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Running title: ABL1 regulates ABA and auxin responses

For whom correspondence should be addressed.

Prof. Hong-Wei Xue

300 Fenglin Road, 200032 Shanghai, P.R.China

Tel.: +86-21-54924059 Fax: +86-21-54924060 Email: [email protected]

Journal research area: Hormone section

Plant Physiology Preview. Published on May 5, 2011, as DOI:10.1104/pp.111.173427

Copyright 2011 by the American Society of Plant Biologists

www.plantphysiol.orgon October 16, 2020 - Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved.

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TITLE:

Rice ABI5-like1 Regulates ABA and Auxin Responses by Affecting the Expression of

ABRE-Containing Genes

Xi Yang1,#, Ya-Nan Yang1,#, Liang-Jiao Xue1, Mei-Juan Zou2, Jian-Jing Liu2, Fan

Chen2, Hong-Wei Xue1,* 1SIBS (Shanghai Institutes for Biological Sciences)-UC Berkeley (University of

California, Berkeley) Joint Center on Molecular Life Sciences, National Key Laboratory

of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai

Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032,

China 2National Centre for Plant Gene Research, Key Laboratory of Molecular and

Developmental Biology, Institute of Genetics and Developmental Biology, Chinese

Academy of Sciences, P.O. Box 2707, South 1-3, Zhongguancun, Beijing 100080, PR

China

# these authors contribute equally to the study.



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FINANCIAL SOUCE

The study was supported by the National Science Foundation of China (90717001,

30721061), Chinese Academy of Sciences (KSCX2-YW-N-016), Science and

Technology Commission of Shanghai Municipality (08XD14049).



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ABSTRACT

Abscisic acid (ABA) regulates plant development and is crucial for plant responses to

biotic and abiotic stresses. Studies have identified the key components of ABA signaling

in Arabidopsis thaliana, some of which regulate the ABA responses by the

transcriptional regulation of downstream genes. Here we report the functional

identification of rice (Oryza sativa) ABI5-like1 (ABL1), which is a bZIP transcription

factor. ABL1 is expressed in various tissues and is induced by the hormones ABA and

IAA and stress conditions including salinity, drought and osmotic pressure. The ABL1

deficiency mutant, abl1, shows suppressed ABA responses, and ABL1 expression in the

Arabidopsis abi5 mutant rescued the ABA sensitivity. The ABL1 protein is localized to

the nucleus and can directly bind ABRE (G-box) elements in vitro. A gene expression

analysis by DNA chip hybridization confirms that the large proportion of down-regulated

genes of abl1 is involved in stress responses, being consistent with the transcriptional

activating effects of ABL1. Further studies indicate that ABL1 regulates the plant stress

responses by regulating a series of ABRE-containing WRKY family genes. In addition,

the abl1 mutant is hypersensitive to exogenous IAA, and some ABRE-containing genes

related to auxin metabolism or signaling are altered under ABL1 deficiency, suggesting

that ABL1 modulates ABA and auxin responses by directly regulating the

ABRE-containing genes.

Key words: ABI5-like1 (ABL1), ABA-response elements (ABRE), G-box, auxin, rice,

WRKY



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INTRODUCTION

Abscisic acid (ABA) is an important plant hormone that affects many aspects of plant

growth and developmental processes including cell division, seed maturation and

germination, seedling development, stomata opening and leaf senescence (Axel et al.,

2003), and plays a crucial role in plant responses to stresses. ABA serves as an

endogenous signal to initiate the adaptive responses when plants are challenged by

abiotic stress (Zhu, 2002) and biotic stress (Adie et al., 2007).

Insights into the signal transduction of ABA have unfolded dramatically in the past

few years and revealed an unanticipated complexity. In Arabidopsis thaliana, several

candidate receptors have been identified by their high-affinity binding to ABA. Recently,

a 14-member family of intracellular ABA receptors, named PYR/PYL/RCAR (Park et al.,

2009; Ma et al., 2009; Santiago et al., 2009), was identified. However, no ABA receptor

in monocot rice (Oryza sativa) has been reported yet.

Genetics studies have helped to identify the key components of ABA signaling. Five

ABA Insensitive members (ABI1-5) have been identified by genetic screens as the

deficiency of corresponding genes results in the insensitive responses of seed

germination to exogenous ABA. Transcriptome studies by DNA chip analysis revealed

the dramatic changes in the expression of thousands of genes after ABA treatment and

indicated the crucial roles of transcriptional regulation in ABA signaling (Hoth et al.,

2002; Seki et al., 2002). ABI3, ABI4 and ABI5 are all transcription factors

[VIVIPAROUS1 (VP1), APELATA2 (AP2), and basic region/leucine zipper motif (bZIP)

types, respectively] and are involved in the regulation of ABA-mediated gene expression

(Giraudat et al., 1992; Finkelstein et al., 1998; Finkelstein and Lynch, 2000). Arabidopsis

ABI4 and maize (Zea mays) ZmABI4 bind the dehydration-responsive element (DRE)

that acts as a second cis element or ‘coupling element’ (CE) to assist with

ABA-controlled gene expression (Niu et al., 2002; Narusaka et al., 2003). ABI3

represents an accessory enhancer of transcription and forms a complex with ABI5 to

regulate the expression of downstream genes (Nakamura et al., 2001). Arabidopsis ABI5

and rice bZIP transcription factor TRAB1 interact with ABA-responsive elements

(ABREs, ACGT-containing ‘G-boxes’ in the promoter region, Hattori et al., 2002) to

activate a series of downstream events. The activities of ABI5 and TRAB1 are regulated

by phosphorylation (Kagaya et al., 2002) and ABI5 can be stabilized by blocking the ABI



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FIVE BINDING PROTEIN (AFP)-mediated protein degradation through the 26S

proteasome (Lopez-Molina et al., 2003).

Comprehensive analyses employing microarrays are very helpful for studying the

molecular mechanisms, especially when the functional clues are limited. Several groups

have tried to analyze the gene expression profiling using the Arabidopsis abi5 mutant or

ABI5-overexpressing seedlings, including dry seeds (Nakabayashi et al., 2005;

Nakashima et al., 2009) and seedlings after ABA treatment at multiple time points

(Dissertation of Hur, 2007). However, the detailed mechanisms of ABI5 function are still

insufficient.

Plant bZIP transcription factors contain a basic region that binds DNA, a leucine

zipper dimerization motif to create an amphipathic helix, and regulate multiple processes

especially stress responses (Kang et al., 2002). In Arabidopsis there are 75 bZIP members

that can be divided into ten groups according to the sequence similarity of the basic

region (Jakoby et al., 2002). There are seven members of Group-A (AtbZIP39/ABI5,

AtbZIP36/ABF2/AREB1, AtbZIP38/ABF4/AREB2, AtbZIP66/AREB3,

AtbZIP40/GBF4, AtbZIP35/ABF1 and AtbZIP37/ABF3), and studies reveal that most of

them are involved in ABA or stress signaling (Choi et al., 2000; Finkelstein and Lynch,

2000; Uno et al., 2000; Lopez-Molina et al., 2001). In addition, these members are

designated as ABRE-binding factors (ABF) or ABA-responsive element binding proteins

(AREB) because they can bind different ABRE-containing promoters (Choi et al., 2000;

Uno et al., 2000).

There are 89 potential non-redundant bZIP transcription factor genes in rice

(Aashima et al., 2008). We are particularly interested in the VI family (14 members)

because they are highly homologous to the Arabidopsis Group A subfamily (ABI5

subfamily). Until now, only three members of this subfamily have been functionally

characterized. TRAB1 was identified by a yeast two-hybrid screen for isolating the

protein interacting with VP1/ABI3 (Hobo et al., 1999). TRAB1 is localized to the

nucleus and activated by ABA-dependent phosphorylation (Kagaya et al., 2002;

Kobayashi et al., 2005). OsABI5, a transcriptional activator that can bind to ABRE

(G-box), is involved in ABA signal transduction and regulates fertility and stress

responses (Zou et al., 2007, 2008). OsbZIP23 functions as a transcriptional activator to

positively regulate the ABA responses and hence increase the stress resistance in rice

(Xiang et al., 2008). Whether other members of this sub-family have similar functions is



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still unknown.

Studies showed similar inhibitory effects of ABA in germination and root growth of

Arabidopsis and rice, although the sensitivities are much different, especially in seed

germination. Considering most studies at present focus on the ABA functions in stress

responses in Arabidopsis, and the conservation and specificity of the rice ABA signaling

remains largely unknown, studies on rice ABA signaling will be very valuable to

illustrate the functional mechanism of ABA. Here we report the functional identification

of a rice bZIP transcriptional factor, ABI5-like 1 (ABL1), which is involved in the rice

ABA signaling and stress responses by directly binding to ABRE-containing genes,

especially the WRKY-family genes. In addition, ABL1 suppresses auxin signaling by

targeting ABRE-containing genes related to auxin metabolism or signaling, revealing the

central role for ABL1 in the ABA and auxin responses.



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RESULTS

ABL1 is Expressed in Various Tissues and is Induced by Hormones and Stress

Conditions

A rice bZIP transcription factor, bZIP46 (LOC_Os6g10880), which belongs to

subfamily VI of the rice bZIP family and has a close relationship with Arabidopsis ABI5,

was identified and designated as ABI5-like 1 (ABL1). A comparison of the protein

sequence revealed that several members of this subgroup, including ABL1, contain

conserved motifs that have been identified in members of Arabidopsis ABI5 and rice

TRAB1 (Hobo et al., 1999). Besides the conserved basic leucine zipper motif, ABL1 also

contains phosphorylation sites, including the potential casein kinase II (CKII)

phosphorylation site (S/TxxD/E, where x represents any amino acid) and a

Ca2+-dependent protein kinase phosphorylation site (R/KxxS/T), implying the possible

regulation of ABL1 by various protein kinases.

An analysis of the expression pattern by quantitative real-time RT-PCR (qRT-PCR)

analysis or promoter-reporter gene (GUS) fusion studies revealed that ABL1 is expressed

in various tissues, with a relatively higher expression in the leaves and stems (Figure 1A,

upper panel). Further detailed histochemical analyses of the GUS activities of

pABL1:GUS lines showed that ABL1 is mainly expressed in the coleoptiles and primary

roots at the seedling stage (Figure 1A, bottom panel, 1-3), particularly in the root

vascular bundles (Figure 1A, bottom panel, 4). ABL1 is mainly expressed in the midvein

of the leaves of adult plants (Figure 1A, bottom panel, 5), suggesting the possible role of

ABL1 throughout the development process, primarily in the vegetative growth stage.

Some Arabidopsis bZIP transcription factor-encoding genes, AREB1/ABF2,

AREB2/ABF4 and ABF3/DPBF5, exhibit ABA-, drought-, and high-salinity-inducible

expression (Fujita et al., 2005), and expression of rice bZIP23 is induced by ABA or

under different stresses such as drought and salinity (Xiang et al., 2008), which is

consistent with the hypothesis that ABA stimulates the expression of target transcription

factors. To explore the possible involvement of ABL1 in the regulation of ABA and

abiotic stress-related genes, the expression of ABL1 under exogenous hormones (ABA,

BR, IAA and GA) and abiotic stress conditions was examined by qRT-PCR. The results

showed that ABL1 was strongly induced by ABA treatment and slightly stimulated by

IAA and GA, but not BR (Figure 1B, upper panel). Similarly, ABL1 was induced by



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drought, high-salinity and polyethylene glycol (PEG) (Figure 1B, bottom panel), and the

most dramatic induction was under the 12-h PEG treatment. These results are consistent

with the previous reports by microarray hybridization that indicated ABL1 is up-regulated

by dehydration and salt, but not cold (Nijhawan et al., 2008).

Deficiency of ABL1 by T-DNA Insertion Results in the Suppressed ABA responses

A search of the Shanghai T-DNA insertion population (http://ship.plantsignal.cn, Fu et

al., 2009) resulted in the identification of a putative mutant line in which the insertion is

located at the first intron of ABL1 (Figure 2A, upper panel). An analysis by PCR

amplification confirmed the single-copy integration of T-DNA (Supplementary Figure 1),

and deficiency of ABL1 in the homozygous lines by qRT-PCR analysis (Figure 2A,

bottom panel), indicating that abl1 is a knockout mutant.

The Arabidopsis abi5 mutant is insensitive to exogenous ABA, and the deficiencies

of rice ABI5 and bZIP23, which belong to the same subfamily of ABL1, also display the

insensitive responses to ABA (Carles et al., 2002; Xiang et al., 2008; Zou et al., 2008).

Similarly, phenotypic observation and statistical analysis of root and seedling growth of

WT and abl1 under ABA treatment showed that the deficiency of ABL1 results in

suppressed responses to exogenous ABA. abl1 shows only a 9% inhibition of the root

length under the 0.5 µM ABA treatment, whereas the WT has an approximately 30%

inhibition (Figure 2B). Unlike in Arabidopsis, both phenotypic observation and

calculation of the seed germination frequency revealed that there is no significant

difference of abl1 in ABA-inhibited seed germination when compared to WT.

To confirm that the altered ABA response of abl1 is indeed due to the deficiency of

ABL1, a complementation study was performed. The full-length cDNA of ABL1 under

the CaMV35S promoter was transformed into the abl1 mutant. An analysis of the

transgenic lines with the complementary expression of ABL1 (Figure 2A, bottom panel)

revealed a normal ABA responses in seedling and root growth under the ABA treatment

(Figure 2B), demonstrating the effects of ABL1 in mediating ABA response and

signaling.

The full-length cDNA of ABL1 was transformed into the Arabidopsis abi5-1 mutant

to examine the conserved function of ABA signaling in rice and Arabidopsis. The

analysis of the transgenic Arabidopsis lines (homozygous lines at the T3 generation)

expressing ABL1 (Supplementary Figure 2) showed that, in comparison to that abi5-1



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can grow well and is insensitive to ABA treatment, the ABA inhibition of seed

germination and seedling growth under 3 µM ABA were rescued by expressing ABL1

(Figure 2C), indicating that the rice ABL1 has a function that is conserved from

Arabidopsis ABI5.

ABL1 Localizes to the Nucleus and Binds to the ABRE cis-element

A nuclear localization signal is detected in ABL1. Observation of the green

fluorescence by transient expression of the ABL1-GFP fusion protein in onion epidermal

cells revealed the specific nuclear localization of ABL1 (Figure 3A), implying the role of

ABL1 as a transcription factor.

Previous studies have demonstrated the binding activity of the rice bZIP VI

subfamily members to the ABRE cis-element containing a 5’-CACGTG-3’ core

sequence (G-box) (Xiang et al., 2008; Zou et al., 2008). We then examined the

specificity of the binding activity of ABL1 to the G-box cis-element with a yeast

one-hybrid system. As shown in Figure 3B, a yeast strain (YM4271) transformed with

G-box and expressing the ABL1 fusion protein can grow on the SD medium

(-His/-Ura/-Leu, supplemented with 30 mM 3-AT) and β-Gal activity test of selected

transformants indicated that ABL1 binds to the G-box to activate the expression of the

reporter gene. Further analysis with site-mutation of G-box (ABRE/C-box or DRE

element) indicated the defective growth of yeast cells and deficiency of β-Gal activity,

confirming that OsABL1 specifically binds to the ABRE/G-box. These results suggest

that ABL1 might act as a transcription factor by binding to the ABRE cis-element

(G-box).

ABL1 Is Involved in ABA and Stress Responses by Regulating the ABRE

(G-box)-Containing Genes

To study the functional mechanism of ABL1 and identify the potential targets, the

genome-wide expression profile was analyzed by DNA chip hybridization using roots as

the sample material as the phenotype of insensitive response to ABA was found notably

in roots. Total RNAs of roots of 14-day-old abl1 and WT seedlings under ABA treatment

(100 μM, 4 hour) were used and biological replicates were performed for the

hybridization. Analysis of the Pearson's correlation coefficient for the replicates showed

that they were highly correlated (>0.98), indicating good reproducibility of the chip



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hybridization. With a threshold of 1.5-fold change (FDR P<0.01), a total of 373 genes

(excluding different probes of same transcript) were down-regulated (Supplementary

Table 1) and 304 genes were up-regulated (Supplementary Table 2) in the ABL1 deficient

plant.

Because ABL1 binds to the ABRE (G-box) cis-element to regulate the gene

expression, the cis-elements in the promoter regions of the altered genes were analyzed

in detail to further identify the targets whose promoters might be bound by ABL1. The

DNA sequence up to 3000 bp upstream of the start codon ATG of each selected gene was

extracted, and the analysis showed that several ABRE core motifs were enriched in the

regulated genes (Table 1), particularly in the down-regulated ones. These results indicate

that ABL1 may bind the ABRE (G-box) elements of the promoters of these target genes

to activate their transcription. Although some ABRE (G-box) motifs are detected as

enriched in the up-regulated genes, the majority of the motifs are not the most

representative putative binding elements of this bZIP subfamily (Nijhawan et al., 2008).

Further, expressions of several down-regulated genes that contain at least four G-box

elements were selected for qRT-PCR analysis in the abl1 mutant and WT in the presence

or absence of ABA. As shown in Figure 4A, all five of the selected genes are

differentially up-regulated after ABA treatment in WT, while are uniformly greatly

reduced in abl1 in comparison with WT, although the absolute values of the fold changes

showed some variations for some genes between the microarray and qRT-PCR analyses.

Based on the results that ABL1 binds to the ABRE cis-element, these observations

further suggested that the down-regulated G-box containing genes were potential target

genes of ABL1. Indeed, gene LOC_Os01g50100 was selected and proved to be directly

bound by ABL1 by an electrophoretic mobility shift assay (EMSA) using promoter

region containing at least two ABRE core cis-element (ACGT) (Figure 4B), confirming

that ABL1 could specifically and directly bind to ABRE core cis-element

(ACGT)-containing segment of promoter of regulated genes.

Interestingly, the GO-process analysis presented a notable difference between the up-

and down-regulated genes. Over one-third (P<0.2) of the down-regulated genes are

annotated as being involved in the responses to abiotic or biotic stresses, including cold,

nitrogen starvation and water deprivation, or the hormones SA, JA, ABA (Supplementary

Table 3). In contrast, the up-regulated genes showed diverse functions and fewer of them

are involved in the responses to stresses, though some stress pathways are still enriched,



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including oxidative stress and defense responses (Supplementary Table 4). These results

suggest that ABL1 plays a critical role in ABA signaling and ABA-triggered stress

responses. In addition, although some GO stress response processes were enriched in

both the down- and up-regulated genes under the ABL1 deficiency, it is reasonable to

suppose that the regulation of these genes was an indirect effect of ABL1.

ABL1 is Involved in Stress Responses by Regulating ABRE (G-box)-Containing

WRKY Family Genes

Many plant transcription factors serve as targets of other upstream factors to

elaborate developmental regulation or signal magnification. Analysis of the microarray

data showed that there were thirty-two transcription factor encoding genes among the

down-regulated genes and only nine among the up-regulated ones. Surprisingly, eight

WRKY family genes were uniformly and significantly down-regulated. To clarify the

activating effects of ABL1 on these WRKY genes, the expression of these WRKY genes

in abl1 and WT in the absence or presence of ABA was verified. As shown in Figure 4C,

under ABA treatment, all eight WRKY genes were dramatically up-regulated in WT

plants, while this stimulation was significantly suppressed in abl1, implying that ABL1

mediates the stimulation of these WRKY genes by ABA. Further EMSA analysis of the

binding activity of ABL1 to the promoter region of putative target WRKY genes

confirmed, as expected, the direct binding of ABL1 to the promoter region of one

WRKY gene LOC_Os08g29660 (Figure 4D).

Interestingly, the analysis of the promoter regions of these eight WRKY family

genes showed that they also contain multiple ABRE cis-elements. Previous studies

showed that the transcription of WRKY genes was strongly and rapidly up-regulated in

response to wounding, pathogen infection or abiotic stresses in numerous plant species

(Eulgem et al., 2000), strongly supporting the assumption that ABL1 may mediate

ABA-stimulation of these WRKY genes through direct activation. In addition, it is also

noted that genes containing the cis-elements possibly directly bound by WRKY

transcription factors were enriched in the down-regulated genes in ABL1 deficient plants

(Table 2), which suggests that ABL1 triggered a chain of WRKY-regulated

transcriptional events.

Additionally, we examined the tolerance of abl1 mutant plants to high salinity and

PEG treatment. The results showed that the ABL1 deficiency did not result in obvious



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growth differences under various concentrations of salt (150, 180, or 200 mM), whereas

abl1 plants had a much lower resistance to the PEG (15%) treatment (grew slowly and

weak, and more leaves turned yellow, Figure 4E, left panel). Further measurement of the

leaf relative water content (RWC) (according to Guo and Qian, 2003) under PEG

treatment showed the hypersensitivity of abl1 mutant to drought stress in comparison to

WT (reduced RWC), whereas the complementary expression of ABL1 rescued the

suppressed growth of abl1 under PEG treatment (Figure 4E, right panel). These

suggested a primary role for ABL1 in the abiotic stress response.

ABL1 Modulates the Auxin Responses

It is interesting that ABL1 is induced by exogenous IAA (Figure 1B), which

suggests a possible role of ABL1 in mediating the cross-talk of ABA and IAA. The

examination of IAA sensitivity by measuring the primary root length under different

concentrations of IAA showed that abl1 exhibits much more inhibition, indicating a

hypersensitive response of abl1 to exogenous IAA (Figure 5A). Consistently,

examination of the transcription of auxin-responsive genes IAA1, IAA9 and IAA24 (three

pronounced auxin stimulation Aux/IAA genes) in abl1 confirmed the significantly

enhanced expression under exogenous IAA and hence the increased auxin responses

(Figure 5B).

DNA chip hybridization analysis (this study and a public database of

Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/, GSE5167) of the genes

with altered expression under ABL1 deficiency showed that some altered genes shared a

similar regulation to the IAA treatment, and most of the genes contain the concentrated

ABRE (G-box) cis-elements in their promoter regions (Table 3). Two up-regulated and

three down-regulated auxin-related genes that contain multiple ABRE cis-elements were

then selected for analyzing the transcription under IAA treatment. The results showed the

same tendency after IAA treatment both in the WT and abl1 mutant except for one gene

(LOC_Os06g46160), and some genes presented more significant increases in abl1

(Figure 5C), confirming the hypersensitive response of abl1 to exogenous IAA.

In addition, a detailed GO process analysis of the altered genes in abl1 showed that

six genes are related to IAA, and five of them have enriched ABRE elements (Table 4).

However, not all of the regulated genes contained ABRE cis-elements, implying that

indirect regulation of ABL1 in IAA signaling might exist.



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DISCUSSION

Crucial Roles of Rice ABL1 in ABA and Stress Response

Although ABL1 could partially rescue the insensitive response to ABA of

Arabidopsis abi5-1 mutant, functional difference exists in both Arabidopsis ABI5 and

rice ABL1. Seeds of abl1 do not show altered germination under ABA treatment which

is particularly obvious in Arabidopsis abi5. The tissue expression pattern of Arabidopsis

ABI5 is much more abundant in developing siliques than in vegetative tissue, while ABL1

is transcribed throughout the developmental processes primarily in the vegetative phase.

This indicates that Arabidopsis ABI5 and rice ABL1 still have functional diversity that

may be achieved by different regulatory networks.

Most members of the rice bZIP subfamily VI (10 of 14) are involved in ABA

signaling and abiotic stress responses (Xiang et al., 2008; Zou et al., 2008), especially

stress-related AREB/ABF factors of both rice and Arabidopsis (Yoshida et al., 2010).

Phylogenetic analysis showed that ABL1 is closest homologous to rice bZIP23 and

TRAB1 (a functional unknown OsbZIP72) and Arabidopsis AREB1, AREB2, ABF3

(Supplementary Figure 3). The three Arabidopsis AREBs have been proved to interact

with SRK2D/SnRK2.2, a SnRK2 protein kinase that was identified as a regulator of

AREB1. Previous study showed that all these rice homologues require ABA for full

activation as Arabidopsis AREB (Yoshida et al., 2010), suggesting that rice ABL1,

bZIP23 and bZIP72 (TRAB1) may function downstream of SnRK2 to activate the target

genes in ABA signaling.

Based on the previous studies showing that rice bZIP23, TRAB1 and ABI5 also

function in stress-related responses, it is supposed that the common stress-related effects

of rice ABL1 are masked in the abl1 mutant due to the redundant functions and

expressions of other homologs such as OsZIP23. Examination of the expression level of

other homologs of ABL1 in abl1 mutant and WT under the absence or presence of

exogenous ABA revealed that although the ABL1 homologs are down-regulated in abl1

mutant, expressions of them are restored to the normal expression level under ABA

treatment, which indicated that the observed phenotype related to stress in abl1 mutant is

mainly owing to the deficiency of ABL1. Generation of multiple mutants of genes

including rice bZIP23, ABL1, TRAB1, and bZIP72 might help to study the functional

redundancy and synergetic regulation of them, as what has been done in Arabidopsis



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using the triple mutant areb1 areb2 abf3 (Yoshida et al., 2010). Whether these rice

AREBs function cooperatively as master transcription factors to regulate the

ABRE-dependent ABA signaling and to involve in drought stress tolerance need further

studies.

ABL1 Regulates Abiotic Stress Responses by Modulating the Expression of

ABRE-containing Genes, Especially the WRKY Family Genes

Analysis of the transcriptome of rice seedlings exposed to cold, drought, high salinity,

or application of ABA revealed that many transcription factors are involved in

ABA-mediated stress responses (Rabbani et al., 2003). ABL1 localizes to the nucleus

and suppressed expression of ABRE-containing genes under ABA treatment in abl1

plants indicates a primary role for ABL1 in ABA-triggered transcriptional regulation. In

addition, the GO analysis revealed that most of the altered genes in abl1 plants were

closely correlated to the response to abiotic stresses, implying the important role of

ABL1 in ABA-mediated stress responses.

It has been reported that overexpression of rice bZIP proteins such as OsbZIP23

resulted in the enhanced response to the abiotic stresses, further comparison of the up- or

down-regulated genes in abl1 and OsbZIP23-overexpressing plants revealed the specific

characters of ABL1. There were 795 or 318 up-regulated and 1017 or 390

down-regulated genes respectively in OsbZIP23-overexpressing or abl1 plants, while

only 6 and 7 genes in common in up- and down- regulated genes (Supplementary Table

5). The very limited regulated genes in common suggested that the function of ABL1 in

transcriptional regulation in ABA responses is different from other bZIP family members.

Compared to that of OsbZIP23-overexpressing plants (Xiang et al., 2008), there is only

one lipid transfer (LTP) protein encoding gene and no dehydrin family and late

embryogenesis abundant (LEA) gene is down-regulated in abl1, though lots of genes

related to stress response are down-regulated. Meanwhile, much more stress-related

regulatory factors were down-regulated in abl1 than OsbZIP23-overexpressing plants

including protein kinase and transcription factors, suggesting the differential regulation

between ABL1 and OsbZIP23 in stress responses.

The WRKY gene family encodes a large group of TFs and the WRKY domain can

bind to the W-box or SURE (sugar-responsive cis-element) elements in the promoter

regions of target genes to regulate their transcription (Rushton et al., 1995; Sun et al.,



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2003). Interestingly, a series of ABRE-containing WRKY family genes, which are

involved in abiotic stress responses, are transcriptionally regulated by ABL1, and their

induced expression under ABA treatment is evidently suppressed in ABL1 deficient

plants. WRKY genes are up-regulated by multiple stress conditions and hormone

treatments (Supplementary Table 6, Ramamoorthy et al., 2008), which is consistent with

ABL1 being a transcriptional activator that is greatly up-regulated by drought, salinity

and IAA treatments and indicates that ABL1 regulates the abiotic stress responses by

modulating the expression of ABRE-containing WRKY family genes (Figure 6).

Most of the down-regulated genes in abl1 are stimulated by abiotic stress conditions.

An analysis of the rice gene profile under drought, salinity and cold conditions

(microarray data were obtained from the public database under GEO Accession

No.GSE6901) and the comparison with the altered genes in abl1 showed that 58% of

down-regulated genes (Supplementary Table 7) and 49% of up-regulated genes

(Supplementary Table 8) in abl1 are also regulated under stress conditions (at least one

of the three conditions). A further analysis on the promoter region of these commonly

altered genes revealed the presence of the W-box cis-element, which is particularly

enriched in both the up- or down-regulated genes with higher fold changes. Considering

the role of WRKY transcription factors serving as either transcriptional activators or

suppressors, it is thus assumed that under abiotic stresses, ABL1 is up-regulated (possibly

mediated by ABA) and stimulates the down-stream ABRE-containing genes to regulate

the responses or ABA signaling. Importantly, ABL1 stimulates a series of

ABRE-containing WRKY genes that activate or suppress the W-box-containing genes to

promote stress responses (Figure 6).

ABL1 Mediates the Cross-talk of ABA and Auxin

Previous studies revealed the cross-talk of ABA and auxin on different levels.

PGGT I encodes the β-subunit of protein geranylgeranyltransferase type I and negatively

regulates ABA signaling in guard cells. The deficiency of PGGT I results in the

increased lateral root formation in response to exogenous auxin (Johnson et al., 2005),

indicating synergistic cross-talk of ABA and auxin. Detailed studies on the

transcriptional profiling of genes assigned to the ABA and auxin biosynthetic pathways

by GO annotation revealed the complex interaction of ABA and auxin signaling with

ambiguous outcomes (Nemhauser et al., 2006).



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Cross-talk between the ABA- and auxin-dependent responses takes place mainly during

seed germination and early seedling development in which the ABA-dependent

repression of growth is potentiated by auxin (Ni et al., 2001; Nakabayashi et al., 2005;

Liu et al., 2007). Recent studies showed that ABA and auxin have synergic functions in

repressing embryonic axis elongation in Arabidopsis (Belin et al., 2009), which involves

the transcriptional repression of the Aux/IAA genes, AXR2/IAA7 and AXR3/IAA17, by

ABI5 and ABI3 binding to the particular ABRE elements. The hypersensitive responses

to IAA treatment in the root length by ABL1 deficiency (Figure 5A) was somehow in

contradiction to ABA repression of embryonic axis elongation in Arabidopsis through

enhanced IAA signaling. However, when considering the physiological effects, there is a

consistent tendency for ABA to suppress the embryo axis elongation or seedling growth

by regulating auxin signaling. ABI5 stimulates auxin signaling to suppress the embryo

axis elongation and ABL1 suppresses auxin signaling and hence seedling growth,

revealing an interesting and complex cross-talk between ABA and auxin. In addition,

auxin-induced lateral root formation is completely suppressed by ABA in

35S-VP1/abi3-6 plants, indicating that VP1 mediates the interaction between ABA and

auxin signaling (Suzuki et al., 2001). Considering the interaction of TRAB1 and VP1, we

ponder whether the role of ABL1 in auxin signaling was related with ABI3 orthologs

in rice. It is assumed that the cross-talk of ABA and auxin, especially the suppression of

auxin by ABA, is correlated to the specific growth and developmental processes such as

embryo axis elongation or seedling growth. ABL1 could directly or indirectly target the

ABRE-containing genes that encode the proteins involved in the auxin response,

metabolism or signaling, providing new clues of cross-talk between ABA and auxin

signaling and functions (Figure 6).

Additionally, coordination of the GA- and ABA-dependent signals is starting to be

unveiled in Arabidopsis (Okamoto et al., 2010; Piskurewicz et al., 2009). ABL1 could be

up-regulated by GA (Figure 1B) and analysis of the microarray data showed that some

genes related to GA were altered, and all of them contained ABRE cis-elements in the

promoter regions (Supplementary Table 9). One of the down-regulated genes possibly

encodes GA ent-kaurene synthase (KS), and another is possibly involved in converting

ent-kaurene to GA12. Two genes whose products catalyzed the penultimate steps of GA

activation were up-regulated in the abl1 mutant. According to the previous finding that

GA influences GA metabolism by a feedback regulation and most of the GA20ox genes



18

are down-regulated by applied bioactive GAs (GA1and GA4, Hedden and Phillips, 2000),

the down-regulation of early GA-synthesis genes and up-regulation of GA metabolism

genes implies a hypersensitive GA response of the abl1 mutant and antagonistic

relationship between GA and ABA.

In sum, our studies demonstrated that ABL1 is involved in the ABA and stress

responses, and mediates the cross-talk of ABA and auxin by directly regulating a series

of ABRE-containing downstream genes, providing new insights into the ABA signaling

and ABA-auxin interactions.



19

MATERIALS AND METHODS

Chemicals and Plant Materials

Indole acetic acid (IAA, I2886), 24-epi-brassinolide (24-epi-BL, E1641), abscisic

acid (ABA, A1049), and gibberellin (GA, G7645) were purchased from Sigma-Aldrich

(Germany).

For rice growth, seeds are germinated in sterilized water and then grown in pots in

phytotron with a 12h-light (26ºC) / 12h-dark (18ºC) cycle.

To detect the transcription levels of relevant genes, rice wild type (Zhonghua 11,

japonica variety) and abl1 plants are grown in the greenhouse with a 12h-light / 12h-dark

cycle, and 12-day-old seedlings are treated with hormones (1 µM BR, 10 µM IAA, 10

µM GA, or 0.1 mM ABA) followed by sampling at 0, 1, 3 and 8 h. For abiotic stress

treatment, 12-day-old seedlings are irrigated with 20% PEG 6000 (followed by sampling

at 1, 5, and 12 h) and 200 mM NaCl solution (sampling at 0, 5, 12, and 24 h). Drought

stress is achieved by leaving the intact seedlings in the air without water supply and

seedlings are sampled at 0, 3, 5, 12 h. Total RNAs are extracted from total seedlings after

treatment and used for further analysis.

Seeds of Arabidopsis wild-type Wassilewskija (WS-2, stock number CS22659) and

abi5-1 (stock number CS8105), which has a mutation in the Arabidopsis ABI5 gene

(AT2G36270), are obtained from the Arabidopsis Biological Resource Center (ABRC) at

Ohio State University (Columbus, OH, USA).

Sub-cellular Localization Studies of ABL1

The whole ABL1 coding region is amplified with primes ABL1-G-S

(5’-GAAGATCTAATGGAGTTGCCGGCGGATG-3’, added BglII site underlined) and

ABL1-G-A (5’-GACTAGTGCATGGACCAGTCAGTGTTC-3’, added SpeI site

underlined), and sub-cloned into the binary vector pCAMBIA1302 (Cambia, Canberra,

Australia), resulting in an C-terminal fusion to GFP. The resultant construct

p35S:ABL1-EGFP is sequenced to confirm the in-frame fusion of ABL1 and GFP, and

used for transient expression in onion (Allium cepa) epidermal cells. Transient expression

of GFP fusion protein was performed with a model PDS-1000/He Biolistic particle

delivery system (Bio-Rad, CO, USA). Five micrograms of purified plasmids was coated

with 0.6-1 μm gold particles and bombardment was performed with the following

parameters: 1100-p.s.i. rupture disc; 27-inch Hg vacuum; 6-cm distance from the



20

stopping screen to the target tissues. After bombardment, onion epidermal tissue was

incubated in MS medium at 25°C in darkness for at least 24 h. Cell plasmolysis was

achieved by incubating the onion epidermal tissue in sucrose solution (1 M) for 10 min.

The green fluorescence was observed through a confocol laser scanning microscopy

(FITC488, Zeiss LSM500; Herts, UK) with an argon laser excitation wavelength (488

nm GFP). Onion cells harboring vector pCAMBIA1302 and expressing unique EGFP

was observed as control.

RT-PCR and Quantitative Real-time RT-PCR (qRT-PCR) Analysis

Total RNAs are extracted from 12-day-old seedlings of WT (Zhonghua 11) and abl1

mutant using TRIzol reagent (Invitrogen) and reverse-transcribed according to the

manufacturer’s instructions (ReverTra Ace-a-first-strand synthesis kit; Toyobo). For

RT-PCR analysis of ABL1 transcription, equal amounts of first strand cDNAs are used as

templates for PCR amplification using primers (5’-GCGGCAGAGGCGGATGAT-3’

and 5’-GTTCGTCGGAGGCAAAATCT-3’). The rice ACTIN gene (Os03g50890) is

amplified by primers (5’-CCTTCAACACCCCTGCTATG-3’ and

5’-TGAGTAACCACGCTCCGTCA-3’) and used as an internal positive control for

quantification of the relative amounts of cDNA.

For qRT-PCR analysis, the resultant 1st strand cDNA is used as template to

quantitatively analyze the gene expressions uisng the Rotor-Gene real-time thermocycler

R3000 (Corbett Research) with real-time PCR Master Mix (Toyobo). For the analysis, a

linear standard curve is generated using a series of dilutions for each PCR product and

the levels of the transcript in all unknown samples are determined according to the

standard curve. The rice ACTIN gene is amplified and used as an internal standard to

normalize the expression of ABL1 and the other tested genes. The primers used are as

listed in supplementary Table 10.

Constructs and Rice Transformation

The full-length cDNA of ABL1 is isolated from KOME AK105312 by digestion with

EcoRI and XbaI, and sub-cloned into the binary vector pCAMBIA2300S (Cambia,

Australia) precut with KpnI and XbaI. The resultant construct is transferred into the

Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993) by electroporation and

used for transformation of abl1 mutant using immature embryos as materials.



21

Promoter-reporter Gene Fusion Studies

A 1.9-kb genomic DNA fragment containing the promoter region of the ABL1 gene is

amplified by PCR using primers

(5’-CCCAAGCTTTATCCCTCTGTAACCAAACCAAAC-3’, added HindIII site

underlined; and 5’-CGGGATCCTCCCACCTCAAAATCCTTCAAATC-3’, added

BamHI site underlined) and then sub-cloned into modified vector

pCAMBIA1300+pBI101.1 (Liu et al., 2003), resulting in the fusion of the ABL1

promoter and GUS reporter gene. The construct is then transformed into rice plants as

described above and ~30 independent transgenic lines are obtained. GUS activities are

histochemically detected as described (Jefferson et al., 1987).

DNA Binding Assay

DNA binding assay is performed according to the methods described by Zou et al.

(2008). The yeast reporter strain YM4271 which carries the dual reporter genes HIS3 and

lacZ with a trimmer of 27 bp DNA fragments including ABA-responsive cis-acting

elements (ABRE/G-box composed as follows:

5’-agctAGCCACGTGTCGGACACGTGGCA-3’, G-box positions underlined) upstream

of the TATA element is used as an assay system. ABRE/C-box

(5’-agctAGCGACGTCTCGGAGACGTCGCA-3’, site-mutated positions underlined)

and DRE (5’-agctAGCTACCGACATTCGGATACCGACATGCA-3’, site-mutated

positions underlined ) element were constructed as the site-mutation and used to examine

the specific binding activity of ABRE/G-box. The full-length cDNA of ABL1 is

sub-cloned into the vector pAD-Gal4-2.1 and the resultant construct was then

transformed into yeast reporter strains YM4271. The transformed cells were incubated on

medium lacking histidine and supplemented with 30 mM 3-aminotriazole (3-AT, a

competitive inhibitor of HIS3 gene product) for 3 days, and then the cell growth was

observed. β-gal activity of selected different transformants was examined after 6 hours.

Electrophoretic Mobility Shift Assay (EMSA)

Recombinant ABL1 protein was expressed in E. coli and used in the EMSA assay.

Coding region of ABL1 was amplified via PCR using primers ABL1-pET32a-S

(5’-CATGCCATGGAGATGGAGTTGCCGGCGGAT-3’, added NcoI site underlined)



22

and ABL1-pET32c-A (5’-GGAATTCGCATGGACCAGTCAGTGT-3’, added EcoRI

site underlined), and then subcloned into pET-32a(+) (Novagen, Madison, WI). The

resultant construct was sequenced to confirm the in-frame fusion and then transformed

into the E. coli strain BL21 for expression of the recombinant proteins. The recombinant

proteins were purified with Ni-NTA agarose (30210, Qiagen, Hilden, Germany).

The DNA fragment of the promoter region of LOC_Os01g50100 (position -523 bp

to -265 bp) and LOC_Os08g29660 (position -1023 bp to -872 bp) were amplified with

the primers (5’-TTGACCGTTGGTATCTGCATCA-3’ and

5’-CGTACTGCTGTTCCTTTTCTGC-3’; and

5’-TAGAAATGGACGTAGATCTCAACGC-3’ and

5’-CCAAGATCCTGGCACCTACCT-3’) by using rice genome-DNA as the template,

sequenced, sub-cloned, and PCR-labeled with digoxigenin according to the

manufacturer’s instructions (Roche, Mannheim, Germany).

Labeled DNA was incubated with or without unlabeled DNA and recombinant

proteins (10-160 ng) in binding buffer (75 mM HEPES, 175 mM KCl, 5 mM EDTA,

40% glycerol, 5 mM DTT, 1 mM MgCl2). The reactions were incubated at room

temperature for 20 min, then run on a 0.5X 5% polyacrylamide gel and followed by

transmembrane, cross-linking, blocking, and antibody incubation, then detected with

disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2´-(5'-chloro)tricyclo

[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD, 11655884001, Roche, Mannheim,

Germany) according to the manufacturer’s instructions.

Expression of ABL1 in Arabidopsis abi5-1 Mutant and ABA Sensitivity Assay

The ABL1 cDNA was sub-cloned into the vector pCAMBIA1301 and the resultant

construct was transformed into the Arabidopsis abi5-1 mutant by the Agrobacterium

tumefaciens floral dip method (Clough et al., 1998). T1 seeds from the infiltrated plants

were screened on MS medium containing 25 μg/L hygromycin (Roche, Basel,

Switzerland) and the homozygous lines (T3 generation) were used for ABA sensitivity

analysis.

The seeds of Arabidopsis WS-2, abi5-1 mutant, and transgenic abi5-1 plants

expressing ABL1 were placed on 1/2 MS medium supplemented with 3 µM ABA, chilled

for 2 days at 4°C in darkness, and grown at 22°C (16h-light/8h-dark cycle) for 7 or 14

days, then observed.



23

GeneChip Analysis Using Affymetrix Array and Data Statistical Analysis

GeneChip® rice genome array (Affymetrix, Santa Clara, CA) is used to study the

gene expressions under ABL1 deficiency. abl1 and WT seedlings were grown in the

phytotron with a 12-h-light/12-h-dark cycle and 14-day-old seedlings were treated in

water with ABA (100 μM, 4 h). Total RNAs were extracted from roots using TRIzol

reagent (Invitrogen Life Technologies). 2 μg total RNA was used as starting material for

hybridization, and each sample had two biological repeats. Washing, staining, and

scanning of chips were performed as described by the supplier’s protocol.

The hybridization signals were normalized using Affymetrix’s MAS5.0 methods

from Bioconductor Suite (www.bioconductor.org) of tools for the statistical package R

(http://www.R-project.org). All the data can be accessed at GEO under the accession

number GSE18529. Least-square linear regression for each sample (Schmid et al., 2005)

was carried out to check the replicate quality of microarray. The R2 statistic of each

sample were higher than 0.98. TIGR Mev (version 4.0, http://www.tm4.org/mev.html) is

used to identify the genes differentially expressed in abl1. FDRs for various P value

thresholds are later determined by the method of Benjamini and Yekutieli (2001). The

changed genes in the abl1 mutant under threshold of 1.5-fold change (FDR P<0.01) were

selected for further analysis. The expression data of GSE6901 were downloaded from

NCBI GEO.

GO Process Analysis

Up to date, the integrity of gene ontology information in Arabidopsis is much better

than in rice, so the rice genes are firstly identified by using BLASTp (E value<1e-5) as

described by TIGR (http://www.tigr.org) and then the functions of genes in Arabidopsis

genes were assigned to their rice homolog. To determine the significance of

over-representation of selected genes for each pathway in GO catalog, a Chi-square test

is performed to test the null hypothesis or the alternative hypothesis defined as H0:p0=p1;

H1:p0≠p1; where p0=m/M, p1=n/N (m is the number of selected genes mapped to the

pathway, M is the number of genes on the microarray that are mapped to that particular

pathway; n is the total number of selected genes mapped to all pathways, and N is the

total number of genes on the microarray mapped to all pathways).



24

Cis-element Analysis

DNA sequence up to 3000-nt upstream of ATG of each selected gene was extracted

and used for cis-elements analysis. Known cis-elements were analyzed by using the

program of Plant Cis-acting Regulatory DNA Elements (PLACE,

http://www.dna.affrc.go.jp/PLACE/) and the ratio of each cis-element in regulated genes

in abl1 mutant with that in the whole genome was followed by performing Chi-square

test. FDRs for various P value thresholds were determined as 0.06 (Table1) and 0.02

(Table 2). If a known cis-element is enriched with a low FDR P value, this cis-element is

selected.



25

ACKNOWLEDGEMENTS

We greatly thank Ms. Shu-Ping Xu for rice transformation.



26

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FIGURE LEGENDS

Figure 1. Expression Pattern Analysis of ABL1.

A The quantitative real-time PCR (qRT-PCR) analysis of ABL1 expression in various

tissues (upper panel). Seven-day-old seedlings were used to harvest the samples of

the seedling (shoot and leaf) and roots (primary and adventitious roots). Leaf and

stem samples were harvested from the plants before heading. Bottom panel: The

promoter-GUS fusion studies revealed the expression of ABL1 in the seed (1),

seedling (2, 3), primary root of the seedling (4), leaf (5) and flower (6). Bars = 2 mm

(1, 2, 5, 6), 5 mm (3) or 50 µm (4).

B qRT-PCR analysis of ABL1 expression under ABA, BR, IAA or GA treatments (upper

panel) or under drought, high-salinity or PEG treatments (bottom panel).

Twelve-day-old seedlings under different treatments were used for the analysis, and

the expression of ABL1 without treatment was set at 1.0. Biological replicates of the

experiments were performed, and the data are presented as the average of three

independent experiments.

Figure 2. The Deficiency of ABL1 Caused by a T-DNA Insertion Resulted in

Suppressed ABA Responses.

A The schematic exon-intron structure of the ABL1 gene and the position of the T-DNA

insertion (1st intron). Red boxes represent the exons (upper panel). Bottom panel:

qRT-PCR analysis of the ABL1 expression in homozygous mutant and transgenic

lines with complementary expression of ABL1 (L3 and L5). The expression of ABL1

in the WT is normalized to 1.0. RNAs extracted from rice leaves were used for

analysis and the rice ACTIN gene was used as the internal control.

B The phenotypic observation (upper panel, bar = 1 cm) and calculation (bottom panel)

of the root growth revealed that abl1 had suppressed responses to the ABA treatment,

whereas abl1 seedlings with complemented expression of ABL1 (representative lines

L3 and 5) had normal responses to the ABA treatment. The root lengths of 6-day-old

seedlings (after germination) were measured, and the relative root lengths were

calculated (the root lengths of untreated seedlings were set at 100%). The data are

presented as the average ± SD (n>20). A statistical analysis with a one-tailed

Student’s t-test indicates the significant differences (**, P<0.01).



33

C ABL1 rescues the ABA insensitivity of Arabidopsis abi5-1. The seeds of WS-2,

abi5-1 and transgenic abi5-1 lines carrying p35S:ABL1 were germinated on 1/2 MS

medium supplemented with 3 µM ABA for 7 days or 14 days.

Figure 3. ABL1 Localizes to the Nucleus and Binds G-box/ABRE Elements.

A A nuclear green fluorescence after transient expression of ABL1 fused to EGFP in

onion epidermal cells revealed that ABL1 was localized to the nucleus. Onion cells

expressing EGFP alone (left panel) were used as the control. Bar=100 µm.

B The yeast-based one-hybrid assay showed the binding specificity of ABL1 to the

ABRE/G-box element. The yeast strain YM4271 was used and transformed yeast

cells were spot with serial dilutions of cells (OD600) on SD/-His/-Ura/-Leu selective

medium for 3 days. Proteins TRAB1 (BAA83740) and RF2a (AF005492) were used

as the positive and negative controls, respectively; ABRE/C-box and DRE element

were used as the site-mutation of ABRE/G-box. Transformants of different

concentrations were dropped onto SD/-His/-Ura/-Leu+30 mM 3-AT or selected by

β-Gal activity.

Figure 4. Suppressed Expression of G-box/ABRE-Containing Genes, Especially

WRKY Family Members, in ABL1 Deficient Plants.

A qRT-PCR analysis of genes containing multiple G-box elements in the absence or

presence of ABA (0.1 mM). Expression of the corresponding genes in the WT in the

absence of ABA is normalized to 1.0, and relative gene expression is shown. The

experiments were biologically repeated, and the data are presented as the average of

three independent experiments (same for C).

B Electrophoretic mobility shift assay (EMSA) confirms the binding of ABL1 to the

promoter regions of putative ABRE-containing gene (LOC_Os01g50100). The

recombinant expressed ABL1 protein was used to test the binding capacity to the

dig-labeled DNA fragment of the promoter region. Unlabeled DNAs (3 or 12 times)

were used as the cold competitor for assays. Free probes are indicated with star and

shift bands are indicated with arrows.

C The G-box/ABRE-containing WRKY family genes were down-regulated in abl1

under the ABA treatment. Twelve-day-old WT or abl1 seedlings in the absence or

presence of ABA (0.1 mM) were harvested and used for the qRT-PCR analysis. The



34

expression of the corresponding genes in the WT in the absence of ABA was set at

1.0.

D EMSA confirms the binding of ABL1 to the promoter regions of one

ABRE-containing WRKY gene (LOC_Os08g29660). The experiments were

performed as described in B. Unlabeled DNAs (3 to 24 times) were used as the cold

competitor for assays.

E abl1 was more sensitive to the PEG treatment (left panel, bar = 1 cm).

Twenty-day-old WT and abl1 seedlings were exposed to 15% PEG for 10 days. The

relative leaf water content after PEG treatment for 3d was measured, revealing that

abl1 was hypersensitive to drought stress in comparison to WT. Complementary

expression of ABL1 rescued the suppressed growth of abl1 (right panel).

Seven-day-old seedlings were exposed to 20% PEG for 3d. Statistical analysis by a

one-tailed Student’s t-test indicated significant differences (**, P<0.01).

Figure 5. ABL1 is Involved in the IAA Responses.

A The measurement and calculation of the relative root elongation reveal the

hypersensitive responses of abl1 to exogenous auxin. Five-day-old WT and abl1

seedlings (after germination) grown on medium supplemented with various

concentrations of IAA were analyzed. The root length without IAA treatment was set

at 100%. The data are presented as the average ± SD (n>20). Statistical analysis by a

one-tailed Student’s t-test indicated significant differences (*, P<0.05; **, P<0.01).

B qRT-PCR analysis showed that the IAA-responsive marker genes (IAA1, 9, 24) were

increased to a higher level in abl1 under the IAA treatment than those of the WT.

Twelve-day-old WT or abl1 seedlings were treated with IAA (10 μM) for 6 h and

then harvested for analysis. The expression of the corresponding genes of the WT and

abl1 in the absence of IAA was set at 1.0. The experiments were repeated, and the

data are presented as the average of three independent experiments (same for C).

C The altered genes in the abl1 mutant that share a similar response to the IAA

treatment (up- or down-regulation) were selected, and their expression in the absence

or presence of IAA (10 μM) was examined by qRT-PCR analysis.

Figure 6. A Hypothetical Working Model of ABL1 Function in Stress Response and

Auxin/ABA Cross-Talk.



35

ABL1 expression is stimulated by various environmental stresses, which may be

mediated by ABA, and then activates the expression of a series of ABRE

(G-box)-containing genes, especially those involved in ABA signaling and stress

responses through direct binding to the ABRE motifs. In addition, ABL1 stimulates the

expression of ABRE-containing WRKY members, which differentially regulate the

down-stream W-box-containing genes to regulate the biotic/abiotic responses and ABA

signaling. Meanwhile ABL1 is induced by IAA and negatively regulates auxin signaling

by regulating the expressions of ABRE (G-box)-containing genes related to auxin

metabolism or signaling.



36

Supplemental Material

Supplementary Figure 1. Analysis of abl1 Mutant.

The position of T-DNA insertion of abl1 mutant and confirmation of the homozygous

lines by PCR amplification. The positions of used primers are marked (upper panel). For

PCR amplification, A, primers ABL1-T1 and LP are used; B, primers ABL-T1 and

ABL-T2 are used. Line 1, 2, 3, 8 are homozygous lines of abl1.

Supplementary Figure 2. Expression Analysis of ABL1 in Arabidopsis abi5-1 Mutant

Transformed with ABL1.

ABL1, driven by CaMV35S promoter (p35S:ABL1), is transformed into Arabidopsis

abi5-1 mutant, and evident expression of which in transformants is confirmed by PCR

amplification. Three dependent homozygous lines are analyzed.

Supplementary Figure 3. Phylogenetic analysis of the rice VI bZIP subfamily

members.

Alignment of the conserved bZIP protein sequences and phylogenetic analysis were

performed by using the program ClustalX 1.83 and Treeview, respectively. Members of

bZIP family were designated according to Nijhawan et al. (2008). OsbZIP09,

LOC_Os01g59760; OsABI5, LOC_Os01g64000; OsbZIP12, LOC_Os01g64730;

OsbZIP23, LOC_Os02g52780; OsbZIP24, LOC_Os02g58670; OsbZIP29,

LOC_Os03g20650; OsbZIP40, LOC_Os05g36160; OsbZIP42, LOC_Os05g41070;

ABL1, LOC_Os06g10880; OsbZIP62, LOC_Os07g48660; TRAB1, LOC_Os08g36790;

OsbZIP69, LOC_Os08g43600; OsbZIP72, LOC_Os09g28310; OsbZIP77,

LOC_Os09g36910; AtAREB1, AT1G45249.1; AtAREB2, AT3G19290.1; AtABF3,

AT4G34000.1; AtABI5, AT2G36270.1. A schemic domain structure of ABL1 is shown

(bottom panel). Motif 1 and 2 represents the potential casein kinase II (CKII)

phosphorylation site (S/TxxD/E) or Ca2+-dependent protein kinase phosphorylation site

(R/KxxS/T), respectively. “x” represents any amino acid.

Supplementary Figure 4. The expression level of ABL1 homologs genes in abl1

mutant.

Twelve-day-old WT or abl1 seedlings in the absence or presence of ABA (0.1 mM) were



37

harvested and used for the qRT-PCR analysis. The expression of the corresponding genes

in the WT in the absence of ABA was set to 1.0. The experiments were biologically

repeated, and the data are presented as the average of three independent experiments.

Supplementary Table 1. Down-regulated genes in abl1 mutant by GeneChip analysis.

Total RNAs extracted from roots of 14-day-old abl1 and WT seedlings under ABA

treatment (100 μM, 4 hour) are used for analysis (same for supplementary tables 2-7).

Numbers indicate the regulation ratio.

Supplementary Table 2. Up-regulated genes in abl1 mutant by GeneChip analysis.


Supplementary Table 3. The down-regulated genes in abl1 involve in abiotic and biotic

responses by GO process analysis (P<0.2). 35.4% of the down-regulated genes

response to abiotic and biotic stimuli.

Supplementary Table 4. The up-regulated genes in abl1 involve in abiotic and biotic

responses by GO process analysis (P<0.2). 15.5% of the up-regulated genes response

to abiotic and biotic stimuli.

Supplementary Table 5. Genes in common of up- and down-regulated genes in abl1

mutant and OsbZIP23-overexpression plants.

Supplementary Table 6. WRKY transcription factors stimulated by ABL1 are

up-regulated under stress conditions and hormone treatments.

Supplementary Table 7. Down-regulated genes in abl1 mutant and their expressions

under drought, high-salinity and cold stress by GeneChip analysis. 58% of

down-regulated genes are also regulated under stress conditions (at least one of the

three conditions).

Supplementary Table 8. Up-regulated genes in abl1 mutant and their expressions under

drought, high-salinity and cold stress by GeneChip analysis. 49% of up-regulated

genes are also regulated under stress conditions (at least one of the three conditions).

Supplementary Table 9. Genes changed in abl1 mutant and are related to GA by GO

process analysis.

Supplementary Table 10. Primers used for qRT-PCR analysis.



38

Table 1. Cis-elements those have been demonstrated being involved in ABA responses

are enriched in down- or up-regulated genes under ABL1 deficiency (FDR p<0.06). Cis elements p-value Description Sequence

Down-regulated genes

ACGTTBOX 8.33E-05 T-box; ACGT element AACGTT

ACGTABOX 8.04E-04 A-box; ACGT element; sugar TACGTA

DPBFCOREDCDC3 2.05E-03 Dc3; LEA class gene; ABA; ABI5 ACACNNG

ABRERATCAL 2.07E-03 ABRE; calcium MACGYGB

ABRELATERD1 2.57E-02 ABRE; etiolation; ERD ACGTG

ACGTATERD1 3.16E-02 ACGT; etiolation; ERD ACGT

CE3OSOSEM 4.98E-02 CE3; ABA; VP1; TRAB1; bZIP AACGCGTGTC

HY5AT 5.95E-02 bZIP; HY5; stimulus-response TGACACGTGGCA

Up-regulated genes

ACGTABOX 2.16E-02 A-box; ACGT element; sugar TACGTA

RYREPEATBNNAPA 2.31E-02 RY repeat; RY/G box CATGCA

ABRE3HVA1 2.91E-02 ABRE; ABA; HVA1 GCAACGTGTC



39

Table 2. Cis-elements possibly directly bind by WRKY transcription factors are enriched

in down-regulated genes under ABL1 deficiency (FDR p<0.02). Cis elements p-value Description Sequence

WBOXATNPR1 5.46E-03 NPR1; WRKY18; disease resistance; W box TTGAC

WBOXNTCHN48 9.80E-03 W box; WRKY CTGACY

WBBOXPCWRKY1 1.10E-02 W box; WRKY TTTGACY

WBOXNTERF3 1.63E-02 W box; ERF3; wounding TGACY

WBOXHVISO1 1.66E-02 Sugar; WRKY TGACT



40

Table 3. Genes changed in abl1 mutant and present similar response to IAA treatment.

Genes containing at least three ABRE cis-elements in the promoter region are marked

(*). Numbers indicate the regulation ratio.

Gene Ratio in abl1 IAA regulation Description

Up-regulated

LOC_Os07g36430 1.8 5.9 Expressed protein

LOC_Os02g56700* 1.8 5.3 Dehydrogenase, putative

LOC_Os06g12500 1.6 2.9 Membrane associated DUF588 domain

containing protein, putative

LOC_Os01g67950* 1.9 2.2 Ubiquitin family protein, putative

LOC_Os10g18870* 1.7 2.2 DIRIGENT, putative

Down-regulated

LOC_Os06g37300* -2.4 -2.0 Cytochrome P450, putative

LOC_Os06g46160* -2.1 -2.1 Expressed protein

LOC_Os07g03590* -1.8 -2.1 SCP-like extracellular protein

LOC_Os09g34160* -2.1 -2.2 Resistance protein, putative

LOC_Os11g03290* -2.1 -2.3 Nucleoside-triphosphatase, putative

AK059453 -1.8 -2.4

LOC_Os01g68740* -1.9 -2.4 Keratin, type I cytoskeletal 9, putative

LOC_Os06g33100* -2.3 -3.4 Peroxidase precursor, putative

LOC_Os02g44130* -2.1 -5.3 ZOS2-14 - C2H2 zinc finger protein



41

Table 4. Genes changed in abl1 mutant and are related to IAA by GO process analysis.

Genes containing at least three ABRE cis-elements in the promoter region are marked (*).


Gene Ratio Description Function

LOC_Os03g62060* 2.2 Hydrolase, putative Auxin metabolic process

LOC_Os04g56690* 2.0 OsSAUR23 - Auxin-responsive SAUR

gene family member

Response to auxin stimulus

LOC_Os05g04820 1.6 MYB family transcription factor, putative Response to auxin stimulus

LOC_Os01g57470* -1.6 EF hand family protein, putative Response to auxin stimulus

LOC_Os02g37000* -1.6 Mitochondrial prohibitin complex protein

1, putative

Response to auxin stimulus

LOC_Os07g14610* -1.7 IAA-amino acid hydrolase ILR1-like 6

precursor, putative

Auxin metabolic process



02468

10

12

Rel

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010

20

30

40

50

60

70

Flower Seed Seedling Root Leaf Stem

Rel

ativ

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s

BR ABA IAA GA

0 h1 h3 h8 h

Rel

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s

0

4

8

12

16

20

Drought NaCl PEG

Yang et al., Figure 1

A

B

0 3 5 12 0 5 12 24 0 1 5 12 (h)

1 2 3 4 5 6

Figure 1. Expression Pattern Analysis of ABL1.

A. The quantitative real-time PCR (qRT-PCR) analysis of ABL1 expression in various tissues (upper panel). Seven-day-old seedlings were used to harvest the samples of the seedling (shoot and leaf) and roots (primary and adventitious roots). Leaf and stem samples were harvested from the plants before heading. Bottom panel: The promoter-GUS fusion studies revealed the expression of ABL1 in the seed (1), seedling (2, 3), primary root of the seedling (4), leaf (5) and flower (6). Bars = 2 mm (1, 2, 5, 6), 5 mm (3) or 50 µm (4).

B. qRT-PCR analysis of ABL1 expression under ABA, BR, IAA or GA treatments (upper panel) or under drought, high-salinity or PEG treatments (bottom panel). Twelve-day-old seedlings under different treatments were used for the analysis, and the expression of ABL1 without treatment was set at 1.0. Biological replicates of the experiments were performed, and the data are presented as the average of three independent experiments.



T-DNA

WT L3 L5 abl1

1 μM ABA0

abl1 p35S:ABL1WT L3 L5 abl1

abl1 p35S:ABL1

abi5-1 p35S:ABL1 WS-2

abi5-1

WTabl1abl1 p35S:ABL1-L3abl1 p35S:ABL1-L5

0

20

40

60

80

100

120

0 0.1 0.5 1 2 µM ABA

****

****

Rel

ativ

e ro

ot le

ngth

(%)


A

B

C

7 days

14 days

Figure 2. The Deficiency of ABL1 Caused by a T-DNA Insertion Resulted in Suppressed ABA Responses.

A. The schematic exon-intron structure of the ABL1 gene and the position of the T-DNA insertion (1st intron). Red boxes represent the exons (upper panel). Bottom panel: qRT-PCR analysis of the ABL1 expression in homozygous mutant and transgenic lines with complementary expression of ABL1 (L3 and L5). The expression of ABL1 in the WT is normalized to 1.0. RNAs extracted from rice leaves were used for analysis and the rice ACTIN gene was used as the internal control.

B. The phenotypic observation (upper panel, bar = 1 cm) and calculation (bottom panel) of the root growth revealed that abl1 had suppressed responses to the ABA treatment, whereas abl1 seedlings with complemented expression of ABL1 (representative lines L3 and 5) had normal responses to the ABA treatment. The root lengths of 6-day-old seedlings (after germination) were measured, and the relative root lengths were calculated (the root lengths of untreated seedlings were set at 100%). The data are presented as the average ± SD (n>20). A statistical analysis with a one-tailed Student’s t-test indicates the significant differences (**, P<0.01).

C. ABL1 rescues the ABA insensitivity of Arabidopsis abi5-1. The seeds of WS-2, abi5-1and transgenic abi5-1 lines carryingp35S:ABL1 were germinated on 1/2 MS medium supplemented with 3 µM ABA for 7 days or 14 days.

0

0.5

1

1.5

2

Rel

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WT abl1 L3 L5abl1 p35S:ABL1

**

**

3 μM ABA




A

B

Figure 3. ABL1 Localizes to the Nucleus and Binds G-box/ABRE Elements.

A. A nuclear green fluorescence after transient expression of ABL1 fused to EGFP in onion epidermal cells revealed that ABL1 was localized to the nucleus. Onion cells expressing EGFP alone (left panel) were used as the control. Bar = 100 μm.

B. The yeast-based one-hybrid assay showed the binding specificity of ABL1 to the ABRE/G-box element. The yeast strain YM4271 was used and transformed yeast cells were spot with serial dilutions of cells (OD600) on SD/-His/-Ura/-Leu selective medium for 3 days. Proteins TRAB1 (BAA83740) and RF2a (AF005492) were used as the positive and negative controls, respectively; ABRE/C-box and DRE element were used as the site-mutation of ABRE/G-box. Transformants of different concentrations were dropped onto SD/-His/-Ura/-Leu+30 mM 3-AT or selected by β-Gal activity.

OsABL1/DRE

OsABL1/G-boxTRAB1/G-box

RF2a/G-box

OsABL1/C-box

OD600 1.8 10-1 100-1 1.8 10-1 100-1 1.8 10-1 100-1

SD/-His/-Ura/-Leu SD/-His/-Ura/-Leu+30 mM 3-AT

β-Gal assay

p35S:EGFP p35S:ABL1-EGFP




Competition

B

12x 3x

*

ABL1 protein 0 0.01 0.02 0.04 0.08 0.02 0.02 (μg)

A

2

4

6

8

10

0

WT-mockWT-ABA

abl1-mockabl1-ABA

Os03g2

1710

Os06g4

4010

Os08g2

9660

Os05g2

5770

Os05g4

6020

Os05g0

9020

Os11g2

9870

Os05g3

9720

Rel

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s

C

Os01g5

0100

Os05g4

3390

Os06g4

6160

Os12g2

8137

Os12g3

6940

Figure 4. Suppressed Expression of G-box/ABRE-Containing Genes, Especially WRKY Family Members, in ABL1 Deficient Plants.

A. qRT-PCR analysis of genes containing multiple G-box elements in the absence or presence of ABA (0.1 mM). Expression of the corresponding genes in the WT in the absence of ABA is normalized to 1.0, and relative gene expression is shown. The experiments were biologically repeated, and the data are presented as the average of three independent experiments (same for C).

B. Electrophoretic mobility shift assay (EMSA) confirms the binding of ABL1 to the promoter regions of putative ABRE-containing gene (LOC_Os01g50100). The recombinant expressed ABL1 protein was used to test the binding capacity to the dig-labeled DNA fragment of the promoter region. Unlabeled DNAs (3 or 12 times) were used as the cold competitor for assays. Free probes are indicated with star and shift bands are indicated with arrows.

C. The G-box/ABRE-containing WRKY family genes were down-regulated in abl1 under the ABA treatment. Twelve-day-old WT or abl1 seedlings in the absence or presence of ABA (0.1 mM) were harvested and used for the qRT-PCR analysis. The expression of the corresponding genes in the WT in the absence of ABA was set at 1.0.

D. EMSA confirms the binding of ABL1 to the promoter regions of one ABRE-containing WRKY gene (LOC_Os08g29660). The experiments were performed as described in B. Unlabeled DNAs (3 to 24 times) were used as the cold competitor for assays.

E. abl1 was more sensitive to the PEG treatment (left panel, bar = 1 cm). Twenty-day-old WT and abl1seedlings were exposed to 15% PEG for 10 days. The relative leaf water content after PEG treatment for 3d was measured, revealing that abl1 was hypersensitive to drought stress in comparison to WT. Complementary expression of ABL1 rescued the suppressed growth of abl1 (right panel). Seven-day-old seedlings were exposed to 20% PEG for 3d. Statistical analysis by a one-tailed Student’s t-test indicated significant differences (**, P<0.01).

Os01g50100

Competition 3x 6x 12x 24x

*

ABL1 protein 0 0.04 0.08 0.16 0.04 0.04 0.04 0.04 (μg)

Os08g29660

WT abl1

D

24 h 48 h 72 h

Rel

ativ

e w

ater

con

tent

(%)

0

20

40

60

80

100

120

WT-mockabl1 p35S:ABL1-L3abl1-mockWT-PEGabl1 p35S:ABL1-L3abl1-PEG

**

**

E

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0123456

WT-mockWT-ABA

abl1-mockabl1-ABA



Rel

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20

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0 0.01 0.1 1 10 µM IAA

WTabl1

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*


A

B

C

Up-regulation Down-regulation

01234567

IAA1 IAA9 IAA24

Rel

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1

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Os1g67950

Os2g56700

Os2g44130

Os6g37300

Os6g46160

Rel

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n WT-mockWT-IAAabl1-mockabl1-IAA

Figure 5. ABL1 is Involved in the IAA Responses.

A. The measurement and calculation of the relative root elongation reveal the hypersensitive responses of abl1 to exogenous auxin. Five-day-old WT and abl1seedlings (after germination) grown on medium supplemented with various concentrations of IAA were analyzed. The root length without IAA treatment was set at 100%. The data are presented as the average ± SD (n>20). Statistical analysis by a one-tailed Student’s t-test indicated significant differences (*, P<0.05; **, P<0.01).

B. qRT-PCR analysis showed that the IAA-responsive marker genes (IAA1, 9, 24) were increased to a higher level in abl1 under the IAA treatment than those of the WT. Twelve-day-old WT or abl1 seedlings were treated with IAA (10 μM) for 6 h and then harvested for analysis. The expression of the corresponding genes of the WT and abl1 in the absence of IAA was set at 1.0. The experiments were repeated, and the data are presented as the average of three independent experiments (same for C).

C. The altered genes in the abl1 mutant that share a similar response to the IAA treatment (up- or down-regulation) were selected, and their expression in the absence or presence of IAA (10 μM) was examined by qRT-PCR analysis.



ABL1

ABA

Drought, Salt, Cold …

IAA


ABRE-containing genes

W-box-containing genes

WRKY genes

Biotic/abiotic responses

Auxin signaling

Figure 6. A Hypothetical Working Model of ABL1 Function in Stress Response and Auxin/ABA Cross-Talk.

ABL1 expression is stimulated by various environmental stresses, which may be mediated by ABA, and then activates the expression of a series of ABRE (G-box)-containing genes, especially those involved in ABA signaling and stress responses through direct binding to the ABRE motifs. In addition, ABL1 stimulates the expression of ABRE-containing WRKYmembers, which differentially regulate the down-stream W-box-containing genes to regulate the biotic/abiotic responses and ABA signaling. Meanwhile ABL1 is induced by IAA and negatively regulates auxin signaling by regulating the expressions of ABRE (G-box)-containing genes related to auxin metabolism or signaling.

ABA signalingResponsive genes

Direct bindingDire

ct

bind

ing



running title abl1 regulates aba and auxin responses€¦ · 05/05/2011 · five aba insensitive...

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