The Plant Journal 77, 604–615 doi: 10.1111/tpj.12407 ... · to the response of plants towards drought stress (Estavillo et al., 2011). The polyamines are especially important for

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

mailto:[email protected]

mailto:[email protected]

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).


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).



***P < 0.001, **P < 0.01 or *P < 0.05. Values

represent means � standard deviation (SD).



(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).



*P < 0.05. Values represent means � standard

deviation (SD).



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.



(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


single asterisk. Values represent means � stan-

dard deviation (SD) (n = 3).



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).



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



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



www.sigmaaldrich.com

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.

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The Plant Journal 77, 604–615 doi: 10.1111/tpj.12407 ... · to the response of plants towards drought stress (Estavillo et al., 2011). The polyamines are especially important for