Article

Reciprocal Regulation of t

he TOR Kinase and ABAReceptor Balances Plant Growth and StressResponse

Graphical Abstract

Highlights

d The TOR kinase phosphorylates ABA receptor PYLs at a

conserved serine residue

d PYLs phosphorylation inhibits stress responses by

abolishing PYLs activities

d Stress- and ABA-activated SnRK2s phosphorylate Raptor

and inhibit TOR activity

d TOR and ABA signaling balance plant growth and stress

responses

Wang et al., 2018, Molecular Cell 69, 100–112January 4, 2018 ª 2017 Elsevier Inc.https://doi.org/10.1016/j.molcel.2017.12.002

Authors

Pengcheng Wang, Yang Zhao,

Zhongpeng Li, ..., W. Andy Tao,

Yan Xiong, Jian-Kang Zhu

Correspondenceyanxiong@sibs.ac.cn (Y.X.),jkzhu@sibs.ac.cn (J.-K.Z.)

In Brief

Wang et al. reveal that the TOR kinase

phosphorylates ABA receptors to repress

stress responses under unstressed

conditions and to promote growth

recovery once environmental stresses

subside. Under stress conditions,

SnRK2s phosphorylate Raptor, a

regulatory component in the TOR

complex, to prevent growth by inhibiting

TOR activity.

Molecular Cell

Article

Reciprocal Regulation of the TOR Kinase and ABAReceptor Balances Plant Growth and Stress ResponsePengcheng Wang,1,2,5 Yang Zhao,1,5 Zhongpeng Li,1,5 Chuan-Chih Hsu,3,5 Xue Liu,1 Liwen Fu,1 Yueh-Ju Hou,2,4

Yanyan Du,1 Shaojun Xie,2 Chunguang Zhang,2 Jinghui Gao,2 Minjie Cao,1 Xiaosan Huang,2 Yingfang Zhu,1,2 Kai Tang,1,2

Xingang Wang,2 W. Andy Tao,3 Yan Xiong,1,* and Jian-Kang Zhu1,2,6,*1Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences,Shanghai 200032, China2Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA3Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA4Present address: Division of Biological Sciences, University of California, San Diego, San Diego, CA 92037, USA5These authors contributed equally6Lead Contact

*Correspondence: yanxiong@sibs.ac.cn (Y.X.), jkzhu@sibs.ac.cn (J.-K.Z.)

https://doi.org/10.1016/j.molcel.2017.12.002

SUMMARY

As sessile organisms, plants must adapt to varia-tions in the environment. Environmental stress trig-gers various responses, including growth inhibition,mediated by the plant hormone abscisic acid (ABA).The mechanisms that integrate stress responseswith growth are poorly understood. Here, we discov-ered that the Target of Rapamycin (TOR) kinasephosphorylates PYL ABA receptors at a conservedserine residue to prevent activation of the stressresponse in unstressed plants. This phosphoryla-tion disrupts PYL association with ABA and withPP2C phosphatase effectors, leading to inactiva-tion of SnRK2 kinases. Under stress, ABA-activatedSnRK2s phosphorylate Raptor, a component of theTOR complex, triggering TOR complex dissocia-tion and inhibition. Thus, TOR signaling repressesABA signaling and stress responses in unstressedconditions, whereas ABA signaling represses TORsignaling and growth during times of stress. Plantsutilize this conserved phospho-regulatory feedbackmechanism to optimize the balance of growth andstress responses.

INTRODUCTION

Upon sensing environmental stresses, plants sacrifice growth

and activate protective stress responses. The phytohormone ab-

scisic acid (ABA) plays a critical role in integrating awide range of

stress signals and controlling downstream stress responses (An-

toni et al., 2011; Assmann and Jegla, 2016; Chater et al., 2015;

Cutler et al., 2010; Hubbard et al., 2010; Lumba et al., 2014;

Raghavendra et al., 2010; Umezawa et al., 2010; Zhu, 2016).

ABA reduces transpiration and photosynthesis (Munemasa

et al., 2015; Roelfsema et al., 2012), reprograms metabolism to

100 Molecular Cell 69, 100–112, January 4, 2018 ª 2017 Elsevier Inc

accumulate osmolytes, inhibits growth (Julkowska and Tester-

ink, 2015), and promotes dormancy and senescence to adapt

to and survive severe stress (Zhao et al., 2016).

In response to environmental stresses such as drought, ABA

accumulates rapidly and binds receptors in the PYR1/PYL/

RCAR family of proteins (herein referred to as PYLs) (Cutler

et al., 2010). The ABA-PYL receptor complex subsequently

inhibits downstream protein phosphatases in clade A of the

PP2C family, which includes ABI1, ABI2, HAB1, HAB2, PP2CA,

and AHG1 (Fujii et al., 2009; Gonzalez-Guzman et al., 2012; Ma

et al., 2009; Park et al., 2009). PP2C inhibition releases sucrose

non-fermenting-1 (SNF1) related protein kinase-2 s (SnRK2s),

which phosphorylate downstream effectors to initiate protective

responses such as stomatal closure and gene expression re-

programming (Geiger et al., 2009; Grondin et al., 2015; Umezawa

et al., 2013; Wang et al., 2013). ABA also has important roles in

regulating plant growth and development (Cutler et al., 2010;

Humplık et al., 2017). ABA promotes seeds dormancy and

inhibits growth of young seedlings by reducing polar auxin

transport (Shkolnik-Inbar and Bar-Zvi, 2010) or by affecting the

expression of auxin-responsive genes (Liu et al., 2013; Zhao

et al., 2014). ABA signaling also cross-talks with brassinosteroid

signaling in controlling seed germination (Hu and Yu, 2014;

Zhang et al., 2009; Cai et al., 2014), growth, and grain filling

(Gui et al., 2016; Clouse, 2016). Under stress conditions, ABA

signaling interacts with gibberellin and auxin signaling pathways

and controls lateral root development (De Smet et al., 2003; Gou

et al., 2010; Duan et al., 2013; Zhao et al., 2014). ABA also

promotes the senescence and abscission in adult plants by

inducing ethylene biosynthesis and by ethylene-independent

mechanisms (Liu et al., 2016; Zhao et al., 2016).

There are 14 members in the PYL family in Arabidopsis.

Although PYR1 and PYL1–4 interact with and inhibit PP2Cs

only in the presence of ABA, the remaining PYLs can interact

with and inhibit PP2Cs in vitro even without ABA (Fujii

et al., 2009; Hao et al., 2011). It is not known how these latter

PYLs, with ABA-independent activities, are kept inactive in

plants to prevent ABA responses in the absence of stress.

Although single-gene mutations in most PYLs do not

.

significantly compromise ABA signaling, mutations in multiple

PYL genes lead to strong ABA-resistant phenotypes (Gonza-

lez-Guzman et al., 2012; Park et al., 2009), suggesting that

the PYLs have redundant functions. Several PYLs, particularly

PYL3, can also sense the ABA catabolite phaseic acid (Weng

et al., 2016).

Plant growth is inhibited under stress to maximize stress

responses and ensure survival. Upon cessation of environmental

stress, plants must quickly deactivate protective stress re-

sponses and stimulate growth recovery mechanisms. How

plants switch between growth processes and stress responses

is a long-standing and major question in plant biology (Achard

et al., 2006). Although the mechanism underlying this switch is

still unclear, it is known to involve the intricate regulation of key

factors for growth control.

Target of Rapamycin (TOR) is an evolutionarily conserved

master regulator that integrates energy, growth, hormone, and

stress signaling to promote growth in all eukaryotes (Dobrenel

et al., 2016; Laplante and Sabatini, 2012; Shimobayashi and

Hall, 2014; Xiong et al., 2013). In plants, the TOR complex has

essential roles in regulating cell proliferation, cell size, develop-

ment, protein synthesis, transcription, and metabolism (Bogre

et al., 2013; Caldana et al., 2013; Ren et al., 2012; Schepetilnikov

et al., 2013, 2017; Xiong et al., 2013; Xiong and Sheen, 2012).

TOR controls translational initiation by phosphorylating the

translation initiation factor elF3h and 40S ribosomal protein S6

kinases (S6Ks) (Mahfouz et al., 2006; Schepetilnikov et al.,

2011, 2013). The activation of both the root and shoot meristems

requires TOR kinase (Li et al., 2017; Xiong et al., 2013). In the root

meristem, TOR phosphorylates the E2Fa transcription factor

to regulate the expression of genes involved in metabolism,

cell cycle, transcription, signaling, transport, and protein folding

(Xiong et al., 2013). The plant growth hormone auxin stimulates

TOR activity through a physical interaction between TOR and

auxin-activated Rho-like GTPase 2 (ROP 2) to promote the acti-

vation of shoot meristem (Li et al., 2017; Schepetilnikov et al.,

2017). TOR also mediates the crosstalk between sugar signaling

and brassinosteroid (BR) signaling by regulating the stability of

BR signaling key transcription factor BZR1, thus allowing carbon

availability to control growth-promotion hormonal programs to

ensure supply-demand balance in plant growth (Zhang et al.,

2016). Thus, TOR represents a potential regulator of the switch

between the stress response and growth.

Here, we report that TOR kinase phosphorylates PYL ABA

receptors in the absence of stress. This phosphorylation nega-

tively regulates PYL activity to inhibit ABA binding and its inter-

action with downstream PP2C enzymes. Our results suggest

that this negative regulation is important for preventing ABA

and stress signaling by ABA-independent PYLs when there is

no stress. Under stress, ABA triggers PYL-mediated activation

of SnRK2s, which phosphorylate the TOR regulator Raptor and

thereby inhibit TOR activity to contribute to ABA- and stress-

induced growth inhibition. When stress subsides, TOR phos-

phorylation of PYLs is critical for growth recovery. Our results

uncover a phosphorylation-dependent regulatory loop between

ABA core signaling and the TOR complex. Plants utilize this

conserved mechanism to repress stress and ABA responses

under unstressed conditions, to inhibit growth under stress,

and to promote growth recovery once environmental stresses

subside.

RESULTS

Phosphorylation of PYLs at a Conserved Serine ResidueAbolishes Their ActivityTo test whether phosphorylation regulates the activity of PYL

ABA receptors, we performed a large-scale phosphoproteomic

comparison of Arabidopsis seedlings treated with or without

ABA. After protein extraction and digestion, phosphopeptides

were enriched using Fe-IMAC StageTips (Tsai et al., 2014). Our

phosphoproteomics data uncovered a PYL4 phosphopeptide

containing phosphorylated Ser114 in control seedlings, but

not in ABA-treated seedlings (Figure 1A, ProteomeXchange:

PXD003746). To investigate phosphorylation at this site in other

PYL proteins, we uncovered additional phosphopeptides by

fractionating enriched phosphopeptides with basic pH reverse

phase StageTips (Dimayacyac-Esleta et al., 2015). We identified

a total of 47,126 phosphopeptides corresponding to 37,387

phosphorylation sites (22,346 class I sites) using MaxQuant

software. ABA treatment abolished phosphorylation of Ser119

in PYL1 and Ser94 in PYL9 (Figure S1A), which correspond

to PYL4 Ser114. However, ABA treatment did not substantially

affect phosphorylation of Ser9 in PYL1 (Figure S1B). We

alsomeasured the phosphorylation of PYL1 immunoprecipitated

from transgenic pyr1pyl1pyl2pyl4 (pyr1pyl124) quadruple-

mutant plants carrying native promoter-driven, wild-type PYL1-

Myc. Similar to PYL4 (Figure 1A), ABA treatment abolished the

Ser119 phosphorylation in PYL1-Myc (Figure S1C). The residue

corresponding to PYL1 Ser119/PYL4 Ser114 is located within

the ABA binding pocket and is conserved within all 14 PYLs in

Arabidopsis; this residue promotes binding of the ABA dimethyl

group (Melcher et al., 2009; Miyazono et al., 2009).

To determine the role of this conserved serine in the PYL

family, we generated a series of PYL mutants for testing in a re-

porter assay in protoplasts that monitors PYL-mediated inhibi-

tion of PP2Cs (Fujii et al., 2009). Specifically, we mutated the

serine (S) to a similarly sized, non-phosphorylatable and neutral

alanine (A) or cysteine (C), to a larger, non-phosphorylatable,

neutral leucine (L) or glutamine (Q), or to a negatively charged,

phosphomimic aspartic acid (D) or glutamic acid (E). As reported

previously (Fujii et al., 2009), ABA relieved ABI1-mediated

repression of SnRK2.6 in protoplasts expressing wild-type

PYL1 or PYL4 and induced the ABA-responsive reporter

RD29B-LUC (Figure 1B, lanes 3 and 6). We found that ABA

also induced RD29B-LUC in protoplasts expressing PYL4S114A,

PYL1S119A (Figure 1B, lanes 4 and 7), or PYL1S119C (Figure S1D),

but not in protoplasts expressing the phosphomimic mutants

PYL1S119D and PYL4S114D, or the larger side-chain mutants

PYL1S119L and PYL1S119Q (Figure 1B, lanes 5 and 8, and Fig-

ure S1D). In contrast, a phosphomimic substitution at Thr118

in PYL1 did not affect ABA-dependent induction (Figure S1B).

These data implied that a small, uncharged side chain (S, A, C)

at position 119 is required for ABA binding. Phosphorylation of

Ser119, which enlarges and negatively charges the side chain,

or substitution with larger side chain and/or charged amino acids

(L, Q, D, E) block the ABA binding pocket and decrease ABA

Molecular Cell 69, 100–112, January 4, 2018 101

A B C

D E F

G H I

Figure 1. PYL Phosphorylation and the Effects of Non-Phosphorylatable and Phosphomimic Mutations on PYL Function In Vitro

(A) Quantitative analysis of phosphopeptide containing Ser114 in PYL4 in seedlings with or without ABA treatment. A phosphopeptide containing phosphorylated

Thr481 in AT3G02750 was used as a control. N.D., not identified in the samples. Error bars, SEM (n = 3).

(B) Phosphomimic mutations of PYL1 and PYL4 abolished their activity in transient reporter gene expression assays in protoplasts. RD29B::LUC and

ZmUBQ::GUSwere used as the ABA-responsive reporter and internal control, respectively. After transfection, protoplasts were incubated for 5 hr under light and

in the absence of ABA (open bars) or in the presence of 5 mMABA (filled bars). Error bars, SEM (n = 3). Anti-His tag immunoblot shows the levels of histidine-tagged

wild-type and mutated PYL1 and PYL4 proteins in the protoplasts.

(C) PYL1S119D is an inactive ABA receptor and cannot inhibit ABI1 in an in vitro reconstitution of the ABA signaling pathway. The autoradiography (upper panel) and

Coomassie staining (lower panel) show ABF2 fragment phosphorylation and protein loading, respectively.

(D) Phosphomimic mutants of PYL1 and PYL4 cannot inhibit ABI1 phosphatase activity. Error bars, SEM (n = 3).

(E) PYL1 and PYL1S119A but not the phosphomimic mutant PYL1S119D interact with ABI1 in a yeast two-hybrid assay.

(F) Dose-dependent curves show the effect of mutations at Ser119 on ABA binding affinity of PYL1 in TSA assay. Data shown are representative of three

independent experiments.

(G–I) Binding isotherms showing the ABA binding affinity of wild-type PYL1 (G), PYL1S119A (H), and PYL1S119D (I) in MST assay. Equilibrium dissociation

constant (KD) used to indicate the binding affinity between PYL protein and ABA. Data shown are representative of two independent experiments.

See also Figure S1.

binding affinity. In addition, PYL4S114D also abolished ABA-

dependent inhibition of other clade A PP2C phosphatases,

including HAB1, PP2CA, HAI3, and AHG1 (Figure S1E). Further,

phosphomimic substitutions at the conserved serine in all other

PYLs eliminated or substantially reduced ABA-dependent induc-

tion of RD29B-LUC (Figure S1E). These results suggest that

phosphorylation of the conserved serine alters the ABA binding

pocket and negatively regulates PYLs function in vivo.

We then examined how phosphorylation affects PYL activity

using a modified in vitro system that reconstitutes the core

signaling pathway ABA-PYL-ABI1-SnRK2.6-ABF2 (Abscisic

Acid Responsive Elements-Binding Factor 2) (Fujii et al., 2009).

In this system, phosphorylation of the well-characterized

102 Molecular Cell 69, 100–112, January 4, 2018

SnRK2 substrate ABF2 is monitored to evaluate SnRK2.6 activ-

ity. Recombinant GST-ABI1 inhibited SnRK2.6 activity and ABF2

phosphorylation (Figure 1C, lane 3), and addition of recombinant

wild-type PYL1 reversed ABI1-mediated inhibition of SnRK2.6 in

an ABA-dependent manner (Figure 1C, lane 5). PYL1S119A also

triggered ABA-dependent inhibition of ABI1 and induced ABF2

phosphorylation, similar to wild-type PYL1 (Figure 1C, lane 7).

In contrast, PYL1S119D lost ABA-dependent inhibition of ABI1,

and ABF2 was not phosphorylated (Figure 1C, lane 9). Consis-

tent with this finding, PYL1S119D could not inhibit ABI1 phospha-

tase activity in vitro, even in the presence of ABA (Figure 1D).

These data further suggest that phosphorylation of PYL1 at the

conserved serine prevents ABA-dependent inhibition of PP2Cs.

A B C

D E F

Figure 2. Phosphomimic Mutations Abolish PYL Activities

(A) Phosphomimic mutants of PYL10 cannot inhibit the phosphatase activity of HAB1. Error bars, SEM (n = 3).

(B and C) Phosphomimic mutants of PYL10 cannot interact with HAB1 (B) or ABI1 (C) in alpha screen assay. Error bars, SEM (n = 3).

(D) Dose-dependent curves showing the effect ofmutations at Ser88 on the binding affinity of PYL10 for ABA in TAS assay. Data shown are representative of three

independent experiments. Error bars, SEM (n = 3).

(E and F) Binding isotherms showing the ABA binding affinity of wild-type PYL10 (E) and PYL10S88D (F) in MST assay. Data shown are representative of two

independent experiments.

See also Figure S2.

To determine whether the phosphomimic mutations at the

conserved serine affect the interaction between the ABI1 and

PYL proteins, we used a yeast two-hybrid assay. We found

that the phosphomimic mutation in PYL1 abolished the ABA-

dependent interaction between ABI1 and PYL1 (Figure 1E).

The phosphomimic mutations also eliminated the ABA-depen-

dent interactions between ABI1 and PYR1, PYL2, PYL3, and

PYL4, and the ABA-independent interactions between ABI1

and PYL7, PYL9, PYL11, PYL12 (Figure S1F). Interestingly, the

ABA-independent interactions between ABI1 and PYL5, PYL6,

PYL8 and PYL10 were only partially inhibited or unaffected

by these phosphomimic mutations in the yeast two-hybrid assay

(Figure S1G). However, phosphomimic mutated forms of PYL10

prevented HAB1 inhibition (Figure 2A). We used an alpha screen

assay to re-investigate the physical interaction between PYL10

and HAB1 and ABI1 (Melcher et al., 2009) and found that the

S88D and S88E mutations abolished PYL10 interactions with

these PP2C phosphatases (Figures 2B and 2C). Overall, these

results suggest that phosphorylation of the conserved serine in

the PYLs abolishes the ABA-dependent and ABA-independent

physical interactions with PP2Cs.

Phosphomimic Mutations of Ser119 in PYLs Inhibit ABABindingTo determine how the phosphomimic substitutions abolish PYL

function, we evaluated the ABA binding activity of the PYL pro-

teins by a thermal stability shift assay (TSA) and a microscale

thermophoresis (MST) assay. We found that PYL1S119D did not

bind ABA in the TSA assay, whereas PYL1S119A bound ABA,

albeit with lower affinity than wild-type PYL1 (Figure 1F). This

finding is consistent with the reduced capacity of PYL1S119A to

inhibit ABI1 and activate the reporter in the protoplast assay

(Figure 1B). The MST assay revealed that wild-type PYL1 and

PYL1S119A bind ABA with affinities of 63.7 and 219 mM, respec-

tively (Figures 1G and 1H). In contrast, the phosphomimic

mutated PYL1S119D did not show any ABA binding activity (Fig-

ure 1I). Similarly, PYL10S88D and PYL10S88E did not show any

ABA binding activity in the TSA and MST assays (Figures

2D–2F). These results suggest that phosphorylation of PYLs at

the conserved serine residue negatively regulates ABA percep-

tion and signaling by inhibiting ABA binding. This negative regu-

lation applies not only to the PYLs that are fully dependent on

ABA, but also to the PYLs that have ABA-independent activities.

To determine whether ABA receptor phospho-regulation is

conserved, we performed an in silico homology search in land

plants. We found that the serine residue corresponding to

Ser119 in PYL1 is conserved across all 121 PYLs from 12

different species (Figures S2A and S2B). This finding suggests

that phosphorylation-based inhibition of the PYLs-ABA interac-

tion is conserved across land plants.

Phosphorylation of PYLs Ser119 Promotes GrowthRecovery after StressNext, we evaluated how PYL phosphorylation affects the

response to ABA in planta. We generated transgenic plants

carrying native promoter-driven wild-type PYL1, PYL1S119A,

and PYL1S119D in pyr1pyl1pyl2pyl4 (pyr1pyl124) quadruple-

mutant plants. Transgenic expression of wild-type PYL1 or

PYL1S119A complemented the ABA-hyposensitive phenotypes

of the quadruple mutant in germination, root elongation, and

seedling growth (Figures 3A, 3B, and S3A–S3E). In contrast,

transgenic expression of PYL1S119D did not complement the

Molecular Cell 69, 100–112, January 4, 2018 103

A B

C D E

Figure 3. Effects of Non-Phosphorylatable and Phosphomimic Mutations on PYL1 Function In Vivo

(A) Photographs of seeds after 5 days of germination and growth on 1/2 Murashige-Skoog (MS) medium containing 3 mM ABA.

(B) 10-day-old seedlings grown on 1/2 MS medium with 10 mM ABA (right panel) or without ABA (left panel).

(C) Photographs of seedlings after 5 days of growth recovery. The seedlings grown on 1/2MSmedium containing 10 mMABAwere transferred to 1/2 MSmedium

without ABA. Data shown are representative of six independent experiments.

(D) Fresh weight of seedlings after 3 days recovery growth on 1/2 MS medium. Error bars, SEM (n = 6). *p < 0.05, Student’s t test.

(E) Rosetta diameter of seedlings after indicated time of recovery growth on 1/2 MS medium. Error bars, SEM (n = 6). *p < 0.05, Student’s t test.

See also Figure S3.

ABA-insensitive phenotypes of pyr1pyl124 mutant plants (Fig-

ures 3A, 3B, and S3A–S3E), even though transgene expression

levels were similar to wild-type PYL1 or PYL1S119A plants (Fig-

ure S3F). These data from transgenic plants together with the

in vitro assay results demonstrate that phosphomimic mutated

PYLs are non-functional receptors.

We further investigated how loss of Ser119 phosphorylation af-

fects growth and stress responses in transgenic plants carrying

PYL1S119A and wild-type PYL1. Both pyr1pyl124-PYL1S119A

104 Molecular Cell 69, 100–112, January 4, 2018

and pyr1pyl124-PYL1 showed arrested seedling growth and

yellow leaves when grown on medium containing 10 mM ABA

(Figures 3B and S3C–S3E). Upon transfer to fresh medium

without ABA, pyr1pyl124-PYL1 seedlings lost the ABA-induced

leaf yellowing after 5 days of recovery. In contrast, the greening

of yellow leaves was abolished in pyr1pyl124-PYL1S119A seed-

lings (Figure 3C). The pyr1pyl124-PYL1S119A seedlings also

showed delayed recovery from osmotic stress induced by

mannitol treatment, when transferred from medium containing

A B C

D E

Figure 4. TOR Kinase Phosphorylates PYLs

(A) Immunoprecipitated TOR kinase phosphorylates

recombinant PYL1 and TOR inhibitor PP242 inhibits

the PYL1 phosphorylation. Autoradiograph (upper

panel) and Coomassie staining (lower panel) show

phosphorylation and loading of purified PYL1.

(B) Phosphorylation of recombinant wild-type PYL1

and PYL1S119A by immunoprecipitated TOR ki-

nase. Autoradiograph (upper panel) and Coomassie

staining (lower panel) show phosphorylation and

loading of purified PYL1 and PYL1S119A.

(C) Phosphorylation by TOR kinase inhibits the

activity of PYL1 but not PYL1S119A. Inhibition of

ABI1 and PP2CA phosphatase activity was used to

indicate the PYL1 activity. Error bars, SEM (n = 3).

*p < 0.05, Student’s t test.

(D) Quantitative analysis of PYL4 phosphopeptide

in wild-type and raptor1-2 seedlings. A phos-

phopeptide containing phosphorylated Thr481 in

AT3G02750 was used as a control. N.D., not iden-

tified in the samples. Error bars, SEM (n = 3).

(E) Co-immunoprecipitation assay showing the

interaction between TOR and PYL1 in pyr1pyl124-

PYL1-Myc transgenic plants.

See also Figure S4.

300 mM mannitol to fresh medium without mannitol (Figures 3D

and 3E). Thus, pyr1pyl124-PYL1S119A seedlings show delayed

growth recovery compared to pyr1pyl124-PYL1 seedlings.

TOR Kinase Phosphorylates PYLs and NegativelyRegulates ABA Signaling in the Absence of StressOur findings suggest that phosphorylation of Ser119 in PYL1

negatively regulates ABA and stress signaling, and positively

regulates recovery. Therefore, we hypothesized that the kinase

targeting this site should promote plant growth. To test this hy-

pothesis, we screened protein kinases that interact with PYL1,

that are co-expressed with PYL1, or that were reported to

promote plant growth. We screened over 30 protein kinases

using an in vitro kinase assay and found that several kinases,

such as CKL3 and AT1G51850, could phosphorylate PYLs

(Figure S4A). However, only the immunoprecipitated TOR

kinase could phosphorylate recombinant PYL1 at Ser119

in vitro (Figure 4A). Only one phosphopeptide, which contains

Ser119, from PYL1 was detected by mass spectrometry in the

TOR kinase reaction products after trypsin digestion (Fig-

ure S4B). Furthermore, the S119A substitution within PYL1 sub-

stantially reduced phosphorylation by TOR kinase, but not the

phosphorylation by CKL3 or AT1G51850 (Figures 4B, S4A, and

S4C). Immunoprecipitated TOR complex phosphorylated all 11

of the recombinant PYL proteins that we tested, whereas the

TOR inhibitors PP242 and Torin2 substantially reduced PYL

phosphorylation (Figures 4A and S4D). It is notable that the

PYL phosphosite regions do not contain any known TOR recog-

nition motifs (Hsu et al., 2011). Plant TORs might recognize

a unique phosphorylation motif in the PYLs that differs from

animal TORs.

The activity of clade A PP2Cs is inhibited by PYL1 in the pres-

ence of ABA.We found that immunoprecipitated TOR kinase and

ATP prevented the ABA-dependent inhibition of PP2CA and

ABI1 by PYL1, but not by the non-phosphorylatable PYL1S119A

(Figure 4C). Further, Torin2 abolished the suppressive effect

of TOR in this assay (Figure 4C). These data suggest that

phosphorylation of PYL by TOR suppresses PYL-mediated inhi-

bition of PP2Cs.

To examine the relationship between TOR kinase activity and

PYL phosphorylation in vivo, we measured the phosphorylation

status of PYLs in a raptor1-2 mutant plant by quantitative phos-

phoproteomics. These mutant plants lack the Regulatory-asso-

ciated protein of mTOR (RaptorB), and display reduced TOR

kinase activity, based on the phosphorylation status of T449 in

S6 protein kinase 6 (S6K1), a well-characterized TOR kinase

target site, in vivo (Figure S4E).We detected the phosphopeptide

QVHVVSGLPAASpSTER from PYL4, which contains the phos-

phosite Ser114, in wild-type seedlings, but not in raptor1-2

mutant seedlings (Figure 4D). In addition, TOR co-immunopre-

cipitated with PYL1-Myc from pyr1pyl124-PYL1-Myc seedlings

(Figure 4E), supporting a PYL-TOR physical interaction. These

data strongly suggest that PYL ABA receptors are TOR kinase

substrates in vivo, with the conserved serine residues corre-

sponding to Ser119 in PYL1 as TOR kinase targets.

We hypothesized that TOR kinase may negatively modulate

ABA signaling and stress responses, since TOR phosphorylation

at the conserved serine in PYLs negatively regulates PYL func-

tion. To test this hypothesis, we measured ABA responses in

an estradiol inducible TOR RNAi line, es-tor (Xiong and Sheen,

2012), and in the raptor1-2 mutant plants. After ABA treatment,

estradiol-treated es-tor seedlings and raptor1-2 seedlings lost

more chlorophyll than wild-type seedlings (Figures 5A and 5B).

The raptor1-2 mutants also showed ABA hypersensitivity in

germination and early seedling growth (Figure 5C). Thus, deple-

tion of TOR or loss of RaptorB triggers ABA hypersensitive phe-

notypes compared to wild-type plants.

Next, we determined whether loss of TOR or RaptorB function

affects ABA signaling. To detect ABA-induced SnRK2 activation

in seedlings, we used an in-gel assay to monitor SnRK2 activity

Molecular Cell 69, 100–112, January 4, 2018 105

A B C

FED

G H

Figure 5. TOR Kinase Inhibition Enhances ABA Signaling

(A) Chlorophyll content of wild-type (WT) and es-tor seedlings 6 days after growing in a liquid medium or a medium supplemented with different ABA concen-

trations. Error bars, SEM (n = 6). *p < 0.05, Student’s t test.

(B) Total chlorophyll content of the seedlings 8 days after transfer to the medium containing 10 mM ABA. Error bars, SEM (n = 6). *p < 0.05, Student’s t test.

(C) Photographs of WT and raptor1 plants after 5 days of germination and growth on 1/2 MS medium or 1/2 MS medium containing 0.5 mM ABA.

(D) In-gel kinase assay showing SnRK2 activity in the es-tor line, raptor1 mutant, or wild-type after 0, 10, or 30 min of 10 mM ABA treatment. The position of

SnRK2.6 is indicated by an arrowhead. Radioactivity levels of the band (arrowhead) were normalized using wild-type after 10-min treatment. Error bars, SEM

(n = 3). *p < 0.05, Student’s t test. Anti-TOR and anti-SnRK2.6 immunoblots show TOR and SnRK2 protein levels in the samples.

(E) In-gel kinase assay showing ABA-induced SnRK2 activity in wild-type seedlings preincubated with DMSO, Rapamycin or PP242. The position of SnRK2.6 is

indicated by an arrowhead. Radioactivity levels of the band (arrowhead) were normalized usingwild-type after 10-min treatment. Error bars, SEM (n = 3). *p < 0.05,

Student’s t test. Anti-TOR and anti-SnRK2.6 immunoblots show TOR and SnRK2 protein levels in the samples.

(F) In-gel kinase assay showing the activities of SnRK2s in the es-tor seedlings with 3 days of DMSO or estradiol incubation. Arrowhead indicates the position of

SnRK2.6. Radioactivity of the band indicated by arrowhead was normalized by comparing the radioactivity band in the DMSO-treated sample. Error bars, SEM

(n = 3). *p < 0.05, Student’s t test. Anti-TOR and anti-SnRK2.6 immunoblots show TOR and SnRK2 protein levels in the samples.

(G) ABA-responsive gene expression in the es-tor and raptor1 seedlings with 3 days of DMSO or estradiol treatment or with 6 hr treatment with rapamycin. Error

bars, SEM (n = 3). *p < 0.05, Student’s t test.

(H) Osmotic stress sensitivity as measured by electrolyte leakage in wild-type, es-tor, and raptor1 plants treated with 30% PEG. Error bars, SD (n = 6).

See also Figure S5.

on histone substrates (Fujii et al., 2007). After ABA application,

estradiol-treated es-tor and raptor1-2 seedlings showed higher

levels of SnRK2 activity than wild-type (Figure 5D, upper and

middle panels). Similarly, pretreatment with the TOR kinase

inhibitors rapamycin or PP242 also increased ABA-induced

SnRK2 activity compared to control seedlings (Figure 5E).

106 Molecular Cell 69, 100–112, January 4, 2018

Further, when we increased loading of the protein extract to

200 mg/lane, a 10-fold loading increase relative to that used

in Figure 5D and 5E, we detected SnRK2 activity in the es-tor

seedlings even without ABA treatment (Figure 5F, lane 2),

whereas no SnRK2 activity was detected in the wild-type (Fig-

ure 5F, lane 1).

Next, we analyzed the transcriptome data of estradiol-treated

es-tor plants generated by Xiong et al. (2013) to obtain a tran-

scriptomic profile. Out of the 1,833 upregulated genes in the

es-tor samples (fold change >2, p < 0.001, Pearson’s chi-square

test) (Table S1), 1,043 genes are known to be regulated by

abiotic stress (Table S2), and 246 genes are known to be induced

by ABA treatment (Tables S3 and S4). Compared to the entire

transcriptome, stress- and ABA-responsive genes were en-

riched in the subset of genes that were upregulated in estra-

diol-treated es-tor plants (p < 0.001, Pearson’s chi-square test)

but not in the subset that were downregulated (Figure S5).

Real-time qPCR results verified that the transcript levels of

several ABA marker genes, such as RD29A and RD22, were

slightly elevated in estradiol-treated es-tor lines, the raptor1-2

mutant, and in wild-type seedlings treated with rapamycin,

even in the absence of ABA (Figure 5G). Further, when exposed

to a high concentration of PEG, the estradiol-treated es-tor and

raptor1-2 mutant seedlings show much less electrolyte leakage

compared to wild-type seedlings (Figure 5H), indicating less

stress damage in the es-tor line and raptor1 mutant. Together,

these data are consistent with the partial activation of ABA

and stress responses in these mutant plants. Thus, inhibition of

TOR kinase can partially activate ABA signaling, resulting in

SnRK2 activation, induction of stress-responsive gene expres-

sion, and increased stress tolerance. The results suggest that

TOR kinase phosphorylation of PYLs prevents ABA signaling

and stress responses in the absence of stress.

ABA Represses TOR Kinase Activity throughSnRK2-Mediated Phosphorylation of RaptorBOur results indicate that TOR kinase plays a critical role to pre-

vent stress signaling under favorable conditions by phosphory-

lating PYL receptors. Interestingly, PYL receptor phosphoryla-

tion was quickly reversed after ABA treatment (Figures 1A and

S1A), which suggests that ABA also inhibits TOR. Consistent

with this observation, we found that ABA treatment decreased

the phosphorylation of recombinant PYL1 and PYL4 by immuno-

precipitated TOR in vitro (Figure 6A). ABA treatment also signif-

icantly reduced phosphorylation of Thr449 in the TOR substrate

S6K1 (Figure 6B). Similar to ABA, osmotic stress induced by

mannitol treatment also repressed phosphorylation of Thr449

in S6K1 (Figure 6C).

To study how ABA and stress inhibit TOR kinase activity, we

first determined the phosphorylation status of the TOR target

S6K1 in mutants lacking components of the ABA core signaling

pathway. We expressed S6K1-HA in wild-type protoplasts, as

well as in snrk2.2/3/6 triple- and the pyr1pyl12458 sextuple-

mutant protoplasts. We found ABA treatment reduced S6K1

Thr449 phosphorylation in wild-type protoplasts, and this was

almost totally abolished in both the snrk2.2/3/6 triple and the

pyr1pyl12458 sextuple mutants (Figure 6D). These data suggest

that TOR inhibition induced by ABA depends on the ABA core-

signaling pathway.

In yeast and animals, the Target of Rapamycin complex

(TORC) regulatory component Raptor can be phosphorylated

by multiple protein kinases, such as AMPK1, GSK3, and NLK,

which thenmodulates TORC activity (Gwinn et al., 2008; Stretton

et al., 2015; Yuan et al., 2015). We hypothesized that RaptorB

may be a direct target of SnRK2s, given that SnRK2s are related

to AMPK1 (Zhu, 2016) and that multiple phosphosites were

detected in Arabidopsis RaptorB in vivo (ProteomeXchange:

PXD003746). To test this hypothesis, we investigated a potential

interaction between RaptorB and SnRK2s by the yeast two-

hybrid assay. We found that RaptorB physically interacts with

SnRK2.2, SnRK2.3, SnRK2.6, and SnRK2.8 (Figure S6A). We

also found that SnRK2.6, but not a randomly selected protein

kinase cyclin-dependent protein kinase CDKF, could phosphor-

ylate a recombinant fragment of RaptorB (Figure 6E). These re-

sults suggest that RaptorB is a direct substrate of SnRK2s.

To further confirm the relationship between SnRK2 activity and

Raptor phosphorylation in vivo, we measured the phosphoryla-

tion status of RaptorB protein in wild-type and snrk2 decuple-

mutant plants (Fujii et al., 2011) by performing MS-based

quantitative phosphoproteomics. We found that ABA treatment

of wild-type seedlings substantially increased the levels of a

RaptorB phosphopeptide containing phosphorylated Ser897,

but not another phosphopeptide containing phosphorylated

Ser941 (Figure 6F). However, Ser897 phosphorylation was not

detected under either condition in the snrk2 decuple mutant,

which lacks all ten members in SnRK2 family (Figure 6F).

Moreover, a Ser-to-Ala substitution at Ser897 dramatically

reduced the phosphorylation by SnRK2.6, when compared to

the wild-type RaptorB fragment (Figure 6G). The phosphoryla-

tion of Ser897, along with 7 other serine residues, could be

detected by MS in the kinase reaction products after trypsin

digestion (Figures S6B and S6C). These data suggest that

S897 is a major, though perhaps not a unique, SnRK2 phos-

phorylation site, and the phosphorylation of S897 is dependent

on ABA activated SnRK2s in vivo.

Next, we investigated whether ABA signaling might regulate

TOR activity by influencing the association of TOR with RaptorB.

We increased the level of RaptorB in wild-type protoplasts by

introducing a 35S promoter-driven RaptorB construct. We found

that increased RaptorB levels impaired ABA-induced TOR inhibi-

tion (Figures 6H and 6I, n = 3, p < 0.05, Student’s t test). In addi-

tion, we immunoprecipitated the TOR complex from seedlings

that were treated with ABA or left untreated. We found that

ABA treatment reduced the interaction between RaptorB and

TOR, detected by co-immunoprecipitation (Figures 6J and

S6D, n = 3, p < 0.001, Student’s t test). Our results suggest

that ABA-mediated activation of SnRK2.6 leads to phosphoryla-

tion of RaptorB to promote disassociation of RaptorB from the

TOR complex and inhibit TOR kinase activity.

DISCUSSION

In this study, we discovered that TOR kinase regulates ABA

perception through phosphorylation of a conserved serine in

the ABA receptor PYLs. This phosphorylation abolishes the

ABA binding activity of PYLs. Our results suggest that TOR ki-

nase-mediated PYL phosphorylation represents a conserved

mechanism to prevent stress signaling under non-stress condi-

tions and desensitizes stress signaling after stress subsides. In

addition, our results suggest that ABA and stress inhibit plant

growth by activating SnRK2 kinases to phosphorylate RaptorB,

a regulatory component of the TOR complex.

Molecular Cell 69, 100–112, January 4, 2018 107

A B C

D

F GE

H I J

Figure 6. ABA and Stress Inhibit TOR Kinase Activity

(A) In vitro kinase assay showing ABA inhibition of PYL1 and PYL4 phosphorylation. TOR was immunoprecipitated from 7-day-old seedlings incubated with or

without ABA and incubated with recombinant His-Sumo-PYL1 and His-Sumo-PYL4 proteins as substrates. TOR protein levels are indicated by the anti-TOR

immunoblot. PYL1/PYL4 protein levels are indicated by Coomassie-stained gel. Band radioactivity levels were normalized to control without ABA and Torin2

treatment. Error bars, SD (n = 3).

(B) ABA represses phosphorylation of S6K1 at T449 site in seedlings. S6K1-HA was overexpressed in the seedlings to facilitate S6K1 detection using anti-HA

immunoblot.

(C) Phosphorylation level of T449 of S6K1 was decreased by mannitol treatment in seedlings. S6K1-HA was overexpressed in the seedlings to facilitate S6K1

detection using anti-HA immunoblot.

(D) ABA inhibition on TOR kinase activity was almost abolished in pyr1pyl12458 sextuple and snrk2.2/3/6 triple mutants. S6K1-HA was expressed in protoplasts

made from WT, pyr1pyl12458 sextuple, and snrk2.2/3/6 triple mutants.

(E) SnRK2.6 but not CDKF phosphorylates a Raptor fragment in vitro. Recombinant GST-Tag-fused SnRK2.6 and CDKF were used to phosphorylate AtRaptorB

fragment (aa 487–1057) expressed and purified in E. coli in the presence of [g-32P]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation

and loading of purified SnRK2.6, CDKF, and RaptorB. Asterisk indicates partially degraded AtRaptorB.

(F) Quantitative analysis of phosphopeptides containing S897 and S941 in wild-type and snrk2 decuple-mutant seedlings, with or without ABA treatment. The

relative abundance of individual phosphopeptides was normalized to the amount of S941 phosphopeptide in the snrk2 decuple-mutant samples with ABA

treatment. N.D., not identified in the samples. Error bars, SD (n = 3).

(G) Phosphorylation of recombinant wild-type and S897Amutated AtRaptorB fragments by recombinant SnRK2.6 protein kinase. Autoradiograph (left panel) and

Coomassie staining (right panel) show phosphorylation and loading of purified SnRK2.6 and RaptorB fragments. Asterisk indicates partially degraded AtRaptorB.

(H and I) ABA inhibitory effect on TOR kinase can be suppressed by RaptorB overexpression. S6K1-Flag and RaptorB-HA were expressed in protoplasts made

from raptor1-2. (H) Immunoblot results are representative of three independent experiments. (I) Band intensities of ABA-treated samples were normalized to

samples without ABA treatment. Error bars, SD (n = 3). *p < 0.05, Student’s t test.

(J) Less RaptorB co-immunoprecipitated with TOR after ABA treatment. CoIP assays were performed using ABA treated or untreated seedlings. Band intensities

from ABA-treated samples were normalized to samples without ABA treatment. Error bars, SD (n = 3). ***p < 0.001, Student’s t test.

See also Figure S6.

108 Molecular Cell 69, 100–112, January 4, 2018

Figure 7. Model Illustrating How TOR Kinase

and PYL Phosphorylation Balances Growth

and Stress Response in Plants

TOR kinase is an evolutionarily conserved regulator of growth

in eukaryotes. We found that plants with reduced TOR activity

are hypersensitive to ABA and display ABA treatment-related

phenotypes, including growth arrest, SnRK2 activation, and

expression of ABA-responsive genes, even in the absence of

ABA (Figure 5). These data support a critical role for TOR kinase

in suppressing ABA and stress signaling in the absence of stress.

Some PYLs in Arabidopsis (PYL5 to PYL12) can bind and inhibit

PP2Cs even in the absence of ABA (Fujii et al., 2009; Hao et al.,

2011). However, the activation of ABA signaling in the absence of

ABA is not observed in wild-type seedlings. We found that the

phosphomimic mutations at the conserved serine within PYL10

abolishes its ABA-independent interactionwith PP2Cs (Figure 2).

We propose that TOR-mediated phosphorylation serves as a

mechanism to inhibit the activation of the ABA-independent

PYLs under non-stress conditions.

Under stress conditions, activated SnRK2s phosphorylate

RaptorB to promote the disassociation of the TOR complex.

This might represent a mechanism to rapidly amplify ABA

signaling. In the presence of ABA, a small fraction of unphos-

phorylated PYLs bind to ABA first and then activate SnRK2s.

SnRK2s inhibit TOR kinase by phosphorylating RaptorB

to release the majority of PYLs from phosphorylation. These

unphosphorylated PYLs can then bind ABA to rapidly amplify

ABA signaling. In addition, an unknown phosphatase may also

be activated under stress and contribute to the dephosphoryla-

tion of PYLs. TOR kinase inhibition by ABA and osmotic stress

also suggest a mechanism through which environmental

stresses and ABA inhibit plant growth. Osmotic stress represses

TOR-mediated S6K1 phosphorylation (Figure 6C) and thereby

inhibits S6K1 activity (Mahfouz et al., 2006). Because TOR-medi-

ated phosphorylation activates S6K1 and the initiation of trans-

Mole

lation (Holz et al., 2005), our findings sug-

gest a role for SnRK2s in reprogramming

translation in response to ABA and os-

motic stress. The inhibition of TOR may

also cause repression of BR signaling

(Xiong et al., 2017; Zhang et al., 2016)

and impairment of auxin signaling (Li

et al., 2017; Schepetilnikov et al., 2017).

The DELLA family of growth inhibitory nu-

clear proteins play important roles in medi-

ating growth inhibition by salt stress, ABA,

and ethylene (Achard et al., 2006). Future

research will determine how ABA inhibition

of TOR kinasemay be coordinated with the

stabilization of DELLA proteins to inhibit

plant growth during stress.

As a key regulator of energy,metabolism,

and cell division and growth, TOR kinase

activity is tightly controlled when plants

respond to both internal and environmental

stimuli. Raptor is a regulatory component in the TOR complex in

both animal and yeast cells. Upon energy stress, AMPK phos-

phorylates Raptor and in turn inhibits the mTORC1 pathway and

suppresses cell growth and biosynthetic processes (Gwinn

et al., 2008). Nemo-like kinase (NLK), an osmotic and oxidative

stress activated protein kinase, also represses TORC1 through

Raptor phosphorylation (Yuan et al., 2015). In addition, ERK1/2,

GSK3, Intestinal Cell Kinase (ICK), and S6K1 phosphorylate

Raptor tomodulate TORactivity (Carriere et al., 2008, 2011; Stret-

ton et al., 2015;Wu et al., 2012). However, the functional relation-

ship between Raptor phosphorylation and TORC regulation in

plants had remainedunclear. Here,wediscovered that plant-spe-

cific, stress-activated protein kinases, SnRK2s, phosphorylate

Raptor (Figure 6E) to cause TOR inhibition, possibly by promoting

the disassociation of the TOR complex in plants (Figure 6J). Our

results suggest that Raptor phosphorylation by stress-activated

protein kinases is a conserved mechanism for the regulation of

TOR and growth in eukaryotic organisms.

TOR phosphorylation of PYLs prevents activation of stress

responses when stress is absent. On the other hand, Raptor

phosphorylation by stress- and ABA-activated SnRK2s is a

mechanism that prevents plant growth under unfavorable condi-

tions to conserve energy and ensure survival. The phosphoryla-

tion of PYLs can also switch off ABA signaling during stress re-

covery in plants. We observed a defect in growth recovery in

transgenic plants carrying a non-phosphorylatable PYL1S119A

(Figure 3). As the serine corresponding to Ser119 in PYL1 is

conserved in all 121 PYLs identified in 12 different species (Fig-

ure S2), the phospho-regulation of PYLs appears to be highly

conserved in land plants. We propose that the phosphorylation

loop between the ABA core signaling components and the

TOR complex represents a critical mechanism that balances

cular Cell 69, 100–112, January 4, 2018 109

stress and growth responses for plants to adapt to continuously

changing environments (Figure 7).

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d METHOD DETAILS

110

B Generation of the es-tor Mutant

B Germination or Growth under ABA and Mannitol

Treatment

B Plasmid Constructs

B Yeast Two Hybrid Assay

B Protoplast Isolation and Transactivation Assay

B Measurement of Chlorophyll Content

B In-gel Kinase Assay

B In vitro Kinase Assays and Immunoprecipitation (IP)

TOR Kinase Assays

B Co-Immunoprecipitation (CoIP) Assays

B Protein Extraction and Digestion

B Phosphopeptide Enrichment

B High-pH Reverse Phase StageTip Fractionation

B LC-MS/MS Analysis

B Proteomics Data Analysis

B ABA Binding Assay

B Phosphatase Activity Assay

B Alpha Screen Assay

B RNA Extraction and Real Time PCR

B Electrolyte Leakage Assay

B Analysis of Microarray Data

B Sequence Alignment and Phylogenetic Tree Gen-

eration

d QUANTIFICATION AND STATISTICAL ANALYSIS

d DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures and six tables and can be found

with this article online at https://doi.org/10.1016/j.molcel.2017.12.002.

ACKNOWLEDGMENTS

This work was supported by the Chinese Academy of Sciences, including

its Strategic Priority Research Program (Grant No. XDPB0404), NIH Grants

R01 GM059138 (to J.-K.Z.) and 1R01GM111788 (to W.A.T.), and National Sci-

ence Foundation Grant CH1506752 (to W.A.T.). We are grateful to Prof. Pedro

Rodriguez of Universidad Politecnica de Valenci, Spain for kindly providing the

pyr1pyl12458 mutant seeds, to Prof. Christian Meyer of Institut Jean-Pierre

Bourgin, France for kindly providing raptormutants and to the National Centre

for Protein Science Shanghai (Protein Expression and Purification system) for

their instrument support and technical assistance. We would like to thank Life

Science Editors for editorial assistance.

AUTHOR CONTRIBUTIONS

P.W., Y.X., and J.-K.Z. designed research. P.W., Y. Zhao, Z.L., C.-C.H., X.L.,

L.F., Y.-J.H., Y.D., C.Z., J.G., M.C., X.H., and Y .Zhu performed experiments;

Molecular Cell 69, 100–112, January 4, 2018

P.W., Y. Zhao, Z.L., C.-C.H., S.X., K.T., X.W., W.A.T., Y.X., and J.-K.Z.

analyzed the results; P.W., Y.X., and J.-K.Z. wrote the manuscript.

DECLARATION OF INTEREST

The authors declare no competing interests.

Received: April 13, 2017

Revised: July 19, 2017

Accepted: December 1, 2017

Published: December 28, 2017

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Anti-Myc Antibody Millipore 05-724

SnRK2.6 Agrisera AS13 2635

HIS Sigma-Aldrich H1029

TOR Xiong and Sheen, 2012 N/A

Anti-HA tag antibody Abcam ab9110

S6K1 p-T449 Agrisera AS13 2664

Anti-FLAG Sigma-Aldrich F1804

Anti-GFP Sigma-Aldrich 11814460001

Anti-Actin antibody Abcam ab197345

Anti-RatporB This study N/A

Chemicals, Peptides, and Recombinant Proteins

Abscisic Acid Sigma-Aldrich A1049

Luciferase Assay Reagent Promega E1483

Phosphatase substrate Sigma-Aldrich P4744

D-Mannitol Sigma-Aldrich M4125

PP242 Sigma-Aldrich P0037

Torin2 Selleckchem S2817

estradiol Sigma-Aldrich E8875

Rapamycin Sigma-Aldrich R8781

Phosphatase inhibitor 3 Sigma-Aldrich P0044

SnRK2.6 phosphopeptide Fujii et al., 2009 HSQPKpSTVGTP

Recombinant DNA

Recombinant DNAs provided in Table S5

Deposited Data

Raw Imaging Files This study, Mendeley Data https://doi.org/10.17632/2zchtvbv58.1

Phosphoproteomic This study ProteomeXchange: PXD003746

Experimental Models: Organisms/Strains

pyr1pyl12458 Gonzalez-Guzman., 2012 N/A

pyr1pyl124 Park et al., 2009 N/A

pyr1pyl124-PYL1-myc This study N/A

pyr1pyl124-PYL1S119A-myc This study N/A

pyr1pyl124-PYL1S119D-myc This study N/A

snrk2.2/2.3/2.6 Fujii et al., 2009 N/A

snrk2.1/2/3/4/5/6/7/8/9/10 Fujii et al., 2011 N/A

es-tor Xiong and Sheen 2012 N/A

SnRK2.6-GFP Wang et al., 2015 N/A

S6K1-HA Xiong et al., 2013 N/A

S6K1-FLAG Xiong and Sheen 2012 N/A

raptor1-1 Deprost et al., 2005 SALK_078159

raptor1-2 Deprost et al., 2005 SALK_006431

Oligonucleotides

Primers provided in Table S6

(Continued on next page)

Molecular Cell 69, 100–112.e1–e6, January 4, 2018 e1

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

Software and Algorithms

MaxQuant Cox and Mann, 2008 http://www.coxdocs.org/doku.php

SEQUEST Thermo Fisher Scientific

GraphPad Prism GraphPad Software Inc https://www.graphpad.com/

Image Lab Bio-Rad Laboratories N/A

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to andwill be fulfilled by the LeadContact, Jian-Kang

Zhu (jkzhu@sibs.ac.cn).

METHOD DETAILS

Generation of the es-tor Mutantes-tor seeds were geminated in liquid medium (1/2 MS, 1% glucose) under 75 mmol m-2 s-2 light intensity. After germination, estradiol

was added to the medium at a final concentration of 10 mM to induce silencing of the TOR gene in es-tor transgenic plants (Xiong and

Sheen., 2012). Three days post-induction, ABA was added at the indicated final concentrations for the indicated lengths of time.

Germination or Growth under ABA and Mannitol TreatmentSeedswere surface-sterilized for 10min in 20%bleach, and then rinsed four times in sterile-deionized water. For germination assays,

sterilized seeds were grown on medium containing 1/2 MS nutrients (PhytoTech), 1% sucrose, pH 5.7, with or without the indicated

concentration of ABA, and kept at 4�C for 3 days. Radicle emergence was analyzed 72 hours after placing the plates in a Percival

CU36L5 incubator at 23�C under a 16 hours light/8 hours dark photoperiod. Photographs of seedlings were taken at indicated times

after taken to light. For growth assays, sterilized seeds were grown vertically on 0.6% Phytagel containing 1/2 MS, 1% sucrose,

pH5.7, and kept at 4�C for 3 days. Seedlings were grown vertically for 3-4 days and then transferred to medium with or without

10 mM ABA. Root length, fresh weight and chlorophyll content were measured at the indicated days. For recovery growth, vertically

grown seedlings were transferred fromplates with 10 mMABA, or 300mMmannitol to 0.6%Phytagel containing 1/2MS, 5%sucrose,

pH5.7, and were grown vertically for 5 days.

To detect the effect of ABA and mannitol on TOR kinase activity, 7-day-old S6K1-HA transgenic seedlings grown in 6-well plates

containing 1ml liquid medium (1/2 MS, 0.5% sucrose, pH5.7) were treated with 50 mM ABA for the indicated times or indicated

concentrations of mannitol for 6 hours.

Plasmid ConstructsFor the transactivation assay, ZmUBQ::GUS, RD29B::LUC; ABF2-HA, SnRK2.6-FLAG, PP2Cs, and His-PYLs were the same as

previously reported (Fujii et al., 2009). The plasmids carrying wild-type pHBT95-PYLs were used for site-directed mutagenesis using

specific primers (Table S6).

pBD-GAL4-PYLs vectors for the yeast two-hybrid (Y2H) assay were the same as reported (Park et al., 2009; Zhao et al., 2013), and

were used for site-directed mutagenesis using specific primers (Table S6). To generate the vector for Y2H, RaptorBCDSwas cloned

into the pGADT7 vector between EcoRI and XhoI sites with RaptorB specific primers (Table S6). To generate the pENTR-PYL1

genomic construct, the genomic sequence of PYL1 was amplified using pPYL1pF and pPYL1pR primers (Table S6), and cloned

into pENTR vector using pENTR/D-TOPO Cloning Kit (Invitrogen). The genomic sequence of PYL1 genomic was cloned into the

pGWB16 binary vector through an LR reaction (Invitrogen).

To generate the construct for RaptorB protein expression in E. coli, the RaptorB CDS fragment (bp 1459 to 3171) was cloned into

the pGEX-4T-1vector between EcoRI and SalI sites with gene specific primers (Table S6).

Yeast Two Hybrid AssayTo detect protein interactions between ABI1 and PYLs, pGADT7 plasmids containing ABI1 were co-transformed with wild-type or

mutated pGBKT7-PYLs into Saccharomyces cerevisiae AH109 cells. To detect protein interactions between RaptorB and SnRK2s,

pGADT7 plasmids containing RaptorB were co-transformed with wild-type or mutated pGBKT7-PYLs into Saccharomyces

cerevisiae AH109 cells. Successfully transformed colonies were identified on yeast SD medium lacking Leu and Trp. Colonies

were transferred to selective SD medium lacking Leu, Trp, His in the absence or presence of 10 mM ABA as indicated. To determine

the intensity of protein interaction, saturated yeast cultures were diluted to 10�1, 10�2 and 10�3 and spotted onto selection medium.

Photographs were taken after 4 days incubation at 30�C.

e2 Molecular Cell 69, 100–112.e1–e6, January 4, 2018

Protoplast Isolation and Transactivation AssayProtoplast isolation and transactivation assays were performed as previously described (Fujii et al., 2009). In brief, 4-week-old plants

grown under a short photoperiod (10 hours light at 23�C/14 hours dark at 20�C) were used for protoplast isolation. Strips of young

rosette leaves were treated with enzyme solution containing cellulase R10 (Yakult Pharmaceutical Industry) and macerozyme R10

(Yakult Pharmaceutical Industry) in the dark. After being diluted with equal volume of W5 solution (2 mM MES, pH 5.7, 154 mM

NaCl, 125mMCaCl2, and 5mMKCl), the protoplasts were filtered through a nylonmesh and pelleted at 100 x g for 2min. Protoplasts

were resuspended in W5 solution and kept for 30 min. Protoplasts (100 mL) in MMg solution (4 mMMES, pH 5.7, 0.4 Mmannitol, and

15 mM MgCl2) were mixed with the plasmid mix, and incubated for 5 min with 110 mL PEG solution (40% w/v PEG-4000, 0.2 M

mannitol, and 100 mM CaCl2). The protoplasts were washed twice with 1 mL W5 solution. After transfection, protoplasts were

incubated for 5 hours under light in washing and incubation solution (0.5 Mmannitol, 20 mM KCl, 4 mMMES, pH 5.7) with or without

5 mM ABA. All steps were performed at room temperature. The RD29B promoter fused to the LUC coding sequence was used as an

ABA-responsive reporter gene (7 mg of plasmid per transfection). ZmUBQ::GUS was included in each sample as an internal control

(1 mg per transfection). For ABF2-HA, SnRK2.6-FLAG, wild-type and mutated PYLs and PP2C plasmids, 3 mg per transfection was

used. After transfection, protoplasts were incubated for 5 hours under light in washing and incubation solution (0.5 M mannitol,

20 mM KCl, 4 mM MES, pH 5.7) with or without 5 mM ABA.

To detect the effect of ABA and RaptorB overexpression on TOR kinase activity in protoplasts, protoplasts were co-transfected

with constructs encoding S6K1 and other proteins (as indicated), incubated for 3 hours under light and then treated with 5 mM

ABA for 2 hours before harvest.

Measurement of Chlorophyll ContentSeedlings were weighed, quickly frozen in liquid nitrogen, ground in liquid nitrogen, homogenized in extraction buffer (containing

ethanol, acetone, and H2O in a ratio of 5:5:1), incubated at 37�C for 4-h, and finally centrifuged at 13000 rpm for 5 min. The

absorbance of the supernatant was measured at 645 nm and 663 nm using Wallac VICTOR2 plate reader (Perkin Elmer) with the

642 nm and 665 nm filters, respectively.

In-gel Kinase AssayFor in-gel kinase assays, 20 or 200 mg extract of total proteins was used for SDS/PAGE analysis with histone embedded in the gel

matrix as the kinase substrate. After electrophoresis, the gel was washed three times at room temperature with washing buffer

(25 mM Tris-Cl, pH 7.5, 0.5 mM DTT, 0.1 mM Na3VO4, 5 mM NaF, 0.5 mg/mL BSA, and 0.1% Triton X-100) and incubated at 4�Covernight with three changes of renaturing buffer (25 mM Tris-Cl, pH 7.5, 1 mM DTT, 0.1 mM Na3VO4, and 5 mM NaF). The gel

then was incubated at room temperature in 30 mL reaction buffer (25 mM Tris-Cl, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT,

and 0.1 mM Na3VO4) with 200 nM ATP plus 50 mCi of [g-32P]ATP for 90 min. The reaction was stopped by transferring the gel

into 5% (w/v) trichloroacetic acid and 1% (w/v) sodium pyrophosphate. The gel was washed in the same solution for at least 5 hours

with five changes fresh wash solution. Radioactivity was detected with a Personal Molecular Imager (Bio-Rad). The radioactivity from

each band was calculated by Image Lab software (Bio-Rad).

In vitro Kinase Assays and Immunoprecipitation (IP) TOR Kinase Assays1 mg wild-type or mutated GST-PYL1 proteins were incubated with 0.005 mg GST-ABI1 with or without 5 mMABA in 20 mL of reaction

buffer (50 mMTris-HCl pH 7.0, 20mMMgCl2, 0.1 mMEGTA and 0.1% b-mercaptoethanol), the reactions without PYL1 were used as

control. After 15minutes incubation, 1 mgMBP-SnRK2.6 was added to the reaction and incubated for additional 15minutes. After the

incubation step, a mixture containing 1 mM ATP, 5 mCi [g-32P]ATP, 4 mg GST-ABF2 fragment, and phosphatase inhibit cocktail 3

was add to the reaction to total volume of 25 mL. The reaction mixtures were incubated for 30 minutes at 25�C. The reaction was

stopped by adding SDS sample buffer, and the mixtures were then subjected to SDS-PAGE. Radioactivity was detected with a

Personal Molecular Imager (Bio-Rad).

The immunoprecipitation kinase assays of TOR kinase were performed as previously described (Xiong et al., 2013). 10 mL

immunoprecipitated TOR kinase was incubated with 1 mg recombinant PYLs proteins in 25 mL kinase buffer (25 mM HEPES,

pH 7.4, 20 mM KCl, 10 mM MgCl2, 1 mM cold ATP, 5 mCi [g-32P]ATP, with or without 100 mM PP242 or 25 mM Torin 2) for 30 min

at 25�C. The reaction was stopped by adding of SDS sample buffer. After separation on 12%SDS-PAGE and subsequent gel drying,

radiolabeled PYLs proteins were detected by the Personal Molecular Imager (Bio-Rad). A parallel reaction using g-[18O4]-ATP (Xue

et al., 2014) and GST-PYL1 was subjected to trypsin digestion, phosphopeptides enriched, and LC-MS/MS analysis.

For detection of RaptorB phosphorylation by SnRK2.6, 1 mgGST-RaptorB-M proteins were incubated with 0.2 mgGST-SnRK2.6 in

20 mL of kinase buffer and 1 mM cold ATP, 5 mCi [g-32P]ATP for 60 min at 25�C. The reaction was stopped by adding SDS sample

buffer, and the mixtures were then subjected to SDS-PAGE. Radioactivity was detected with a Personal Molecular Imager (Bio-Rad).

Co-Immunoprecipitation (CoIP) AssaysTo detect the interaction between TOR and PYLs, 7-day old PYL1-Myc transgenic orWT seedlings from one 6-well plate were ground

in liquid nitrogen and lysed in 1000 mL Co-IP buffer (400 mM HEPES pH 7.4, 2 mM EDTA, 10 mM pyrophosphate, 10 mM glycerol

phosphate, 0.3% CHAPS and 1 3 cocktail inhibitors [Roche]) for 1.5 hours at 4�C. After centrifuging at 14,000 g for 10 min at

Molecular Cell 69, 100–112.e1–e6, January 4, 2018 e3

4�C, the supernatant of each sample was mixed with or without anti-TOR antibody and rotated at 4�C for 2 hours and additional

2 hours after adding 15 mL pre-balanced protein G Sepharose beads (GE healthcare). The immunoprecipitated proteins were

washed three times with low salt wash buffer (400 mM HEPES pH 7.4, 100 mM NaCl, 2 mM EDTA, 10 mM pyrophosphate,

10mMglycerol phosphate, 0.3%CHAPS) before SDS-PAGE separation and protein blot analyses. To detect the interaction between

TOR and RaptorB, 7-day old WT seedlings treated with or without 50 mM ABA for 2 hours were used and anti-RaptorB antibody

(Rabbit polyclonal antibody, Synthetic peptide sequence: c-HLEASRPSDPQPEP, Antigen affinity purified) were used for detection

of RaptorB protein after immunoprecipitation.

To detect the phosphopeptide of PYL1 protein, 10-day old PYL1-Myc transgenic or WT seedlings were ground in liquid nitrogen

and lysed in 1000 mL IP buffer (25 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 10 mM glycerol

phosphate, 1 mM leupeptin, 1 mM aprotinin, 1 3 Phosphatase inhibitor cocktail [Sigma], 0.1% Triton X-100). After centrifuging at

14,000 g for 10 min at 4�C, the supernatant of each sample was mixed with anti-Myc antibody and rotated at 4�C for 4 hours and

additional 2 hours after adding 15 mL pre-balanced protein G Dynabeads (Thermofisher). The immunoprecipitated proteins were

washed three times with wash buffer (25 mM Tris-Cl pH 7.5, 300 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 10 mM glycerol phosphate,

1 mM leupeptin, 1 mM aprotinin, 1 3 Phosphatase inhibitor cocktail [Sigma]) before digestion and phosphopeptide enrichment.

Protein Extraction and DigestionGround Arabidopsis tissues were lysed in 8 M urea in 50 mM triethylammonium bicarbonate. Proteins were reduced and alkylated

with 10 mM tris-(2-carboxyethyl) phosphine and 40 mM chloroacetamide at 37�C for 30 min. Protein amount was quantified by BCA

assay (Thermo Fisher Scientific). Protein extracts were diluted to 4 M urea and digested with Lys-C in a 1:100 (w/w) enzyme-to-

protein ratio for 3 hours at 37�C, and further diluted to a 1 M urea concentration. Trypsin was added to a final 1:100 (w/w)

enzyme-to-protein ratio overnight. Digests were acidified with 10% trifluoroacetic acid (TFA) to a pH �3, and desalted using a

100 mg of Sep-pak C18 column (Waters).

Phosphopeptide EnrichmentPhosphopeptide enrichment was performed according to the reported IMAC StageTip protocol with some modifications (Tsai et al.,

2014). The in-house-constructed IMAC tip was made by capping the end with a 20 mm polypropylene frits disk (Agilent). The tip was

packed with 5 mg of Ni-NTA silica resin by centrifugation at 200 3 g for 1 min. Ni2+ ions were removed by 100 mL of 100 mM EDTA

solution. The tip was then activatedwith 100 mL of 100mMFeCl3 and equilibrated with 100 mL of loading buffer (6% (v/v) acetic acid at

pH 3.0) prior to sample loading. Tryptic peptides (200 mg) were reconstituted in 100 mL of loading buffer and loaded onto the IMAC tip.

After successive washes with 200 mL of washing buffer (4% (v/v) TFA, 25% acetonitrile (ACN)) and 100 mL of loading buffer respec-

tively, the bound phosphopeptides were eluted with 150 mL of 200 mM NH4H2PO4. The eluted phosphopeptides from six IMAC

StageTips were directly loaded on a C18 StageTip to perform high-pH reverse phase (Hp-RP) fractionation.

High-pH Reverse Phase StageTip FractionationHigh-pHReverse Phase fractionation was performedwith somemodifications to a previousmethod (Dimayacyac-Esleta et al., 2015).

2 mg of the C18-AQ beads (5mm particles) were suspended in 100 mL of methanol and loaded into a 200 mL of StageTip with a 20 mm

polypropylene frit. The C18 StageTips were activated with 100 mL of 40 mM NH4HCO2, pH 10, in 80% ACN, and equilibrated with

100 mL of 200 mM NH4HCO2, pH 10. After loading with eluted phosphopeptides from the IMAC StageTips, the C18 StageTips

were washed with 100 mL of 200 mM NH4HCO2, pH 10. The bound phosphopeptides were fractionated from the StageTip with

50 mL of 8 different ACN concentrations: 4%, 7%, 10%, 13%, 16%, 19%, 22%, and 80% of ACN in 200 mM NH4HCO2, pH 10.

The eluted phosphopeptides were dried and stored at �20�C.

LC-MS/MS AnalysisThe phosphopeptides were dissolved in 5 mL of 0.25% formic acid (FA) and injected into an Easy-nLC 1000 (Thermo Fisher Scientific).

Peptides were separated on a 45 cm in-house packed column (360 mmOD3 75 mm ID) containing C18 resin (2.2 mm, 100A, Michrom

Bioresources). The mobile phase buffer consisted of 0.1% FA in ultra-pure water (Buffer A) with an eluting buffer of 0.1% FA in 80%

ACN (Buffer B) run over a linear 60 min gradient of 6%–30% buffer B at flow rate of 250 nL/min. The Easy-nLC 1000 was coupled

online with a Velos LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in the

data-dependent mode in which a full-scan MS (from m/z 350-1500 with the resolution of 60,000 at m/z 400) was followed by top

10 collision-induced dissociation (CID) MS/MS scans of the most abundant ions with dynamic exclusion for 60 s and exclusion

list of 500. The normalized collision energy applied for CID was 35% for 10 ms activation time.

Proteomics Data AnalysisThe raw files were searched directly against Arabidopsis thaliana database (TAIR10) with no redundant entries using SEQUEST HT

algorithm in Proteome Discoverer version 2.1 (Thermo Fisher Scientific) andMaxQuant software (Cox andMann, 2008). Peptide pre-

cursor mass tolerance was set at 15 ppm, and MS/MS tolerance was set at 0.6 Da. Search criteria included a static carbamidome-

thylation of cysteines (+57.0214 Da) and variable modifications of (1) oxidation (+15.9949 Da) on methionine residues, (2) acetylation

(+42.011 Da) at N terminus of protein, and (3) phosphorylation (+79.996 Da) on serine, threonine or tyrosine residues were searched.

e4 Molecular Cell 69, 100–112.e1–e6, January 4, 2018

For 18O-phosphopeptides, heavy phosphorylation (+85.979 Da) was set as a variable modification. Search was performed with full

tryptic digestion and allowed a maximum of two missed cleavages on the peptides analyzed from the sequence database. Relaxed

and strict false discovery rates were set for 0.05 and 0.01, respectively. PhosphoRS (Taus et al., 2011) was utilized for localization of

phosphorylation sites. Label-free quantitation was performed using Maxquant (version 1.5.0.28). All the mass spectrometric data

have been deposited to the ProteomeXchange Consortium (Vizcaıno et al., 2014) via the PRIDE partner repository with the dataset

identifier PXD003746 (https://www.ebi.ac.uk/pride).

ABA Binding AssayThe interaction between ABA and the recombinant SUMO-HIS tagged PYL1, PYL1S119A, PYL1S119D, PYL10, PYL10S88A, PYL10S88D,

and PYL10S88E was measured by Thermal Stability Shift Assay (TSA) or Microscale Thermophoresis (MST). MST assays were per-

formed as previously described (Parker and Newstead, 2014). Briefly, wild-type andmutated PYL1 and PYL10 proteins were labeled

in assay buffer (50 mMHEPES, pH 7.5, 100mMNaCl) using the Protein Labeling Kit RED-NHS (NanoTemper Technologies). ABA in a

range of concentrations (0.0001 mM to 4 mM) was incubated with 2 mM of labeled proteins for 1 hour at 4�C. The samples were then

loaded into the NanoTemper glass capillaries (NanoTemper Technologies) andmicrothermophoresis was performed using 20% LED

power and 80%MST. TheKD values for ABA-PYLs binding were calculated using themass action equation through the NanoTemper

software (NanoTemper Technologies).

For the TSA assay, 5 mM of purified recombinant proteins were mixed with a range of concentration of ABA (0.4 mM to 6.25 mM),

and then incubated with SYPRO Orange (Sigma-Aldrich) for 1 hour on ice. All reactions were performed in final volumes of 10 mL in

384-well plates on a LightCycle 480II real-time PCR system (Roche). The mixtures were heated from 25�C to 90�C with a ramp rate

of 0.06�C/s, and fluorescence signals were recorded. Data were analyzed using the Protein Thermal Shift Software (Thermo Fisher

Scientific), and the melting temperature (Tm) values were calculated.

Phosphatase Activity AssayFor PYL1 and PYL4, phosphatase activity was measured using the colorimetric substrate pNPP (Sigma-Aldrich). Reactions were

performed in a reaction buffer containing 50 mM Tris-HCl, pH 7.5, 25 mM Mg(OAc)2, 2 mM MnCl2, 0.5 mM EGTA, 0.5% b-mercap-

toethanol, and 0.5% BSA. ABA and PYLs were added as indicated. The concentrations of recombinant ABI1 and PYLs was 0.1 and

0.5 mM, respectively. Reactions were started by the addition of pNPP to a final concentration of 5 mM. The hydrolysis of pNPP was

measured by reading the absorbance at 405 nm (A405).

For PYL10, the phosphatase activity assay used SnRK2.6 phosphopeptide (Fujii et al., 2009) as a substrate. The concentrations of

recombinant HAB1, PYL10 and SnRK2.6 phosphopeptide were 0.1, 0.5 mM and 0.1 mM, respectively. Colorimetric dye (BioVision)

was used to stop the reaction and to determine the released phosphate.

For measurement of the effect of TOR phosphorylation on PYL1 activity, 0.1 mM instead of 0.5 mM GST-PYL1 were used in the

in vitro TOR kinase assay and subsequent phosphatase activity assay.

Alpha Screen AssayInteractions between PYL10 and HAB1/ABI1 were assessed by luminescence-based AlphaScreen technology (Perkin Elmer) as

previously described (Melcher et al., 2010). All reactions contained 100 nM recombinant H6-SUMO-PYL10 proteins bound to

nickel-acceptor beads and 100 nM recombinant biotin-MBP-HAB1 or biotin-MBP-ABI1 bound to streptavidin-acceptor beads in

the presence and absence of ABA.

RNA Extraction and Real Time PCRTotal RNA was extracted from raptor1-2, es-tor transgenic seedlings treatment with estradiol, control seedlings with or without

3 hours rapamycin treatment (10 mM). Total RNAwas isolated using TRIzol (Thermo Fisher Scientific) according to themanufacturer’s

instructions. For real-time PCR assays, reactions were set up with iQ SYBR Green Supermix (Bio-Rad). A CFX96 Touch Real-Time

PCR Detection System (Bio-Rad) was used to detect amplification levels (initial step at 95�C for 2 min, followed by 40 cycles of 5 s

at 95�C, 15 s at 56�C and 20 s at 72�C). Quantification was performed using three independent biological replicates.

Electrolyte Leakage AssayTomeasure ion leakage in seedlings induced by PEG treatment, 5-day-old wild-type, raptor1-2 and estradiol treated es-tor seedlings

were rinsed briefly in distilled water, and placed in a solution containing 30% PEG for 5 hours. After treatment, seedlings were rinsed

briefly in distilled water and placed immediately in a tube with 5 mL of water. The tube was agitated gently for 3 hours before the

electrolyte content was measured. Six replicates of each treatment were conducted.

Analysis of Microarray DataGEO2R was used to identify differentially expressed genes between es-tor mutant and wild-type requiring adjusted P value < 0.01

and at least 2 fold changes. Data were deposited under accession number of GSE40245 (Xiong et al., 2013). Only data from

experiments with glucose supplement were used in this analysis.

Molecular Cell 69, 100–112.e1–e6, January 4, 2018 e5

To identify stress responsive genes, we used three databases to obtain a list of genes responsive to salt, drought, osmotic and ABA

stress: 1) The RIKEN Arabidopsis Genome Encyclopedia (RARGE) database; For RARGE database, we extracted genes that

have a R 2.5-fold expression change at any time course under different stresses (drought, NaCl, and ABA, and rehydration after

dehydration 2 h). 2) Database Resource for the Analysis of Signal Transduction In Cells (DRASTIC). 3) Microarray data deposited

in GEO datasets were used to identify stress responsive genes using GEO2R.

Sequence Alignment and Phylogenetic Tree GenerationTo obtain different PYLs homologs in different species, proteins from a particular species were used as queries to blast against

14 PYLs. The proteins that can hit all 14 Arabidopsis PYLs (E value cutoff: 0.00001) were extracted. Proteins that have ‘‘specific’’

PYLs like domain use Batch CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) were retained as PYLs

homologs. Multiple alignment results were generated using MUSCLE (Edgar, 2004). View of multiple aligned sequences and

secondary structure using Espript interface (http://espript.ibcp.fr/ESPript/ESPript) (Gouet et al., 2003). The crystallographic structure

of PYR1 (Protein databank code: 3K90) was taken. A phylogenetic tree of PYLs from 12 different plant species was generated using

NCBI Taxonomy (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi).

QUANTIFICATION AND STATISTICAL ANALYSIS

Band intensity quantification of in-gel kinase assay results was performed using the Image Lab.

Statistical significance of relative luciferase activity, phosphatase activity, fresh weight, Rosetta diameter, relative intensity, ion

leakage, gene expression was examined by Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001).

DATA AND SOFTWARE AVAILABILITY

All the mass spectrometric data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the

dataset identifier PXD003746 (https://www.ebi.ac.uk/pride)

Raw image files are deposited on Mendeley Data (https://data.mendeley.com/datasets/2zchtvbv58/1).

e6 Molecular Cell 69, 100–112.e1–e6, January 4, 2018