Over-expression of microRNA169 confers enhanced drought tolerance to tomato.

Abstract

Plant miRNA regulates multiple developmental and physiological processes, including drought responses. We found that the accumulation of Sly-miR169 in tomato (Solanum lycopersicum) was induced by drought stress. Consequently, Sly-miR169 targets, namely, three nuclear factor Y subunit genes (SlNF-YA1/2/3) and one multidrug resistance-associated protein gene (SlMRP1), were significantly down-regulated by drought stress. Constitutive over-expression of a miR169 family member, Sly-miR169c, in tomato plant can efficiently down-regulate the transcripts of the target genes. Compared with non-transgenic plants, transgenic plants over-expressing Sly-miR169c displayed reduced stomatal opening, decreased transpiration rate, lowered leaf water loss, and enhanced drought tolerance. Our study is the first to provide evidence that the Sly-miR169c negatively regulates stomatal movement in tomato drought responses.

Full text

Page 1

ORIGINAL RESEARCH PAPER
Over-expression of microRNA169 confers enhanced drought
tolerance to tomato
Xiaohui Zhang•Zhe Zou•Pengjuan Gong•
Junhong Zhang•Khurram Ziaf•Hanxia Li•
Fangming Xiao•Zhibiao Ye
Received: 29 August 2010/Accepted: 21 September 2010/Published online: 20 October 2010
? Springer Science+Business Media B.V. 2010
Abstract
mental and physiological processes, including drought
responses. We found that the accumulation of
Sly-miR169 in tomato (Solanum lycopersicum) was
induced by drought stress. Consequently, Sly-miR169
targets, namely, three nuclear factor Y subunit genes
(SlNF-YA1/2/3) and one multidrug resistance-associ-
ated protein gene (SlMRP1), were significantly
down-regulated by drought stress. Constitutive over-
expressionofamiR169familymember,Sly-miR169c,
in tomato plant can efficiently down-regulate the
transcripts of the target genes. Compared with non-
transgenic plants, transgenic plants over-expressing
Plant miRNA regulates multiple develop-
Sly-miR169c displayed reduced stomatal opening,
decreased transpiration rate, lowered leaf water loss,
and enhanced drought tolerance. Our study is the first
to provide evidence that the Sly-miR169c negatively
regulates stomatal movement in tomato drought
responses.
Keywords
Stomatal opening ? Tomato
Drought tolerance ? microRNA169 ?
Introduction
miRNAs are a class of 18–24 nucleotide small RNAs
produced from hairpin pre-miRNA structures tran-
scribed by endogenous RNA polymerase II (Voinnet
2009). MiR169 is a large miRNA family that is
widespread in the plant kingdom (Li et al. 2010), and
plays important roles in plant development and
responses to environmental cues (Combier et al.
2006). In rice (Oryza sativa), some members of the
miR169 family were induced by drought (Zhao et al.
2007) and salt stress (Zhao et al. 2009). In contrast to
findings on rice, miR169a/c in Arabidopsis was
down-regulated by drought and transgenic plants
over-expressing miR169a were more prone to
drought stress (Li et al. 2008). Most of the proved
target genes of miR169 belong to the Nuclear factor
YA family (Combier et al. 2006).
Nuclear factor Y (NF-Y) is a CCAAT-box-binding
transcription factor complex consisting of NF-YA
Xiaohui Zhang and Zhe Zou contributed equally to this work.
Electronic supplementary material
this article (doi:10.1007/s10529-010-0436-0) contains
supplementary material, which is available to authorized users.
The online version of
X. Zhang ? Z. Zou ? K. Ziaf ? Z. Ye (&)
National Key Laboratory of Crop Genetic Improvement,
Huazhong Agricultural University, Wuhan, China
e-mail: zbye@mail.hzau.edu.cn
P. Gong ? J. Zhang ? H. Li ? Z. Ye
Key Laboratory of Horticultural Plant Biology,
Ministry of Education, Huazhong Agricultural University,
Wuhan, China
F. Xiao
Department of Microbiology, Molecular Biology
and Biochemistry, University of Idaho, Moscow,
ID 83844-3052, USA
123
Biotechnol Lett (2011) 33:403–409
DOI 10.1007/s10529-010-0436-0

Page 2

(also known as CBF-B or HAP2), NF-YB (CBF-A or
HAP3), and NF-YC (CBF-C or HAP5) subunits,
which control the expression of about 25% of
eukaryotic genes (Testa et al. 2005). The NF-YA
genes play central roles in plant development and
responses to environmental stresses (Kumimoto et al.
2008). Over-expression of NF-YA5 (a target of
miR169) and NF-YB1 in Arabidopsis can improve
plant drought tolerance (Li et al. 2008; Nelson et al.
2007).Multidrugresistance-associated
(MRPs or ABCCs), a subfamily of the ATP-binding
cassette (ABC) superfamily, are involved in cellular
detoxification by transporting toxic compounds from
the cytosol into the vacuole (Klein et al. 2006).
Recent studies show that MRPs also regulate stomatal
movements (Gaedeke et al. 2001; Klein et al. 2004).
Tomato is an important vegetable crop grown
worldwide, including in arid and semi-arid regions
that are prone to water shortage. An urgent goal for
breeders and plant molecular biologists is to develop
drought-tolerant cultivars that can efficiently use
water. In this study, the role of miR169 and its target
genes in tomato are investigated in response to
drought stress.
proteins
Materials and methods
Plant material, growth conditions and stress
treatment
Tomato (Solanum lycopersicum cv. Ailsa Craig) was
used in this study. Plants were grown in pots filled
with a mixture of soil and compost (3:1 w/w), and
maintained in a greenhouse under a 16/8 h light/dark
regime at 25–30?C. For drought stress assays, the
seedlings were thoroughly watered by placing the
pots in a water-filled shallow plastic basin overnight,
and then subjected to drought stress.
Construction of plant expression vector
and tomato transformation
A 300 bp DNA fragment harboring the Sly-mir169c
hairpinstructure(SupplementaryFigureS1)wasPCR-
amplified using the primers Sly-mir169c-Fw (50-A
TAGCTACATGAAACTTGTGATCCATAATAC-30)
and Sly-mir169c-Rv (50-TTAATTGGGTTGTGATT
GCATGTG-30), as well as tomato genomic DNA as
template. The DNA fragment was inserted into
pMD18-T vector (Takara, Japan) for sequencing
confirmation,andthensub-clonedintotheplantbinary
vectorpBI121generatingthepBI-Slymir169cplasmid
driven by the CaMV 35S promoter. The plasmid was
transformed into tomato mediated by Agrobacterium
tumefaciens strain C58 (Fillatti et al. 1987).
RNA isolation and Northern blot analysis
Total RNA was extracted from plant tissues using
TRIzol reagent (Invitrogen, USA). For the analysis of
the accumulation of Sly-miR169c transcript, 30 lg
total RNA was separated on a 15% denaturing
polyacrylamide/1X TBE (8.9 mM Tris/HCl, 8.9 mM
boric acid, 20 mM EDTA)/8 M urea gel, and subse-
quently transferred onto Hybond-N?
(Amersham Biosciences). DNA oligonucleotides of
miR169R (50-TCGGCAAGTCATCCTTGGCTG-30),
the exact reverse-complementary sequence to Sly-
miR169c, were end-labeled with [c-32P] ATP and T4
polynucleotide kinase (New England Biolabs, USA)
to generate a highly specific probe. The DNA probe
specific to U6 was synthesized with DNA oligonu-
cleotideU6(50-TGTATCGTTCCAATTTTATCG
GATGT-30) and used as loading control. Hybridiza-
tion was performed overnight at 37?C in 5 ml
hybridization buffer containing 7% (v/v) SDS in
0.2 M sodium phosphate buffer (pH 7.0). Membranes
were then washed twice at the hybridization temper-
ature with 3X SSC containing 0.1% SDS for 10 min,
and exposed to Kodak MS film for 7–10 days.
membrane
Real-time and semi-quantitative RT-PCR
For real-time RT-PCR analysis of the target genes of
Sly-miR169c, 3 lg total plant RNA was pretreated
with DNase I (Promega) and reversely transcribed
using ReverTra Ace (TOYOBO, Japan) and 5 pmol
of oligonucleotide (dT)18. The resultant cDNA was
diluted to 100 ng/ll with RNase free water, and 5 ll
was used as template in a 20 ll PCR reaction. PCR
was performed in the LightCycler 480 system
(Roche, American) using SYBR Green I Master
(Roche, American). The comparative Ct method was
used for data analysis. For semi-quantitative RT-PCR
detection of pre-mir169c transcript, the RT-PCR
products were viewed in a 1% ethidium bromide/
agarose gel. Gene-specific primers are shown in
404Biotechnol Lett (2011) 33:403–409
123

Page 3

Supplementary Table S1. The b-actin transcripts
were used as internal controls.
Stomatal aperture measurement
The mature leaves of transgenic plants (2 month old)
and the non-transgenic plants grown in the field were
analyzed for stomatal aperture measurement. After
exposure to light for 3 h, the abaxial surfaces of
leaves from intact plants were smeared with clear nail
varnish and exposed for a 2 min air hardening. The
impression material was detached and transferred into
a drop of floating solution on a glass slide for
microscopical analysis. The width and length of the
stomatal pores were measured. The stomatal aperture
index (width/length ratio) was calculated with more
than 90 stomatal pores for each line.
Stomatal conductance and transpiration
measurements
Fully expanded leaves of 2-month-old plants were
sampled for the measurement of the stomatal conduc-
tanceandtranspirationrateusingtheCIRAS-2portable
photosynthesis system (PP Systems, UK) equipped
with an LED light source fixed on top of the Auto PLC
leaf cuvette (PLC5 Broad 2.5, PP Systems, UK).
The flow rate through the leaf chamber was kept at
200 lmol s-1and the chamber was at 30?C. Nine
independent plants of each line were analyzed.
Measurement of water loss from detached leaves
For the measurement of water loss from detached
tomato leaves, mature leaves of the same size were
excised from 12 different transgenic plants of each
transgenic line, or from non-transgenic plants grow-
ing under the same conditions. Leaves were placed on
a shelf under fluorescent light at 25?C, and the fresh
weight of the excised leaves was determined at
assigned time intervals (Fig. 5d). Water loss was
represented as the percentage of initial fresh weight at
each time point.
Measurement of water uptake into detached
shoots
For the measurement of water uptake into detached
shoots, eight side shoots of similar size (*9 g per
shoot) were detached from eight plants of each
transgenic line and from the non-transgenic control
line growing under the same conditions. The
detached shoots were dipped in flasks containing
100 ml of tap water and maintained in a culture room
at a 16/8 h light/dark regime at 25?C. The volume
of the remaining water was determined at each time
interval (Fig. 5e). Three flasks without shoots were
used to monitor evaporation. The water uptake was
represented as water volume reduced minus volume
of water decreased due to evaporation.
Results and discussion
MiR169s and their targets in tomato
In our preliminary microarray analysis of genes
regulated by drought treatment [TOM2 microarray
analysis using M82 and two introgression lines, IL5-2
and IL9-1, of Solanum pennellii with the M82
background (http://ted.bti.cornell.edu/cgi-bin/TFGD/
miame/experiment.cgi?ID=E041)],
NF-YA genes were down-regulated in response to
drought stress. Because NF-YA genes are targets of
miR169 in rice and Arabidopsis (Li et al. 2008; Zhao
et al. 2009), we investigated the role of miR169 and
its targets in tomato drought responses. Four putative
target genes with less than four mismatches to
miR169 were found in a search against the SGN
unigene database using miRU2 (http://bioinfo3.noble.
org/miRU2/index.php?function=function1).
them, three belonged to the NF-YA transcription
factor family genes (SlNF-YA1, SGN-U322766;
SlNF-YA2, SGN-U323102 and SlNF-YA3, SGN-
U323737), and one was an MRP-type ABC trans-
porter gene (SlMRP1, SGN-U343564) (Fig. 1a). The
NF-YA genes have been reported as targets of
miR169 and are engaged in plant drought resistance
(Li et al. 2008); in this study, however, SlMRP1 was
the first ABC transporter gene predicted as the target
of miRNA169.
several tomato
Among
Expression pattern of miR169 targets
The expression pattern of the predicted miR169
target genes in different plant tissues was investigated
by real-time RT-PCR analysis. The transcripts of
SlNF-YA1 and SlNF-YA2 were expressed in all the
Biotechnol Lett (2011) 33:403–409405
123

Page 4

plant tissues tested, but at a slightly lower level in
stem and bud tissues (SlNF-YA1), as well as in roots
(SlNF-YA2) compared with other tissues tested
(Fig. 1b). SlNF-YA3 and SlMRP1 were highly
expressed in mature leaves and flowers (Fig. 1b).
MiR169 is induced and the targets are down-
regulated by drought treatment
Tomato seedlings at the four-leaf stage were stressed
by withholding water for seven days, with the non-
treated seedlings used as control. When the leaves of
the stressed plants started wilting, leaf tissues were
sampled for RNA extraction, and the plants were
watered. After five hours of rehydration, RNA was
again extracted from the leaves. Expression levels of
miR169 were monitored by Northern blot analysis. As
shown in Fig. 2a, miR169 was induced more strongly
in the drought-stressed plants compared with the non-
stressed control plants. The transcripts of the putative
targets were also analyzed by real-time RT-PCR. All
four target genes were down-regulated by drought
treatment at different degrees of severity (Fig. 2b).
After five hours of recovery with re-watering, SlMRP1
expression was significantly restored (Fig. 2b). These
results imply that miR169 and its target genes play
an important role in response to drought stress in
tomato. Moreover, SlMRP1 was found as a battlefront
drought-responsive gene that responded rapidly to
fluctuations of water potential in plants.
Over-expression of miRNA169c down-regulates
SlNF-YA1/2/3 and SlMRP1 in tomato
To test whether these putative targets are truly
regulatedby miR169,
expressed in tomato plant. The PCR and Southern
sly-miR169cwas over-
Fig. 1 Expression
a Sequence alignment of the Sly-miR169c and its binding
sites in SlNF-YA1/2/3 and SlMRP1. The matched base pairs are
indicated with colons, and the GU match is indicated with a
dot. b Tissue-specific expression pattern of miR169 target
genes by real-time RT-PCR analysis. Error bars represent
standard error for three replicates. YL Young Leaf, ML Mature
Leaf, S Stem , B Bud, F Flower, R Root
patterns ofmiR169target genes.
Fig. 2 Expression of miR169 and its target genes in response
to drought stress. a Northern blot analysis of miR169
expression under drought stress and normal growth conditions.
U6 RNA was used as loading control. b Real-time RT-PCR
analysis of the expression of four targets under Control (Con),
Drought (Dro), and Recovery (Rec) conditions. Error bars
represent SE for three replicates
406 Biotechnol Lett (2011) 33:403–409
123

Page 5

blot analysis of the transgenic plants were shown in
Supplementary Figure S2. Three transgenic lines (42,
43, and 45) expressing pre-miR169c at a high level
(Fig. 3a) were chosen for further analysis. Northern
blot analysis also verified the over-expression of
miR169c in transgenic plants (Fig. 3b). Subse-
quently, the real-time RT-PCR results show that the
transcript abundance of all the four putative targets
declined dramatically in young or mature leaves
(Fig. 3c), indicating that the four genes were truly
regulated by miR169c.
Transgenic plants show enhanced drought
tolerance
To test whether over-expression of miR169c can
confer drought tolerance, transgenic and wild-type
plants were subjected to drought stress for seven
days. As shown in Fig. 4, the non-transgenic plants
showed obvious dehydration symptoms, such as
turgor loss and wilting, whereas the transgenic plants
remained turgid. We investigated the effect of
miR169c on the regulation of the size of stomatal
opening in the drought-tolerant transgenic lines
because the MRP gene was reported to control
stomatal movement (Klein et al. 2004). When grown
in a pot and kept in a culture room under artificial
light, the average stomatal aperture index was
0.18–0.23 for transgenic lines, which was about
35–49% less than that of the non-transgenic plants
under the same growth conditions (Fig. 5a). Consis-
tent with these observations, the stomatal conduc-
tance of transgenic lines was reduced to 33–45%
(Fig. 5b). The transpiration rate of the transgenic
lines was also reduced to 45–62% (Fig. 5c), and the
leaf water loss was lower in transgenic lines
compared with the wild-type plants (Fig. 5d). More-
over, the capacity of water uptake in the detached
shoots was also significantly reduced in transgenic
lines (Fig. 5e). Furthermore, all water deficiency-
associated features, such as stomatal aperture, con-
ductance, transpiration, water loss, and water uptake
were negatively correlated with the expression of
miR169c (Figs. 3a, 5), strongly suggesting that
tomato miR169c confers drought tolerance by con-
trolling stomatal movement.
Fig. 3 Expression analysis of the transgenic plants. a RT-PCR
analysis of the pre-mir169c transcripts in three 35S:miR169c
lines (42, 43, and 45) and the non-transgenic (Wt) plant. b-actin
was used as internal control. b Northern blot analysis of the
accumulation of mature miR169c RNA in 35S:miR169c
(mixture of lines 42, 43, and 45) and non-transgenic plants.
U6 RNA was used as loading control. c Real-time RT-PCR
analysis of the expression of four targets of miR169c in
35S:miR169c (mixture of lines 42, 43, and 45) and non-
transgenic (Wt) plants. Error bars represent SE for three
replicates. YL Young Leaf, ML Mature Leaf
Fig. 4 Enhanced
35S:miR169c transgene in tomato. The image was taken seven
days after withholding water
droughttoleranceconferred bythe
Biotechnol Lett (2011) 33:403–409407
123

Page 6

Conclusion
This study is the first to report that over-expression of
miR169c can improve plant drought tolerance. Four
putative target genes, especially SIMRP1 as a poten-
tially new target gene, have been shown to be
regulated by miR169 in this study. However, whether
these genes are directly controlled by miR169 by
dicing needs more extensive studies such as cleavage
assays and mutagenesis complementation experi-
ments. Stomatal movement is an important plant
biophysical activity controlling gas exchange. Under
the constitutive over-expression of miR169c, the
stomatal conductance and transpiration rate were
significantly reduced in transgenic plants. However,
the transgenic plants showed no noticeable defect in
biomass and fruit productivity under field conditions
(data not shown), suggesting that stomatal opening is
flexible and redundant for plant growth in normal
environments. Thus, improving crop water-use effi-
ciency is possible by manipulating miRNAs, such as
miR169, to regulate genes responsible for drought-
stress responses.
Acknowledgments
the 973 Program (No. 2009CB119000) and NSFC (No.
30671417), and the 863 Program (No. 2007AA10Z131), as
well as the earmarked fund for Modern Agro-industry
Technology Research System (Nycytx-35-gw02).
This work was supported by grants from
References
Combier JP, Frugier F, de Billy F et al (2006) MtHAP2–1 is a
key transcriptional regulator of symbiotic nodule devel-
opment regulated by microRNA169 in Medicago trun-
catula. Genes Dev 20:3084–3088
Fillatti J, Kiser J, Rose R et al (1987) Efficient transfer of a
glyphosate tolerance gene into tomato using a binary
Agrobacterium tumefaciens
5:726–730
Gaedeke N, Klein M, Kolukisaoglu U et al (2001) The
Arabidopsis thaliana ABC transporter AtMRP5 controls
root development and stomata movement. EMBO J
20:1875–1887
Klein M, Geisler M, Suh SJ et al (2004) Disruption of
AtMRP4, a guard cell plasma membrane ABCC-type ABC
transporter, leads to deregulation of stomatal opening and
increased drought susceptibility. Plant J 39:219–236
Klein M, Burla B, Martinoia E (2006) The multidrug resis-
tance-associated protein (MRP/ABCC) subfamily of ATP-
vector.Nat Biotechnol
Fig. 5 Stomatal opening analysis. a Measurement of stomatal
aperture in three 35S:miR169c transgenic lines and non-
transgenic plants. Data are mean ratios of width to length ± SE
for 90 stomatal pores. b Average stomatal conductance and
c transpiration rate measured on intact leaves from three
35S:miR169c lines and non-transgenic plants. Error bars
represent SE (n = 9). d Water loss in leaves detached from
three 35S:miR169c lines and non-transgenic plants. Water loss
is represented as the percentage of initial fresh weight at each
time point. Values are means of 12 leaves per line for each of
the three independent experiments. e Water uptake by shoots
detached from three 35S:miR169c lines and non-transgenic
plants. Data indicate mean ± SE (n = 8)
408Biotechnol Lett (2011) 33:403–409
123

Page 7

binding cassette transporters in plants. FEBS Lett
580:1112–1122
Kumimoto RW, Adam L, Hymus GJ et al (2008) The Nuclear
Factor Y subunits NF-YB2 and NF-YB3 play additive
roles in the promotion of flowering by inductive long-day
photoperiods in Arabidopsis. Planta 228:709–723
Li WX, Oono Y, Zhu J et al (2008) The Arabidopsis NFYA5
transcription factor is regulated transcriptionally and post-
transcriptionally to promote drought resistance. Plant Cell
20:2238–2251
Li Y, Fu Y, Ji L et al (2010) Characterization and expression
analysis of the Arabidopsis mir169 family. Plant Sci
178:271–280
Nelson DE, Repetti PP, Adams TR et al (2007) Plant nuclear
factor Y (NF-Y) B subunits confer drought tolerance and
lead to improved corn yields on water-limited acres. Proc
Natl Acad Sci USA 104:16450–16455
Testa A, Donati G, Yan P et al (2005) Chromatin immuno-
precipitation (ChIP) on chip experiments uncover a
widespread distribution of NF-Y binding CCAAT sites
outside of core promoters. J Biol Chem 280:13606–13615
Voinnet O (2009) Origin, biogenesis, and activity of plant
microRNAs. Cell 136:669–687
Zhao B, Liang R, Ge L et al (2007) Identification of drought-
induced microRNAs in rice. Biochem Biophys Res
Commun 354:585–590
Zhao B, Ge L, Liang R et al (2009) Members of miR-169
family are induced by high salinity and transiently inhibit
the NF-YA transcription factor. BMC Mol Biol 10:29
Biotechnol Lett (2011) 33:403–409409
123