A dual role for MYB60 in stomatal regulation and root growthof Arabidopsis thaliana under drought stress

Jee Eun Oh • Yerim Kwon • Jun Hyeok Kim •

Hana Noh • Suk-Whan Hong • Hojoung Lee

Received: 7 October 2010 / Accepted: 16 May 2011 / Published online: 3 June 2011

� Springer Science+Business Media B.V. 2011

Abstract In response to environmental challenges, plant

cells activate several signaling pathways that trigger the

expression of transcription factors. Arabidopsis MYB60

was reported to be involved in stomatal regulation under

drought conditions. Here, two splice variants of the MYB60

gene are shown to play a crucial role in stomatal move-

ment. This role was demonstrated by over-expressing each

variant, resulting in enhanced sensitivity to water deficit

stress. The MYB60 splice variants, despite the fact that one

of which lacks the first two exons encoding the first MYB

DNA binding domain, both localize to the nucleus and

promote guard cell deflation in response to water deficit.

Moreover, MYB60 expression is increased in response to a

low level of ABA and decreased in response to high level

of ABA. At initial stage of drought stress, the plant system

may modulate the root growth behavior by regulating

MYB60 expression, thus promotes root growth for

increased water uptake. In contrast, severe drought stress

inhibits the expression of the MYB60 gene, resulting in

stomatal closure and root growth inhibition. Taken

together, these data indicate that MYB60 plays a dual role

in abiotic stress responses in Arabidopsis through its

involvement in stomatal regulation and root growth.

Keywords Arabidopsis thaliana � Auxin �Drought stress � Guard cell �MYB60

Introduction

The world population has been steadily growing since early

1400s. According to projections, the population will con-

tinue to increase until approximately 2050, resulting in

various environmental problems such as global warming.

Temperature rise is expected to cause severe drought

stresses in crops and thus, eventually results in food

shortages. Hence, it is believed that elucidation on and

manipulation of drought stress tolerance in plants may

alleviate human suffering in the face of agricultural stres-

ses. Various studies in Arabidopsis have implicated that a

large proportion of the genome responds to drought

(Shinozaki and Yamaguchi-Shinozaki 2000; Shinozaki

et al. 2003) and high salinity stresses (Xiong et al. 2002;

Zhu 2002).

Guard cells, responsible for controlling the stomatal

openings in the epidermis of leaves and other tissues,

represent the primary plant defense against drought stress.

However, several factors also play important roles in the

regulation of stomatal movements including light, carbon

dioxide concentration, phytohormones and water avail-

ability. Among these, the role of abscisic acid (ABA) has

been well documented in the control of guard cell move-

ments. ABA inhibits guard cell Kin channels while acti-

vating Ca2?-permeable channels and anion channels for

anion efflux. These changes lead to guard cell deflation

J. E. Oh and Y. Kwon authors contributed equally.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-011-9796-7) contains supplementarymaterial, which is available to authorized users.

J. E. Oh � Y. Kwon � J. H. Kim � H. Lee (&)

College of Life Sciences and Biotechnology, Korea University,

1, 5-ka Anam-dong, Sungbuk-ku, Seoul 136-713,

Republic of Korea

e-mail: lhojoung@korea.ac.kr

H. Noh � S.-W. Hong

Department of Molecular Biotechnology, College of Agriculture

and Life Sciences, Bioenergy Research Institute, Chonnam

National University, Gwangju, Korea

123

Plant Mol Biol (2011) 77:91–103

DOI 10.1007/s11103-011-9796-7

(Blatt 2000; Schroeder et al. 2001; Hetherington 2001;

Pandey et al. 2007). This inhibits stomatal opening, pro-

motes stomatal closure, and reduces plant water loss

(Sirichandra et al. 2009). In contrast, the transcriptional

control of stomatal movement has not been studied, in part

due to the belief that alterations in gene expression are

temporally incompatible with a prompt drought stress

response by guard cells. Therefore, it was considered more

plausible that cell volume changes are more rapidly

achieved by altering membrane transport via channels or

pumps. However, the level of PP2CA transcripts, encoding

a type 2C protein phosphatase, were found to increase in

Arabidopsis guard cell protoplasts upon treatment with

ABA (Leonhardt et al. 2004). The idea that stomatal

movement could be regulated by changes in gene expres-

sion was further supported by the observation that several

transcription factors can modulate stomatal movements

when plants are exposed to different environmental stimuli.

To date, a few MYB transcription factors have been

implicated in the control of stomatal movement. The MYB

factors, comprising about 196 family members in Arabid-

opsis thaliana, are classified into four different subgroups

(1R-, R2R3-, 3R- and 4R-MYB) according to their

arrangement of MYB domains (Chen et al. 2006; Dubos et al.

2010). This domain may consist of one to four imperfect

repeats and each of which includes about 50–53 amino acid

sequences forming three a-helices thus, building a helix-

turn-helix DNA-binding motifs (Rosinsky and Atchley

1998; Jia et al. 2004). The characterization of MYB60, an

Arabidopsis R2R3-MYB gene, revealed a guard cell-specific

expression pattern that is reduced in response to drought

stress (Cominelli et al. 2005). Plants containing a MYB60

null mutation were shown to exhibit a constitutive reduction

in stomatal opening, resulting in a decreased wilting

response under water stress conditions. Additionally, over-

expression of another guard cell-specific MYB family

member, MYB44, leads to enhanced ABA sensitivity and a

more rapid stomatal closure response in comparison to wild-

type plants (Jung et al. 2008). Thus, the reduced expression

of genes encoding type 2C protein phosphatases, known to

be negative regulators, partly explains the increased toler-

ance of MYB44 over-expressing plants to abiotic stresses.

Another MYB superfamily member, MYB61, also belongs to

the R2R3-MYB family and mediates responses leading to

stomatal closure (Liang et al. 2005). More recently, the

MYB96-mediated ABA signals were shown to be integrated

into an auxin signaling pathway (Seo et al. 2009). MYB96 is

induced by ABA while MYB60 is repressed by ABA. This

indicates that the expressions of different MYB genes are

carefully controlled in their unique and specific manner. This

notion was supported by the observation that MYB96-over-

expressing Arabidopsis plants exhibited enhanced drought

resistance with reduced lateral roots (Seo et al. 2009).

To understand the role of MYB60 in drought stress, two

splice variants were identified from the TAIR database.

One of these variants was initially suspected to have an

alternative role in stomatal movements due to the absence

of the first two exons, which encode a R2-type repeat of

R2R3-MYB domain found in full MYB60 sequence. The

results presented here demonstrate that both MYB60 splice

variants confer sensitivity to drought stress conditions

when overexpressed. Microarray analysis also detected

decreased levels of several aquaporin transcripts in plants

overexpressing the MYB60.2 splice variant under drought

stress, confirming the previous hypothesis reported by

Cominelli et al. (2005). Furthermore, it is clarified that

MYB60 transcripts are expressed in roots as well as in

guard cells. This result correlates with reduced expression

levels of the auxin-responsive DR5::GUS (beta-glucuroni-

dase) construct and an increased number of lateral roots in

MYB60-overexpressing plants. Taken together, these

results support that MYB60.1 and MYB60.2 are essential

transcriptional regulators involved in monitoring the status

of water availability and fine-tuning of the plant responses

to drought stress by regulating guard cell movements and

root growth.

Materials and methods

Plant materials and growth conditions

Arabidopsis thaliana, ecotype Columbia-0, was used for all

experiments. To examine soil growth phenotypes, seeds

were allowed to germinate and grow at 23�C with a 16 h

light/8 h dark cycle at a relative humidity of 70% following

3 days of stratification at 4�C. To examine plant growth

and developmental phenotypes on sterilized medium, seeds

were surface-sterilized and sown in Murashige and Skoog

(MS) agar supplemented with 2% sucrose. After 3 days of

stratification at 4�C, seeds were germinated at 23 ± 1�C

with a 16 h light/8 h dark cycle. Five-day-old seedlings

were transferred to test medium containing MS basal salts,

2% (w/v) sucrose and 1.2% phytoagar (Duchefa Biochemie

B.V., The Netherlands), and grown vertically.

Transgenic plants

Since the Arabidopsis Information Resource (TAIR,

www.arabidopsis.org) indicated that At1g08810 has two

transcripts, two sets of primers were designed as follows:

for the longer transcript, MYB60.1, forward 50-ATGGGTA

GGCCTCCATGC-30 and reverse 50-GAGCTCTCTAATA

TGCTTTAA-30, and for the shorter transcript, MYB60.2,

forward 50-ATGTGTCCTGATGTTTGTATCTTA-30 and

reverse 50-GAGCTCTCTAATATGCTTTAA-30. To amplify

92 Plant Mol Biol (2011) 77:91–103

123

the promoter region, the reverse primer MYB60proR

(50-CTCTCTGAGTCTTATGCTCTC-30) was used in com-

bination with MYB60proF1 (50-GCTTGTGACTCTTCT

TCCA-30), resulting in the synthesis of a *1.2 kb fragment,

with MYB60proF2 (50-CACATCCTTCACGTAGATGAC-

30), resulting in the synthesis of a *800 bp fragment, or

with MYB60proF3 (50-GTCTCTTCTTACGTTCCCTTC-30),resulting in the synthesis of a *400 bp fragment. For

overexpressing lines, DNA fragments were introduced into

the binary vector pBI121 (Promega Corp., WI, USA) under

the control of 35S CaMV promoter. Agrobacterium tum-

efaciens strain GV3101 was used to transform Arabidopsis

plants by the floral dipping method (Clough and Bent 1998).

Microscopy

Arabidopsis Col-0 wild-type and transgenic plants were

grown on moist soil until the rosettes were fully developed.

A month after germination, leaves were excised, allowed to

dry for 5 h, and monitored by inverted fluorescence

microscopy (Carl-Zeiss, Germany) to determine stomatal

opening. For confocal microscopy, green fluorescence

images were obtained with a Confocal Laser Scanning

Microscope (Bio-Rad, UK) using a 510–580 nm emission

setting. GFP was excited with a 568 nm krypton laser and

confocal images were collected using a 600–620 nm

emission setting. FM4-64 was excited using the 488 nm

line from an argon ion laser, and emitted fluorescence was

monitored using the 650LP filter.

RNA gel blot analysis

Wild-type and transgenic plants grown for 2 weeks on MS

medium were treated with hormones and chemicals as

indicated in the figures. Total RNA was extracted from

whole seedlings as previously described (Lee et al. 2002).

20 lg of total RNA was subjected to electrophoresis on

1.5% (w/v) agarose gels containing formaldehyde and then

transferred to a HybondTM-XL membrane (Amersham

Biosciences, NY, USA). The membranes were pre-

hybridized for 1 h and hybridized overnight at 65�C with

probes labeled with 32P-dCTP using the Random Primers

DNA Labeling System (Invitrogen Corp., CA, USA) fol-

lowing the manufacturer’s instruction.

qRT–PCR

Total RNA and the subsequent first-strand cDNA were pre-

pared from 2 week-old Col-0 and overexpressor seedlings.

RNA was extracted using Trizol reagent (Invitrogen, CA,

USA) and cDNA was synthesized using M-MLV Reverse

Transcriptase (iNtRON Biotechnology, Korea) according to

the manufacturer’s instructions. qRT-PCR reactions (total

volumes of 20 ll) were conducted using iCycler iQ

(Bio-Rad, CA, USA). Specific primer sets were designed as

follows. Primer sets to specifically detect MYB60.1 or

MYB60.2 transcripts, forward 50-ATGGGTAGGCCTCCA

TGC-30 and reverse 50-TTGTTACCCAATAAGGCTTGC-

30 or forward 50-ATGGTTGTGGTAAGATGGG-30 and

reverse 50- GCTTGAAGCATAGGTAGATC-30 were used

respectively. For qRT-PCR to quantify flavonoid biosyn-

thetic genes, primer pairs were as follows: PAP1(50-AT-

GGAGGGTTCGTCCAAAG-30 and 50-CAAGGTGCTCC

CCTTTTCTGTT-30) and F3H (50- ATGGCTCCAGGAA

CTTTGAC-30 and 50- AGCGAAGATTTGGTCGACAG-

30). Data represent the average of three independent

experiments.

Microarray analysis

The transcript levels of flavonoid-related genes in desic-

cation-treated Col-0 and MYB60.2-ox plants were obtained

by microarray analysis. four-week-old soil grown Col-0

and MYB60.2-ox plants were dehydrated for 3 days prior to

sampling for microarray analysis. Total RNA from the

rosette leaves of 1-month-old plants, grown on soil and

dried for 3 days, was purified using the RNeasy Plant Mini

kit (Qiagen, MD, USA). 5 lg of fibroblast total RNA was

used for probe-labeling and hybridization, detection and

scanning were completed according to the manufacturer’s

standard protocols, Affiymetrix, Inc. Using One-Cycle

cDNA Synthesis Kit (Affymetrix, CA, USA), single-

stranded cDNA was synthesized from total RNA and

double-stranded cDNA was obtained using DNA ligase,

DNA polymerase I, RNase H, and T4 DNA polymerase.

In vitro transcription was performed with ds-cDNA for in

vitro transcription using the GeneChip IVT Labeling Kit

(Affymetrix). After cleaning up with Sample Cleanup

Module (Affymetrix), 10–15 lg of labeled cRNA was

fragmented from 35 to 200 bp and hybridized to Human

Genome U133A 2.0 gene chips (Affymetrix)) at 45�C for

16 h. Then the arrays were washed in a GeneChip Fluidics

Station 450 with wash buffers according to the Affymetrix

standard protocol. The arrays were then stained and the

intensities were measured with GeneChip scanner 3000

(Affymetrix) by GCOS Affymetrix software.

Histochemical GUS assays

Two- or six-week-old Arabidopsis plants were subjected to

various abiotic stresses as indicated in the figures. The

GUS assay was performed by incubating whole plant tissue

in GUS assay solution at 37�C overnight. Chlorophyll was

then removed with 70% ethanol at 70�C prior to observa-

tion as described (Jefferson et al. 1987).

Plant Mol Biol (2011) 77:91–103 93

123

Statistical analysis

Data are expressed as means ± standard error (SE).

Comparisons were made between groups with the Stu-

dent’s t tests.

Results

The Arabidopsis MYB60 gene has two splice variants

Initially, the role of MYB60 as a negative regulator

against plant drought stress was investigated. Two splice

variants of MYB60, specifying cDNAs of 843 and 594 bp,

were identified from the TAIR database. These variants

were designated MYB60.1 and MYB60.2, respectively

(Fig. 1a). MYB60 is also classified as a member of the

R2R3-type MYB subfamily which contains 125 addi-

tional genes. However, the first R domain is absent in the

MYB60.2 transcript and its translation initiates with an

ATG in the latter part of the second intron (Fig. 1a). As

the MYB domain plays a crucial role in the recognition

of cis-acting elements in DNA, the individual role of the

MYB60.2 transcript was scrutinized and characterized to

determine whether it had a role distinct from that of

Fig. 1 Sequence analysis of Arabidopsis MYB60. a AtMYB60 is

alternative spliced during post-transcriptional processing, resulting in

two non-canonical transcripts. The longer transcript contains 843 bp

and the shorter transcript contains 594 bp. The shorter transcript

initiates translation with an ATG codon located within the second

intron of the gene. Probe locations for MYB60.1 or MYB60.2 are as

indicated. b Sequence alignment of MYB60.1 and MYB60.2 with

other R2R3-MYB family members of Arabidopsis. Among 126

R2R3-MYBs, AtMYB30, AtMYB94, and AtMYB96 share the

highest similarity. Interestingly, the shorter transcript lacks the first

R2 domain. c MYB60.1 and MYB60.2 transcripts were cloned into

Col-0 plants. The transcript expression levels from T3 generation of

each homozygous transgenic plant line were confirmed via northern

blot analysis and quantified

94 Plant Mol Biol (2011) 77:91–103

123

MYB60.1. Based on DNA sequence alignment, MYB60.1

and MYB60.2 were found to have high sequence

homology and to contain several conserved domains (or

blocks) also found in MYB30, MYB96 and MYB94.

Amino acid sequence homology between MYB60.1 and

MYB60.2 is 69.3% as determined by protein blast anal-

ysis while their sequences are 99% identical within the

shared region as determined by using Blastp Suit-2.

Moreover, the amino acid sequence of MYB60.1 shares

89, 78, and 76% homology with MYB30, MYB96 and

MYB94, respectively (Fig. 1b).

To examine potential hormone-responsive elements in

the MYB60 promoter, three fragments of the MYB60 pro-

moter were fused to a GUS-encoding sequence in the

vector pBI121. The resulting constructs, containing

*1.2 kb, *800 bp, and *400 bp of sequence upstream of

the start codon, were named MYB60proF1, MYB60proF2,

and MYB60proF3, respectively (Fig. S1). As shown in Fig.

S1, the 1.2 kb element resulted in the highest GUS

expression levels. GUS staining was observed in the sto-

mata of leaves, stems, and inflorescences.

Soil-grown MYB60.1-ox and MYB60.2-ox plants are

more susceptible to drought stress conditions

MYB60.1 and MYB60.2 transcripts were overexpressed to

further examine their roles in environmental stress

responses. The MYB60 transcript levels of MYB60.1-ox and

MYB60.2-ox seedlings were detected by northern analysis

(Fig. 1c). To specifically detect the MYB60.1 transcript, a

DNA fragment encompassing the first and second exons,

which are absent in AtMYB60.2, was used as a probe.

Substantial levels of MYB60 expression were detected in

the MYB60.1-ox lines #9 and 16, and in MYB60.2-ox lines

#3, 7 and 8 (Fig. 1c). As MYB60 is predicted to encode a

transcription factor with an R2R3-type MYB DNA-binding

domain, the protein was suspected to localize to the

nucleus. To investigate this hypothesis, MYB60.1::GFP or

MYB60.2::GFP construct was separately inserted into

onion epidermal cells by particle bombardment. As

expected, green fluorescence of GFP-fused each MYB

transcripts were precisely observed in the nucleus (Fig. 2).

When MYB60.1-ox and MYB60.2-ox seedlings were

grown under well-controlled humid conditions, no pheno-

typic alterations were observed (data not shown). However,

when 1-month-old plants grown in pots were dehydrated, a

clear phenotypic difference was apparent between the wild-

type and transgenic plants. After 10 days of drought stress,

the MYB60.1-ox and MYB60.2-ox plants started to wilt. By

day 14, the transgenic plants, especially the MYB60.2-ox

plants, were clearly distinguished from wild-type by their

fatality. On the contrary, the wild-type Col-0 plants remained

alive due to altered stomatal regulation (Figs. 3, 4). To

characterize the stomatal response, leaves of the wild-type,

MYB60.1-ox and MYB60.2-ox plants were detached and

placed into petri-dishes for drought stress treatment. Sto-

matal aperture was determined 5 h after detachment using a

microscope. As shown in Fig. 4, the stomata of MYB60.1-ox

and MYB60.2-ox plants are less responsive to drought stress

when compared to wild-type plants. The root growth of the

transgenic plants in MS agar medium supplemented with

mannitol (Fig. 5a) was also analyzed. Surprisingly, the 4

day-old seedlings of MYB60.1-ox and MYB60.2-ox plants

grown in control MS medium developed a greater root mass

upon transfer to medium containing mannitol than wild-type

plants. Characterization of the MYB60.1 and MYB60.2

transcript levels in response to ABA (10 lM) and salt (NaCl

200 mM) are shown in Fig. 5d. MYB60.1 and MYB60.2

transcripts accumulated to higher levels upon treatment with

10 lM ABA in Col-0 seedlings.

Since auxin plays a fundamental role in root growth,

MYB60 expression levels following auxin treatment were

investigated. As shown in Fig. 6a, auxin slightly induced

the accumulation of MYB60. Moreover, expression of

Fig. 2 Subcellular localization of MYB60.1::GFP and MYB60.2::

GFP fusion proteins. Constructs containing MYB60.1 fused to GFP

(MYB60.1::GFP) or MYB60.2 fused to GFP (MYB60.2::GFP) were

inserted into onion cells via gold-particle bombardment. Control

experiment with GFP protein alone is shown in the left panel

Plant Mol Biol (2011) 77:91–103 95

123

MYB60 was detected in the root tissues of wild-type plants

upon 10 lM of auxin treatment (Fig. 6a). Additionally,

GUS staining confirmed that the MYB60 promoter in the

roots and guard cells of the transgenic plants was sensitive

to IAA, although patch-type expression was detected in the

root tissue (Fig. 6b).

Possible target genes of MYB60 under drought stress

To comprehensively analyze the regulatory role of MYB60

and to gain more insights into the coordination of drought

stress responses with developmental and environmental

signaling systems, microarray analysis was conducted

using Col-0 and MYB60.2-ox plants (Tables 1, 2). Four-

week-old soil-grown Col-0 and AtMYB60.2-ox plants were

dehydrated for 3 days prior to sampling for microarray

analysis. Large numbers of drought-regulated genes,

including many novel genes, were detected to be altered.

The interactions between drought, rehydration, plant hor-

mones, and other environmental factors were investigated

using microarray analysis and in silico comparisons. Many

flavonoid biosynthetic genes were up-regulated in MYB60-

ox plants. As flavonoids are known to interfere with polar

auxin transport, plant phenotypes in response to auxin were

characterized. DR5::GUS plants were crossed with

MYB60.1-ox and MYB60.2-ox plants, and their progenies

were screened to identify double homozygous lines.

Fig. 3 Drought tolerance of MYB60.1-ox and MYB60.2-ox plants.

Col-0, MYB60.1-ox and MYB60.2-ox plants were seeded on soil.

1 month after germination, water was withheld for up to 2 weeks

prior to image collections

Fig. 4 Stomatal response of MYB60.1-ox and MYB60.2-ox plants

under drought stress. Col-0, MYB60.1-ox and MYB60.2-ox plants

were seeded on soil. To determine stomatal responses, the leaves of

wild-type, MYB60.1-ox and MYB60.2-ox plants at 1 month after

germination were detached and placed in a petri-dish (without the lid)

for drought stress treatment. 5 h after detachment, the stomatal

aperture was determined under the microscope (a) and pictures were

taken (b). The data represents the average of three independent

experiments with 100 stomata evaluated for each determination.

Lower case letters (a, b, c, d) indicate differences that are statistically

significant (t test P \ 0.05–0.01, comparison made between Col-0

and different overexpressing lines in the indicated treatment). Verticalbars indicate SE (n = 3)

96 Plant Mol Biol (2011) 77:91–103

123

Fig. 5 Drought-related

phenotypes of MYB60.1-ox and

AtMYB60.2-ox plants. a Root

architecture of Col-0, MYB60.1-

ox and MYB60.2-ox plants

grown on media containing

mannitol. Main root lengths

b and root mass c were

measured from each line. Seeds

were germinated on MS

medium and 5-day-old

seedlings were transferred to

medium supplemented with

mannitol and allowed to grow

for 2 additional weeks. The data

represents the average of three

independent experiments with

20 seedlings evaluated for each

determination. Lower caseletters (a, b, c, d) indicate

differences that are statistically

significant (t test

P \ 0.05–0.01, comparison

made between Col-0 and

different overexpressing lines in

the indicated treatment).

Vertical bars indicate SE

(n = 3). d For qRT-PCRanalysis, wild-type seedlings

were grown on growth medium

for 2 weeks and then transferred

to medium supplemented with

ABA (10 lM) or NaCl

(200 mM) for 3 or 6 h as

indicated prior to RNA

extraction. Transcript levels of

MYB60.1and MYB60.2 are

shown. Actin transcript levels

were used as an internal control

Plant Mol Biol (2011) 77:91–103 97

123

DR5::GUS x AtMYB60.1-ox F3 plants, DR5::GUS x At-

MYB60.2-ox F3 plants, and DR5::GUS plants were grown

in control MS-agar medium for 2 weeks and then trans-

ferred to media supplemented with 0, 0.1 or 10 lM IAA

for 6 h. As shown in Fig. 7, GUS expression was signifi-

cantly decreased in the roots and leaves of both DR5::GUS

x AtMYB60.1-ox F3 and DR5::GUS x AtMYB60.2-ox F3

plants.

Anthocyanin, a subgroup of plant secondary metabolite

flavonoid, was considered as a potential regulator of polar

auxin transport in plant tissues. Anthocyanin levels were

determined in the wild-type, MYB60.1-ox lines #9 and 16,

and MYB60.2-ox lines #7 and 8 (data not shown). However,

the differences in anthocyanin accumulation levels

between the control and overexpressor plants were

insignificant. Since some flavonoid biosynthetic genes,

such as PAP1 and PAP2, are reported to express in

response to sucrose (Teng et al. 2005), the transcript level

of MYB60 was examined by northern analysis after sucrose

treatments. As shown in Fig. 8, the MYB60 transcript level

was increased strongly after 3 or 6 h of incubation in

medium containing 4, 6, or 8% sucrose.

Interestingly enough that number of flavonoid biosyn-

thetic genes were determined to be highly expressed in

MYB60.2-ox plants after drought treatment as listed by

microarray analysis data (Table 2). The PAP1 and flava-

none-3ß-hydroxylase (F3H) expression levels were quan-

tified with quantitative RT-PCR to assess the relative

transcript levels of the flavonoid biosynthetic genes such as

PAP1 and F3H in MYB60-ox plants in Fig. 9. The tran-

scription level of PAP1and F3H were increased upon 8%

sucrose treatment.

Discussion

Drought and highly saline soils are among the most serious

challenges to crop production in the world today. Both

traditional breeding and genetic engineering of crop plants

have been utilized to improve drought and high salinity

tolerance or resistance with the goal of increasing agri-

cultural productivity in affected regions. An understanding

of the plant responses to abiotic stress at the genomic level

is expected to provide an essential foundation for future

breeding and genetic engineering strategies. Thus, to fulfill

the ultimate aim of crop-yield improvement, the function

of MYB60, which is involved in abiotic stress response, was

investigated in detail. MYB60 is a member of the R2R3-

type MYB subfamily. In the Arabidopsis Information

Resources (TAIR), MYB60 is listed as encoding two splice

variants designated MYB60.1 and MYB60.2. Interestingly,

MYB60.2 lacks the first R region and is apparently trans-

lated from an ATG located in the latter part of the second

intron (Fig. 1a). Each MYB repeat is known to play a

crucial role in the recognition of cis-acting elements in the

DNA. Hence, this study investigated whether these two

transcripts have roles that are distinct from or redundant to

one another. Full MYB60.1 protein was located in the

nucleus (Fig. 2) as predicted from its biomolecular func-

tion as a transcription factor. Moreover, the MYB60.2

variant, which is missing the first and second exons of

MYB60 (Fig. 1), also localizes to the nucleus (Fig. 2)

supporting that it may act as a transcriptional regulator

similar to MYB60.1.

The two splice variants of MYB60, MYB60.1 and

MYB60.2, alter plant responses to drought stress by con-

trolling stomatal movement (Figs. 3, 4). The regulatory

mechanism of guard cells as drought-adaptive plant

Fig. 6 MYB60 is induced by auxin. a The MYB60 transcript was

monitored in whole or root tissues isolated from three-week-old wild-

type, MYB60.1-ox and myb60-ko plants by northern analysis. Seeds

were germinated on MS medium and subsequent seedlings were

transferred to liquid medium supplemented with 10 lM IAA for the

indicated time period. The rRNA gene is shown as a loading control.

b To determine whether the MYB60 gene promoter is responsive to

IAA, a plasmid containing the promoter region (*1.2 kb) of MYB60fused to the coding region of the reporter gene GUS was constructed.

Histochemical assays were conducted to visualize the location of

b-glucurodinase (GUS) activity in transgenic plants at 0, 6 or 24 h

after incubation with 10 lM IAA. Two-week-old plants were stained

with GUS solution overnight, and chlorophyll was removed by

treatment with 70% ethanol

98 Plant Mol Biol (2011) 77:91–103

123

response was recently reviewed (Sirichandra et al. 2009).

As the epidermis is covered with an impermeable wax

layer, stomata play a crucial role in gas exchange between

plants and the atmosphere. Stomatal movement appears to

be controlled by an interaction between the activities of ion

channels and vacuolar and membrane transporters, and by

metabolite conversions (Roelfsema and Hedrich 2005;

Wasilewska et al. 2008). Additionally, the importance of

MYB60 in modulating stomatal movements has been

established (Cominelli et al. 2005). However, previous

reports did not realize the presence of a splice variant.

Recognizing its presence, this variant is the focus of this

study, as splice variants have been shown to sometimes

exhibit opposing functions. For instance, SULF1, a recently

discovered member of the sulfatase gene family from quail

embryo, has one splice variant (SULF1B) that appears to

oppose the functional activity of a second splice variant

(SULF1A) (Sahota and Dhoot 2009). Because ectopic

expression of either MYB60 variant confers greater sus-

ceptibility to drought stress, MYB60.1 and MYB60.2 appear

to play similar roles in terms of stomatal movement

(Figs. 3, 4). In contrast to myb60 knock-out plants (Fig.

S2), MYB60.1-ox and MYB60.2-ox plants developed

greater root mass than wild-type plants in sealed growth

medium (Fig. 5). This finding is surprising because previ-

ous results revealed that the MYB60.1-ox and MYB60.2-ox

plants withered earlier than wild-type plants when grown

under drought conditions in soil. The difference between

the two experimental results (Figs. 3, 5) may occur from a

higher level of humidity in the sealed plate shown in Fig. 5.

Apparently, ectopic expression of MYB60 was not able to

enhance evaporation through the stomata of transgenic

plants, causing faster mortality rate, although their guard

cells were constantly wide open in the sealed plate.

In addition to the fact that MYB60 expression was lim-

ited in guard cells in aerial parts, MYB60 was also

Table 1 Microarray analysis of flavonoid-related genes in drought-treated Col-0 and MYB60.2-ox plants

Probe sets Col-

0

60.2oX P value Log2

ratio

Target description MIPS Gene

symbol

Up-regulated genes

260140_at 20.2 239.8 4.34E-06 3.57 AtMYB90; production of anthocyanin pigment 2 Atlg66390 PAP2

254283_s_at 41.4 199.6 4.91E-05 2.27 Putative leucoanthocyanidin dioxygenase; putative

anthocyanidin synthase

At4g22870 –

250207_at 130.3 565.0 1.06E-05 2.12 Chalcone synthase (TT4) At5g13930 CHS

249215_at 19.0 79.1 4.09E-05 2.06 Dihydroflavonol 4-reductase (TT3) At5g42800 DFR

245628_at 21.4 88.9 3.68E-05 2.06 AtMYB75; production of anthocyanin pigment 1 Atlg56650 PAPI

252123_at 101.9 299.9 0.000348 1.57 Flavanone 3-hydroxylase (TT6) At3g51240 F3H

250533_at 79.6 226.3 3.67E-05 1.51 Flavonol synthase At5g08640 FLS1

261804_at 67.1 189.2 0.000244 1.50 Flavonoid 3-o-rhamnosyltransferase Atlg30530 UGT78D1

253222_at 19.4 53.9 0.00063 1.49 Chalcone and stilbene synthase family protein At4p34850 –

267147_at 40.4 101.2 7.48E-05 1.33 Putative anthocyanidin synthase; 2oG-Fe(II) oxygenase

family protein

At2g38240 –

255773_at 81.0 189.2 0.000275 1.22 Putative flavonol 40-sulfotransferase; sulfotransferase 17 Atlg18590 SOT17

261907_at 33.8 75.3 2.71E-05 1.15 4-coumarate:CoA lipase 3 Atlg65060 4CL3

251827_at 97.3 211.2 1.57E-05 1.12 Chalcone flavanone isomerase (TT5) At3g55120 CHI

265091_s_at 35.8 75.9 0.0008 1.03 Similar to anthocyanin 5-aromatic acyltransferase Atlg03495 –

245624_at 22.0 45.3 0.005175 1.02 Anthocyanidin 5-o-glucosyl transferase At4gl4090 –

Down-regulated genes

257288_at 155.6 76.6 0.001297 -1.02 Similar to malonyl-CoA; isoflavone 7-o-glucoside-6,-o-

malonyltransferase

At3g29670 –

267337_at 768.4 367.4 2.45E-06 -1.06 Putative anthocyanin 5-aromatic acyltransferase At2g39980 –

254835_s_at 220.7 105.4 0.000183 -1.07 Flavonoid 3,5-hydroxylase -like; member of CYP706A At4g12310 CYP706A5

252534_at 74.9 32.5 0.006755 -1.21 AtMYB 48; production of flavonol glycoside 3 At3g46130

249494_at 174.8 67.1 2.23E-05 -1.38 Anthocyanin 5-aromatic acyltransferase; transferase family

protein

At5g39050 –

249493_at 149.0 46.4 0.000188 -1.69 Anthocyanin 5-aromatic acyltransferase; transferase family

protein

At5g39080 –

Transcript levels of flavonoid-related genes were analyzed in desiccation-treated Col-0 and MYB60.2-ox plants by microarray analysis. Four-

week-old plants grown in soil were dehydrated for 3 days prior to sampling for microarray analysis. 60.2ox stands for MYB60.2 overexpressor

Plant Mol Biol (2011) 77:91–103 99

123

specifically expressed at a low level in roots upon auxin

exposure (Fig. 6). Microarray analysis revealed that several

genes involved in flavonoid biosynthesis and auxin sig-

naling were altered in MYB60.2-ox plants (Tables 1, 2). In

accordance with this molecular phenotype, overexpression

of the splice variants resulted in reduced DR5::GUS

expression in response to auxin in the roots (Fig. 7). Ini-

tially, it was suspected that MYB60 overexpression would

bring about an altered phenotype in transgenic plants.

However, no alterations were detected under normal

growth conditions. As a result, microarray analysis was

utilized to explore the possible targets of MYB60 under

drought conditions (Table 1). The main focus was placed

on the enhanced levels of several flavonoid biosynthetic

genes that are induced in MYB60.2-ox plants experiencing

drought stress (Table 1). From this analysis, it appeared

that flavonoid accumulation in MYB60.2-ox plants were

arising via independent manner from the action of MYB60

in guard cells. One flavonoid synthetic enzyme, chalcone

synthase (CHS), is detected in epidermal cells but not in

guard cells (Zobel and Hrazdina 1995). This indicates that

MYB60 may function in the root where its expression was

observed in response to auxin (Fig. 6). It is well established

that flavonoids function in the modulation of polar auxin

transport as well as in auxin-dependent tropic responses

(Peer and Murphy 2007). Reduced levels of auxin-

responsive DR5 activity and increased expression of flavo-

noid biosynthetic genes were observed in the MYB60.1-ox

and MYB60.2-ox plants (Fig. 7). However, there was no

drastic elevation in anthocyanin accumulation levels

among Col-0, MYB60.1-ox and MYB60.2-ox plants in

response to auxin. Key flavonoid biosynthetic genes, such

as PAP1 or PAP2, have higher levels of transcription when

the plants are exposed to sucrose (Teng et al. 2005). As

demonstrated in Fig. 8, the level of the MYB60 transcript

increased strongly when the plants were exposed to high

levels of sucrose, suggesting that this gene may be involved

in sucrose-induced flavonoid biosynthesis or that a high

concentration of sucrose might cause enhanced osmotic

stress in seedlings. Indeed, MYB60.1 and MYB60.2 tran-

scripts were accumulated in response to 10 lM ABA

(Fig. 5d) whereas MYB60 has been reported to be

decreased by ABA treatment (Cominelli et al. 2005) to the

contrary. It should be noted that these authors used 100 lM

ABA in their experiments while this study used a much

lower concentration (10 lM) of ABA for 3 or 6 h. In this

study, incubation for greater than 24 h with 10 lM ABA

also resulted in decreased levels of MYB60 transcripts (data

not shown). As a major flavonoid biosynthetic gene PAP1

is induced by sucrose and number of flavonoid biosynthetic

genes are up-regulated in MYB60-ox plants (Table 2), a

keen observation of MYB60 regulation in response to

Table 2 Microarray analysis of phytohormone-related genes in drought-treated Col-0 and MYB60.2-ox plants

Probe sets Col-0 60.2OX P value Log2 ratio Description MIPS code

Auxin

250294 at 15.13 64.61 3.98 E-05 2.098 Auxin-reponsive-like protein At5gl3380

245244_at 196.77 620.54 1.29E-06 1.658 IAA-amino acid hydrolase Atlg44350

256178_s_at 332.23 985.95 0.00126 1.572 IAA-amino acid conjugate hydrolase subfamily (ILL5) Atlg51780

234809_at 63.74 186.68 4.9E-05 1.552 Auxin-responsive family protein Artgl2410

251291_at 36.79 17.89 0.007468 -1.069 Auxin-responsive family protein At3g61900

257690_at 615.81 154.71 1.9E-05 -1.997 Auxin-responsive family protein At3gl2830

254323_at 168.31 35.33 3.95E-05 -2.261 Auxin-responsive family protein At4g22620

264014_at 59.25 10.80 6.3E-05 -2.447 Putative auxin-regulated protein At2g21210

GA

266613_at 122.14 403.53 1.14E-05 1.724 Gibberellin-regulated family protein At2gl4900

260141_at 148.72 398.23 4.82E-05 1.424 Negative regulator of GA responses (RGLl); GRAS family Atlg66350

261768_at 71.21 30.05 0.000649 -1.236 Gibberellin 3 beta-hydroxylase (GA3oX1) Atlgl5550

ABA

248227_at 11.02 31.30 0.001641 1.492 ABA-inducible protein-like; similar to pollen coat protein At5g53820

Ethylene

266821_at 319.39 998.82 5.95E-06 1.645 ERF (ethylene response factor) subfamily B-3 (ERF13) At2g44840

253259_at 1598.74 4300.10 1.86E-06 1.427 ERF (ethylene response factor) subfamily B-3 (RRTFl) At4g34410

Cytokinin

253696_at 221.09 80.40 1.35E-05 -1.460 Cytokinin oxidase (CKX4) At4g29740

Transcript levels of phytohormone-related genes were analyzed in Col-0 and MYB60.2-ox plants after drought treatment by microarray analysis.

Four-week-old Col-0 and MYB60.2-ox plants grown in soil were dehydrated for 3 days previous to sampling for microarray analysis

100 Plant Mol Biol (2011) 77:91–103

123

sucrose would provide interesting results. Thus, the tran-

scription level of PAP1 and F3H before and after 8%

sucrose treatment in Col-0 and MYB60 overexpressor

plants were quantified and determined to be elevated upon

stress treatment. However, the role of MYB60 in sucrose

signaling was not extensively investigated in this study. It

is hypothesized that the effect of sucrose may be similar to

osmotic stress.

Robust root growth is an important determinant of plant

proliferation during abiotic stress challenges due to the

total root mass affecting the efficiency of water uptake

(Peret et al. 2009). Many factors regulate root development

with auxin levels playing a critical role. Therefore, regu-

lating auxin distribution to ensure that appropriate levels

are reached in the root tissues is a central aspect of auxin

homeostasis control (Petersson et al. 2009; Ikeda et al.

2009). Based on the observations reported here, a

hypothesis is proposed for the role of MYB60 (Fig. S3).

Under normal conditions, MYB60 is expressed at sub-

stantial levels and mediates stomatal opening. However,

under severe drought conditions, MYB60 is down-regu-

lated in the guard cells. In this case, stomatal opening is no

longer activated, and stomatal closure is triggered by

increased ABA levels (Cominelli et al. 2005). During mild

drought stress, a low level of ABA leads to an increase in

Fig. 7 Histochemical GUS

assays of DR5::GUS x

AtMYB60-ox F3 plants. a GUS

activity in DR5::GUS x

AtMYB60.1-ox and b DR5::GUSx AtMYB60.2-ox seedlings

treated with IAA. DR5::GUS is

included as a control, and GUS

staining was detected in leaves

and roots. Histochemical assays

were conducted to visualize

expression of b-glucurodinase

(GUS) activity in transgenic

plants 24 h after incubation with

0, 0.1, 1, 5 or 10 lM IAA.

Three week-old plants were

stained with GUS solution

overnight, and chlorophyll was

removed by treatment with 70%

ethanol

Fig. 8 MYB60 is responsive to sucrose. For northern analysis, wild-

type seedlings were grown on MS medium for 2 weeks and then

transferred to medium supplemented with 0, 4, 6 or 8% sucrose as

indicated prior to RNA extraction. Transcript levels of MYB60 are

shown. The rRNA is shown as a loading control

Plant Mol Biol (2011) 77:91–103 101

123

MYB60 transcript levels. This alters local flavonoid levels,

leading to the modulation of polar auxin transport, thus

affecting overall root growth (Fig. S3). In this study, an

increase in the flavonoid level of MYB60.1-ox and

MYB60.2-ox plants was not detected. This finding may be

due to the restriction of flavonoid biosynthesis to specific

tissues, or due to low levels of local flavonoid accumula-

tion. Additionally, another type of flavonoid may accu-

mulate in MYB60.1-ox and MYB60.2-ox plants. This

possibility was not investigated in the current study.

Therefore, during the initial stages of stress, the plant

system may modulate root growth behavior by regulating

MYB60 expression, which would result in the growth of

roots with an increased capacity for water uptake. In con-

trast, severe drought stress blocks MYB60 expression,

resulting in stomatal closure and root growth inhibition.

The dual role of MYB60 in stomatal function and root

growth reflects the possibility that MYB60 may target and

regulate different downstream genes in different tissues, as

evidenced in this study. However, the potential alternative

roles of the MYB60 splice variants remain to be fully

explored. Realizing that MYB60 overexpression results in

enhanced leaf withering in response to water deficit, the

engineering of MYB60 to enhance plant stress tolerance

may be a challenge. This is despite the knowledge that

MYB60 improves root growth under the same abiotic

stresses in the presence of high humidity. Analysis of the

fusion of MYB60 to a root-specific promoter will provide

insights into its role in water uptake and potentially

contribute to strategies for responding to unfavorable

environmental stresses.

Acknowledgments This work was supported by a grant from the

Korea Research Foundation to Hojoung Lee (2009; Grant #2011-

0003726) in part by a grant from the Korea Research Foundation to

Suk-Whan Hong (grant #2011-0003259 and #2011-0018393). The

authors express their gratitude to Dr. Cominelli for providing seeds of

the myb60 knock-out line.

References

Blatt MR (2000) Cellular signaling and volume control in stomatal

movements in plants. Annu Rev Cell Dev Biol 16:21–241

Chen Y, Yang X, He K, Liu M, Li J, Gao Z, Lin Z, Zhang Y, Wang X,

Qiu X, Shen Y, Zhang L, Deng X, Luo J, Deng XW, Chen Z, Gu

H, Qu LJ (2006) The MYB transcription factor superfamily of

Arabidopsis: Expression analysis and phylogenetic comparison

with the rice MYB family. Plant Mol Biol 60:09–126

Clough SJ, Bent AF (1998) Floral dip: a simplified method for

Agrobacterium-mediated transformation of Arabidopsis thali-ana. Plant J 16:35–43

Cominelli E, Galbiati M, Vavasseur A, Conti L, Sala T, Vuylsteke M,

Leonhardt N, Dellaporta S, Tonelli C (2005) A guard-cell

specific MYB transcription factor regulates stomatal movements

and plant drought tolerance. Curr Biol 15:196–1200

Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L

(2010) MYB transcription factors in Arabidopsis. Trends Plant

Sci 15:73–581

Hetherington AM (2001) Guard cell signaling. Cell 107:711–714

Ikeda Y, Men S, Fischer U, Stepanova AN, Alonso JM, Ljung K,

Grebe M (2009) Local auxin biosynthesis modulates gradient-

Fig. 9 Expression levels of flavonoid biosynthetic genes in MYB60-

ox seedlings after sucrose treatment. qRT-PCRs were done to assess

the relative mRNA levels of the major flavonoid biosynthetic genes,

such as a PAP1, and b F3H, determined to be highly expressed in

MYB60-ox plants according to the microarray analysis in Table 2.

Analysis of upregulated flavonoid biosynthetic genes upon 8%

sucrose treatment was done and actin was used as internal control.

Col-0, MYB60.1-ox#16 and MYB60.2-ox#7 seeds were germinated on

MS-agar medium and 2 week-old seedlings were treated with

sucrose-containing MS liquid media for 5 h. Error bars represent

the standard deviation

102 Plant Mol Biol (2011) 77:91–103

123

directed planar polarity in Arabidopsis. Nat Cell Biol

11:731–738

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-

glucuronidase as a sensitive and versatile gene fusion marker in

higher plants. EMBO J 20:901–3907

Jia L, Clegg MT, Jiang T (2004) Evolutionary dynamics of the DNA-

binding domains in putative R2R3-MYB genes identified from

rice subspecies indica and japonica genomes. Plant Physiol

134:75–585

Jung C, Seo JS, Han SW, Koo YJ, Kim CH, Song SI, Nahm BH, Choi

YD, Cheong JJ (2008) Overexpression of AtMYB44 enhances

stomatal closure to confer abiotic stress tolerance in transgenic

Arabidopsis. Plant Physiol 146:23–635

Lee H, Guo Y, Ohta M, Xiong L, Stevenson B, Zhu JK (2002) LOS2,

a genetic locus required for cold-responsive gene transcription

encodes a bifunctional enolase. EMBO J 21:692–2702

Leonhardt N, Kwak JM, Robert N, Waner D, Leonhardt G, Schroeder

JI (2004) Microarray expression analyses of Arabidopsis guard

cells and isolation of a recessive abscisic acid hypersensitive

protein phosphatase 2C mutant. Plant Cell 16:596–615

Liang YK, Dubois C, Dodd IC, Holroyd GH, Hetherington AM,

Campbell MM (2005) AtMYB61, an R2R3-MYB transcription

factor controlling stomatal aperture in Arabidopsis thaliana.

Curr Biol 15:201–1206

Pandey S, Zhang W, Assmann SM (2007) Roles of ion channels and

transporters in guard cell signal transduction. FEBS Lett

581:325–2336

Peer WA, Murphy AS (2007) Flavonoids and auxin transport:

modulators or regulators? Trends Plant Sci 12:56–563

Peret B, Rybel BD, Casimiro I, Benkova E, Swarup R, Laplaze L,

Beeckman T, Bennett MJ (2009) Arabidopsis lateral root

development: an emerging story. Trends Plant Sci 14:399–408

Petersson SV, Johansson AI, Kowalczyk M, Makoveychuk A, Wang

JY, Moritz T, Grebe M, Benfey PN, Sandberg G, Ljung K (2009)

An auxin gradient and maximum in the Arabidopsis root apex

shown by high-resolution cell-specific analysis of IAA distribu-

tion and synthesis. Plant Cell 21:1659–1668

Roelfsema MRG, Hedrich R (2005) In the light of stomatal opening:

new insights into ‘the Watergate’. New Phytol 165:65–691

Rosinsky JA, Atchley WR (1998) Molecular evolution of the MYB

family of transcription factors: evidence for polyphyletic origin.

J Mol Evol 46:4–83

Sahota AP, Dhoot GK (2009) A novel SULF1 splice variant inhibits

Wnt signalling but enhances angiogenesis by opposing SULF1

activity. Exp Cell Res 315:752–2764

Schroeder JI, Kwak JM, Allen GJ (2001) Guard cell abscisic acid

signalling and engineering of drought hardiness in plants. Nature

410:27–330

Seo PJ, Xiang F, Qiao M, Park JY, Lee YN, Kim SG, Lee YH, Park

WJ, Park CM (2009) The MYB96 transcription factor mediates

abscisic acid signaling during drought stress response in

Arabidopsis. Plant Physiol 151:275–289

Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to

dehydration and low temperature: differences and cross-talk

between two stress signaling pathways. Curr Opin Plant Biol

3:17–223

Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory

network of gene expression in the drought and cold stress

responses. Curr Opin Plant Biol 6:410–417

Sirichandra C, Wasilewska A, Vla F, Valon C, Leung J (2009) The

guard cell as a single-cell model towards understanding drought

tolerance and abscisic acid action. J Exp Bot 60:1439–1463

Teng S, Keurentjes J, Bentsink L, Koornneef M, Smeekens S (2005)

Sucrose-specific induction of anthocyanin biosynthesis in Ara-

bidopsis requires the MYB75/PAP1 gene. Plant Physiol

139:1840–1852

Wasilewska A, Vlad F, Sirichandra C, Redko Y, Jammes F, Valon C,

Frey N, Leung J (2008) An update on abscisic acid signaling in

plants and more. Mol Plant 1:198–217

Xiong L, Lee H, Ishitani M, Zhu JK (2002) Regulation of osmotic

stress-responsive gene expression by the LOS6/ABA1 locus in

Arabidopsis. J Biol Chem 277:8569–8588

Zhu JK (2002) Salt and drought stress signal transduction in plants.

Ann Rev Plant Biol 53:247–273

Zobel AM, Hrazdina G (1995) Chalcone synthase localization in early

stages of plant development. I. Immunohistochemical use of

plasmolysis for localizing the enzyme in epidermal cell

cytoplasm of illuminated buckwheat hypocotyls. Biotech Histo-

chem 70:1–6

Plant Mol Biol (2011) 77:91–103 103

123