RESEARCH ARTICLE

Transcriptome Profiling of the Potato

(Solanum tuberosum L.) Plant under Drought
Stress and Water-Stimulus Conditions
Lei Gong1, Hongxia Zhang2, Xiaoyan Gan1, Li Zhang1, Yuchao Chen1, Fengjie Nie1,
Lei Shi1, Miao Li1, Zhiqian Guo3, Guohui Zhang3, Yuxia Song1*
1 Ningxia Key Laboratory for Agrobiotechnology, Agricultural Bio-Technology Center, Ningxia Academy of
Agriculture and Forestry Science, Yinchuan, Ningxia Hui Nationality Autonomous Region, China, 2 National Key
Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy
of Sciences, Shanghai, China, 3 Guyuan sub-centers of National Potato Improvement Center, Ningxia
Academy of Agriculture and Forestry Science, Guyuan, Ningxia Hui Nationality Autonomous Region, China

* [email protected]

Abstract
Drought stress can seriously affect tuberization, yield and quality of potato plant. However,
OPEN ACCESS the precise molecular mechanisms governing potato stolon’s response to drought stress
Citation: Gong L, Zhang H, Gan X, Zhang L, Chen Y, and water supply are not very well understood. In this work, a potato (Solanum tuberosum
Nie F, et al. (2015) Transcriptome Profiling of the
L.) variant, Ningshu 4, was subjected to severe drought stress treatment (DT) and re-water-
Potato (Solanum tuberosum L.) Plant under Drought
Stress and Water-Stimulus Conditions. PLoS ONE ing treatment (RWT) at tuber bulking stage. Strand-specific cDNA libraries of stolon materi-
10(5): e0128041. doi:10.1371/journal.pone.0128041 als were constructed for paired-end transcriptome sequencing analyses and differentially
Academic Editor: Binying Fu, Institute of Crop expressed gene (DEG) examination. In comparison to untreated-control (CT) plants, 3189
Sciences, CHINA and 1797 DEGs were identified in DT and RWT plants and 4154 solely expressed DEGs
Received: January 12, 2015 were screened out from these two comparison groups. Interestingly, 263 genes showed op-
posite expression patterns in DT and RWT plants. Among them, genes homologous to Pro-
Accepted: April 21, 2015
tein Phosphatase 2C (PP2C), Aspartic protease in guard cell 1 (ASPG1), auxin-responsive
Published: May 26, 2015
protein, Arabidopsis pseudo response regualtor 2 (APRR2), GA stimulated transcripts in
Copyright: © 2015 Gong et al. This is an open Arabidopsis 6 (GASA6), Calmodulin-like protein 19 (CML19), abscisic acid 8'-hydroxylases
access article distributed under the terms of the
and calcium-transporting ATPase, et al. were related with drought-stress and water stimulus
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any response. Sixteen DEGs involved in starch synthesis, accumulation and tuber formation ex-
medium, provided the original author and source are hibited significantly different expression upon re-watering. In addition, 1630, 1527 and 1596
credited. transcription factor encoding genes were detected in CT, DT and RWT. DEGs of ERF,
Data Availability Statement: All relevant data are bHLH, MYB, NAC, WRKY, C2H2, bZIP and HD-ZIP families accounted for 50% in three
within the paper and its Supporting Information files. comparison groups, respectively. Furthermore, characteristics of 565 gene ontology (GO)
Funding: This work has been jointly supported by and 108 Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were analyzed with
the following grants: The China Agriculture Research the 4154 DEGs. All these results suggest that the drought- and water-stimulus response
System (CARS-10-ES30), The Mega Crop Breeding
could be implemented by the regulated expression of metabolic pathway DEGs, and these
Project of Ningxia District-Engineering of New Potato
Cultivars, Natural Science Foundation of Ningxia genes were involved in the endogenous hormone biosynthesis and signal transduction path-
(NZ13098) and The Scientific and Technological ways. Our data provide more direct information for future study on the interaction between
Innovation Priority Research Program of Ningxia key genes involved in various metabolic pathways under drought stress in potato.
Academy of Agriculture and Forestry Science
(NKYG-13-05, NKYG-14-05 and NKYG-15-06).

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 1 / 20

 Transcriptome Profiling of Potato

Competing Interests: The authors have declared Introduction

that no competing interests exist.
Global climate change is reducing the reliability of rainfall, the availability of soil-water, and
consequently, limiting plant production. Potato (Solanum tuberosum L.) ranks as the fourth
most predominant non-grain food crop in the world. Due to their shallow root system, which
limits water extraction from soil, potato plants are sensitive to drought stress. Leaf size, photo-
synthesis rate, tuber number, yield and quality were all severely limited when grown under a
drought stress condition [1, 2]. Therefore, in order to breed new potato varieties with improved
drought resistance, the molecular mechanisms governing water use, tuber starch synthesis and
accumulation under drought conditions must be defined.
In higher plants, drought stress can induce a wide range of physiological and biochemical
responses regulated by genes encoding functional and/or regulatory proteins, to maintain a
normal homeostasis and avoid wilting death [3]. Fast and efficient recovery from water stress
will allow agricultural crops to adapt to the changes of meteorological conditions and maximize
their growth and production during drought stress. In a diploid potato population, 47 quantita-
tive trait loci were identified, out of them, 28 were drought-specific, and 17 were specifically ex-
pressed during recovery from drought stress [4]. Recently, the published potato genome [5]
and transcriptome [6] with corresponding gene structure, location, and functional annotation
provide powerful resources for the understanding of the complex responses to drought stress
in potato. However, data currently available on transcriptomic changes upon rehydration in
potato is insufficient, and the precise pathways involved in drought responses and recovery
from water deficit still remain to be deduced.
Transcriptome sequencing technologies provide a framework dataset for researches related to
construction of transcriptome map, determination of metabolic pathways, clarification of gene
expression patterns, and mining of new genes. To explore the molecular regulation mechanisms
of plants in response to different stresses, transcriptome analysis has been performed on a variety
of crops, including sunflower (Helianthus annuus) [7], grape (Vitis vinifera) [8], maize (Zea
mays) [9] and sorghum (Sorghum bicolor L. Moench) [10]. Data reported in these studies have
provided information on the networks of regulatory and functional genes at different levels.
Upon the completion of whole genome and transcriptome sequencing of potato DM1-3
516R44 [5, 6], a number of genes related to drought-stress response and water use efficiency
have been identified by quantitative trait locus mapping [2, 4], and transcriptome analyses of
stress response in potato leaves has been performed with plants grown under a single condi-
tion. However, molecular mechanism of potato plant, especially the tuber or stolon responsing
to water-stimulus are not very well understood with the results available now. Tubers are the
main “sink” organ and it also the most important agronomic trait of potato plants. In addition,
the tuber bulking stage is a critical period of time for potato tuberization, during which plants
are extremely sensitive to water deficit. Therefore, we explored the molecular mechanisms un-
derpinning drought stress responses in the stolon tissue of a potato variety. Plant cultivated
under severe drought stress condition during tuber bulking stage were used for next-generation
transcriptome sequencing to fully resolve gene expression profiles in response to water deficit
and re-watering.

Materials and Methods

Plant materials and drought treatments
The potato (Solanum tuberosum L.) strain Ningshu 4 was from our own lab. All plant materials
were collected from our Guyuan Experimental Field (Guyuan, Ningxia Hui Autonomous Re-
gion, China), which does not involve any endangered or protected species. Before tuber bulking

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 2 / 20

 Transcriptome Profiling of Potato

stage, 40 healthy and uniform ball-plants were transplanted into buckets and cultivated indoors
at room temperature under 12-hr light/dark cycles at 4,000 lux. Experimental soil was collected
from a test site in Guyuan City, Ningxia Hui Autonomous Region, with the maximum field
water-holding capacity of 21%.
At flowering stage, the above plants were split in to three groups with ten plants in each
group. The first group of plants was exposed to severe drought stress treatment (DT) for three
days, during which relative soil moisture was controlled at 35–40% of the maximum field water-
holding capacity. The second group of plants was exposed to severe drought stress treatment for
3 days, followed by re-watering to the control level and grown for 3 days (RWT). And the last
group of plants was maintained under normal watering condition throughout the time period as
control (CT). Then, stolon tips from three randomly selected plants of each group were pooled
together, immediately grounded into fine powder in liquid nitrogen for sequencing analysis.

Total RNA extraction

Total RNA was extracted from plant materials using a TransZol Plant kit (ET121-01, Transgen
Biotech, Beijing, China) according to the manufacturer’s instruction. Digestion of gDNA in
each sample was conducted using DNase I (2212, TaKaRa, Dalian, China). The integrity and
purity of RNA was confirmed using 1.2% non-denaturing agarose gel electrophoresis and the
Agilent 2100 RNA 6000 Kit (Agilent Technologies, Santa Clara, CA, USA).

Strand-specific cDNA library construction and sequencing

From each sample, 3 μg of total RNA was taken to construct strand-specific cDNA libraries
using the NEBNext Ultra RNA Library Prep Kit (New England labs, Ipswich, MS, USA). The
quality of cDNA libraries was tested, and quantified cDNA was subjected to purification, elu-
tion, end repair, poly(A) addition and adaptor ligation. Then, agarose gel electrophoresis was
used for size selection of gene fragments, followed by PCR amplification. The established
cDNA libraries were sequenced using the Illumina Hiseq 2000 platform (Illumina Inc., San
Diego, CA, USA) to generate 100-bp paired-end reads.

Sequence analysis
Image data obtained from cDNA sequencing was converted to the corresponding nucleotide
sequence data. Raw reads were firstly processed through in-house perl scripts. In this step,
clean data (clean reads) were obtained by removing reads containing adapter, reads containing
ploy-N and low quality reads from raw data. At the same time, Q20, Q30, GC content and se-
quence duplication level of the clean data were calculated. All the downstream analyses were
based on the clean data with high quality. Then, index of the reference genome
(PGSC_DM_v3_2.1.10) was built using Bowtie v2.1.0 and paired-end clean reads were aligned
to the reference genome using TopHat v2.0.9 (Broad Institute, Boston, MA) [11]. Next, HTSeq
v0.5.3 was used to count the reads numbers mapped to each gene. And RPKM [12] of each
gene was calculated based on the length of the gene and reads count mapped to this gene. All
the clean data have been deposited in the Short Read Archive (SRA) at the NCBI database with
the project accession number SRP056128.

Screening, clustering and functional annotation of differentially

expressed genes (DEGs)
DEGs between different treatments were identified based on the RPKM value calibrated with
the edgeR program [13]. DEG analysis was conducted using the DEGSeq R package, as

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 3 / 20

 Transcriptome Profiling of Potato

described previously. A q value <0.005 and a fold change |log2| 1 were used as the screening
cutoffs for extremely significant differential gene expression between two samples.
Following pairwise comparison of samples, gene ontology (GO) classification of DEGs was
performed using the GO seq software [14] with corrected p-Value <0.05 as the screening cutoff
for enriched GO terms. GO enrichment was analyzed in the categories of molecular function,
cellular component and biological process. Enrichment analysis of Kyoto Encyclopedia of
Genes and Genomes (KEGG) metabolic pathways was performed using KOBAS [15].

Quantitative real-time PCR (qRT-PCR) verification of DEGs

To test the validity and accuracy of transcriptome sequencing data, 10 genes were randomly se-
lected from sequencing data for fluorescent qRT-PCR. Primers were designed using Primer Ex-
press software (v3.0, Applied Biosystems), and primer sequences were shown in S1 Table. The
elongation factor 1-α (ef1α) was used as a reference gene [16]. The 20 μl PCR reaction buffer
contains: 2 μl diluted cDNA, 1 μl each of forward and reverse primers (10 μmol
L-1), 10 μl
qPCR Master Mix (ABI, Foster City, CA, USA) and 6 μl ddH2O. Amplification of genes was
performed on a LightCycler 480 Real-Time PCR machine (Roche, Basel, Switzerland) under
the following conditions: 95°C for 3 min, followed by 40 cycles at 95°C for 15 sec and 60°C for
40 sec. Detection of each gene was repeated 3 times. The 2-(ΔΔCt) method [17] was used to cal-
culate and calibrate the expression level of target genes in different treatments.

Results
Total RNA quality testing
The quality of total RNA extracted from the collected stolons of CT, DT and RWT plants was
tested using agarose gel electrophoresis and an Agilent 2100 Bioanalyzer. All the three samples
had 28S:18S ratios in a range of 1.8–2.0 with intact 28S, 18S and 5S RNA bands, high RNA pu-
rity, and a mean RNA integrity number (RIN) >8.0, which met the requirements for library
construction and sequencing.

Sequencing data and DEG analysis

After transcriptome sequencing, a total of 78448498, 74764759, and 71081020 raw reads were
obtained from CT, DT, and RWT cDNA libraries, respectively. From these reads, 21.74 G
clean bases were obtained by filtering impurities, and 76.1% of them fell into the published po-
tato genome. Sequencing analysis of CT, DT and RWT cDNA libraries produced 46302, 47166
and 46341 genes, and 75924, 76516 and 74678 transcripts, respectively (Table 1).

Table 1. Statistical analyses of cDNA libraries from potato stolons under different growth conditions.

CT (Control) DT (Drought stress treatment) RWT (Re-watering after drought stress treatment)
Raw reads 78448498 74764759 71081020
Clean reads 74826108 71748174 68670240
Total mapped 56185839 (75.09%) 52476932 (76.42%) 56769533 (76.8%)
Clean bases (G) 7.48 6.86 7.4
Error rate (%) 0.03 0.03 0.03
Q30 (%) 92.63 92.63 92.51
GC content (%) 42.07 42.33 42.45
Gene number 46302 47166 46341
Transcripts number 75924 76516 74678
doi:10.1371/journal.pone.0128041.t001

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 Transcriptome Profiling of Potato

Table 2. List of genes showing significantly different expression in the comparison DT vs. CT.

Gene ID Readcount DT Readcount CT Log2 Fold change q-value Blast Swiss prot
PGSC0003DMG400012174 399.16 0 11.01 9.15E-55 -//-
PGSC0003DMG400011197 58.91 0 10.57 5.53E-09 Heat shock 70 kDa protein
PGSC0003DMG400026969 21.73 0 9.13 0.00023524 Probable aquaporin TIP3-2
PGSC0003DMG400026590 82.9 0 8.74 6.59E-16 Expansin-like B1
PGSC0003DMG400032771 40.67 0 8.04 1.29E-08 Bidirectional sugar transporter
PGSC0003DMG400015495 130.51 0 8.02 8.49E-27 -//-
PGSC0003DMG400008517 57.07 0 7.72 3.01E-12 Bidirectional sugar transporter
PGSC0003DMG400018766 28.23 0 7.51 2.09E-06 -//-
PGSC0003DMG400015275 11.96 0 6.68 0.0036329 Probable aquaporin TIP3-2
PGSC0003DMG400019320 46.12 0 6.63 1.12E-10 Premnaspirodiene oxygenase
PGSC0003DMG400002085 1.05 601.17 -9.17 7.90E-111 Pectinesterase/pectinesterase inhibitor 18
PGSC0003DMG400026575 0 80.14 -9.26 7.81E-15 Cationic peroxidase 1
PGSC0003DMG400022295 0 35.91 -9.68 1.52E-06 Putative lipid-transfer protein DIR1
PGSC0003DMG400005273 0 36.53 -9.71 1.26E-06 Peroxidase 3
PGSC0003DMG400002074 0 77.78 -9.8 2.12E-13 Pectinesterase/pectinesterase inhibitor 18
PGSC0003DMG400002108 0 163.19 -9.87 1.90E-27 -//-
PGSC0003DMG400010771 0 118.03 -10.4 1.94E-18 Probable pectate lyase 5
PGSC0003DMG400026463 0 356.71 -10.41 4.82E-55 Aquaporin TIP2-1
PGSC0003DMG400030587 0 126.28 -11.5 1.68E-16 Non-specific lipid-transfer protein 2
doi:10.1371/journal.pone.0128041.t002

Then, data obtained from the three samples were subjected to pairwise comparisons, and
DEGs were identified using the preset cutoffs. A total of 9216 DEGs (including 5604 solely ex-
pressed DEGs) were identified. When DT was compared to CT, 1227 genes were up-regulated,
and 1962 genes were down-regulated. When RWT was compared to CT, 658 genes were found
to be up-regulated and 1139 gene down-regulated. Different from the above two comparison
groups, 2352 genes were up-regulated, and 1878 genes showed down-regulated mode when
RWT was compared to DT. Down-regulated transcripts accounts for 62%, 63% and 44% of the
total DEGs in above three comparison groups, respectively (S2 Table).
To screen the DEGs, two different analysis methods were applied in our study. First, we identi-
fied 20 up- and down-regulated genes whose expression differed significantly (fold change |log2|
1, corrected p-value <0.005) in DT vs. CT (Table 2), RWT vs. CT (Table 3) and RWT vs. DT
(Table 4). Expression of heat shock protein (PGSC0003DMG400011197), aquaporins
(PGSC0003DMG400026969, PGSC0003DMG400015275 and PGSC0003DMG400026463), bidi-
rectional sugar transporters (PGSC0003DMG400032771 and PGSC0003DMG400008517) and
lipid-transfer proteins (PGS C0003DMG400022295 and PGSC0003DMG400030587) differed sig-
nificantly between DT and CT (Table 2). Whereas, expression of starch synthesis and accumula-
tion, and water stimulus-responsive genes such as those encoding protein EARLY FLOWERING
(PGSC0003DMG400001221), glucose-6-phosphate/phosphate translocator
(PGSC0003DMG400005269), serine protease inhibitors (PGSC0003DMG400010128 and
PGSC0003DMG400009512), granule-bound starch synthase (PGSC0003DMG400012111), ethyl-
ene-responsive transcription factor (PGSC0003DMG400012527), and gibberellin 3-beta-dioxy-
genase (PGSC0003DMG400005698) differed most significantly between RWT and CT (Table 3).
Homologous gene of Cytochrome P450 (PGSC0003DMG400006368), Galactinol synthase
(PGSC0003DMG400015490), bidirectional sugar transporter SWEET12
(PGSC0003DMG400032771), heat shock protein (PGSC0003DMG400011197), lipid-transfer
proteins (PGSC0003DMG400030587), ASPG1 (PGSC0003DMG400037894) and BURP14

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 5 / 20

 Transcriptome Profiling of Potato

Table 3. List of genes showing significantly different expression in the comparison RWT vs. CT.

Gene ID Readcount Readcount Log2 Fold change q-value Blast swiss prot
RWT CT
PGSC0003DMG400005269 1267.78 2.35 9.08 4.9453E-227 Glucose-6-phosphate/phosphate translocator 2,
chloroplastic
PGSC0003DMG400025158 35.07 0 7.86 2.5423E-07 9-divinyl ether synthase
PGSC0003DMG400030925 22.28 0 6.20 0.000033552 -//-
PGSC0003DMG400009512 370.40 6.51 5.83 9.8857E-83 Serine protease inhibitor 1
PGSC0003DMG400022807 15.04 0 5.63 0.0012378 -//-
-129
PGSC0003DMG400010128 578.44 12.41 5.54 6.6347E Serine protease inhibitor 7
PGSC0003DMG400011323 41.67 1.02 5.35 2.2123E-09 Putative lipid-transfer protein DIR1
PGSC0003DMG400012111 1346.25 38.70 5.12 6.3024E-296 Granule-bound starch synthase 1, chloroplastic/
amyloplastic
PGSC0003DMG400013516 41.31 1.48 4.81 5.0155E-09 Polygalacturonase At1g48100
PGSC0003DMG400028535 136.29 5.07 4.75 4.4972E-29 -//-
-14
PGSC0003DMG400012494 0 59.92 -6.74 3.0461E PGR5-like protein 1B, chloroplastic
PGSC0003DMG401026984 0 15.93 -7.05 0.00058233 BTB/POZ and TAZ domain-containing protein 1
PGSC0003DMG400012527 0 31.89 -7.32 1.9372E-07 Ethylene-responsive transcription factor ERF025
PGSC0003DMG400005698 0 16.61 -7.70 0.0006312 Gibberellin 3-beta-dioxygenase 3
PGSC0003DMG400007672 1.20 283.08 -7.88 3.4095E-61 -//-
PGSC0003DMG401028252 0 30.42 -8.57 2.5024E-06 Beta-fructofuranosidase, insoluble isoenzyme 1
Novel00218 0 34.77 -8.76 4.9726E-07 Phenylalanine N-monooxygenase
PGSC0003DMG400013481 0 40.59 -8.99 6.1882E-08 Probable inactive poly [ADP-ribose] polymerase
SRO2
PGSC0003DMG400032247 0 67.34 -9.13 9.8393E-13 Vicilin-like antimicrobial peptides 2–1
PGSC0003DMG400001221 0 46.95 -10.20 9.4907E-08 Protein EARLY FLOWERING4
doi:10.1371/journal.pone.0128041.t003

(PGSC0003DMG400027019) which related to stress signal transduction or carbohydrate synthe-

sis and metabolism were significantly different expressed between RWT and DT groups (Table 4).
Second, of the identified DEGs, 263 of them exhibited opposite expression patterns between
the two comparison treatments CT vs. DT and CT vs. RWT (S3 Table), that is, transcripts that
were regulated in an opposite direction in DT and RWT compared to CT. The most significant
20 DEGs (with the greatest positive or negative changes) were genes homologous to those re-
sponsible for drought-stress response and regulation, such as TAS14
(PGSC0003DMG400003530), PP2C5 (PGSC0003DMG400027174), ASPG1
(PGSC0003DMG400037894) and BURP14 (PGSC0003DMG400027019) (Table 5). Addition-
ally, prominent changes were also observed in the expression of some other stress response
genes, such as those encoding WRKYs (PGSC0003DMG400011633,
PGSC0003DMG400016769, PGSC0003DMG400028520 and PGSC0003DMG400021 895),
ERFs (PGSC0003DMG400025797, PGSC0003DMG400008248 and PGSC0003D
MG400008265), bHLHs (PGSC0003DMG400006394 and PGSC0003DMG400000599), cyto-
chrome P450 (PGSC0003DMG401031520), auxin-responsive protein
(PGSC0003DMG400006093), APRR2 (two-component response regulator-like,
PGSC0003DMG400026587), GASA6 (gibberellin-regulated protein 6,
PGSC0003DMG401019533), CML19 (putative calcium-binding protein,
PGSC0003DMG400002993), SAMDC (S-adenosylmethionine decarboxylase proenzyme,
PGSC0003DMG400010051), Abscisic acid 8'-hydroxylases (PGSC0003DMG402018475 and
PGSC0003DMG400001 960), calcium-transporting ATPase (PGSC0003DMG400018942), F-
box protein (PGSC0003DMG400026346) and ZOG1 (zeatin O-glucosyltransferases

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 6 / 20

 Transcriptome Profiling of Potato

Table 4. List of genes showing significantly different expression in the comparison RWT vs. DT.

Gene ID Readcount Readcount Log2 Fold q-value Blast Swiss prot

change
DT RWT
PGSC0003DMG400012174 378.09 0 -12.22 2.2E-43 -//-
PGSC0003DMG400015495 123.62 0 -10.61 5.56E-19 -//-
PGSC0003DMG400014293 282.00 0 -10.48 1.21E-43 sp|Q04980|LTI65_ARATH Low-temperature-induced 65 kDa
protein OS = Arabidopsis thaliana GN = LTI65 PE = 2 SV = 2//
2.5331e-35
PGSC0003DMG400026590 78.53 0 -8.95 2.17E-15 sp|O23547|EXLB1_ARATH Expansin-like B1 OS = Arabidopsis
thaliana GN = EXLB1 PE = 2 SV = 2//5.27796e-07
PGSC0003DMG400011197 55.80 0 -8.46 9.9E-12 sp|P26413|HSP70_SOYBN Heat shock 70 kDa protein
OS = Glycine max GN = HSP70 PE = 3 SV = 1//4.92362e-50
PGSC0003DMG400032771 38.52 0 -7.60 5.85E-09 sp|O82587|SWT12_ARATH Bidirectional sugar transporter
SWEET12 OS = Arabidopsis thaliana GN = SWEET12 PE = 2
SV = 1//1.31834e-26
Novel05792 60.63 0 -7.41 5.24E-14 sp|Q9M439|BCAT2_ARATH Branched-chain-amino-acid
aminotransferase 2, chloroplastic OS = Arabidopsis thaliana
GN = BCAT2 PE = 1 SV = 1//3.37023e-175
PGSC0003DMG400015490 22.81 0 -7.17 9.98E-06 sp|C7G304|GOLS2_SOLLC Galactinol synthase 2 OS = Solanum
lycopersicum GN = GOLS2 PE = 2 SV = 1//1.32863e-42
PGSC0003DMG400028543 578.87 4.91 -6.88 4.7E-135 -//-
PGSC0003DMG400006368 12.36 0 -6.70 0.001778 sp|P24465|C71A1_PERAE Cytochrome P450 71A1 OS = Persea
americana GN = CYP71A1 PE = 1 SV = 2//1.28232e-112
PGSC0003DMG400014850 0 245.89 9.95 1.16E-39 sp|P14009|14KD_DAUCA 14 kDa proline-rich protein DC2.15
OS = Daucus carota PE = 2 SV = 1//2.41216e-33
PGSC0003DMG400008980 0 85.25 10.01 6.01E-14 sp|P13917|7SB1_SOYBN Basic 7S globulin OS = Glycine max
GN = BG PE = 1 SV = 2//4.91172e-60
PGSC0003DMG400014690 0 46.19 10.13 1.09E-07 -//-
PGSC0003DMG400027019 0 233.51 10.14 1.14E-36 sp|Q6K2M1|BURPE_ORYSJ BURP domain-containing protein 14
OS = Oryza sativa subsp. japonica GN = BURP14 PE = 2
SV = 2//1.71576e-37
PGSC0003DMG400022295 0 61.94 10.55 1.59E-09 sp|Q8W453|DIR1_ARATH Putative lipid-transfer protein DIR1
OS = Arabidopsis thaliana GN = DIR1 PE = 1 SV = 1//5.41356e-
08
PGSC0003DMG400018917 0 153.88 10.86 8.22E-22 sp|Q08303|PPOA_SOLLC Polyphenol oxidase A, chloroplastic
OS = Solanum lycopersicum PE = 3 SV = 2//0
PGSC0003DMG400037894 0 178.61 11.08 2.16E-24 sp|Q9LS40|ASPG1_ARATH Protein ASPARTIC PROTEASE IN
GUARD CELL 1 OS = Arabidopsis thaliana GN = ASPG1 PE = 1
SV = 1//1.31192e-38
PGSC0003DMG400030587 0 111.53 11.40 1.24E-14 sp|P82353|NLTP2_PRUAR Non-specific lipid-transfer protein 2
OS = Prunus armeniaca PE = 1 SV = 1//1.21051e-14
PGSC0003DMG400008444 0 918.71 12.44 1.02E-97 -//-
PGSC0003DMG400005273 0 236.39 12.48 3.16E-25 sp|O23044|PER3_ARATH Peroxidase 3 OS = Arabidopsis
thaliana GN = PER3 PE = 2 SV = 1//4.47243e-66
doi:10.1371/journal.pone.0128041.t004

PGSC0003DMG400000432 and PGSC0003DMG400028331). In addition to the above genes,

sixteen genes involved in starch accumulation and synthesis and tuber formation exhibited sig-
nificantly different expression (S4 Table). Among them, six genes PHYB
(PGSC0003DMG400027211), LOX (PGSC0003DMG400032155), gibberellin 2-beta-dioxygen-
ase (PGSC0003DMG400026762), gibberellin 20 oxidase (PGSC0003DMG400001249) and
aquaporin-encoding genes (PGSC0003DMG400007134 and PGSC0003DMG400011875) only
showed differential expression between DT and CT, and two genes LOX-encoding gene
(PGSC0003DMG400022894) and GASA6 (PGSC0003DMG 401019533) exhibited opposite
expression patterns.

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 7 / 20

 Transcriptome Profiling of Potato

Table 5. Twenty differentially expressed genes with most significant positive or negative changes.

Gene ID Log2 Fold change Log2 Fold change Range Blast swiss prot
(DT/CT) (RWT/CT)
Novel05792 1.76 -5.68 7.44 Branched-chain-amino-acid aminotransferase 2,
chloroplastic
PGSC0003DMG400028543 2.97 -3.94 6.91 -//-
PGSC0003DMG400002161 2.23 -3.39 5.62 -//-
PGSC0003DMG400000339 1.07 -4.54 5.60 Beta-galactosidase 1
PGSC0003DMG400016931 3.54 -1.88 5.42 -//-
PGSC0003DMG400003530 2.26 -3.04 5.30 Abscisic acid and environmental stress-inducible protein
TAS14
PGSC0003DMG401033888 1.87 -3.25 5.12 Universal stress protein A-like protein
PGSC0003DMG400014487 2.19 -2.70 4.89 MOUSE RING finger and CHY zinc finger domain-
containing protein 1
PGSC0003DMG400001518 2.12 -2.57 4.69 -//-
PGSC0003DMG400027174 3.27 -1.18 4.45 Probable protein phosphatase 2C 30
PGSC0003DMG400020481 -6.70 1.94 -8.63 14 kDa proline-rich protein DC2.15
PGSC0003DMG400026540 -7.90 1.04 -8.95 Uncharacterized protein At5g22580
PGSC0003DMG400006162 -5.95 3.03 -8.99 Fruit-specific protein
PGSC0003DMG400013680 -7.05 2.75 -9.80 Probable pectinesterase/pectinesterase inhibitor 12
PGSC0003DMG400014850 -6.91 3.01 -9.92 14 kDa proline-rich protein DC2.15
PGSC0003DMG400027019 -7.52 2.60 -10.11 BURP domain-containing protein 14
PGSC0003DMG400018917 -7.50 3.33 -10.83 Polyphenol oxidase A, chloroplastic
PGSC0003DMG400037894 -8.61 2.44 -11.05 Protein ASPARTIC PROTEASE IN GUARD CELL 1
PGSC0003DMG400008444 -9.09 3.32 -12.41 -//-
PGSC0003DMG400005273 -9.71 2.74 -12.45 Peroxidase 3
doi:10.1371/journal.pone.0128041.t005

qRT-PCR verification
To verify data validity, ten genes, including the target genes of interest, were selected from se-
quencing data, and qRT-PCR analyses were performed (S5 Table). In the comparison of DT
and RWT, the correlation coefficients of gene expression trends in sequencing data and
qRT-PCR results were 0.9267 and 0.5543, respectively (Fig 1). In particular, significant expres-
sion difference occurred in drought stress- and water stimulus-responsive genes before and
after the treatment, including genes encoding MYB60 (PGSC0003DMG400033043), WRKY33
(PGSC0003DMG400011633) and NAC072 (PGSC0003DMG400015342), thus supporting the
validity of DEG data obtained.

GO classification and KEGG pathway annotation analysis of DEGs

In order to investigate their functions and highlight metabolic pathways potentially related to
drought tolerance, GO classification and KEGG pathway annotation analyses were performed
on the identified DEGs. A total of 639 unique GO functional annotation terms were assigned
to the identified DEGs (Fig 2). Of them, 979 were within the biological process category, ac-
counting for 73.4% of the total GO functional annotation categories, and the rest were within
the cellular component (217) and molecular function categories (137). Intra-group analysis
(Table 6) revealed that in the comparison of CT and DT, the top 3 significantly enriched GO
functional annotation categories were stress response (GO:0006950), macromolecular complex
(GO:0032991) and structural constituent of ribosome (GO:0003735). These included 635
DEGs, of which 454 were down-regulated. In the comparison of CT and RWT, the top 3

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 8 / 20

 Transcriptome Profiling of Potato

significantly enriched GO functional annotation categories were translation (GO:0006412), ri-

bosome (GO:000 5840) and structural constituent of ribosome (GO:0003735). These included
274 DEGs, of which, 256 were up-regulated genes. Clearly, DEGs were mainly down-regulated
in DT and up-regulated in RWT. Similar to the RWT vs. CT group, the top 3 significantly en-
riched GO functional annotation categories in the comparison of DT and RWT were transla-
tion (GO:0006412), ribosome (GO:0005840) and structural constituent of ribosome
(GO:0003735) either. However, each of the GO category include much more DEGs. Totally,
this comparison group includes 713 DEGs, of which, 675 were up-regulated genes.
A directed acyclic graph (DAG) was used to illustrate the affiliation and functional scope of
the 10 most significantly enriched GO categories. In the comparison of CT and DT (Fig 3A),
DEGs were intensively enriched in the following five GO categories: metabolic process
(GO:0008152), stress response (GO:0006950), organic substance metabolic process
(GO:0071704), primary metabolic process (GO:0044238) and cellular macromolecular com-
plex assembly (GO:0034622), which included 4076 DEGs. In the comparison of CT and RWT
(Fig 3B), the top 3 significantly enriched GO categories were metabolic process (GO:0008152),
cellular metabolic process (GO:0044237) and translation (GO:0006412).

Fig 1. Verification of differentially expressed genes in DT vs. CT group (A) and in RW vs. CT group (B)
by qRT-PCR.
doi:10.1371/journal.pone.0128041.g001

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 Transcriptome Profiling of Potato

Fig 2. Gene Ontology (GO) categories of assembled differentially expressed genes in the comparison
groups.
doi:10.1371/journal.pone.0128041.g002

To further identify the key regulatory genes related to drought- and water-stimulus re-
sponses, as well as genes involved in starch and sucrose metabolism, we focused on DEGs of
the same GO terms but showed opposite expression regulation patterns between the two com-
parison treatments, that is, transcripts that were regulated in an opposite direction in DT and
RWT compared to CT. A total number of 5 GO terms were related to starch and sucrose me-
tabolism, and 3 GO terms were related to drought-stress and water-stimulus responses (S6
Table). Among these 8 GO terms, 60 DEGs showed opposite regulation patterns between DT
and RWT. Further analysis revealed that 41 genes were down-regulated in DT and up-regulat-
ed in RWT, of which 37 were related to starch and sucrose metabolism, including homologous

Table 6. Top 3 significantly enriched GO functional annotation categories.

GO accession Description Term type Corrected p-value DEG item Up Down

DT vs. CT GO:0006950 response to stress biological process 6.85E-16 263 89 174
-20
GO:0032991 macromolecular complex cellular component 1.65E 292 85 207
GO:0003735 structural constituent of ribosome molecular function 9.41E-10 80 7 73
Total 635 181 454
RWT vs. CT GO:0006412 translation biological process 1.46E-19 104 91 13
GO:0005840 ribosome cellular component 9.04E-24 88 85 3
GO:0003735 structural constituent of ribosome molecular function 3.52E-22 82 80 2
Total 274 256 18
RWT vs. DT GO:0006412 translation biological process 8.82E-64 255 235 20
GO:0005840 ribosome cellular component 2.76E-97 236 226 10
GO:0003735 structural constituent of ribosome molecular function 1.68E-91 222 214 8
Total 713 675 38
doi:10.1371/journal.pone.0128041.t006

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 Transcriptome Profiling of Potato

Fig 3. GO analysis of DT vs. CT group (A) and RWT vs. CT group (B) illustrated by Directed Acyclic
Graph. A, *significantly enriched GO categories, including GO:0008152 (metabolic process, corrected p-
value 2.45E-15), GO:0006950 (response to stress, corrected p-value 6.85E-16), GO:0071704 (organic
substance metabolic process, corrected p-value 5.13E-15), GO:0044238 (primary metabolic process,
corrected p-value 2.45E-15) and GO:0034622 (cellular macromolecular complex assembly, corrected p-value
1.91E-13). B, *significantly enriched GO categories, including GO: 0008152 (metabolic process, corrected p-
value 5.97E-16), GO:0044237 (cellular metabolic process, corrected p-value 3.7E-16) and GO:0006412
(translation, corrected p-value 1.46E-19).
doi:10.1371/journal.pone.0128041.g003

genes encoding beta-galactosidase 1 (BGAL1), UDP-glucose 4-epimerase and 6-phosphogluco-

nate dehydrogenase. Additionally, 19 genes were up-regulated in DT and down-regulated in
RWT, of which 13 were related to drought-stress and water-stimulus responses.
In the pathway annotation analysis, data was compared with the published KOBAS data-
base. A total number of 4549 DEGs were enriched in 314 (114 unique) pathways. Intra-group

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 11 / 20

 Transcriptome Profiling of Potato

analysis of each comparison was then performed to identify the 10 most significantly enriched
pathways (Fig 4). Revealing DEGs were intensively enriched in 14 pathways. The metabolic
pathways with the most significant DEGs enrichment were metabolic pathways (sly01100),
biosynthesis of secondary metabolites (sly01110), ribosome (sly03010) and carbon cycle me-
tabolism (sly01200), which included 868, 446, 297, and 143 DEGs, respectively.
Furthermore, we performed a detailed classification of KEGG pathways for the 263 genes
which showed opposite expression regulation patterns between the two comparison treatments,
revealing 66 DEGs annotated to KEGG pathways (S7 Table) including 16 metabolic pathways
(Table 7). Of them, the most frequently associated pathways were metabolic pathways (21), fol-
lowed by ribosome (18). Only 3 enriched DEGs were associated with carotenoid biosynthesis
and plant hormone signal transduction pathways (S1A and S1B Fig). These included
PGSC0003DMG402018475 (encoding abscisic acid 8'-hydroxylase 1-like, CYP707A2),
PGSC0003DMG400001960 (encoding abscisic acid 8'-hydroxylase 1-like) and
PGSC0003DMG400028897 (encoding beta-carotene hydroxylase) involved in carotenoid bio-
synthesis pathway, and PGSC0003DMG400008011 (encoding AREB-like protein, AREB),
PGSC0003DMG40 1025624 (encoding two-component response regulator ARR2-like) and
PGSC0003DM G400006093 (encoding auxin-responsive protein, AUX) associated with plant
hormone signal transduction pathway. The above results suggest that DEGs with opposite ex-
pression regulation patterns were mainly involved in drought- and water-stimulus response and
regulation via the carotenoid biosynthesis pathway, which affect the synthesis and signal trans-
duction pathways of other endogenous hormones (e.g., abscisic acid and indole acetic acid).

Drought stress and water stimulus responsive TFs

TF-encoding genes are key factors in the process of stress signal perception and transduction.
Based on the sequencing analyses, a total number of 1630, 1527 and 1596 TF-encoding genes
were detected in CT, DT and RWT, respectively (Table 8). In the comparison to the control
treatment, both drought and re-watering treatments reduced the number of TF-encoding
genes detected. In the comparison between DT and RWT with CT, a total number of 1019 and
866 TF-encoding DEGs were identified, respectively.
Further analyses revealed that in the comparison between DT and CT, the identified 1019
DEGs fell into 50 TF families, and most of DEGs (268) belong to bHLH, ERF and MYB families
(S8 Table). Similarly, in the comparison between RWT and CT, the identified DEGs were in-
volved in a total of 52 TF families, and most of them (230) also belong to ERF, bHLH, and MYB
families. When it comes to RWT vs. DT, 1018 DEGs were involved in a total of 51 TF families.
bHLH (97), ERF (90), C2H2 (57) and MYB (57) were the top 4 families with the most differently
expressed genes. Overall (S8 Table, Fig 5A, 5B and 5C), significant changes occurred in the tran-
scripts of the following eight TF families: bHLH (282 DEGs), ERF (281 DEGs), MYB (169
DEGs), C2H2 (147), NAC (147 DEGs), WRKY (145 DEGs), HD-ZIP (123) and bZIP (84 DEGs).
DEGs belonging to the above eight transcription factor families accounted for almost 50% of the
total differentially expressed TFs detected in the DT and RWT treatments, respectively.
We also analyzed the quantity relationship of up- and down-regulated TFs in 3 comparison
groups (S8 Table). In the comparison between DT and CT, the number of down-regulated
DEGs was approximately 2-fold that of the up-regulated ones among the 1019 differentially ex-
pressed TFs. The largest number of down-regulated DEGs was found in the bHLH family,
while that of up-regulated DEGs was mostly found in the ERF family. Similar with comparison
between DT and CT, the number of down-regulation DEGs in RWT vs. DT exhibited almost
twice as much as the number of up-regulation TFs. The bHLH family contained 69 decreased
expression of TFs and the ERF family included 31 increased expression of DEGs. In the

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 Transcriptome Profiling of Potato

Fig 4. Top 10 statistically enriched KEGG pathways in the comparison groups. sly01100: Metabolic
pathways, sly01110: Biosynthesis of secondary metabolites, sly03010: Ribosome, sly01200: Carbon
metabolism, sly00190: Oxidative phosphorylation, sly04141: Protein processing in endoplasmic reticulum,
sly00195: Photosynthesis, sly04075: Plant hormone signal transduction, sly01230: Biosynthesis of amino
acids, sly00500: Starch and sucrose metabolism, sly04626: Plant-pathogen interaction, sly00010:
Glycolysis/Gluconeogenesis, sly00710: Carbon fixation in photosynthetic organisms, sly03040:
Spliceosome.
doi:10.1371/journal.pone.0128041.g004

comparison between RWT and CT, the number of up- or down-regulated DEGs was about the
same among the 866 differentially expressed TFs. The largest number of down-regulated DEGs
was found in the ERF family, whereas the largest number of up-regulated ones was found in
the bHLH family.

Table 7. The number of differentially expressed genes with opposite expression patterns annotated
to KEGG pathways.

KEGG pathway item DEGs number

Metabolic pathways 21
Ribosome 18
Plant-pathogen interaction 4
Carotenoid biosynthesis 3
Plant hormone signal transduction 3
Stilbenoid, diarylheptanoid and gingerol biosynthesis 2
Ubiquitin mediated proteolysis 2
Sulfur metabolism 2
Galactose metabolism 2
Steroid biosynthesis 2
mRNA surveillance pathway 2
Phagosome 1
Vitamin B6 metabolism 1
Biosynthesis of secondary metabolites 1
Photosynthesis 1
Ribosome biogenesis in eukaryotes 1
Total 66
doi:10.1371/journal.pone.0128041.t007

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 Transcriptome Profiling of Potato

Table 8. Transcription factors (TFs) in sequencing libraries.

Normal control (CT) Drought stress (DT) Re-watering after drought stress (RWT)
TFs 1630 1527 1596
DT vs. CT RWT vs. CT RWT vs. DT
Total differentially expressed TFs 1019 866 1018
Up-regulated differentially expressed TFs 336 442 648
Down-regulated differentially expressed TFs 683 424 370
doi:10.1371/journal.pone.0128041.t008

Fig 5. Top 10 families of differentially expressed transcription factors in DT vs. CT group (A), in RWT
vs. CT group (B) and RWT vs. DT (C).
doi:10.1371/journal.pone.0128041.g005

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 Transcriptome Profiling of Potato

Discussion
Rapid growth of global population and decrease of arable land have presented a serious chal-
lenge for food production. Potato is a non grain crop and one of the most important sources
for food and industry uses worldwide. One of the most essential approaches is to breed new
plant species which can grow on uncultivated lands. Understanding how plants including pota-
to respond to water-deficit stress will facilitate the breeding of drought tolerant plants. In the
present study, we employed high-throughput sequencing technology to profile the transcrip-
tome changes in the stolon tissues of potato undergoing water-deficit stress and re-watering
treatment. A total number of 3189, 1797 and 4230 DEGs (S2 Table), including 1630, 1527 and
1596 transcriptional factor-encoding DEGs (Table 8), were identified in the comparisons with-
in CT, DT and RWT, respectively. These DEGs were classified into 639 unique gene ontology
functional annotation terms and were involved in 114 Kyoto Encyclopedia of Genes and
Genomes pathways.
In terms of the current reports, cDNA microarrays and target metabolite analysis were per-
formed on leaves of two different drought-tolerance potato clones (Solanum tuberosum subsp.
andigenum) [2]. Gene expression analysis revealed that photosynthesis, photorespiration and
carbohydrate-related gene, coupled with SPS and SS, showed more drastically down regulated
in drought-tolerance clone than the expression in drought-sensitive clone. Zhang et al. [18] re-
ported transcriptome dynamics of potato leaves under drought stress with high throughput se-
quencing platform and according to their report the DEGs were mainly annotated as genes
coding for metabolism alteration, osmolite adjustment, cell rescue and gene regulation. Periph-
eral-type benzodiazepine receptor and polygalacturonase noncatalytic subunit AroGP3 were
the one of the ten most up-regulated and the ten most down-regulated genes, respectively. Un-
like previous studies, we took stolon tissues, one of the major agronomic traits, as sequencing
material to examine transcriptome changes under two treatments and identified more signifi-
cantly altered DEGs associated with drought stress (Tables 2, 3 and 4), starch synthesis and
transfer (S4 Table), and water stimulus response (S6 Table). The difference of DEGs identified
in these studies could be due to the different material properties, treatments, sampling strate-
gies and genetic backgrounds of plants.
Many functional and regulatory genes are responsive to water deficit stress [19, 20]. The
main role of these functional genes is to maintain intracellular water and ion homeostasis, mem-
brane structural stability, and reconstruction of primary and secondary metabolism [21], where-
as regulatory genes (e.g., calcium ions, receptor protein kinases, and TFs) are involved in stress
response by regulating signal transduction and metabolic pathways [22, 23]. Heat shock respon-
sive genes was one of the functional gene that facilitate protein refolding and stabilize polypep-
tides and membranes under stress condition by preventing protein from aggregation, protecting
non-native enzymes from degradation and assisting protein refolding [24, 25]. Expression in-
duction of these genes under drought stress has been reported in barley [26] and rice [27]. Our
data showed different expression of heat shock protein-encoding genes (Tables 2 and 3), indicat-
ing that they were also directly involved in regulating drought-stress responses in potato.
Aquaporins, which facilitate transmembrane transport of small molecules such as water,
have been showed to be involved in response to drought and salt stress, and to ABA treatment
[28, 29]. In our data, aquaporin genes (Tables 2 and 3, S4 Table) were significantly expressed in
Ningshu 4 stolons under drought stress condition. Similar expression pattern was also ob-
served in Arabidopsis [28], chickpea (Cicer arietinum L.) [30], foxtail millet (Setaria italica L.)
[31] and maize [32]. Additionally, ScPIP2a, an aquaporin, was reported to act in photoperiod
perception, generating flower-inducing signals in the leaves [33]. The existing researches indi-
cated these proteins could play an important role in water stimulus response and the

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 15 / 20

 Transcriptome Profiling of Potato

maintenance of intracellular osmotic potential stability and organelle integrity. Therefore, we

hypothesize that aquaporin(s) may play a similar regulatory role in potato tuber bulking and
growth under drought stress.
Potato tuber formation is a complex biological process governed by both environmental fac-
tors and genes [34]. Cell division and expansion, together with the associated starch and pro-
tein accumulation, are the major events of tuber bulking in potato [35]. Photoperiod
perception in leaves initiates tuberization in the subapical region of the underground stolons
[36]. Dioxygenases, which catalyze the oxygenation of polyunsaturated fatty acids (LOXs) such
as linoleic and linolenic acids, contribute to 9(S)-hydroperoxy linolenic acid (9(S)-HPOT) and
13(S)-hydroperoxy linolenic acid (13(S)-HPOT) accumulation [37]. Jasmonic acid is synthe-
sized from 13(S)-HPOT and then metabolized to tuberonic acid and tuberonic acid glucoside,
both of which induce tuber formation [38]. In this study, expression of PHYB, GA and LOX-
encoding gene (S4 Table) was significantly different in drought and re-watering plants, suggest-
ing that these genes could function in regulating starch accumulation and tuber formation of
potato plants.
As an important plant hormone, ABA plays important roles in plant response to drought
stress by inducing the expression of TF-, heat shock protein-, transporter-, and osmotic regula-
tor-encoding genes downstream of stress signaling pathways in both ABA-dependent and
ABA-independent manners [19]. We observed that expression of ABA anabolism- or signal
transduction-related genes such as abscisic acid 8'-hydroxylase 1-like, Beta-carotene hydroxy-
lase, AREB, and PP2C5 was all significantly altered during drought stress and re-watering treat-
ment (Table 5, S3 Table), indicating that ABA and anabolism related genes were also
important regulators for drought-stress and re-watering stimulus-response in potato. This hy-
pothesis is also supported by previous findings in corn (Zea mays) and cotton (Gossypium hir-
sutum) [24, 32]. In addition, expression of some gene involved in auxin, cytokinin and
gibberellin anabolism was significantly affected (S3 Table, S1 Fig), which, together with ABA,
jointly regulates the response of potato stolons to drought stress and re-watering. Similar re-
sults were also found in Chinese cabbage (Brassica rapa ssp. pekinensis) [39] and barley (Hor-
deum vulgare) [40]. Collectively, our findings demonstrate the existence of a synergistic and
antagonistic cross talk at endogenous hormone level in potato during the response to drought
stress and re-watering treatment.
Many TFs, such as ERF [41], MYB [42], WRKY [43] and bHLH, act as key regulators in sig-
nal transduction pathways involved in plant response to drought stress [19, 20]. Consistently,
we observed four TF-encoding genes, WRKY, bHLH, ERF and MYB, were differentially ex-
pressed under drought stress and re-watering conditions (Tables 2 and 3, S3 Table). In particu-
lar, genes encoding WRKY, bHLH, and ERF showed opposite expression alterations under
either condition, indicating that TF-encoding genes may have important functions in regulat-
ing the response to drought stress and re-watering stimulus in potato. Meanwhile, expression
of genes mediated by them, such as those encoding transporter, osmotic adaptor, and those in-
volved in fatty acid metabolism pathway (Tables 2 and 3, S3 Table), was also up- or down-regu-
lated. Therefore, we postulate that drought stress response in potato stolons is similar to that in
cotton as proposed by Padmalatha et al [24]. That is, potato stolons mediate the synthesis and
signal transduction of ABA and the other endogenous hormones, and thereby influence the ex-
pression of downstream genes related to heat shock proteins, fatty acid metabolism and starch
anabolism. Consequently, this mechanism further realizes drought- and water-stimulus re-
sponse and regulation.
Based on DEGs detection, enriched biological processes and metabolism pathways analysis,
our results provide a probable insight into the molecular mechanism in potato tuber response
to drought stress and water-stimulus conditions. Our data shown in this work will provide

PLOS ONE | DOI:10.1371/journal.pone.0128041 May 26, 2015 16 / 20

 Transcriptome Profiling of Potato

more direct evidence and information for future study on the interaction between key genes in-
volved in various metabolic pathways under drought stress condition. More detailed studies on
the components involved in these pathways will help to understand the precise regulation pro-
cess and mechanism related to drought stress response in potato, as well as for the molecular
breeding of drought tolerant plants.

Supporting Information
S1 Fig. Enriched differentially expressed genes in the metabolic pathway of carotenoid bio-
synthesis (A) and plant hormone signal transduction (B) in the comparison groups.
(TIF)
S1 Table. Primers used for qRT-PCR.
(XLS)
S2 Table. The number and regulation pattern of differentially expressed genes.
(XLS)
S3 Table. Genes with opposite expression patterns between the comparisons DT vs. CT and
RWT vs. CT.
(XLS)
S4 Table. Differentially expressed genes related to starch synthesis and accumulation and
tuber formation.
(XLS)
S5 Table. qRT-PCR verification data.
(XLS)
S6 Table. Genes of the same GO annotation items with opposite differential expression pat-
terns between comparison groups DT vs. CT and RWT vs. CT.
(XLS)
S7 Table. Significantly differentially expressed genes annotated to KEGG pathways.
(XLS)
S8 Table. Transcription factor (TF) families related to differentially expressed genes in
comparison groups.
(XLS)

Acknowledgments
We are grateful to Mr. Jiehui Guanzhang (University of Toronto, Canada) for the critical read-
ing and comments on this manuscript.

Author Contributions
Conceived and designed the experiments: LG YXS. Performed the experiments: LG LS XYG LZ
YCC FJN ML. Analyzed the data: LG HXZ YXS. Contributed reagents/materials/analysis tools:
ZQG GHZ. Wrote the paper: YXS LG.

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