3,
isoform 3 of four AO present in Arabidopsis), which
catalyzes the final step of ABA synthesis, and requires
ABA3-bound sulfurated molybdenum co-factor (S-Moco)
(Schwarz et al., 2009). The three other AOs (AAO1, AAO2
and AAO4) accept other substrates, are not involved in
ABA biosynthesis and do not contain the S-Moco factor
**
**
**
*
** * ******
* *****
***
*
(a)
(b)
(c)
(d)
(e)
Figure 2. Cys availability affects abscisic acid
(ABA) steady-state levels in seedlings and
seeds.
(a) ABA levels of 2-week-old wild-type (Col-0)
and SULTR3;1 loss-of-function mutants
(sultr3;1-2 and sultr3;1-4) in absence and pres-
ence of 0.5 mM Cys for 6 h (n = 3).
(b) ABA contents of 1-week-old wild-type (Col-
0) and SiR knock-down mutant (sir1-1) (n = 3).
(c) ABA levels of 2-week-old wild-type (Col-0)
and loss-of-APR2 mutant (apr2-2) under differ-
ent sulfate levels ranging from 0–1500 lM as
indicated (n = 3).
(d) Cysteine contents of 2-week-old wild-type
(Col-0) and loss-of-APR2 mutant (apr2-2) under
different sulfate levels ranging from 0–1500 lMas indicated (n = 3).
(e) Glutathione contents of 2-week-old wild-type
(Col-0) and APR2 loss-of-function mutant (apr2-
2) under different sulfate levels ranging from 0–1500 lM as indicated (n = 3).
Statistically significant difference is determined
according to Student’s t-test and indicated by
***P < 0.001, **P < 0.01 or *P < 0.05. Values
represent means � standard deviation (SD).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
Sulfate availability affects ABA levels in Arabidopsis 607
(reviewed in Bauer et al., 2013). Extractable total AO activi-
ties of sultr3;1 seedlings were decreased significantly in
comparison with that of the wild-type (Figure 3a). The
exogenous feeding of Cys for 6 h increased extractable
AO activity approximately five-fold in wild-type seedlings
(Figure 3a), and demonstrated the significant impact of
Cys on AO maturation in seedlings, even on the wild-type
background. After feeding of Cys to sultr3;1-2 and sultr3;
1-4, both mutants were indistinguishable from the wild-
type in terms of total AO activities; this result provides an
explanation for the restoration of wild-type ABA levels in
both mutants upon this treatment (Figure 2a).
The activities of XDH and NR were determined to further
elucidate which sulfur-requiring step of S-Moco biosynthe-
sis was mainly affected in the sultr3;1 mutants. Specifi-
cally, the activity of the second S-Moco-dependent
enzyme, XDH, and not of the Moco-dependent NR was
affected in both alleles of sultr3;1 (Figure 3b, c). These
results indicated that decreased Cys availability hardly
limited synthesis of Moco per se, but specifically affect
Moco sulfuration by ABA3.
Co-regulation between sulfur and ABA metabolism
Cys availability affected total AO activity (Figure 3a) and
ABA steady-state levels (Figure 2a) in seedlings most prob-
ably by limitation of S-Moco synthesis. We therefore tested
the impact of sulfur availability on expression of ABA3
(AT1G16540) encoding the Moco sulfurase, and of NCED3
(AT3G14440) encoding 9-cis-epoxycarotenoid dioxygenase
that catalyzes the rate-limiting step in ABA precursor bio-
synthesis. Low sulfate supply led to the accumulation of
NCED3 and ABA3 transcript levels in wild-type and sultr3;1
mutants (Figure 4a). The promoters of both genes contain
canonical sulfur-deficiency responsive elements (SURE;
Table S1), which might be responsible for the observed
accumulation. However, up-regulation of ABA biosynthe-
sis-related genes could be also a result of decreased ABA
level during low sulfur supply (Figure 1a).
The decreased ABA synthesis capacity in sultr3;1
mutants (Figure 1b) prompted us to test if ABA regulates
the expression of SULTR3 transporters. Short-term applica-
tion of ABA results in a 35-fold increase of SULTR3;1 tran-
script in wild-type seedlings in a time-dependent manner
(Figure 4b). SULTR3;1 transcript peaked at 6 h after appli-
cation of ABA and stayed high during the treatment.
SULTR3;4 transcript showed a similar pattern of induction
by ABA but with lower amplitude (Figure 4e). Transcript
steady-state levels of SULTR3;2 and SULTR3;3 genes
increased less than two-fold after 6 h of ABA treatment
(Figure 4c,d), while SULTR3;5 and SiR transcript steady-
state levels were not induced by ABA (Figure 4f,g). The
significant induction of SULTR3;1 and SULTR3;4 transcript
levels by ABA was independently verified by northern blot-
ting that also revealed induction of SULTR1;2 and
SERAT2;1 by ABA (Figure S2). Induction of SULTR3;1 and
SULTR3;4 by external application of ABA correlates well
with the significant induction of these transporters in the
early high-salt stress response (Figure 5; 2 h). Both results
indicated the relevance of enhanced sulfate transport into
plastids during abiotic stresses. Most likely, within the
plastids, the sulfate is reduced immediately and incorpo-
rated into cysteine, which serves as a building block of
numerous sulfur-containing stress-defense compounds,
e.g. glutathione GSH (Kruse et al., 2012).
In situ GUS staining of SULTR3;1 promoter activity in
wild-type seedlings that expressed the GUS reporter under
XDHWT
Coomassie blue staining
*
(a)
(b)
(c)
Figure 3. AO and XDH activities are low in
sultr3;1 seedling and can be restored by appli-
cation of Cys.
(a) Total aldehyde oxidase activity of soluble
proteins extracted from 2-week-old wild-type
(Col-0) and SULTR3;1 loss-of-function mutants
(sultr3;1-2 and sultr3;1-4) treated with 0.5 mM
Cys (+) or without Cys (–) for 6 h (n = 4).
(b) In gel staining of xanthine dehydrogenase
activity of soluble protein extracted from the
wild-type (WT) and the two SULTR3;1 knock-
out mutants (sultr3;1-2, sultr3;1-4). Coomassie
blue-stained SDS-PAGE of same samples
served as loading control.
(c) Nitrate reductase activity of 2-week-old wild-
type and SULTR3;1 knock-out mutants (sultr3;1-
2, sultr3;1-4) (n = 6).
Statistically significant difference is determined
according to Student’s t-test and indicated by
*P < 0.05. Values represent means � standard
deviation (SD).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
608 Min-Jie Cao et al.
the control of the SULTR3;1 promoter revealed that
SULTR3;1 was not expressed in roots under non-stressed
conditions, but was induced by exogenous application of
ABA (Figure 4h). This ABA-responsive expression pattern in
roots is consistent with the increase of ABA levels in roots
under stress conditions such as drought and high salt. We
therefore tested transcription induction of SULTR 3 subfam-
ily, SIR1, and APR2 upon short-term high-salt stress in the
wild-type. Transcript levels of SULTR3;1, 3;2, 3;3 and 3;4,
SIR1, and APR2 showed various degrees of induction by
this stress treatment (Figure 5). Consistent with these
results is the presence of ABA- and abiotic stress-respon-
sive cis elements in the promoters of those genes (Table
S1). Taken together these results revealed significant
co-regulation of the ABA biosynthesis pathway and the sul-
fate assimilation pathway at the transcriptional level.
Seed germination of sultr3, sir1, and apr2 mutants are all
sensitive to exogenous ABA application and salt stress
The phytohormone ABA is known to mediate high-salt
stress response and to self-control its own biosynthesis by
positive and negative feedback loops. Germination assays
were conducted to examine whether loss of SULTR3;1
would alter the sensitivity to ABA, due to disturbance of
this complex regulatory network. The seeds of the wild-
type, the sultr3;1-2, the sultr3;1-4 and the 35S-SULTR3;1
complemented sultr3;1-2 mutant were germinated on half-
strength MS medium supplemented with different concen-
trations of ABA. All lines showed similar germination rates
on medium that lacked ABA. In presence of ABA the two
sultr3;1 knock-out mutants showed a dose-dependent
germination delay when compared with the wild-type
ABA3
1500 μM 15 μM
rRNA
[sulfate]
NCED3
Age/days3 6 9 12
-ABA
10 μM
ABA
e f g h
a b c d
*
*
*
* *
*
* *
*
*
*
***
SULTR3;1
SULTR3;4
SULTR3;2
SULTR3;3
SULTR3;5 SIR1
(a)
(b) (c)
(d) (e)
(f)(h) (g)
Figure 4. Co-regulation of abscisic acid (ABA) biosynthesis- and S-metabolism-related genes.
(a) NCED3 and ABA3 are responsive to low sulfur condition. Seedlings were grown on medium with high-sulfur level (15 000 lM sulfate) or low sulfur level
(1500 lM sulfate) for 2 weeks. Total RNA was isolated from sultr3;1-2, sultr3;1-4, the wild-type, and 35S-SULTR3;1 complementation line, respectively, and sub-
jected to northern blot analysis with specific probes for NCED3 and ABA3. Ethidium bromide-stained RNA gel shows equal loading.
(b–g) Relative transcript levels of SULTR3;1 (b), SULTR3;2 (c), SULTR3;3 (d), SULTR3;4 (e), SULTR3;5 (f),and SiR1 (g) in 1-week-old wild-type seedlings treated
with 50 lM ABA for indicated time. Relative transcript level of the respective gene before the application of ABA was set to 1 in order to allow comparison of
ABA impact on expression level. Statistically significant difference is determined according to Student’s t-test and indicated by single asterisk. Values represent
means � standard deviation (SD) (n = 3).
(h) In situ staining of SULTR3;1 promoter-driven transcription in wild-type plants that harbor a SULTR3;1 promoter–GUS fusion construct (Cao et al., 2013).
Seedlings were grown on half-strength MS medium for 3 (a, e), 6 (b, f), 9 (c, g), and 12 days (d, h) and then treated for 5 h with water (a–d) or 10 lM ABA (e–h)before GUS staining.
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
Sulfate availability affects ABA levels in Arabidopsis 609
(Figure 6). This delay could be reverted to the wild-type
germination rate by ectopic expression of SULTR3;1 in the
sultr3;1 mutant background (35S-SULTR3;1, Figure 6). On
the medium that contained 0.5 lM ABA, 100% of the seeds
of the wild-type, the 35S-SULTR3;1 line, and the two
knock-out mutant germinated at day 6 (Figure 6). When the
ABA concentration in the medium was increased to 2 lM;however, fewer than 10% of the seeds of both sultr3;1
mutants germinated while more than 80% of the seeds of
the wild-type and 35S-SULTR3;1 line germinated
(Figure 6).
In order to prove the relevance of SULTR3;1 in an abiotic
stress response that is regulated in higher plants by ABA,
the same lines were assayed for tolerance against high-salt
stress by germination on MS medium that contained
150 mM NaCl or 200 mM NaCl (Figure 6). The germination
rate of sultr3;1-2 and sultr3;1-4 seeds decreased signifi-
cantly when grown on the medium that contained NaCl
compared with that of the wild-type and the complementa-
tion line. The germination assays revealed that decrease of
plastid sulfate uptake in the sultr3;1 mutant background
disturbed ABA-mediated dormancy of seeds and resulted
in a high-salt-sensitive stress phenotype.
To confirm this result, we tested knock-out mutants of
some other genes involved in sulfur assimilation, including
mutants of other members in the SULTR3 subfamily
(Figure 7), sir1-1 and apr2 mutants (Figures S3 and S4). All
tested mutants also showed dose-dependent sensitivity to
both ABA and salt stress. These results showed that altera-
tions in sulfate transport and/or sulfate assimilation can
affect the sensitivity to ABA and salt stress, at least at the
germination stage.
DISCUSSION
Sulfate supply significantly affects ABA synthesis
S-metabolism has been linked to many biotic (Kruse et al.,
2007) and abiotic stress responses of the plant including
high-salt stress (Barroso et al., 1999) and drought (Chan
et al., 2013). Many of these stress responses are accompa-
nied by the formation of ROS, which in turn directed the
investigations of S-metabolism during these stress
responses frequently to focus on analyses of GSH synthe-
sis or cellular reduction/oxidation (redox) state. GSH is a
significant sink for reduced sulfur under non-stress and
stress conditions and acts as a redox buffer that is relevant
(a) (b)
(c) (d)
(e) (f)
Figure 5. Salt stress induces elevated transcript
levels of multiple sulfur assimilation-related
transcripts.
Relative transcript levels of SULTR1;2 (a),
SULTR 3;1 (b), SULTR 3;4 (c), SULTR 4;1 (d),
APR2 (e), and SiR1 (f) in 1-week-old wild-type
seedlings treated with 150 mM NaCl for indi-
cated time, with COR47 (I) used as control. Rel-
ative transcript level of the respective gene
before the application of NaCl was set to 1. Sta-
tistically significant difference was determined
according to Student’s t-test and indicated by
single asterisk. Values represent means � stan-
dard deviation (SD) (n = 3).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
610 Min-Jie Cao et al.
Control 0.5 μM ABA 1 μM ABA
2 μM ABA 160 mM NaCl 200 mM NaCl
Figure 6. Germination of sultr3;1 seedlings is delayed by application of abscisic acid (ABA) and salt stress.
Seeds of the two SULTR3;1 loss-of-function mutants (sultr3;1-2, sultr3;1-4), the wild-type (Col-0), and the complementation line (35S-SULTR3;1) were germinated
on half-strength MS (1% sucrose) medium that contained different concentrations of ABA (0, 0.5, 1 or 2 lM) or NaCl (150 or 200 mM). Seeds were evaluated at
indicated times and considered germinated when the radicles penetrated the seed coat (n = 240, for each genotype grown on four individual plates for each con-
dition). Values represent mean � standard deviation (SD).
MS 0.3 μM ABA 0.5 μM ABA
1 μM ABA 150 mM ABA 180 mM ABA
Figure 7. Germination response of sultr3;2, sultr3;3, sultr3;4, and sultr3;5 mutants to exogenous abscisic acid (ABA) and salt stress.
Seeds of the knock-out mutants (sultr3;2-2, sultr3;3-5, sultr3;4, sultr3;5) and Col-0 were sown on half-strength MS (1% sucrose) medium that contained 0, 0.3,
0.5, 1 lM ABA, or 180, 200 mM NaCl. Seeds were considered germinated when the cotyledon turn green. Germination curves were generated on germination fre-
quency counted for 8 days. Values are the mean germination frequency from three separate plates (60 seeds per plate) for each combination of plant line and
ABA concentration. Error bars indicate standard deviation (SD).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
Sulfate availability affects ABA levels in Arabidopsis 611
to cope with the negative impact of stress-generated ROS
in plant cells (Noctor et al., 2012).
However, recent studies have revealed that stress
responses can also be affected by S-metabolism in a GSH-
independent way. The drought-specific accumulation of
PAP, a byproduct of sulfation reactions in the cytosol and
the endoplasmic reticulum, highlights the importance of
sulfated metabolites in the drought stress response. PAP
has been shown to act in the nucleus as a key signal that
affects expression of drought and high light inducible
genes, most likely by allosteric inhibition of 5′–3′ exoribo-
nucleases (Estavillo et al., 2011). Taken together, these
analyses demonstrated a relevant function of metabolites
related to oxidized or reduced sulfur in the drought stress
response.
However, little information is known about the interac-
tion of S-metabolism with the synthesis of the key regula-
tor of drought stress response, ABA (Chan et al., 2013).
Sulfur deprivation experiments clearly demonstrated that
external sulfur supply limits ABA biosynthesis in wild-type
seedlings, which have an active ABA metabolism (compari-
son of Figure 1b with Figure 1c). By using loss-of-function
mutants for each of the SULTR3 transporters, the sir1-1
mutant, and apr2 mutant (Figures 1 and 2b–d), we pin-
pointed the molecular reason for limitation of ABA biosyn-
thesis by external sulfur supply to the level of Cys
availability, and demonstrated that reduction of sulfate to
sulfide in seedlings is relevant for ABA biosynthesis. This
result was surprising and therefore was confirmed inde-
pendently by restoration of ABA level and total AO activity
(including AAO3 activity) using exogenous application of
Cys to seedlings (Figures 2a and 3a).
The seeds of sultr3;1 display also lowered ABA contents
(Figure 1d). This result further supports the idea that the
availability of Cys is responsible for decreased ABA synthe-
sis, as sultr3;1 seeds have unaffected sulfate and GSH lev-
els but lower Cys contents (Zuber et al., 2010). As a
consequence of the decreased ability to transport sulfate
into the chloroplast, the low ABA (Figure 1b) and the low
Cys contents in seeds of sultr3;1 (Cao et al., 2013), the ger-
minating sultr3;1 seedlings are more sensitive to high-salt
stress, which requires significant production of ABA and
GSH. The germination deficiency of sultr3;1 and other
mutants affected in the sulfur assimilation pathway (sir1-1,
apr2, and sult3;2-sult3;5) could therefore be a result of
decreased GSH or ABA biosynthesis or a combination of
both. Germination of ABA synthesis-deficient mutants was
reported to be insensitive towards high-salt stress (Leon-
Kloosterziel et al., 1996). This finding indicated that
decreased GSH synthesis rather than ABA synthesis under
high-salt conditions (Note: GSH level in sultr3;1 mutant not
changed in normal condition as Zuber et al. described),
which is due to the low Cys level, is responsible for the
observed high-salt germination phenotype. In contrast with
germinating aba3 seeds, 10-day-old aba3 seedlings are
hypersensitive to high-salt application (Xiong et al., 2001);
this finding demonstrates that developmental stage and/or
growth condition can influence significantly the relevance
of ABA in the high-salt stress response. For that reason the
authors do not want to exclude the possibility that altered
ABA biosynthesis by limitation of ABA3 activity can also
contribute to the observed high-salt sensitivity of sultr3;1
in the here applied experimental system. Irrespective of
the reason for the observed high-salt germination pheno-
type, apr2 and sir1-1 show the same phenotype as sultr3;1
to sultr3;5 – furthermore indicating a significant contribu-
tion of SULTR3 group transporters activity to optimal sul-
fate reduction for cysteine production in germinating
seeds.
Cysteine is the substrate of ABA3, a Moco-sulfurylase
that is crucial for AAO3 activity and for ABA synthesis in
plants (Bittner et al., 2001). Under non-stressed conditions
germination the aba3 mutant is hypersensitive to exoge-
nous ABA application (Plessis et al., 2011). The same
hypersensitivity towards ABA was found in sultr3;1
(Figure 6) as well as other mutants with decreased sulfur
assimilation and consequently decreased cysteine synthe-
sis capacity (Figures 7, S3 and S4). The fact that not only
sultr3;1 but all tested sulfur assimilation mutants, including
apr2, sir1-1, show the same ABA-hypersensitive phenotype
than the aba3 mutant, supports the idea that sufficient Cys
synthesis is mandatory for ABA3 activity and consequently
AAO3 activity in germinating seeds. As ABA, Cys and GSH
steady-state levels in seeds of apr2 and sir1-1 are
unknown, the authors do not want to exclude the possi-
bility that decreased translation and/or GSH synthesis
can add to the observed ABA hypersensitivity during
germination.
In summary, several independent lines of evidence indi-
cated that synthesis of the phytohormone ABA in develop-
ing seedlings (see previous section), and most probably
also in germinating seeds, was dependent on sufficient
Cys production/availability and as a consequence affected
by external sulfate supply (Figure 1). To our knowledge,
this report is the first to show that Cys, the end product of
the reductive sulfate assimilation pathway, directly regu-
lates phytohormone metabolism. Independent support for
this unexpected link between Cys and ABA biosynthesis is
provided by treatment of Arabidopsis, V. faba and I. walle-
riana leaves with sulfide, a direct precursor of Cys, which
promotes stomata closure in an ABA-dependent manner
(Garcia-Mata and Lamattina, 2010). Furthermore, stomata
are closed in the cad2 mutant (Okuma et al., 2012) that has
low GSH (30% of wild-type) but high Cys (>200% of wild-
type) levels (Noctor et al., 2012). Surprisingly, closure of
stomata in cad2 could not be attributed to altered ROS
metabolism as expected by the investigators (Okuma et al.,
2012), leaving the molecular link between decreased GSH
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
612 Min-Jie Cao et al.
synthesis and closure of stomata enigmatic. An enhanced
ABA level due to higher Cys availability, as observed in
sir1-1, would provide a solid explanation for the closed
stomata in cad2. Recently, sulfate was identified to be the
chemical that transports the primary stress signal in
the xylem during early stage of water stress, even before
the expression of ABA biosynthesis genes, and promotes
in leaves of Zea mays the effectiveness of ABA in stomata
closure, the most important function of ABA during the
drought stress response (Ernst et al., 2010). A significant
co-regulation of ABA and Cys biosynthesis is also indi-
cated by regulation of cytosolic OAS-TL A (AT4G14880),
the key enzyme for fixation of sulfide into Cys in Arabidop-
sis (Heeg et al., 2008), by ABA during salt stress (Barroso
et al., 1999). However, APR activities or transcripts were
regulated in an ABA-independent manner as demonstrated
by their response to NaCl treatment (Koprivova et al.,
2008). In contrast, APR1 and APR3 are highly responsive to
H2O2 (Xiang and Oliver, 2000) and oxidative stress as
shown by their close correlation with marker transcripts
for H2O2 signaling (Queval et al., 2009). Although APRs do
not respond to ABA, most environmental stresses generate
H2O2 signaling and suggest that APR activity is important
for efficient combating of environmental stresses.
ABA is known to suppress lateral root formation in order
to facilitate growth of primary root to search for water dur-
ing drought (Xiong et al., 2006). In sulfate-deprived plants,
lateral roots are formed closer to the root tip and at
increased density (Lopez-Bucio et al., 2003). This change in
root architecture has been attributed to sulfate starvation-
induced activity of nitrilase isoform 3 (NIT3) that synthe-
sizes indole-3-acetic acid (IAA) (Kutz et al., 2002). However,
decreased ABA level as a result of lowered Cys availability
may contribute to induction of lateral root formation at the
root tip and allows the fine tuning of the IAA-induced
morphogenic response of roots to sulfur deficiency. The
up-regulation of SULTR3;1 transcription by exogenous
application of ABA was most evident in roots (Figure 4h),
although the root is not the bulk site of sulfate reduction in
plants. This result indicates the necessity for a significant
co-regulation of plastid sulfate uptake with ABA biosynthe-
sis in roots. More detailed studies are needed to dissect
the relevance of auxin and ABA signaling for alteration of
sulfate deficiency-induced alteration of root morphology.
Furthermore, the direct link between Cys and ABA syn-
thesis may also stimulate research on sulfur-enhanced
defense (SED) of plants (Kruse et al., 2007). SED describes
the phenomenon that optimal supply of field grown crop
plants with sulfate has a beneficial impact on resistance of
these plants against a broad range of pathogens. The identi-
fication of ABA as an essential signal for plant resistance to
pathogens, which affects jasmonate signaling and activa-
tion of defense genes in Arabidopsis (Adie et al., 2007),
could provide a regulatory connection to SED. Interestingly,
methyl jasmonate does not only induce defense genes but
also regulates a distinct set of primary and secondary sulfur
metabolism-related genes (Xiang and Oliver, 1998; Jost
et al., 2005), this situation adds another layer of regulation
for S-metabolism under stress conditions.
The molecular link between Cys and ABA biosynthesis is
decreased AAO3 activity. The maturation of the AAO3 apo-
enzyme requires ABA3 bound S-Moco, which is produced
by ABA3 upon degradation of Cys (Bittner et al., 2001). The
apparent KM value of ABA3 for Cys is 50 lM (Heidenreich
et al., 2005). This is in the range of the cytosolic Cys con-
centration (approx. 300 lM) in leaves of Arabidopsis plants
that were grown on optimal S-supply (Kr€uger et al., 2009).
Significant decrease of thiols in each tissue of the plants is
the hallmark of the sulfate deficiency response. In Brassica
napus sulfate starvation for 10 days causes 10-fold and
4-fold decrease of thiols in leaves and roots, respectively
(Buchner et al., 2004). A 10-fold decrease of Cys was
observed within 1 day of sulfate starvation in cell suspen-
sion cultures of Arabidopsis (Wirtz et al., 2004), strongly
indicating that decreased cytosolic Cys pool due to limited
sulfate supply can limit ABA3 activity in Arabidopsis. Both
aba3 and sultr3;1 mutants show low ABA level and hyper-
sensitive to exogenous ABA, also indicating the potential
link between Cys and ABA.
Our surprising finding of a direct impact of Cys on ABA
formation together with the herein identified co-regulation
of genes related to ABA synthesis- and S-metabolism
revealed a reciprocal regulatory network with the function
of ensuring optimal supply with Cys for ABA synthesis to
combat environmental stresses.
EXPERIMENTAL PROCEDURES
Plant materials and growth conditions
Arabidopsis thaliana (ecotype Columbia, Col-0) was grown onhalf-strength MS solid medium (Sigma, www.sigmaaldrich.com)that contained 1% (w/v) sucrose at 22°C under 12-h-light/12-h-darkcycles. Sulfur-deficient medium was prepared by replacing sulfatesalts and agar in the MS medium with equivalent chloride saltsand agarose. The plants for isolation of chloroplast and protoplastwere grown on soil for 4 weeks under the same light regime.
Identification of the knock-out mutants and
complementation with 35S-SULTR3;1
The mutants sultr3;1-2, sultr3;1-4, sultr3;2-2, sultr3;3-5, sultr3;4,and sultr3;5 were T-DNA insertion lines (SALK_023190,SALK_127024, SALK_023980, SALK_000822C, CS859766 andSALK_127024 respectively) obtained from the Arabidopsis Biolog-ical Resource Center (ABRC). The homozygotes plants were iden-tified by polymerase chain reaction (PCR) using a commonprimer LBb1 and gene-specific primers 3;1-2-F and 3;1-2-R forsultr3;1-2, 3;1-4-F and 3;1-4-R for sultr3;1-4,3;2-2-Fand 3;2-2-R forsultr3;2-2, 3;3-5-F and 3;3-5-R for sultr3;3-5, 3;4-F and 3;4-R for sultr3;4,and 3;5-F and 3;5-R for sultr3;5, respectively (Table S2). Homozygotelines were confirmed by reverse transcription (RT)-PCR or quantitative
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
Sulfate availability affects ABA levels in Arabidopsis 613
real-time RT-PCR. For complementation analysis, the 35S-SULTR3;1overexpression construct was produced by inserting the codingregion of SULTR3;1 (1977 bp) amplified by PCR using primer pair 3;1attb1-F and 3;1 attb2-R (Table S2) into vector pCB2004 (Lei et al.,2007) via GatewayTM cloning (Invitrogen, www.lifetechnologies.com).The resulting construct was verified by sequencing and transformedinto sultr3;1-2 mutant using the floral-dip method (Bent, 2000). Trans-formants were selected for glufosinate resistance and confirmed byRT-PCR using primers 3;1RT-F and 3;1RT-R and b-tubulin8 was usedas control (Table S2).
Quantitative real-time PCR
Total RNA was extracted from primary root material of 7-day-oldwild-type seedlings using TRIzol reagent (Invitrogen) and reversetranscribed with the TransScript RT kit (Invitrogen) in accordancewith the manufacturer’s instructions. After heat inactivation, a0.5 ll aliquot was used for quantitative RT-PCR. All quantitativeRT-PCR assays were performed using a SYBR� Premix Ex TaqTM IIkit in a One Step real-time PCR system (Applied Biosystem,www.appliedbiosystems.com.cn) as described in the manufac-turer’s protocol. Each assay consisted of three biological replicatesand was performed twice. UBQ5 was used as the control inquantitative RT-PCR (Table S2).
Northern blot analysis
The transcript steady-state levels of sulfur assimilation and ABAbiosynthesis-related genes were determined by the northern blottechnique. After corresponding treatment, total RNA wasextracted as described previously (Xiang and Oliver, 1998), andnorthern blotting was performed in accordance with standard lab-oratory protocols. Probes for SULTR1;2 SULTR3;1, SULTR3;4,SERAT2;1, NCED3 and ABA3 were labeled with 32P using the Ran-dom Primer DNA Labeling Kit (TaKaRa, www.takara.com.cn); theprimer pairs used for templates amplification were 1;2 RT-F and1;2 RT-R, 3;1 RT-F and 3;1 RT-R, 3;4 RT-F and 3;4 RT-R, SERAT2;1RT-F and SERAT2;1 RT-R, NCED3 RT-F and NCED3 RT-R, andABA3 RT-F and ABA3 RT-R, respectively. Ethidium bromide-stained rRNA was used as the loading control.
Determination of ABA and sulfur-related metabolites
Wild-type, sultr3 mutants, sir1-1 and apr2-2 were grown on half-strength MS medium that contained corresponding MgSO4 as sul-fur source. Hydrophilic metabolites were extracted from 2-week-old plants as described in Wirtz and Hell (2007). Measurements ofsulfide and sulfite were performed in accordance with Birke et al.(2012). Thiols were determined as described in Wirtz et al. (2004).ABA was quantified in seeds and seedlings by enzyme linked im-munosorbant assay (ELISA; Sigma) as described in Yang et al.(2001).
Determination of AO, XDH, NR and GUS activity
Two-week-old plants were treated without or with 0.5 mM Cys for6 h. Aldehyde oxidase activity was measured with benzalde-hyde as substrate and O2 as electron donor, as previouslydescribed (Koshiba et al., 1996). Nitrate reductase activity wasdetermined as described (Solomonson and Vennesland, 1972).XDH activity was carried out by native in gel staining with hypo-xanthine as substrate and O2 as electron donor, as described (Hes-berg et al., 2004). The in situ staining of SULTR3,1 promoteractivity was performed as described in Cao et al. (2013) using thesame SULTR3;1 promoter–GUS fusion constructs.
Germination response to exogenous ABA and salt stress
Sterilized seeds were vernalized for 3 days and then plated onhalf-strength MS medium that contained 1% sucrose and ABA orNaCl. Four replicate plates (60 seeds per plate) were used for eachcombination of Arabidopsis lines and ABA level. The plates werekept at 22°C under long-day conditions. Seed germination wasevaluated from day to day and seeds were considered germinatedwhen the radicles penetrated the seed coat.
ACKNOWLEDGEMENTS
This work was supported by grants from NNSFC (90917004,30471038) and the German Research Foundation (DFG, He1848/13-1/14-1). The authors thank ABRC for providing T-DNA insertionlines used in the study and the Hartmut Hoffmann-Berling Interna-tional Graduate School of Molecular and Cellular Biology and theSchmeil Foundation Heidelberg for support of AS.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. Alteration of sulfur-related metabolites in low sulfurcondition.Figure S2. ABA activates genes for sulfate transporters and Cyssynthesis.Figure S3. Germination response of sir1-1 to exogenous ABA andsalt stress.Figure S4. Germination response of apr2 mutant to exogenousABA and salt stress.
Table S1. Predicted ABA- and stress-responsive elements in thepromoters of the genes for sulfur metabolism and sulfur-defi-ciency responsive element in the promoters of the genes for ABAsynthesis.Table S2. Primer Sequences used in the experiment.
REFERENCES
Adie, B.A., Perez-Perez, J., Perez-Perez, M.M., Godoy, M., Sanchez-Serrano,
J.J., Schmelz, E.A. and Solano, R. (2007) ABA is an essential signal for
plant resistance to pathogens affecting JA biosynthesis and the activa-
tion of defenses in Arabidopsis. Plant Cell, 19, 1665–1681.Barroso, C., Romero, L.C., Cejudo, F.J., Vega, J.M. and Gotor, C. (1999)
Salt-specific regulation of the cytosolic O-acetylserine(thiol)lyase gene
from Arabidopsis thaliana is dependent on abscisic acid. Plant Mol. Biol.
40, 729–736.Bauer, H., Ache, P., Lautner, S. et al. (2013) The stomatal response to
reduced relative humidity requires guard cell-autonomous ABA synthe-
sis. Curr. Biol. 23, 53–57.Bent, A.F. (2000) Arabidopsis in planta transformation. Uses, mechanisms,
and prospects for transformation of other species. Plant Physiol. 124,
1540–1547.Birke, H., Haas, F.H., De Kok, L.J., Balk, J., Wirtz, M. and Hell, R. (2012) Cys-
teine biosynthesis, in concert with a novel mechanism, contributes to
sulfide detoxification in mitochondria of Arabidopsis thaliana. Biochem
J. 445, 275–283.Bittner, F. and Mendel, R.R. (2009) Cell Biology of Molybdenum. In Cell Biol-
ogy of Metal and Nutrients, Vol. Plant Cell Monographs (Hell, R. and
Mendel, R.R., eds). Heidelberg, Germany: Springer, pp. 119–144.Bittner, F., Oreb, M. and Mendel, R.R. (2001) ABA3 is a molybdenum
cofactor sulfurase required for activation of aldehyde oxidase and
xanthine dehydrogenase in Arabidopsis thaliana. J. Biol. Chem. 276,
40381–40384.Buchner, P., Stuiver, C.E.E., Westerman, S., Wirtz, M., Hell, R., Hawkesford,
M.J. and De Kok, L.J. (2004) Regulation of sulfate uptake and expression
of sulfate transporter genes in Brassica oleracea as affected by
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
614 Min-Jie Cao et al.
atmospheric H2S and pedospheric sulfate nutrition. Plant Physiol. 136,
3396–3408.Cao, M.J., Wang, Z., Wirtz, M., Hell, R., Oliver, D.J. and Xiang, C.B. (2013)
SULTR3;1 is a chloroplast-localized sulfate transporter in Arabidopsis
thaliana. Plant J. 73, 607–616.Chan, K.X., Wirtz, M., Phua, S.Y., Estavillo, G.M. and Pogson, B.J. (2013)
Balancing metabolites in drought: the sulfur assimilation conundrum.
Trends Plant Sci. 18, 18–29.Ernst, L., Goodger, J.Q.D., Alvarez, S., Marsh, E.L., Berla, B., Lockhart, E.,
Jung, J., Li, P., Bohnert, H.J. and Schachtman, D.P. (2010) Sulphate as a
xylem-borne chemical signal precedes the expression of ABA biosynthet-
ic genes in maize roots. J. Exp. Bot. 61, 3395–3405.Estavillo, G.M., Crisp, P.A., Pornsiriwong, W. et al. (2011) Evidence for a
SAL1-PAP Chloroplast retrograde pathway that functions in drought and
high light signaling in Arabidopsis. Plant Cell, 23, 3992–4012.Garcia-Mata, C. and Lamattina, L. (2010) Hydrogen sulphide, a novel
gasotransmitter involved in guard cell signalling. New Phytol. 188,
977–984.Heeg, C., Kruse, C., Jost, R., Gutensohn, M., Ruppert, T., Wirtz, M. and Hell,
R. (2008) Analysis of the Arabidopsis O-acetylserine(thiol)lyase gene fam-
ily demonstrates compartment-specific differences in the regulation of
cysteine synthesis. Plant Cell, 20, 168–185.Heidenreich, T., Wollers, S., Mendel, R.R. and Bittner, F. (2005) Characteriza-
tion of the NifS-like domain of ABA3 from Arabidopsis thaliana provides
insight into the mechanism of molybdenum cofactor sulfuration. J. Biol.
Chem. 280, 4213–4218.Hell, R. and Wirtz, M. (2011) Molecular biology, biochemistry and cellular
physiology of cysteine metabolism in Arabidopsis thaliana. Arabidopsis
Book, 9, e0154.
Hesberg, C., Hansch, R., Mendel, R.R. and Bittner, F. (2004) Tandem orienta-
tion of duplicated xanthine dehydrogenase genes from Arabidopsis thali-
ana: differential gene expression and enzyme activities. J. Biol. Chem.
279, 13547–13554.Jost, R., Altschmied, L., Bloem, E. et al. (2005) Expression profiling of meta-
bolic genes in response to methyl jasmonate reveals regulation of genes
of primary and secondary sulfur-related pathways in Arabidopsis thali-
ana. Photosynth. Res. 86, 491–508.Khan, M.S., Haas, F.H., Samami, A.A. et al. (2010) Sulfite reductase defines
a newly discovered bottleneck for assimilatory sulfate reduction and is
essential for growth and development in Arabidopsis thaliana. Plant Cell,
22, 1216–1231.Koprivova, A., North, K.A. and Kopriva, S. (2008) Complex signaling net-
work in regulation of adenosine 5′-phosphosulfate reductase by salt
stress in Arabidopsis roots. Plant Physiol. 146, 1408–1420.Koshiba, T., Saito, E., Ono, N., Yamamoto, N. and Sato, M. (1996) Purifica-
tion and properties of flavin- and molybdenum-containing aldehyde oxi-
dase from coleoptiles of maize. Plant Physiol. 110, 781–789.Kr€uger, S., Niehl, A., Martin, M.C.L., Steinhauser, D., Donath, A., Hilde-
brandt, T., Romero, L.C., Hoefgen, R., Gotor, C. and Hesse, H. (2009)
Analysis of cytosolic and plastidic serine acetyltransferase mutants and
subcellular metabolite distributions suggests interplay of the cellular
compartments for cysteine biosynthesis in Arabidopsis. Plant, Cell Envi-
ron. 32, 349–367.Kruse, C., Jost, R., Lipschis, M., Kopp, B., Hartmann, M. and Hell, R. (2007)
Sulfur-enhanced defence: effects of sulfur metabolism, nitrogen supply,
and pathogen lifestyle. Plant Biol (Stuttg), 9, 608–619.Kruse, C., Haas, F.H., Jost, R., Reiser, B., Reichelt, M., Wirtz, M., Gershen-
zon, J., Schnug, E. and Hell, R. (2012) Improved sulfur nutrition provides
the basis for enhanced production of sulfur-containing defense com-
pounds in Arabidopsis thaliana upon inoculation with Alternaria brassici-
cola. J. Plant Physiol. 169, 740–743.Kutz, A., Muller, A., Hennig, P., Kaiser, W.M., Piotrowski, M. and Weiler,
E.W. (2002) A role for nitrilase 3 in the regulation of root morphology in
sulphur-starving Arabidopsis thaliana. Plant J. 30, 95–106.Lei, Z.-Y., Zhao, P., Cao, M.-J., Cui, R., Chen, X., Xiong, L.-Z., Zhang,
Q.-F., Oliver, D.J. and Xiang, C.-B. (2007) High-throughput binary
vectors for plant gene function analysis. J. Integr. Plant Biol. 49,
556–567.Leon-Kloosterziel, K.M., Gil, M.A., Ruijs, G.J., Jacobsen, S.E., Olszewski,
N.E., Schwartz, S.H., Zeevaart, J.A. and Koornneef, M. (1996) Isolation
and characterization of abscisic acid-deficient Arabidopsis mutants at
two new loci. Plant J. 10, 655–661.Lopez-Bucio, J., Cruz-Ramirez, A. and Herrera-Estrella, L. (2003) The role of
nutrient availability in regulating root architecture. Curr. Opin. Plant Biol.
6, 280–287.Mendel, R.R. and Hansch, R. (2002) Molybdoenzymes and molybdenum
cofactor in plants. J. Exp. Bot. 53, 1689–1698.Noctor, G., Mhamdi, A., Chaouch, S., Han, Y., Neukermans, J., Mar-
quez-Garcia, B., Queval, G. and Foyer, C.H. (2012) Glutathione in plants:
an integrated overview. Plant, Cell Environ. 35, 454–484.Okuma, E., Jahan, M.S., Munemasa, S. et al. (2012) Negative regulation of
abscisic acid-induced stomatal closure by glutathione in Arabidopsis. J.
Plant Physiol. 168, 2048–2055.Plessis, A., Cournol, R., Effroy, D. et al. (2011) New ABA-hypersensitive Ara-
bidopsis mutants are affected in loci mediating responses to water deficit
and Dickeya dadantii infection. PLoS ONE, 6, e20243.
Queval, G., Thominet, D., Vanacker, H., Miginiac-Maslow, M., Gakiere, B.
and Noctor, G. (2009) H2O2-activated up-regulation of glutathione in Ara-
bidopsis involves induction of genes encoding enzymes involved in cys-
teine synthesis in the chloroplast. Mol. Plant, 2, 344–356.Sauter, M., Moffatt, B., Saechao, M., Hell, R. and Wirtz, M. (2013) Methio-
nine salvage and S-adenosylmethionine: essential links between sulfur,
ethylene and polyamine biosynthesis. Biochem J. 451, 145–154.Schwarz, G. and Mendel, R.R. (2006) Molybdenum cofactor biosynthesis
and molybdenum enzymes. Annu. Rev. Plant Biol. 57, 623–647.Schwarz, G., Mendel, R.R. and Ribbe, M.W. (2009) Molybdenum cofactors,
enzymes and pathways. Nature, 460, 839–847.Solomonson, L.P. and Vennesland, B. (1972) Properties of a nitrate reduc-
tase of Chlorella. Biochim. Biophys. Acta 267, 544–557.Takahashi, H., Kopriva, S., Giordano, M., Saito, K. and Hell, R. (2011) Sulfur
assimilation in photosynthetic organisms: molecular functions and regu-
lations of transporters and assimilatory enzymes. Annu. Rev. Plant Biol.
62, 157–184.Takahashi, H., Buchner, P., Yoshimoto, N., Hawkesford, M.J. and Shiu,
S.-H. (2012) Evolutionary relationships and functional diversity of plant
sulfate transporters. Fronti. Plant Sci. 2, 19.
Waduwara-Jayabahu, I., Oppermann, Y., Wirtz, M., Hull, Z.T., Schoor, S.,
Plotnikov, A.N., Hell, R., Sauter, M. and Moffatt, B.A. (2012) Recycling of
methylthioadenosine is essential for normal vascular development and
reproduction in Arabidopsis. Plant Physiol. 158, 1728–1744.Wirtz, M. and Hell, R. (2007) Dominant-negative modification reveals the
regulatory function of the multimeric cysteine synthase protein complex
in transgenic tobacco. Plant Cell, 19, 625–639.Wirtz, M., Droux, M. and Hell, R. (2004) O-acetylserine (thiol) lyase: an enig-
matic enzyme of plant cysteine biosynthesis revisited in Arabidopsis tha-
liana. J. Exp. Bot. 55, 1785–1798.Xiang, C. and Oliver, D.J. (1998) Glutathione metabolic genes coordinately
respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell,
10, 1539–1550.Xiang, C.B. and Oliver, D.J. (2000) Multilevel regulation of glutathione
homeostasis in higher plants. In Handbook of Crop and Plant Physiol-
ogy, 2nd edn (Pessarakli, M., ed). New York: Marcel Dekker, Inc., pp.
539–548.Xiang, C., Werner, B.L., Christensen, E.M. and Oliver, D.J. (2001) The biolog-
ical functions of glutathione revisited in Arabidopsis transgenic plants
with altered glutathione levels. Plant Physiol. 126, 564–574.Xiong, L., Ishitani, M., Lee, H. and Zhu, J.K. (2001) The Arabidopsis LOS5/
ABA3 locus encodes a molybdenum cofactor sulfurase and modulates
cold stress- and osmotic stress-responsive gene expression. Plant Cell
13, 2063–2083.Xiong, L., Wang, R.G., Mao, G. and Koczan, J.M. (2006) Identification of
drought tolerance determinants by genetic analysis of root response to
drought stress and abscisic acid. Plant Physiol. 142, 1065–1074.Yang, J., Zhang, J., Wang, Z., Zhu, Q. and Wang, W. (2001) Hormonal
changes in the grains of rice subjected to water stress during grain fill-
ing. Plant Physiol. 127, 315–323.Zuber, H., Davidian, J.C., Aubert, G. et al. (2010) The seed composition of
Arabidopsis mutants for the group 3 sulfate transporters indicates a role
in sulfate translocation within developing seeds. Plant Physiol. 154,
913–926.
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2014), 77, 604–615
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