Arabidopsis AtSUC2 and AtSUC4 , encoding sucrose transporters, are required for abiotic stress...

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Arabidopsis AtSUC2 and AtSUC4, encoding sucrose transporters, are

required for abiotic stress tolerance in an ABA-dependent pathway Xue Gonga,b,†, Mingli Liua,†, Lijun Zhanga,†,Yanye Ruana, Rui Dinga, Yuqi Jic, Ning

Zhanga, Shaobin Zhanga, John Farmerd and Che Wanga,*

aCollege of Biological Science and Technology, Shenyang Agricultural University, Shenyang, 100866,

China bCorn Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China cCollege of Agriculture and Biotechnology, China Agricultural University, Beijing 100094, China dCollege of Land and Environment, Shenyang Agricultural University, Shenyang, 100866, China

*Corresponding author, e-mail: [email protected] †These authors contributed equally to this work.

Sucrose transporters (SUCs or SUTs) play a central role, as they orchestrate sucrose allocation both

intracellularly and at the whole plant level. Previously, we found AtSUC4 mutants changing sucrose

distribution under drought and salt stresses. Here, we systematically examined the role of Arabidopsis

AtSUC2 and AtSUC4 in response to abiotic stress. The results showed that significant induction of

AtSUC2 and AtSUC4 in salt, osmotic, low temperature and exogenous ABA treatments by public

microarray data and real-time quantitative reverse transcription PCR (qRT-PCR) analyses. The

loss-of-function mutation of AtSUC2 and AtSUC4 led to hypersensitive responses to abiotic stress and

ABA treatment in seed germination and seedling growth. These mutants also showed higher sucrose

content in shoots and lower sucrose content in roots, as compared to that in wild-type plants, and

inhibited the ABA-induced expression of many stress-responsive genes and ABA-responsive genes,

especially ABFs and ABF-downstream and upstream genes. The loss-of-function mutant of AtSUC3, a

unique putative sucrose sensor, reduced the expression of AtSUC2 and AtSUC4 in response to abiotic

stresses and ABA. These findings confirmed that AtSUC2 and AtSUC4 are important regulators in

plant abiotic stress tolerance by the use of an ABA signaling pathway, which may be crossed with

sucrose signaling.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppl.12225


Abbreviations – Abscisic acid, ABA; ABSCISIC ACID-INSENSITIVE2, ABI2; Arabidopsis

Biological Resource Center, ABRC; ABA repressor 1, ABR1; High-performance liquid

chromatography, HPLC; Impaired sucrose induction, isi; Left genomic primer, LP; Right genomic

primer, RP; Real-time quantitative reverse transcription PCR, qRT-PCR; Reverse transcription PCR,

RT-PCR; Sucrose, Suc; Sieve elements, SEs; Sugar insensitive, sis; Sucrose non-fermenting 1-related

protein kinase, SnRKs; Sucrose phosphate synthase, SPS; Sucrose synthase, SuSy; Sucrose

transporters, SUCs or SUTs; Wild type, WT

Introduction

Plant growth and productivity are greatly affected by environmental stresses such as high salinity,

dehydration, and low temperature. The responses and adaptations of plants to these stresses can occur

at the molecular, cellular, physiological, and biochemical levels (Chinnusamy et al. 2005, Urano et al.

2010, Fujii and Zhu 2012). As a metabolite, sucrose plays an important role in stress tolerance,

serving as an osmolyte, a signaling molecule and/or a nutritional substance (Yu 1999, Gupta and Kaur

2005, Ruan et al. 2010). The expressions of some sucrose related genes, such as SuSy (sucrose

synthase) (Baud et al. 2004), SPS (sucrose phosphate synthase) (Pelleschi et al. 1997) and SnRK2s

(sucrose non-fermenting 1-related protein kinase2) (Fujii and Zhu 2012, Zhang et al. 2011) have been

reported to change in response to abiotic stresses.

Sucrose transporters (SUCs or SUTs) are an important exporter of photosynthetically produced

sugar, principally sucrose, from higher plant source leaves to sink tissues. In Arabidopsis, 9 AtSUCs

have been identified and researched on some of their expressions and functions. AtSUC2, is expressed

in companion cells collection and transport phloem in source leaves (Truernit and Sauer 1995,

Martens et al. 2006) and functions in loading sucrose into the phloem sieve elements (SEs), as

determined by tissue-specific complementation of different promoters (Srivastava et al. 2008, 2009a)

and by 14C labelling studies (Gottwald et al. 2000). This protein is also found within the vascular

bundles of sink tissues such as stamens, siliques, and roots (Truernit and Sauer 1995, Stadler and

Sauer 1996), suggesting a role in the retrieval of sucrose that leaks out of sieve elements during

long-distance transport (Stadler and Sauer 1996). When planted without sucrose, Atsuc2 mutant

seedlings are smaller than wild-type (WT) seedlings (Gottwald et al. 2000). AtSUC4 and AtSUC3 are

also expressed in minor veins of source leaves of mature plants (Weise et al. 2000, Barker et al. 2000,

Aoki et al. 2012), suggesting an involvement in phloem loading of sucrose in source organs. The

sucrose/proton symporter, AtSUC4, is also expressed in vacuoles and most likely regulates the

transport and vacuolar storage of photosynthetically derived sucrose (Endler et al. 2006, Schulz et al.

2011).


Sporadic reports have appeared regarding the role of SUTs in plant responses to abiotic stresses.

The expression of AgSUT1, which codes for the high affinity of AgSUT1 sucrose/H+ transporter in

celery (Apium graveolens), was decreased in all plant organs in response to salinity stress—a more

marked decrease in roots may have reflected a decreased metabolic demand for sucrose in response to

stress (Noiraud et al. 2000). Among the five sucrose transporters identified in rice (Oryza sativa cv.

Nipponbare), only OsSUT2 expression was up-regulated following exposure to drought and salinity

treatments (Ibraheem et al. 2011, Lundmark et al. 2006) found a low temperature induced

up-regulation of AtSUC1 and AtSUC2 when analyzing the role of the sucrose-phosphate synthase

(SPSox) in carbon partitioning of Arabidopsis at low temperature. Recent studies indicate that Aspen

(Populus tremula) transformed with PtaSUT4-RNAi wilt when exposed to a short-term drought,

indicating a role for PtaSUT4 in drought tolerance (Frost et al. 2012). Although these results indicate

that SUCs are involved in plant response abiotic stress, it is not clear that the potential stress tolerance

functions of the SUCs that control SUC distribution at the whole plant level.

The plant hormone-abscisic acid (ABA) regulates many plant responses to environmental stimuli

(Raghavendra et al. 2010, Seo et al. 2012). Many ABA-regulated genes, such as the ABFs (Choi et al.

2000), CBL9 (Pandey et al. 2004), ABR1 (Kim et al. 2003) and CIPK3 (Pandey et al. 2005), have been

identified in plant tolerance abiotic stresses. At present, many researches indicates that ABA is closely

associated with sugar signaling in plants (Rook et al. 2006, Dekkers et al. 2008). For example, genetic

analysis reveals that several ABA regulated genes and transcription factors are induced by sugar

signals, such as HXK1, HXK2, ABI3, ABI4, ABI5, ABF2, ABF3 and ABF4 (Rolland et al. 2006,

Dekkers et al. 2008, Hanson and Smeekens 2009). Mutant analysis showed that 3 sucrose response

mutants, including sucrose uncoupled (sun), impaired sucrose induction (isi) and sugar insensitive

(sis), were ABA deficient mutants (i.e. aba2/isi4/sis4 and aba3/gin5) and ABA insensitive4

(abi4/sun6/isi3/sis5), which were identified as sugar insensitive (Arenas-Huertero et al. 2000, Huijser

et al. 2000, Laby et al. 2000, Rook et al. 2001). Moreover, ABA can regulate the process of sucrose

loading and unloading (Tanner et al. 1980, Wyse et al. 1980, Berüter 1983, Vreugdenhil 1983,

Schussler et al. 1984). Recently, Peng et al. (2011) found that MdSUT1 (Malus pumila Mill.) in apple

fruit may be a component of ABA signaling pathways associated with the regulation of

photoassimilated transport. However, molecular genetic evidence linking sucrose transporter with

ABA-regulated biological functions under abiotic stresses has been lacking (Peng et al. 2011).

We have identified AtSUC4 mutants that had higher sucrose, fructose and glucose in shoots and

lower those in roots, as compared to that in WT, in salt and drought stresses (Gong et al. 2013a, b). It

is suggested that AtSUCs play an important role in mediating sugar distribution in abiotic stress. To

systematically understand how AtSUCs regulate sucrose distribution in abiotic stress, we carried out

an experiment to analyze gene expression of the AtSUC family by public microarray data and

qRT-PCR in plants exposed to abiotic stresses. We found that AtSUC2 and AtSUC4 are clearly

induced in response to salt, osmotic, drought, low temperature and exogenous ABA treatments.


Mutation of AtSUC2 and AtSUC4 affects the sucrose distribution in shoots and roots, resulting in

hypersensitivity of seed germination and seedling growth to abiotic stresses and to exogenous ABA

treatments. The function of AtSUC2 and AtSUC4 in stress responses may involve in an

ABF-dependent ABA signaling pathway and a sucrose signaling pathway. These findings suggest that

AtSUC2 and AtSUC4 are important regulators of the responses of Arabidopsis to abiotic stresses and

to ABA, and that ABA can regulate sucrose transport and sucrose balance in the plant by controlling

AtSUC2 and AtSUC4, thereby promoting plant stress tolerance.

Materials and methods

The expression of AtSUCs analyzed by public microarray data

The expression of all genes in Arabidopsis under control and stress treatments was available in a

public microarray data set, WeigelWorld (http://weigelworld.org/resources/microarray/AtGenExpress)

(Schmid et al. 2005). Experimental statistical analyses were performed by the original authors or the

database providers. The expression of AtSUCs was determined at different stress treatments (salt,

mannitol, drought and cold) at 0, 0.5, 1, 3, 6, 12 and 24 h, or different hormone treatments at 0, 0.5, 1

and 3 h, respectively (Schmid et al. 2005). We calculated the ratio of AtSUCs expression in the treated

plants to that in the control plants (setting the control value to 1) to identify the key AtSUCs that

responded to the different stress and hormone treatments.

The expression of genes analyzed by qRT-PCR

To assay the expression levels of key AtSUCs genes (AtSUC2 and AtSUC4) in WT after stress and

ABA treatments, real-time quantitative reverse transcription PCR (qRT-PCR) was performed with the

RNA samples isolated from 14-day-old seedlings at the indicated times after the different treatments.

We adopted the consistent treatment methods with public microarray data. For drought stress, the

plants were stressed by 15 min dry air stream (clean bench) until 10% loss of fresh weight, and then

incubation in closed vessels back in the illuminating incubator. For other stresses and ABA treatment,

seedlings were incubated at 4°C for cold stress, or added to a concentration of 150 mM NaCl, 300

mM mannitol, and 10 µM ABA in the solution. About 80 mg seedlings in whole plant were sampled

at 0, 1, 3, 6, and 12 h time points. Total RNA was isolated with RNasy plant mini kit (Qiagen, Hilden,

Germany) supplemented with an on-column DNA digestion (Qiagen RNase-Free DNase set; Hilden,

Germany). A 2 µg aliquot of RNA was reverse transcribed with Superscript II RT kit (Invitrogen,

USA) in a 25 µL reaction volume at 42°C for 1 h. The primers are listed in Table S10. The expression

levels of AtACT1 were served as an internal control. The suitability of the oligonucleotide sequences

in term of efficiency of annealing was evaluated in advance using the Primer 5.0 program. The cDNA

was amplified using SYBR Premix Ex TaqTM (Takara Biotechnology Co., Japan) in 10 µL volume

and analyzed using the System LightCycler480 (Roche Applied Science, Germany). The expression

level of each gene was calculated by delta-delta Ct and Ramakers et al. (2003) methods.


To assay the expression levels of various ABA-responsive genes, ABF-responsive genes and

ABF-regulated genes after ABA treatment in WT, AtSUC2 and AtSUC4 mutants, qRT-PCR analysis

was performed with RNA samples isolated from 14-day-old seedlings harvested at 6 h after the

treatments with or without 40 µM ABA. Total RNA extraction and reverse transcription were

performed as described above. The primers used for qRT-PCR are listed in Table S11 and the

procedures were adopted as described above.

To analyze the stress and ABA-induced expression of AtSUC2 and AtSUC4 in AtSUC3 mutant,

total RNA extraction, reverse transcription and qRT-PCR procedures were performed using

14-day-old Atsuc3-1 and WT seedlings harvested at 0 or 6 h. after the treatments with the different

stresses and ABA. The primers used for qRT-PCR are listed in Table S11. The ratio of AtSUC2 and

AtSUC4 expression was relative to the expression of AtSUC2 and AtSUC4 in Atsuc3-1 compared to

the genes’ expression in WT.

Screening of T-DNA insertion mutants

The T-DNA insertion lines of the AtSUC2 (At1g22710) in the Ws-2 ecotype background, AtSUC3

(At2g02860) and AtSUC4 (At1g09960) in the Col-0 ecotype background were obtained from the

Arabidopsis Biological Resource Center (ABRC). The T-DNA insertion mutant’s lines include

Atsuc2-1 (CS3876; Gottwald et al. 2000), Atsuc2-3 (CS3878; Gottwald et al. 2000), Atsuc3

(SALK_077715), Atsuc4-1 (SALK_100140) and Atsuc4-2 (SALK_021916). The mutant lines were

genotyped by amplifying the genome with the left genomic primer (LP) and right genomic primer

(RP). The primers used for this screen are listed in Table S10. The AtACT1 was used as a quantitative

control. The T-DNA insertion in mutants was confirmed by PCR and DNA gel blot analysis. All of

the PCR procedures were performed using PrimerSTART ® HS DNA polymerase (Takara

Biotechnology Co., Japan) to enhance fidelity.

Growth conditions and phenotype analysis

Plants were grown in a growth chamber at 22°C on half-strength MS medium with 0.7% (w/v) agar

(pH 6) and no exogenous sucrose or 3% exogenous sucrose at about 80 μmol photons m–2 s–1 or in

compost soil at 120 μmol photons m–2 s–1 at 22°C, a 16 h-light /8 h-night dark regime and 75%

relative humidity. For the germination assay, about 100 seeds each from WT and mutants were

sterilized and planted in triplicate on half-strength MS medium. The medium was supplemented with

and or without different concentrations of NaCl (0, 75, and 100 mM), mannitol (0, 250 and 300 mM),

or ABA (0, 1 and 2 µM) at 22, 14 (cold stress) or 10°C (cold stress). The germination (emergence of

radicals) was scored daily at the indicated times. To assay leaf area and primary root length, seeds

each from WT and mutants were planted on half-strength MS medium supplemented with different

concentrations of NaCl (0, 50 and 100 mM), or mannitol (0, 250 and 300 mM), ABA (0, 3, 5 µM or 0,

0.3, 0.5 µM) at 22, 14 or 10°C. We also examined the germination and growth, when WT and mutants


were planted on MS medium supplemented with 3% exogenous sucrose in abiotic stress and ABA

treatments. The seedling were investigated and photographed at 20 d. At least 20 seedlings in each

experiment were observed, and each experiment was repeated three times. The manipulation and

statistical analyses of data were carried out by EXCEL 2003 and SPSS statistical software 13.0.

Extraction of total soluble sugar and sucrose content determinations

Because roots were changed too short in the relatively higher concentration treatments, 20-day-old

seedlings from WT and mutant plants were chosen from the relatively lower treatments (75 mM NaCl,

250 mM mannitol, at 14°C and 0.3 µM ABA) to extract total soluble sugar according to Sahrawy et al.

(2004). Approximately 300 seedlings (3-time replication) were examined in medium with or without

exogenous sucrose in different abiotic stress and ABA treatments. The sucrose concentrations were

determined using high-performance liquid chromatography (HPLC). HPLC analysis was performed

on an Agilent 1100 HPLC system equipped with a refractive index detector (Agilent Technologies,

Santa Clara, CA, USA). The column used was the amino kind (Teknokroma, kromasil, 100-5 NH2

15×0.46 cm2, AKZONOBEL, Sweden), thermostatized at 28°C. The flow rate was set to 1.0 mL min–

1 and injections of 20 µL were made. The ratio of acetonitrile and the deionized water used was 80%

to 20%. Sucrose concentrations were determined and quantified by comparison with known standards.

Three technical replications for three biological replicates were at least performed for each sample.

The numerical data were used for statistical analyses by Microsoft Excel 2003 and SPSS statistical

software 13.0. * and ** indicates significant differences in comparison to WT at P<0.05 and P<0.01,

respectively (Student's t-test).

Results

Expression of AtSUCs in response to salt, osmotic, drought, low temperature and ABA

treatments by public microarray data and qRT-PCR analysis

The changes in expression of 9 AtSUCs family members in shoots and roots of Arabidopsis were

confirmed using a publicly available microarray data set of the AtGenExpress expression atlas in

abiotic stresses. In shoots, salt, osmotic, drought and low temperature stresses promoted the

expression of AtSUC2 and AtSUC4 to levels 1.5 greater than the control at most treatment periods

(Table S2, S4). In some treatments, such as salt stress at 3 h, the expression of AtSUC2 and AtSUC4

increased in excess of 3 fold, compared with the control (Table S2, S4). The expression of AtSUC1

and AtSUC5 decreased below 0.6-fold with most treatments, and especially AtSUC5 decreased below

0.3-fold in most treatments (Table S1, S5). The expressions of AtSUC3, AtSUC6 and AtSUC7 were

also induced by the stresses at specific treatment times, whereas no significant changes in expression

were seen for AtSUC8 and AtSUC9 for any stress treatment (Table S3, S6, S7, S8, S9). In roots,

AtSUC2 and AtSUC4 increased significantly following the four stress treatments at all times, except

cold stress at 0.5, 1, and 3 h. In particular, in salt stress, the expression of AtSUC2 at 24 h increased by


11.86 fold (Table S2, S4). The expression of AtSUC3 increased in excess of 1.5 fold in most

treatments, especially early treatment in salt, drought and cold stress (Table S3). The relative

expression of AtSUC1 fluctuated, showing high expression for some stress treatments and low

expression for others. Few significant changes were noted for the other AtSUCs (Table S1–9).

In general, AtSUC2 and AtSUC4 responded to salt, osmotic, drought and low temperature

stresses by increased expression in both shoots and roots. Interestingly, the changes in expression for

AtSUC2 and AtSUC4 were similar in most of the treatments (Table S2, S4; Fig. 1A, B). For example,

the expression of AtSUC2 and AtSUC4 in shoots under cold stress increased gradually from 0 h to 6 h,

and then decreased at 12 h, followed by a continuous rise until 24 h (Table S2, S4; Fig. 1A). We

further investigated the expression of AtSUC2 and AtSUC4 by qRT-PCR in WT under abiotic stresses.

The expression of AtSUC2 and AtSUC4 responded to almost all treatments and the results analyzed by

qRT-PCR were similar to those found using the microarray data (Fig. 1A–E). Therefore, AtSUC2 and

AtSUC4 may be key genes in response to abiotic stresses and we focused on these two genes for our

subsequent investigations.

Phytohormones are known to play important roles in promoting abiotic stress tolerance. We

investigated the expression of AtSUC2 and AtSUC4 in response to application of five phytohormones:

1-aminocyclopropane-1-carboxylate (ACC), gibberellic acid (GA), indol-3-acetic acid (IAA), ABA,

and zeatin (0, 0.5, 1 and 3 h) by microarray analysis (Table S10). The expressions of AtSUC2 and

AtSUC4 increased significantly in only ABA treatment, and reached a maximum at 3 h, at 3.06 and

5.48 fold respectively, compared with the control (Table S10; Fig. 1F). The changes in AtSUC2 and

AtSUC4 expression were similar (Fig. 1F). The expression of AtSUC2 and AtSUC4 in WT under ABA

treatment (0, 1, 3, 6 and 12 h) was also investigated by qRT-PCR and showed results similar to those

obtained by the microarray analysis: the expression of AtSUC2 and AtSUC4 increased with increasing

ABA treatment time and reached a maximum at 12 h, at 7.86 and 4.66 times, respectively (Fig. 1F).

Identification of T-DNA insertion mutants of AtSUC2, AtSUC3 and AtSUC4

For further examination, we isolated some T-DNA insertion alleles: Atsuc2-1 (CS3876), Atsuc2-3

(CS3878), Atsuc3 (SALK_077715), Atsuc4-1 (SALK_100140), and Atsuc4-2 (SALK_021916) from

the pool of T-DNA insertion mutants in the ABRC. We identified that a homozygous T-DNA

insertion allele, Atsuc2-1, had an insertion within the second exon of AtSUC2 on chromosome 1, and

that Atsuc2-3 contained a single insertion in the second intron on chromosome 1 (Fig. 2A). AtSUC3

mutant was inserted into the promoter at the upstream of the ATG on chromosome 2 (Fig. 7A).

Atsuc4-1 was inserted in the promoter from –799 to –419 bp 5’ upstream of the translation start codon

on chromosome 1 (Fig. 2B). A single copy of T-DNA was inserted into the promoter of the AtSUC4-2

mutant at –326 to –44 bp upstream of the ATG on chromosome 1 (Fig. 2B). We confirmed that

Atsuc2-1, Atsuc2-3, Atsuc3, Atsuc4-1 and Atsuc4-2 were all transcript null mutants by performing

reverse transcription PCR (RT-PCR) analysis with RNA isolated from WT and mutant plants. The


five mutants did not produce their corresponding RT-PCR products under the growth conditions

where WT plants yielded normally AtSUC2, AtSUC3 and AtSUC4 mRNA (Fig. 2C, 7B). In contrast,

there was AtSUC2 transcription in Atsuc4-1 and Atsuc4-2; likewise, there was AtSUC4 transcription in

Atsuc2-1 and Atsuc2-3 (Fig. 2C).

Earlier work described AtSUC2 mutants (Atsuc2-1 and Atsuc2-3) grew smaller than WT and

cannot produce viable seed (Gottwald et al. 2000). However, the developmentally blocked AtSUC2

mutant seedlings can be partially rescued by the addition of sucrose (Gottwald et al. 2000). Recently

Srivastava et al. (2009b) thought that plants can complete their life cycle and produce viable seed in

the absence of AtSUC2, when they grew in 14 h light/10 h dark cycles instead of 24 h illumination

using Gottwald and colleagues, as SUCs play a crucial role in all-day 24 h light, implying continuous

photosynthesis. We also found that shorter light cycles can improve the growth of mutants (data not

shown). Given these findings, we examined growth and seed-setting percentages of AtSUC2 mutants

for a 24-h illumination cycle compared with these percentages for the altered condition of a 16 h

light/8 h dark cycle with extraneous sucrose given in 0 day and 20 day treatments. We found that

AtSUC2 mutants grew well and produced viable seeds in the altered condition, which is the opposite

of the results described by Gottwald (Gottwald et al. 2000) (Table S13; Fig. S3). Moreover, the next

generation of homozygous AtSUC2 mutants was shown to be homozygous plants by PCR (Fig. S4). In

our study, the homozygous AtSUC2 mutants were obtained.

Phenotype of AtSUC2 and AtSUC4 mutants to salt, osmotic, low temperature stresses and

exogenous ABA during seed germination and seedling growth

We investigated seed germination and vegetative growth of WT and mutant plants under various

stress conditions and ABA treatments without exogenous sucrose (0–120 h or 144 h for cold stress,

data after the longest time showed no obvious changes and are not reported). In the controlled

condition, the germination percentage was lower for Atsuc2-1 and Atsuc2-3 seeds compared to WT

seed from 24 h to 120 h. Atsuc4-1 and Atsuc4-2 seeds compared to WT seed germinated lower in 48 h

and 72 h treatments and similar in 24 h and 120 h (Fig. 3A–H). In all treatments, the germination

percentage of different AtSUC2 and AtSUC4 mutant alleles was similar (Fig 3A–H). The difference

between WT and the four mutants was more obvious as the stress strength and treatment

concentrations increased. For example, germination of Atsuc2-1 and Atsuc2-3 was 86.73±2.12% and

83.44±1.98%under the control condition and 54.61±1.17% and 56.53±0.86%with 2 µM ABA

treatments in 120 h, whereas the germination of Atsuc4-1 and Atsuc4-2 changed from 94.93±0.87%

and 95.45±1.65% to 56.45±0.62% and 59.5±0.43% during use of the same treatments (Fig. 3C, D).

The length of the roots and the area of leaves were examined in the different treatments for analyzing

the phenotype of vegetative growth. Under the normal condition, differences were observed between

WT and mutant plants, as the AtSUC2 and AtSUC4 mutant seedlings had shorter roots, and smaller

leaves than WT (Fig. 4A, C). In all treatments, the growth phenotype of different AtSUC2 and


AtSUC4 mutant alleles was similar (Fig 4. A, C). These phenotypic differences became more

significant to abiotic stresses and exogenous ABA (Fig. 4A, C). In particular, root development of

AtSUC2 mutants was seriously inhibited in the media supplemented with 5 µM ABA (Fig. 4A, C).

The original strength of the plant can enhance plant tolerance abiotic stresses. In our result,

obvious differences were observed between WT and mutant plants under the control condition,

mainly in regard to the effect of deletion of SUC under the normal on the transportation of source to

sink. To test whether the original small growth of mutants is the main factor in their hypersensitivity

to abiotic stress and ABA treatments, we added a solution with 3% exogenous sucrose to meet the

demands of sucrose in roots. Our results showed that the mutants grew well and had smaller

differences than WT in a normal condition with 3% exogenous sucrose (Fig. 4B; Fig. S2). However,

in abiotic stress and ABA treatments with exogenous sucrose, the germination and growth of the four

mutants were significantly inhibited (Fig. 4B; Fig. S2), which suggests that the four mutants were

hypersensitive to abiotic stress and ABA treatments though they didn’t grow small under normal

conditions. Especially, the etiolation of AtSUC2 mutants, signifying death, was observed in relatively

higher concentration of salt, mannitol with 3% exogenous sucrose (Fig. 4B). AtSUC2 mutants were

hypersensitive in 0.5 µM ABA treatment with exogenous sucrose, while the hypersensitivity appeared

at a relatively higher concentrate of ABA—5 µM ABA without exogenous sucrose, suggesting

AtSUC2 and AtSUC4 mutants were hypersensitive in ABA treatments with exogenous sucrose than

without it.

Sucrose distribution of AtSUC2 and AtSUC4 mutants in shoots and roots in response to salt,

osmotic, low temperature and exogenous ABA treatments

Our previous data of sucrose content of AtSUC4 mutants was obtained with a stress solution of 3%

exogenous sucrose (Gong et al. 2013a, b). We measured the sucrose contents of WT and the four

mutants following stress and ABA treatments with and also without exogenous sucrose. These results

showed that disruption of AtSUC2 and AtSUC4 affected the sucrose balance between sources in the

normal condition without or with exogenous sucrose, as sucrose contents were higher in shoots and

lower in roots in AtSUC2 and AtSUC4 mutants than WT (Fig. 5 A–D). While the extent of the sucrose

imbalance increased under abiotic stresses and ABA treatments (Fig. 5 A–D). The data suggested that

AtSUC2 and AtSUC4 are required for maintaining the sucrose balance for plant tolerance to abiotic

stresses. Interestingly, there was still an obvious sucrose imbalance in the mutants in abiotic stress

treatments with 3% exogenous sucrose, especially the increasing extent of sucrose contents in shoots

of the mutants in almost all treatments, which should be a main reason of hypersensitivity of mutants

in response to abiotic stress treatments with exogenous sucrose (Fig. 5 A–D).

Expression of stress-responsive and ABA-responsive genes in AtSUC2 and AtSUC4 mutants

The ABA response mechanism that leads to abiotic stress tolerance in Arabidopsis thaliana was


clarified by qRT-PCR analysis of the expression of the following stress-responsive and

ABA-responsive genes in WT and AtSUC2 and AtSUC4 mutants following ABA treatments: ABFs

(ABF1, ABF2/AREB1, ABF3, and ABF4/AREB2) (Choi et al. 2000, Uno et al. 2000); ABI2 (Leung et

al. 1997); RD29A (Yamaguchi-Shinozaki and Shinozaki 1994); MYB2 (Abe et al. 2003); KIN1 and

KIN2 (Kurkela and Borg-Franck 1992); CBL9 (Pandey et al. 2004) and ERD10 (Kiyosue et al. 1994).

In the absence of the ABA treatment, expression of some ABA-responsive genes, including ABF1,

ABF3, ABF4, ABI2 and KIN2 were a bit high in the mutants compared with WT, while CBL9 and

RD29A were lower (Fig. 6). Treatment with ABA for 6 h induced higher expression of almost all of

the genes in WT than in the mutants, except for ABI2 and CBL9 (Fig. 6), which were considered to be

negative ABA signaling regulators. These findings suggested that disruption of AtSUC2 and AtSUC4

inhibited most positive ABA regulators and activated some negative ABA regulators. Notably, the

expression level of the four ABFs clearly decreased in the mutants compared to WT following ABA

treatments (Fig. 6).

Expression of ABF related genes in AtSUC2 and AtSUC4 mutants

The significant inhibition of ABA activated expression of ABFs, by disruption of AtSUC2 and

AtSUC4 led us to further examine whether AtSUC2 and AtSUC4 are involved in ABF-dependent ABA

signaling by analyzing the expression levels of ABF-downstream genes (ABI1, CHS, KIN2, RBCS,

RD29A and RD29B) and -upstream genes in WT and AtSUC2 and AtSUC4 mutants. In the absence of

ABA treatments, expressions of most genes were similar in the mutants compared with WT, except

ABI2, RD29A and KIN2 were higher and RBCS were lower (Fig. 6). The ABA treatments

up-regulated ABI1, CHS, KIN2, RD29A and RD29B genes to a greater degree in WT than in AtSUC2

and AtSUC4 mutants (Fig. 6), which suggested that disruption of AtSUC2 and AtSUC4 inhibited most

ABF-activated genes. However, the expression of RBCS, which is inhibited by ABF2, was

significantly higher in the AtSUC2 and AtSUC4 mutants than in WT following ABA treatments (Fig.

6), suggesting that disruption of AtSUC2 and AtSUC4 restored the expression of the ABF impeded

genes.

We further identified the response mechanism of ABFs by analyzing the expression of

ABF-upstream genes, including SnRK2.2, SnRK2.3 and SnRK2.6 in WT and in AtSUC2 and AtSUC4

mutants. The ABA treatment induced the expression of SnRK2.2, SnRK2.3 and SnRK2.6 in WT but

this expression was significantly lower in the AtSUC2 and AtSUC4 mutants than in WT (Fig. 6),

suggesting that disruption of AtSUC2 and AtSUC4 inhibited ABA induced expression of three SnRK2

genes.

Abiotic stresses and ABA-induced expression of AtSUC2 and AtSUC4 in AtSUC3 mutant

AtSUC3, as a postulated sucrose sensor, interacts with AtSUC2 and AtSUC4 in a split ubiquitin

system (Barker et al. 2000, Reinders et al. 2002, Schulze et al. 2003). We clarified whether AtSUC2


and AtSUC4 were involved in sucrose signaling in response to abiotic stresses and ABA by

investigating AtSUC2 and AtSUC4 expression in Atsuc3 under normal and abiotic stress conditions.

We found that disruption of AtSUC3 weakened the increase in AtSUC2 and AtSUC4 expression

induced by abiotic stresses and ABA. We compared expression levels of AtSUC2 and AtSUC4 in

Atsuc3 to WT under abiotic stress and ABA treatments and found that the ratio of AtSUC2 and

AtSUC4 expression (expression of AtSUC2 or AtSUC4 in the AtSUC3 mutant compared to the

expression in WT) under the normal condition were 1.38±0.03 and 1.32±0.03 respectively (expression

of AtSUC4 in the AtSUC3 mutant relative to the expression in WT) (Fig. 7C), which suggested that

disruption of AtSUC3 increased the relative expression of AtSUC2 and AtSUC4 under the normal

condition. After 6 h of abiotic stress and ABA treatments, the ratios of the two gene expression were

clearly down-regulated compared with the control condition, suggesting that disruption of AtSUC3

decreased the expressions of AtSUC2 and AtSUC4 induced by abiotic stresses and ABA (Fig. 7C).

Especially, ABA treatments significantly reduced ratios of AtSUC2 and AtSUC4 expression compared

with the corresponding ratios under abiotic stresses (Fig. 7C), which suggested that disruption of

AtSUC3 substantially decreased ABA-induced AtSUC2 and AtSUC4 expression.

Discussion

Sucrose transporters (SUCs) play pivotal roles in phloem loading of sucrose in source organs and

unloading of sucrose in sink organs (Kühn and Grof 2010). Some SUCs respond to salinity, drought

and low temperature stresses as indicated by the changes in expression of such genes as AgSUT1

(Noiraud et al. 2000), OsSUT2 in the rice (Oryza sativa) (Ibraheem et al. 2011) and PtaSUT4 (Frost et

al. 2012). Our previous work only examined the change of sucrose distribution in AtSUC4 mutants

under abiotic stresses, suggesting AtSUC4 is involved in response to abiotic stresses (Gong et al.

2013a,b), but the mechanism of AtSUC4 is known little in plant tolerance abiotic stress. In the present

study, the results demonstrate a critical role for a sucrose transporter encoded by AtSUC2 and

AtSUC4 in abiotic stress tolerance and ABA response. The expression of ABFs, key transcriptional

activators for ABA-responsive genes is largely dependent on AtSUC2. Interestingly, the deletion of

AtSUC2 and AtSUC4 caused sucrose accumulation in shoots and less sucrose in roots under abiotic

stresses and ABA treatments, but the medium adding 3% exogenous sucrose didn’t relieve the

mutants’ hypersensitivity to abiotic stress and ABA. These observations suggest that there might be a

complex network of ABA-responsive sucrose transporters involved in stress and ABA responses.

AtSUC2 and AtSUC4 are important for plant tolerance under abiotic stresses

Previous studies found that PtaSUT4 was thought to be responsible for sucrose export from the

vacuole into the cytoplasm, and transformation with PtaSUT4-RNAi leads to a substantial sucrose

accumulation in the vacuole. This then affects the water balance so that PtaSUT4-RNAi plants wilt

even in short-term drought (Frost et al. 2012). Here, we found that sucrose contents were higher in


shoots and lower in roots in AtSUC2 and AtSUC4 mutants than WT under abiotic stress conditions,

and we suggest that disruption of AtSUC2 and AtSUC4 causes sucrose accumulation in source organs,

possibly by interfering with phloem loading of sucrose in source organs. Examination of 9 AtSUCs

showed that AtSUC2, AtSUC3 and AtSUC4 were expressed in the minor veins of leaves of mature

plants, suggesting that they are probably involved in sucrose loading in source leaves (Truernit and

Sauer 1995, Barker et al. 2000, Weise et al. 2000). Among them, AtSUC2 has been confirmed as a

main SUC responsible for phloem loading at the whole plant level under the normal condition

(Srivastava et al. 2008, Gottwald et al. 2000). In contrast, AtSUC4 has not yet been assigned a role in

phloem loading at the whole plant level under the normal condition but it has been confirmed to load

sucrose in yeast (Weise et al. 2000). Weise et al. (2000) considered that the differentially regulated

expression of AtSUC4 and the SUT1-clade of Arabidopsis in SEs could provide a mechanism by

which plants could modulate the rate of export of sucrose from source leaves. We considered that

AtSUC2 and AtSUC4 could be important in phloem loading of sucrose under abiotic stress conditions.

Sucrose transport during stresses may be much more complicated than in normal conditions, as

sucrose transport from source to sink needs to occur promptly, for example, sufficient sucrose can

accumulate in the roots to serve as an osmolyte. Therefore, sucrose transporters with different

characteristics could be involved in plant abiotic stress tolerance. AgSUT1 that belongs to the

SUT1-clade is induced by abiotic stresses, as are OsSUT2 and PtaSUT4, which encode proteins

belonging to the SUT4-clade (Doidy et al. 2012), suggesting that SUCs with different characteristics

are activated under abiotic stress conditions.

AtSUC4 is located on the plasma membrane and/or vacuolar membrane so it may transport

sucrose from the vacuole to the cytoplasm (Weise et al. 2000, Endler et al. 2006). PtaSUT4 plays role

in sucrose export from the vacuole into the cytoplasm. In long-term drought, PtaSUT4 expression

decreased and sucrose levels increased in WT plants, whereas leaf sucrose concentrations in the RNAi

plants nearly matched those in WT plants. Our results showed that AtSUC4 was induced and

disruption of AtSUC4 led to higher leaf sucrose concentrations than in WT, similar to what was seen

in AtSUC2 mutants, following long-term abiotic stresses (Frost et al. 2012). In our opinion, the main

role of AtSUC4, and probably AtSUC2 as well, is to regulate the process of sucrose loading in the

phloem by plasma membrane transport under abiotic stress conditions.

AtSUC2 and AtSUC4 were induced in roots by abiotic stresses (Fig. 1B). Furthermore, the

sucrose contents were significantly lower in the roots of mutants than in WT under stress conditions

(Fig. 5A–F). The results indicated that AtSUC2 and AtSUC4 play roles in roots. At present, a role for

AtSUC2 and AtSUC4 in unloading sucrose in roots has not been established. Recently, sucrose

unloading in planta likely was regulated by SWEET proteins rather than SUCs (Chen et al. 2012).

Therefore, we speculate that AtSUC2 and AtSUC4 probably play a role in retrieval of escaped

sucrose bake into the phloem to regulate sucrose distribution in sink under abiotic stress conditions.

However, when a solution with 3% exogenous sucrose meets the demands of sucrose in roots, the


results showed that the four mutants were more hypersensitive to abiotic stress and ABA treatments

though they didn’t grow small under normal conditions (Fig. 4B; Fig. S2), suggesting AtSUC2 and

AtSUC4 probably play a less important role in roots in abiotic stress.

AtSUC2 and AtSUC4 are involved in an ABF-dependent ABA signaling pathway

Adaptation to the abiotic stresses is regulated by synergistic activity of interconnected

ABA-dependent and ABA-independent signaling pathways (Shinozaki et al. 2003,

Yamaguchi-Shinozaki and Shinozaki 2005). We found that, of the five phytohormones tested (ABA,

ACC, GA, IAA and zeatin) (Supplementary Tab. S1), only ABA could induce a high level of

expression of AtSUC2 and AtSUC4 (Fig. 1D). We also found that AtSUC2 and AtSUC4 mutants are

hypersensitive to ABA (Fig. 3, 4), similar to findings for the AP2-like ABA repressor 1 (ABR1) gene

involved in salt tolerance and CIPK3, a calcium sensor-associated protein kinase gene involved in

cold signal transduction (Kim et al. 2003, Pandey et al. 2005). In addition, our results indicate that the

sucrose distribution in AtSUC2 and AtSUC4 mutants is similar between abiotic stress and ABA

treatments (Fig. 5), so that AtSUC2 and AtSUC4 appear to play roles in plant stress tolerance by

interaction with ABA dependent signaling pathways. Sucrose is metabolized into two

monosaccharides: fructose and glucose (Oliver et al. 2005). Oliver et al. (2007) found that ABA

regulates the process of monosaccharide transport in anthers under cold stress. Our result confirms

that an ABA signaling pathway may be involved in sucrose transport under abiotic stress conditions,

and that AtSUC2 and AtSUC4 are important factors.

The key mechanism for ABA dependent signaling pathways in plants changes in ABA-regulated

gene expression (Dekkers et al. 2008, Abdeen et al. 2010). In this case, plant hypersensitivity to ABA

in seed germination and seedling growth is accompanied by lower expression of most ABA relative

genes such as ABFs, RD29A, KIN1, KIN2 and MYB2 (Fig. 6) under ABA treatment. Similar results

were also reported for ABO3 (Ren et al. 2010).

The ABF gene products in Arabidopsis have been identified as 4 family members: ABF1,

ABF2/AREB1, ABF3 and ABF4/AREB2. Each of these gene products appears to participate in

various ABA-mediated responses to abiotic stresses including cold, salt, heat, drought, and oxidative

stresses (Choi et al. 2000, Uno et al. 2000, Abdeen et al. 2010). In the present study, we found that

many ABF- and stress-responsive genes, such as RD29A, RD29B, ABI1 and CHS, are inhibited in the

AtSUC2 and AtSUC4 mutants compared with WT (Fig. 6). Only RBCS, which is down-regulated by

ABF2 (Kim 2006), is inhibited significantly in WT compared with mutants following ABA treatments

(Fig. 6). We also examined the expression of SnRK2.2, SnRK2.3, and SnRK2.6, as the upstream

regulators of ABFs (Boudsocq et al. 2004, Fujita et al. 2013, Furihata et al. 2006, Yoshida et al. 2006),

in WT and AtSUC2 and AtSUC4 mutants following ABA treatment. The expression of SnRK2.2,

SnRK2.3, and SnRK2.6 under ABA treatment decreased in AtSUC2 and AtSUC4 mutants compared to

WT, suggesting that AtSUC2 and AtSUC4 can affect SnRK2.2, SnRK2.3 and SnRK2.6 involved in


ABA signaling (Fig. 6). SnRK2.6 has a role in stomatal opening and closing in response to ABA

regulation during abiotic stresses (Mustilli et al. 2002, Yoshida et al. 2002, Assmann 2003). SnRK2.6

is expressed in the vascular system of leaves (Zheng et al. 2010), which is the same location as

AtSUC2 and AtSUC4 (Weise et al. 2000, Srivastava et al. 2008), suggesting a potential interaction

with SUCs. Our results showed that the expression of SnRK2.6 was markedly inhibited in the AtSUC2

and AtSUC4 mutants. AtSUC2 and AtSUC4 therefore may have a direct relationship with the role of

SnRK2.6. Therefore, AtSUC2 and AtSUC4 may indirectly control ABFs expression, through other

factors such as SnRK2s or signals.

AtSUC2 and AtSUC4 may be involved in sucrose signaling pathway

Sugars, including sucrose, act as signals in plant stress tolerance (Yu 1999, Gupta and Kaur 2005,

Ruan et al. 2010). Sucrose-specific signaling pathways are involved in controlling sucrose transport

activity (Chiou and Bush 1998). Our results indicated that AtSUC2 and AtSUC4 affected the

expression of SnRKs, which are involved in sucrose signaling in wheat (Coello et al. 2012). Therefore,

AtSUC2 and AtSUC4 may be upstream factors and induced by sucrose signaling. Moreover, AtSUC3

might be responsible for the retrieval of sucrose into the SE along the phloem path (Meyer et al. 2004).

However, it as a unique putative sucrose sensor in Arabidopsis (Barker et al. 2000, Schulze et al.

2003), suggesting that it may act as a sucrose sensor under some environmental signals. The

expression atlas indicated that AtSUC3 in the root is induced by a majority of abiotic stress treatments,

and that the induction of AtSUC3 occurs at an earlier period (Table S3), suggesting that it may

participate in the early events of the adaptation of plant to environment stresses. Because abiotic

stresses can induce changes in sucrose concentrations or in fluxes of sucrose loading, AtSUC3 might

modulate the activity of other sucrose transporters during phloem loading by sensing the changes in

sucrose signals occurring under abiotic stress conditions. So far, only expression of AtSUC2, AtSUC3

and AtSUC4 has been shown in minor veins of source leaves (Barker et al. 2000, Weise et al. 2000,

Srivastava et al. 2008, Gould et al. 2012), and these are co-localized in the same enucleate sieve

element (Reinders et al. 2002). Meanwhile, AtSUC3 interacts with AtSUC2 and AtSUC4 in

yeast-based split ubiquitin system (Reinders et al. 2002, Schulze et al. 2003), suggesting that AtSUC2,

AtSUC3 and AtSUC4 may have a close relationship in phloem loading. Disruption of AtSUC3

inhibits the stress-induced expression of AtSUC2 and AtSUC4. Further, our results showed that the

mutants grew well and had smaller differences than WT under a normal condition with 3% exogenous

sucrose (Fig. 4B; Fig. S2). However, in abiotic stress and ABA treatments with exogenous sucrose,

the germination and growth of the four mutants were significantly inhibited (Fig. 4B; Fig. S2),

AtSUC2 and AtSUC4 are unlikely to play a role as a nutritional regulation in stress tolerance. These

results suggest that stress-induced AtSUC2 and AtSUC4 might be regulated by AtSUC3 and that

AtSUC2 and AtSUC4 are involved in sucrose signaling under abiotic stress conditions.


Acknowledgements – The authors would like to thank the financial support from the National Natural

Science Foundation of China (No. 31170232, 31070157, 31370283 and 31000674), the China

Postdoctoral Science Foundation (No. 2012T5025, 320110490145), the Excellent Talents Foundation

of Liaoning Province (No. LJQ2013073), and the Opening Project of State Key Laboratory of

Shenyang Agricultural University.

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Edited by Z. Gong


Figure legends

Fig. 1. The expression of AtSUC2 and AtSUC4 under abiotic stress and ABA treatments analyzed by

Microarray and qRT-PCR. The expression of AtSUC2 and AtSUC4 in shoots (a) and roots (b) were

analyzed under 4 stresses at 0, 0.5, 1, 3, 6, 12 and 24 h by Microarray. Moreover, the expression of

AtSUC2 and AtSUC4 under abiotic stresses at 0, 1, 3, 6 and 12 h was analyzed in shoots (c), roots (d)

and whole plant (e) by qRT-PCR. The expression of AtSUC2 and AtSUC4 under ABA treatments was

analyzed in whole plant by Microarray and qRT-PCR (f). The expression levels are presented as

relative units with the levels of stress-free treated (0 h) WT seedlings being taken as 1.


Fig. 2. Molecular analysis of AtSUC2 and AtSUC4 T-DNA insertion mutants. (A) T-DNA insertion

sites in Atsuc2-1 and Atsuc2-3. LB, Left border of the T-DNA; RB, right border of the T-DNA; LB

A3, Left border primer for T-DNA; LP1 and RP1, left and right genomic primers for the gene,

respectively. (B) T-DNA insertion sites in Atsuc4-1 and Atsuc4-2. LP3 and RP3 were for Atsuc4-1

primers; P4 and RP4 for Atsuc4-2 primers. (C) RT-PCR analysis of AtSUC2 and AtSUC4 expression

in WT and mutants. The AtACT1 was used as a quantitative control.


Fig. 3. Seed germination of AtSUC2 or AtSUC4 mutants under abiotic stresses and ABA without

exogenous sucrose (A to H). The germination percentage of AtSUC2 mutants (A,C, E, G) and AtSUC4

mutants (B, D, F, H) was recorded during a period from 1 d to 5 d or 6 d (for cold stress) after

stratification under NaCl, Mannitol, or ABA at 22°C or low temperature. Each value is the mean ±SE

from 100 seeds for each of three independent biological determinations. *p < 0.05 versus control; **p

< 0.01 versus control; n = 3.


Fig. 4. Hypersensitive phenotypes of AtSUC2 or AtSUC4 mutants under abiotic stresses and ABA. (A)

Photographs of 20-day-old seedling of AtSUC2 or AtSUC4 mutants were taken under abiotic stress

and ABA treatments without exogenous sucrose. Bar=1 cm.(B) Photographs of 20-day-old seedling of

AtSUC2 or AtSUC4 mutants were taken under abiotic stress and ABA treatments with 3% exogenous

sucrose. Bar=1 cm. (C) Leaf area and root length of AtSUC2 or AtSUC4 mutants were examined

under abiotic stresses and ABA without exogenous sucrose. Each value is the mean ±SE from 100

seeds for each of three independent biological determinations. *p < 0.05 versus control; **p < 0.01

versus control; n = 3.


Fig.5. Changes on sucrose content of AtSUC2 or AtSUC4 mutants under abiotic stresses and ABA.

Sucrose content in shoots (A) or roots (B) of 20-day-old seedling of AtSUC2 mutants was measured

under abiotic stress and ABA treatments with or without exogenous sucrose. Sucrose content in

shoots (C) or roots (D) of 20-day-old seedling of AtSUC4 mutants was measured under abiotic stress

and ABA treatments with or without exogenous sucrose. *p < 0.05 versus control; **p < 0.01 versus

control; n = 3. –Suc, sucrose-free treatment; +Suc, 3% exogenous sucrose treatment.


Fig. 6. qRT-PCR analysis of expression of stress- and ABA-responsive genes in AtSUC2 and AtSUC4

mutants in ABA treatment without exogenous sucrose. The expression levels are presented with the

levels of ABA-free treated WT being taken as 1. –ABA, ABA-free treatment; +ABA, 40 μM ABA.


Fig.7. Disruption of AtSUC3 inhibited increases in AtSUC2 and AtSUC4 expression induced by

abiotic stresses and ABA. (A) T-DNA insertion sites in Atsuc3. LP2 and RP2 were left and right

genomic primers for the gene, respectively. (B) RT-PCR analysis of AtSUC3 expression in WT and

homozygous mutants Atsuc3. The Actin was used as a quantitative control. (C) The ratio of AtSUC2

and AtSUC4 expression in Atsuc3 compared to in WT under abiotic stress and ABA treatments. The

whole plants from 14-day-old WT seedlings were treated with different treatments for 0 and 6 h. The

ratio of AtSUC2 expression was relative expression of AtSUC2 in the AtSUC3 mutant compared to

expression in WT, while the ratio of AtSUC4 expression was relative expression of AtSUC4 in the

AtSUC3 mutant compared to expression in WT. The value obtained from the stress- or ABA-free

treated (0 h) ratio after stratification was taken as 1.

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