Plant Cell Rep (2012) 31:1173–1187

DOI 10.1007/s00299-012-1239-7

ORIGINAL PAPER

Expression profile analysis of the polygalacturonase-inhibiting

protein genes in rice and their responses to phytohormones
and fungal infection
Liaoxun Lu • Fei Zhou • Yong Zhou •
Xiaolei Fan • Shuifeng Ye • Lei Wang •

Hao Chen • Yongjun Lin

Received: 14 December 2011 / Revised: 1 February 2012 / Accepted: 10 February 2012 / Published online: 24 February 2012
Ó Springer-Verlag 2012

Abstract Polygalacturonase-inhibiting proteins (PGIPs) dynamic gene expression pattern, seven Ospgip genes were
are typically leucine-rich repeat (LRR) proteins that can first analyzed using the Affymetrix rice genome array data
inhibit the activity of fungal polygalacturonases (PGs). In from online resource. All of these seven Ospgip genes
this study, two new Ospgip genes, named Ospgip6 and showed variable expression patterns among tissues/organs.
Ospgip7 with consensus sequence of ten imperfect LRR In order to further investigate the potential function of
motif located on rice chromosomes 8 and 9, were identified these Ospgip genes, the responses of Ospgip genes to the
using BLAST analysis. Both of them appear to be extra- treatment of various phytohormones (abscisic acid, bras-
cellular glycoproteins. To have a global view of the sinosteroid, gibberellic acid, 3-indole acetic acid, jasmonic
acid, kinetin, naphthalene acetic acid and salicylic acid) as
Communicated by Y. Lu. well as fungal infection were analyzed by real-time PCR
using time course array. Generally, all the Ospgip genes
Electronic supplementary material The online version of this were slightly up-regulated in the indica rice cultivar
article (doi:10.1007/s00299-012-1239-7) contains supplementary
material, which is available to authorized users.
Minghui 63 under GA3, KT and NAA treatments (except
Ospgip2, which was down-regulated under KT treatment).
L. Lu  F. Zhou  Y. Zhou  X. Fan  L. Wang  H. Chen  In the japonica rice cultivar Zhonghua 11, Ospgip genes
Y. Lin (&) were regulated by most treatments with the response time
National Key Laboratory of Crop Genetic Improvement and
variability. We also analyzed putative cis-elements in the
National Centre of Plant Gene Research, Huazhong Agricultural
University, Wuhan 430070, People’s Republic of China promoter regions of Ospgip genes. This dataset provided a
e-mail: yongjunlin@mail.hzau.edu.cn versatile resource to understand the regulatory network of
L. Lu Ospgip genes during the process of phytohormones treat-
e-mail: luliaoxun@hotmail.com ment and fungal infection in the model monocotyledonous
F. Zhou plant, rice, and could aid in the transgenic breeding against
e-mail: zhoufei@mail.hzau.edu.cn rice fungal diseases.
Y. Zhou Key message All the seven Ospgip genes showed variable
e-mail: zoyon@webmail.hzau.edu.cn expression patterns in Minghui 63 and their expressions
X. Fan were regulated by different phytohormone treatments or
e-mail: fxl731@webmail.hzau.edu.cn fungal infection in Minghui 63 and Zhonghua 11.
L. Wang
e-mail: leiwang@webmail.hzau.edu.cn Keywords Oryza sativa  Polygalacturonase-inhibiting
H. Chen proteins  Expression profile  Biotic treatment  Abiotic
e-mail: hchen@mail.hzau.edu.cn treatment

S. Ye
Abbreviations
Shanghai Agrobiological Gene Center, Shanghai 201106,
People’s Republic of China ABA Abscisic acid
e-mail: ysf@sagc.org.cn BR Brassinosteroid

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GA3 Gibberellic acid Arabidopsis (Ferrari et al. 2003), pea (Richter et al. 2006),
IAA 3-Indole acetic acid tobacco (Joubert et al. 2006, 2007; Manfredini et al. 2005;
JA Jasmonic acid Oelofse et al. 2006) and wheat (Janni et al. 2008) can
KT Kinetin reduce the damage caused by the pathogens. Moreover,
NAA Naphthalene acetic acid antisense expression of Atpgip1 in Arabidopsis thaliana
SA Salicylic acid increased susceptibility to Botrytis cinerea (Ferrari et al.
LRR Leucine-rich repeat 2006). In rice, the results of in vitro tests showed that
OsFOR1 had highly activity against the Aspergillus niger
PG (Jang et al. 2003) and OsPGIP1 was able to inhibit the
PG of Fusarium graminearum (Janni et al. 2006).
The expression of the pgip genes can be regulated by
Introduction both biotic and abiotic stresses, such as fungal infection,
cold, drought and various hormones (De Lorenzo and
Polygalacturonase (PG) is one of the first cell wall- Ferrari 2002; Federici et al. 2006; Juge 2006). In addition,
degrading enzymes secreted when a fungal pathogen some reports showed that pgip genes may be involved in
breaches the plant cell wall (Idnurm and Howlett 2001). It the regulation of plant development. In rice, antisense
cleaves the linkages between D-galacturonic acid residues expression of OsFOR1 increased the number of floral
in homogalacturonan and transiently forms oligogalactu- organs (Jang et al. 2003). In apple, the transcription level of
ronides (OGAs) with degrees of polymerization between 9 pgip changed with the degree of fruit ripeness (Buza et al.
and 15. The OGAs induce defense responses in plants, but 2004). In strawberry, the highest expression of pgip was
PG hydrolyzes the elicitor-active long chain OGAs to detected in mature berries (Mehli et al. 2004).
smaller biologically inactive fragments. Polygalacturonase- In this study, we focused on the expression profiles of
inhibiting proteins (PGIPs) inhibit the hydrolytic activity of Ospgip genes during the entire growth period of rice and
PG and therefore delay the hydrolysis of OGAs, which may their responses to biotic and abiotic stresses. The whole
prolong the defense responses caused by OGAs in the plant dataset will contribute to a better understanding of the
and restrict the growth and invasion of the pathogens biology of Ospgip genes in the process of development of
(D’Ovidio et al. 2004a; De Lorenzo and Ferrari 2002; growth and fungal infection, which may provide important
Gomathi and Gnanamanickam 2004). clues about how to use Ospgip genes to facilitate transgenic
PGIPs are typically leucine-rich repeat (LRR) proteins breeding against rice fungal diseases.
with ten imperfect LRRs (Di Matteo et al. 2003). The
consensus sequence of the conserved LRR motif is
LxxLxxLxxLxLxxNxLxGxIPxx, where L stands for a Materials and methods
conserved Leu and x is any amino acid. This consensus
sequence was reported as LxxLxLxxNxLxGxIPxxLAxLxx Bioinformatic analysis of OsPGIP family
in the first PGIP found in rice (Jang et al. 2003). The LRR
motif has been proven to play an important role in PGIP Ospgip genes and proteins that had been reported previ-
and PG protein–protein interactions (Leckie et al. 1999; ously were used as queries to perform BLAST to search
Shanmugam 2005; Sicilia et al. 2005). PGIPs have been for proteins homologous to PGIPs in three databases:
widely found in dicot plants, but only a few have been National Centre for Biotechnology Information (http://
identified in monocots, including rice (Jang et al. 2003; www.ncbi.nlm.nih.gov/), TIGR rice genome annotation
Janni et al. 2006) and wheat (Janni et al. 2006; Kemp et al. (http://rice.plantbiology.msu.edu/) and KOME—Knowl-
2003). Similar to the pgip gene families characterized in edge-based Oryza Molecular Biological Encyclopedia
other species (D’Ovidio et al. 2004b, 2006; Ferrari et al. (http://cdna01.dna.affrc.go.jp/cDNA/). Number of the LRR
2003; Hegedus et al. 2008; Li et al. 2003), a multigene motifs in the newly identified proteins was also charac-
family with five members was also found in the rice gen- terized. The consensus sequence of the LRR motif was
ome. All five OsPGIPs have ten imperfect LRRs except LxxLxLxxNxLxGxIPxxLxxLxx, which has been reported
OsPGIP1, which lacks the seventh LRR (Jang et al. 2003; in rice (Jang et al. 2003). The signal peptides and glyco-
Janni et al. 2006). sylation sites were predicted in EXPASY (http://www.
The ability of PGIPs to inhibit the growth and coloni- expasy.ch/) by PSORT, NetOGlyc and NetNGlyc. Basic
zation of plant fungal pathogens has been confirmed by information about the OsPGIPs was collected from EXP-
transgenic plants. Over-expression of PGIPs in tomato ASY. The accession numbers of Ospgip1, Ospgip2, Osp-
(Powell et al. 2000), apple (Szankowski et al. 2003), per- gip3, Ospgip4, Osfor1, Ospgip6 and Ospgip7 are shown in
simmon (Tamura et al. 2004), grape (Aguero et al. 2005), Table 1.

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Table 1 Summary of the main attributes of rice Ospgips

Name Accession LOC ID KOME_cDNA No. Signal No. of Prediction pI No. of
number of LRR peptides cysteine of protein potential
residues subcellular glycosylation
localization sites

Ospgip1 AM180652 LOC_OS05g01380 No hits 9 1–17 8 Extracellular 6.6 4

Ospgip2 AM180653 LOC_OS05g01370 No hits 10 1–22 9 Extracellular 4.6 10
Ospgip3 AM180654 LOC_OS05g01430 No hits 10 1–21 9 Extracellular 5.7 7
Ospgip4 AM180655 LOC_OS05g01444 AK108676 10 1–25 9 Extracellular 7.8 5
Osfor1 AF466357 LOC_OS07g38130 AK101897/ 10 1–24 10 Extracellular 7.1 1
AK061685
Ospgip6 NM-001068720 LOC_OS08g39550 AK120908/ 10 1–30 10 Extracellular 5.9 1
AK120866/
AK060670
Ospgip7 AC108762 LOC_OS09g31450 No hits 10 1–31 11 Extracellular 5.9 3

Sequence analysis (secondary branch primordium differentiation stage)

(young panicle 1); (21) young panicle at stage 4 (pistil/
We collected sequences of PGIP proteins derived from stamen primordium differentiation stage) (young panicle
different species that have been previously reported. An 2); (22) young panicle at stage 5 (pollen-mother cell for-
unrooted phylogenetic tree was generated and displayed by mation stage) (young panicle 3); (23) leaf at 4–5 cm young
MEGA4 (Tamura et al. 2007) with the neighbor-joining panicle (leaf 2); (24) sheath at 4–5 cm young panicle
method (Saitou and Nei 1987) and bootstrap analysis (sheath 2); (25) panicle at 4–5 cm young panicle (panicle
(1,000 replicates). 2); (26) hull 1 day before flowering (hull); (27) stamen
1 day before flowering (stamen); (28) spikelet 3 days after
DNA microarray data for expression profile analysis pollination (spikelet); (29) endosperm 7 days after polli-
nation (endosperm 1); (30) endosperm 14 days after pol-
The expression profile data of Ospgip genes were extracted lination (endosperm 2); (31) endosperm 21 days after
from the CREP database (http://crep.ncpgr.cn). The pollination (endosperm 3). Furthermore, the data of the
microarray data was consistent with the real-time PCR three phytohormone treatments, gibberellic acid (GA3),
(RT-PCR) results from previous studies. (Chen et al. 2009; kinetin (KT) and naphthalene acetic acid (NAA) at the
Ma et al. 2009; Nayidu et al. 2008). The rice cultivar trefoil stage of seedlings were collected. Two biological
Minghui 63(indica rice) was used as the plant material for replicates were performed on all the tissues collected
DNA microarray analysis. The following 31 tissues from (except for tissues 1, 10, 20, 21 and 22, which had six
the entire growth period of rice were selected to perform biological replicates).
the expression profile analysis of the Ospgip genes in this
study: (1) embryo and radicle 3 days after sowing (embryo Expression profiling following treatments
and radicle); (2) calli 15 days after subculture (calli 1); (3) with phytohormones and fungal infection
calli screening stage (calli 2); (4) calli 5 days after regen-
eration (calli 3); (5) seed 72 h after imbibition (seed); (6) The plants of Minghui 63 (indica variety) and Zhonghua 11
plumule 48 h after emergence, in dark (plumule 1); (7) (japonica variety) were grown in greenhouse conditions at
radicle 48 h after emergence, in dark (radicle 1); (8) plu- 25–30°C and a photoperiod of 14 h light/10 h darkness. At
mule 48 h after emergence, in light (plumule 2); (9) radicle the trefoil stage, the roots of Minghui 63 plants were
48 h after emergence, in light (radicle 2); (10) seedling at immersed in nutrition solutions which contained gibberel-
trefoil stage (seedling); (11) shoot at seedling with two lic acid, kinetin and naphthalene acetic acid, respectively.
tillers (shoot); (12) root at seedling with two tillers (root); The final concentration of all the hormone solutions was
(13) stem 5 days before heading (stem 1); (14) flag leaf 0.1 mM. 3–5 plants were collected at each time point of 0,
5 days before heading (flag leaf 1); (15) stem at heading 0.5, 1, 2, 4 and 6 h after treatment for RNA isolation.
stage (stem 2); (16) panicle at heading stage (panicle 1); Besides GA3 and KT, five additional treatments (abscisic
(17) flag leaf 14 days after heading (flag leaf 2); (18) leaf at acid, brassinosteroid, 3-indole acetic acid, jasmonic acid
young panicle at stage 3 (leaf 1); (19) sheath at young and salicylic acid, each 0.1 mM) were performed on
panicle at stage 3 (sheath 1); (20) young panicle at stage 3 Zhonghua 11. Roots of Zhonghua 11 plants were immersed

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in the hormone solutions, and 3–5 plants were harvested at analyze the relative changes in the real-time PCR experi-
each time point of 0, 1, 3, 6 and 12 h after treatments for ments (Livak and Schmittgen 2001). The rice actin1 gene
RNA extraction. Zhonghua 11 were inoculated with Rhi- (NCBI accession number: X16280) was used as an
zoctonia solani WH-1 at the booting stage using the bio- endogenous control (McElroy et al. 1990). The primers
assay using detached leaves (BDL) method (Kumar et al. used for the real-time PCR experiments are shown in
2003), and 3–5 flag leaves were collected at each time Table 2.
point of 0, 6, 12, 24, 36, 48, 72 and 96 h after inoculation.
Analysis of cis-elements in the promoter region
Real-time PCR analysis of Ospgip genes in japonica rice cultivar

Total RNA was isolated from the treated seedlings or flag To analyze putative cis-elements in the promoter region of
leaves of Minghui 63 and Zhonghua 11 using TRIzol Ospgip genes, 1,000-bp DNA sequence up-stream the 50 end
reagent (Invitrogen, CA, USA). First strand cDNA was of the cDNAs was extracted from the rice genome annotation
synthesized using the Superscript III reverse transcriptase project database (http://rice.plantbiology.msu.edu/). The
(Invitrogen, CA, USA), according to the manufacturer’s sequences were subjected to the web software plantCARE
instructions. The gene-specific primers (Table 2) were (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)
designed using Primer Express Software version 2.0 (Lescot et al. 2002) to search for the putative cis-elements.
(Applied Biosystems) and Primer 3 (http://redb.ncpgr.cn/
modules/redbtools/primer3.php) with default parameters. Methods for data analysis
The primers were first used as queries to search in NCBI
database and the dissociation curves were analyzed to All the data generated from real-time PCR were analyzed
confirm the specificity in RT-PCR. Each real-time PCR using SPSS software version 16.0 with one-way ANOVA
reaction contained 12.5 ll of 29 SYBR Premix Ex TaqTM, method. The significance level of 0.05 has been indicated
0.5 ll of 509 ROX reference dye II (TaKaRa), 200 nM of with lowercases. A gene was defined as up- or down-reg-
each gene-specific primer and 5 ll cDNA samples in a ulated only if the relative expression level of the gene was
final volume of 25 ll. The thermal cycle was set as fol- more than two-fold and showed significant at 0.05 level
lows: 95°C for 10 s; 45 cycles of 95°C for 5 s, 60°C for compared to control.
34 s using ABI PRISM 7500 real-time PCR system
(Applied Biosystems, CA, USA). The real-time PCR
experiments were performed in triplicates for the two Results
biological replicates. The 2DDCt method was used to
Identification of Ospgip genes in rice
Table 2 Primers of the Ospgip genes and rice actin1 gene used in the
real-time PCR experiments Two new Ospgip genes, LOC_Os08g39550 and LOC_
Os09g31450, were found by screening three databases,
Primer name Primer sequence
TIGR, NCBI and KOME, using the reported Ospgip
Ospgip1RT-F 50 -tgcaggacttcaacgtcagcta-30 sequences as queries. The open reading frames (ORFs) of
Ospgip1RT-R 50 -cttgttgtggaagaaggagtactgatc-30 the LOC_Os08g39550 and LOC_Os09g31450 comprised
Ospgip2RT-F 50 -aacgtcagctacgacaagatgtg-30 1,143 and 1,029 bp encoding 380 and 342 amino acids,
Ospgip2RT-R 50 -cagaggcatttgttgtgttggta-30 respectively. Both new OsPGIP proteins had ten LRRs
Ospgip3RT-F 50 -ggccaacatgacggacatg-30 flanked by short N- and C-terminal regions as well as a
Ospgip3RT-R 50 -tggaagcagtaggcatcgaa-30 typical PGIP structure. Both new OsPGIP proteins con-
Ospgip4RT-F 50 -gcctactgcttccagcacaac-30 tained 30 and 31 amino acid signal peptides and were pre-
Ospgip4RT-R 50 -aggtctggatcaaacatcaatgg-30 dicted to be extracellular proteins by PSORT. In addition, 1
Osfor1RT-F 50 -gcgggtttacgttagtacaaattgtaa-30
and 3 N-linked glycosylation sites and 10 and 11 conserved
Osfor1RT-R 50 -ccctcgtttcgtacaatctgaag-30
cysteine residues were observed, respectively (Fig. 1).
Ospgip6RT-F 50 -caagtgacaaaagcataagaaatgc-30
According to the distribution of Ospgip genes on the rice
chromosomes, the two newly identified Ospgip genes were
Ospgip6RT-R 50 -acctttcaaattgggccaagt-30
named as Ospgip6 (LOC_Os08g39550) and Ospgip7
Ospgip7RT-F 50 -gatctacacagtgtgacaggcattg-30
(LOC_Os09g31450), and Ospgip1, 2, 3, 4 (Janni et al.
Ospgip7RT-R 50 -aagcagtcgacaagttcagatataaca-30
2006) and Osfor1 (Jang et al. 2003) were maintained with
actin1RT-F 50 -tggcatctctcagcacattcc-30
the previously given designation. Detailed information
actin1RT-R 50 -tgcacaatggatgggtcaga-30
about the seven OsPGIPs proteins are shown in Table 1.

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Fig. 2 Phylogenetic relationship among PGIPs from different plant

species. The unrooted tree was generated and displayed using MEGA
version 4.0. The species origin and accession numbers are as follows:
AdPGIP (Actinidia deliciosa, NCBI ID: CAA88846); AtPGIP1-2
(Arabidopsis thaliana, NCBI ID: AAF69827, AAF69828); BnPGIP1-
3, 5-17 (Brassica napus, NCBI ID: ABX46548–ABX46563); CmP-
Fig. 1 Protein structure of OsPGIP6 and OsPGIP7. A The signal GIP (Chamaebatiaria millefolium, NCBI ID: AAK43398); CsPGIP
peptides, B the N-terminal region, C the 10 LRR region, D the (Citrus sinensis, NCBI ID: CAA69910); EgPGIP (Eucalyptus gran-
C-terminal region. The cysteine residues are in bold italics. The dis, NCBI ID: AAF22248); GmPGIP1-4 (Glycine max, NCBI ID:
predicted glycosylation sites are underlined and in italics CAI99392–CAI99395); HaPGIP (Helianthus annuus, NCBI ID:
ABW89508); OsPGIP1-7(Oryza sativa, NCBI ID: CAJ55691,
CAJ55692, CAJ55693, CAJ55694, AAO17320, NP_001062185,
Phylogenetic analysis EEE69955); PaPGIP (Phaseolus acutifolius, NCBI ID: CAR92533);
PcPGIP (Pyrus communis, NCBI ID: AAA33865); PpPGIP (Pyrus
pyrifolia, NCBI ID: ACY56891); PvPGIP1-4 (Phaseolus vulgaris,
Fifty-one PGIP protein sequences from different dicot and NCBI ID: CAH10215–CAH10218); RsPGIP (Rhodotypos scandens,
monocot plants were collected. An unrooted phylogenetic NCBI ID: AAK43455); SbPGIP1-2 (Sorghum bicolor, NCBI ID:
tree was generated by using the alignments of the 51 PGIP XP_002463048, XP_002439097); SlPGIP (Solanum lycopersicum,
sequences (Fig. 2). The results showed that all the selected NCBI ID: AAA53547); SpPGIP (Solanum palustre, NCBI ID:
AAT77428); TaPGIP1-2 (Triticum aestivum, NCBI ID: CAJ55695,
PGIP proteins could be divided into five major groups CAJ55696); VvPGIP (Vitis vinifera, NCBI ID: AAM74142); ZmP-
(Groups A–E). Group (A) contained 13 members, including GIP1-2 (Zea mays, NCBI ID: NP_001147517, NP_001150670)
seven OsPGIP proteins, two TaPGIP proteins, two SbPGIP
proteins and two ZmPGIP proteins. All the plant species classified into any of the above groups and was the sole
belonged to the Gramineae among monocot plants. There member of Group (E). The results of the phylogenetic
were nine members in the Group (B), comprising four analysis demonstrate the conservation of the PGIP proteins
GmPGIP proteins, four PvPGIP proteins and one PaPGIP in the evolution of different plants (Janni et al. 2006).
protein, all of which were in the soybean and bean species.
Group (C) had ten members, including SpPGIP, SlPGIP, Expression profiles of the Ospgip gene family
CsPGIP, CmPGIP, RsPGIP, EgPGIP, PpPGIP, PcPGIP, in different tissues and organs of indica rice Minghui 63
VvPGIP and AdPGIP proteins, all of which were from fruit
or tree plants. Group (D) had 18 members, including 16 To define the expression pattern of the Ospgip genes in
BnPGIP proteins and 2 AtPGIP proteins, all of which were rice, the transcript abundance for all the Ospgip genes in 31
from crucifer plants. The HaPGIP protein could not be different tissues/organs were analyzed using the online

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microarray database. All the Ospgip genes had corre- in the ‘‘Materials and methods’’, and the duration of the
sponding probe sets in the DNA chip data except Ospgip3. treatment was extended to 6 h. In the GA3 treatment, the
Ospgip2 and Ospgip4 had two probe sets, and the one expression levels of all the Ospgip genes were up-regulated.
closer to the 30 end of the mRNA was chosen for analysis. The expression levels of Ospgip1, Osfor1 and Ospgip7
The average signal values for all six of the Ospgip genes started to increase at 0.5 or 1 h after treatment and then
are shown in Online Resource Table 1. slowly decreased. In contrast, the responses of Ospgip2, 3, 4
The results showed that the expression levels of Ospgip and 6 were slow but persistent. The maximum relative
genes in different tissues and organs were divergent expression values of these four genes were shown at 6 h
(Fig. 3). Osfor1 and Ospgip6 were expressed at signifi- (Fig. 4a). Under the KT treatment, Ospgip1, 3, 4, 6, 7 and
cantly higher level in all tissues, with the exception of Osfor1 were up-regulated, but Ospgip2 was down-regu-
much lower expression level in flag leaf at 14 days after lated. The highest expression level of Ospgip1 was detected
heading. Ospgip2 was expressed in root, sheath, and stem at 2 h after treatment and then decreased. In contrast, the
at 5 days before heading and leaf of 4–5 cm young panicle. expression of Ospgip3, 4, 6, 7 and Osfor1 gradually
Ospgip4 was predominantly expressed in calli and seed. increased or maintained the peak of up-regulation without
Transcript abundance for the Ospgip1 and 7 was relatively decrease in transcript abundance (Fig. 4b). Following NAA
low. The hierarchical clustering (Fig. 3) was performed treatment, the expression levels of Ospgip1, 2, 3, 4, 6 and
based on the average signal values of the genes using Osfor1 increased and the peak values of each appeared at
average linkage clustering and Euclidean distance method 6 h after treatments. The transcription level of Ospgip7 was
with the GENESIS software (Sturn et al. 2002). not significantly affected by the NAA treatment (Fig. 4c).
Data about the relative expression levels and the one-way
Expression profiles of the Ospgip gene family ANOVA results of all the seven genes are shown in Online
in response to phytohormone treatments at trefoil stage Resource Table 2. The results of our study were not com-
of indica rice Minghui 63 pletely consistent with the data from the microarrays, due to
the difference of the duration of treatments. Our results
To explore the roles of Ospgip gene family under various showed that most Ospgip genes began to respond to the
hormone (GA3, KT, NAA) treatments in indica rice phytohormones 0.5 or 1 h after treatments, while the sam-
Minghui 63, the online database was analyzed first. In the ples for microarray analyses were mixture samples col-
GA3, KT and NAA treatments of Minghui 63 within 1 h lected within 1 h (at 5, 15, 30 and 60 min after treatments).
(mixture samples collected at 5, 15, 30 and 60 min after
treatments), the expression of Ospgip1, 2, 4 and 7 were Ospgip gene family in japonica rice Zhonghua 11
undetectable. Compared to the untreated samples, no in response to phytohormone treatments
obvious expression level changes were observed in Osp-
gip6 (under GA3, KT and NAA treatments, t test P values As japonica rice Zhonghua 11 was the most frequently
were 0.47, 0.44 and 0.78, respectively). In all of the three used donor for transgenic research in the laboratory, the
treatments, Osfor1 was slightly down-regulated (following mRNA of Zhonghua 11 after various phytohormone
GA3, KT and NAA treatments, t test P values were 0.01, treatments (ABA, BR, GA3, IAA, JA, KT, SA) was ana-
0.00 and 0.01, respectively) (Table 3). lyzed by real-time PCR to investigate the expression profile
To confirm the results of microarray analysis, real-time of Ospgip gene family in response to phytohormone
PCR was performed. Minghui 63 was treated as described treatments (Fig. 5).

Fig. 3 Hierarchical clustering of expression of the Ospgip gene clustering and Euclidean distance methods were used to perform the
family in various tissues and organs throughout the entire life cycle of Hierarchical cluster. The color scale (representing average signal
rice cultivar Minghui 63 (Ospgip3 not included). Average linkage values) is shown below the figure

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MAS 5.0 call: ‘‘A’’ means undetectable; ‘‘P’’ means detectable; ‘‘M’’ means not sure. MAS 5.0 is an abbreviation for ‘‘Affymetrix Microarray Suite Version 5.0’’. It is a statistical algorithm for
Under ABA treatment, the expression of Ospgip1 was

MAS

call
5.0
down-regulated. Ospgip2, 4 and Osfor1 were up-regulated

A
and then repressed after the peak of up-regulation, while

determining the transcripts to be present or absent. The results generate detected P values and assign ‘‘present (detected)’’, ‘‘absent (undetected)’’ or ‘‘marginal’’ calls for transcripts
value ± standard Ospgip3 and 7 were moderately induced without much
Average signal

decreases in transcript abundance after the peak of up-

43 ± 12

50 ± 13

39 ± 27

57 ± 17
regulation. The expression level of Ospgip6 had no obvi-
Ospgip7

ous change (about 1.9-fold up-regulated) after ABA

error

treatment (Fig. 5a). The expression of most of the Ospgip

gene family members was induced in BR- (Fig. 5b) and
MAS

call
5.0

GA3-treated (Fig. 5c) samples, whereas Ospgip6 and 7

P

P
were not affected under GA3 treatment. The expression of
value ± standard

Ospgip1, 3, 4 and Osfor1 decreased after the peak of up-

Average signal

1,264 ± 122

1,448 ± 173

regulation, while Ospgip2, 6 and 7 were not apparently

1,079 ± 23

1,197 ± 56
down-regulated after reaching the peak or reached the
Ospgip6

maximum value until to the end of the course. In IAA

error

treatment, Ospgip1, 4, 6 and Osfor1 were up-regulated and

then decreased after the peak of up-regulation, while the
MAS

call
5.0

expression of Ospgip2, 3 and 7 gradually increased or

P

maintained the peak of up-regulation without decrease in

value ± standard

transcript abundance (Fig. 5d). Under JA treatment, Osp-

Average signal

2,550 ± 115

1,755 ± 126

1,716 ± 130

gip1, 6 and Osfor1 were down-regulated. Ospgip2 was

1,543 ± 2

induced at 6 h after treatment and then repressed. Ospgip4

Osfor1

and 7 were gradually up-regulated. No obvious expression

error

level changes (about 1.9-fold down-regulated) of Ospgip3

were observed (Fig. 5e). Following KT treatment, Ospgip2
MAS

call
5.0

and 3 were up-regulated and then decreased after the peak

A

of up-regulation. The expression of Ospgip1 and 4 were

value ± standard

up-regulated and reached the peak level at 6 and 12 h after

Average signal
Table 3 Microarry data of Ospgip genes in Minghui 63 treated with GA3, KT and NAA

treatment, respectively. Ospgip6 was slightly down-regu-

lated, while there was no apparent change in expression of
26 ± 14

35 ± 11

52 ± 45
Ospgip4

55 ± 0

Osfor1 and Ospgip7 over the entire time course (Fig. 5f).
error

Under SA treatment, the expression of Ospgip1, 2, 3, 4 and

Osfor1 were up-regulated with decreasing transcript
MAS

call

abundance after the peak of up-regulation except Ospgip2.

5.0

The expression of Ospgip6 and 7 were not sharply affected

value ± standard

by SA treatment (Fig. 5g). Data about the relative

Average signal

expression levels and the one-way ANOVA results of all

the seven genes are shown in Online Resource Table 3.
59 ± 20

23 ± 20

37 ± 31
Ospgip2

14 ± 8

By comparing the expression data with the phylogenetic

error

relationship of Ospgip genes in japonica rice (Online

Resource Figure), it was obvious that genes in different
MAS

call

groups exhibited completely different expression patterns

5.0

in response to different stress conditions. For example, the

value ± standard

newly identified Ospgip6 was not clustered with any other

Average signal

Ospgip genes in the phylogenetic tree, and our RT-PCR

results showed that it was down-regulated by JA and KT,
Ospgip1

11 ± 3

19 ± 9

5±1

5±0

slightly up-regulated by BR and IAA (just 2.2-fold), and

error

not induced by ABA, GA3 and SA, while the other Ospgip
genes were up-regulated by most of the treatments. More
(trefoil stage)
phytohormones

NAA (trefoil
GA3 (trefoil

notable, the expression patterns were relatively similar in

Treated with

Treated with

Treated with
KT (trefoil
treated with

Untreated

closely related genes clustered together in the phylogenetic

Seedling

stage)

stage)

stage)
Genes

tree, although expression divergence was observed in

response to different phytohormone treatments. For

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1180 Plant Cell Rep (2012) 31:1173–1187

Fig. 4 Relative expression

levels of Ospgip genes in
Minghui 63 responding to GA3,
KT and NAA treatments: a GA3
treatment; b KT treatment;
c NAA treatment. The samples
were seedlings at trefoil stage
and harvested 0, 0.5, 1, 2, 4 and
6 h after treatments. Error bars
represent the standard
deviations of data

example, Ospgip3 and 4 clustered together in the same after infection. Ospgip7 was slightly down-regulated by
group were both up-regulated by most of the treatments, fungal infection and belonged to Group (4). Data about the
but Ospgip3 was not in response to JA and its response to relative expression levels and the one-way ANOVA results
BR, KT and IAA was less significant and much slower than of all the seven genes are shown in Online Resource
Ospgip4. Table 4. The differences in the responses of Ospgip genes
to fungal infection suggests that they might play different
Ospgip gene family in japonica rice Zhonghua 11 roles in fungal resistance and their expression levels may be
in response to fungal infection regulated by different pathways.

It has been demonstrated that the expression of the pgip Putative cis-elements in the promoter regions of Ospgip
genes could be regulated not only by abiotic stress such as genes in japonica rice cultivar
various hormones, but also by biotic stress such as fungal
infection. To obtain information about responses of the To evaluate the different expression patterns and various
Ospgip gene family in rice to biotic stresses, real-time PCRs responses to stresses of Ospgip genes in japonica rice cul-
were carried out to detect the differences in their expression tivar, putative cis-acting regulatory elements in the pro-
abundance. The responses of Ospgip genes to the fungal moter sequences were analyzed (Table 4). The cis-element
infection could be classified into four major groups (Fig. 6). W-box (Rushton et al. 1996) involved in pathogen response
Group (1) contained Ospgip1 and Ospgip3; the peaks of the was identified in Ospgip1, 4, 7 and Osfor1. Cis-acting ele-
transcript levels of each appeared 48 or 60 h after infection ments ABRE (Shen et al. 1993; Straub et al. 1994) required
and then decreased slowly. There were three genes, Osp- for ABA response were present in the promoters of Osp-
gip2, Osfor1 and Ospgip6, in Group (2). The responses of gip2, 3, 6 and Osfor1. Two elements associated with GA3
these genes were quick and strong (except Ospgip6, which response: GARE (Sutliff et al. 1993) and p-box (Kim et al.
was quick but only about 1.5-fold up-regulated and then 1992) were detected only in Ospgip7 and Ospgip2, 3,
sharply down-regulated), but the responses were not con- respectively. Cis-element AuxRE (Nagao et al. 1993)
tinuous. Group (3) had only one member, Ospgip4. The involved in IAA response was only present in Ospgip7. In
response of Ospgip4 to fungal infection was slow but con- the promoter regions of Ospgip1, 3 and Osfor1, the GCC-
tinuous. The expression level of Ospgip4 started to increase box (Brown et al. 2003) associated with JA response could
at 24 h after infection and its peak value was shown at 96 h be detected. The TCA-element (Goldsbrough et al. 1993)

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Plant Cell Rep (2012) 31:1173–1187 1181

Fig. 5 Relative expression

levels of Ospgip genes in
Zhonghua 11 responding to
phytohormone treatments:
a ABA treatment; b BR
treatment; c GA3 treatment;
d IAA treatment; e JA
treatment; f KT treatment; g SA
treatment. The samples were
seedlings at trefoil stage and
harvested 0, 1, 3, 6 and 12 h
after treatments. Error bars
represent the standard
deviations of data

involved in SA response was identified in all the Ospgip Discussion

genes except Ospgip3 and 6. All the promoters of Ospgip
genes in japonica rice cultivar contain at least one cis-acting In this study, we identified two new Ospgip genes in the
regulatory element associated with pathogen or phytohor- rice genome: Ospgip6 (LOC_OS08g39550) and Ospgip7
mone response. (LOC_OS09g31450). The criteria that we applied in the

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1182 Plant Cell Rep (2012) 31:1173–1187

Fig. 6 Relative expression levels of Ospgip genes in Zhonghua 11 responding to fungal infection. The samples were flag leaves at booting stage
and collected 0, 6, 12, 24, 36, 48, 60, 72 and 96 h after fungal infection. Error bars represent the standard deviations of data

bioinformatic prediction for PGIP proteins are as follows: The differences between PGIP proteins and pgip genes
(1) ten imperfect LRR domains; (2) the signal peptide in monocot and dicot are as follows: (1) the calculated
located to the apoplast; (3) cysteine residues existing in the pI values of dicot PGIPs are much higher. Most of the
mature protein; and (4) potential glycosylation sites in the PGIP proteins in monocot have a pI value \8.0, while this
protein. The newly identified OsPGIP6 is in accordance value is [8.0 in dicot. (2) Some of the pgip genes in dicot
with all of these criteria. There are ten entire imperfect plants have introns. In this study, we found that there was
LRRs in the mature OsPGIP6 protein. The signal peptides one intron in some dicot pgip genes, for example, Atpgip1-
of the OsPGIP6 are 1–30, and extracellular localization of 2, Bnpgip1-17, Hapgip and Rspgip, whereas there were no
OsPGIP6 is predicted by PSORT. The number of cysteine introns in any of the monocot pgip genes. The difference in
residues in OsPGIP6 is ten, with a predicted glycosylation the existence of introns between monocot and dicot pgip
site. There are no differences between OsPGIP6 and genes may provide evidence for the genetic evolution of
OsPGIP7 in the typical characteristics, except the number monocot and dicot plants.
of the cysteine residues and glycosylation sites. The Expression levels of the pgip genes vary in different
number of predicted cysteine residues in OsPGIP7 is 11, tissues and change with the plant development. In dicoty-
and the number of glycosylation sites is 3. In a previous ledon Brassica napus (line DH12075), both Bnpgip1 and
study of OsPGIPs, the author did not consider the Bnpgip2 were strongly expressed in roots and open flowers
LOC_OS09g31450 gene as an Ospgip gene because of the but weakly in stems. Additionally, the transcription level of
11 cysteine residues it contains (Janni et al. 2006). For all Bnpgip1 was much higher than Bnpgip2 in the buds (Li
the OsPGIP proteins reported in rice, the cysteine residue et al. 2003). In strawberry, Fapgip was expressed at various
number was variable (from 8 to 10). For example, OsP- levels in leaves, flowers and fruit at different maturities and
GIP2, OsPGIP3 and OsPGIP4 contained nine cysteine the highest expression level was in the mature berries
residues, while the number of cysteine residues in OsP- (Mehli et al. 2004). In monocotyledon, wheat pgip genes
GIP1 and OsFOR1 were eight and ten, respectively (Jang (Tapigp1 and Tapgip2) were consistently expressed in the
et al. 2003; Janni et al. 2006). Therefore, we assumed that three tissues, spikes, leaves and roots (Janni et al. 2006). In
the number of cysteine residues is not fixed for defining this study, our results showed that expression profiles of the
new PGIPs. six Ospgip genes were variable in different organs and

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 Table 4 Putative cis-elements in the promoter regions of Ospgip genes
Plant Cell Rep (2012) 31:1173–1187

Category Cis-element Sequence Strand/position

Ospgip1 Ospgip2 Ospgip3 Ospgip4 Osfor1 Ospgip6 Ospgip7

Pathogen response W-boxes TTGACC ?540 and -737 ?751 -800 ?407 and -930
ABA response ABRE TACGGTC ?8 ?895
ABRE GCCGCGTGGC -344
ABRE TACGTG -925
ABRE GCAACGTGTC -478
ABRE CGCACGTGTC ?378
ABRE CACGTG ?145 and ?332
GA3 response GARE TAACAAA ?617 and -528 ?73
p-box CCTTTTG ?236 ?239
IAA response AuxRE TGTCTCAATAAG -640
JA response GCC-box GCCGCC -630 ?324 and -588 ?415
and -591 and -719
SA response TCA-element GAGAAGAATA ?377 and ?428
TCA-element CCATCTTTTT -258 -831
TCA-element TCAGAAGAGG ?487
TCA-element CAGAAAAGGA -573 and ?488 ?89
1183

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1184 Plant Cell Rep (2012) 31:1173–1187

developmental stages in indica rice Minghui 63. More would help to uncover the differences of cis-elements in
interestingly, we found that some Ospgip genes showed different cultivars, which will help to analyze the different
different expression patterns in different indica and responses between rice cultivars.
japonica rice cultivars. The transcription analysis of The expression of pgip genes could be regulated by
japonica rice (cv. Roma) pgip genes showed that Ospgip1 pathogen infection. In Arabidopsis, the transcripts of Atp-
and Ospgip4 were clearly expressed in roots, leaves and gip1 and Atpgip2 were accumulated after B. cinerea
flowering spikes (Janni et al. 2006). In our study, in indica infection (Ferrari et al. 2003). In B. napus, only Bnpgip1
rice Minghui 63, the expression of Ospgip1 and 4 were not was up-regulated by Sclerotinia sclerotiorum infection,
detectable in roots, leaves and flowering spikes. On the while the expression of Bnpgip2 was not affected (Li et al.
other hand, Ospgip4 was expressed in calli and seed, but 2003). In M. truncatula, the expression of Mtpgip1 was
not Ospgip1. The expression of Ospgip2 could only be induced at 6 h after infection with Colletotrichum trifolii
detected in roots of japonica rice cultivar Roma (Janni et al. and maintained the high expression levels until over 72 h.
2006), while Ospgip2 expressed not only in roots, but also In contrast, the transcripts of Mtpgip2 could only be
in stem, sheath and leaves in Minghui 63. In japonica rice detected at 24 h post-inoculation (Song and Nam 2005). In
Dongjin, Osfor1 was highly expressed in panicles and calli P. deltoides, the expression of Pdpgip2 was weak, but
and weakly expressed in the seedling roots and mature Pdpgip4 was highly activated by infection of Marssonina
stems; the expression of Osfor1 in seedling shoots, vege- brunnea (Cheng et al. 2008). In Oryza sativa, the responses
tative leaves and flag leaves were undetectable (Jang et al. of Ospgip genes to R. solani infection were various. For
2003). But in Minghui 63, Osfor1 expressed in all the example, the expression of Ospgip2 and Osfor1 was
tissues and organs except flag leaves. Due to lack of the induced to high level immediately after R. solani infection,
genomic sequence of indica rice Minghui 63, it is difficult but decreased in a short time. On the contrary, the high
to explain the results clearly, but the differences exist in the level expression of Ospgip1, Ospgip3 and Ospgip4 could
sequences or cis-elements of the promoter regions may be exist for a long period, but the responses of them to the
the answers to these problems. infection were not very expeditious. In order to maintain a
The expression levels of pgip genes could be regulated high induced expression level and quick response to R.
by different phytohormones. In Arabidopsis, Atpgip1 did solani infection, we could use constitutive promoters, such
not respond to SA or JA, whereas the expression of Atpgip2 as CaMV35s or actin1 promoters, to drive the expression
was only affected by JA (Ferrari et al. 2003). In B. napus, of Ospgip genes in rice. We think this would be helpful for
the transcripts of both Bnpgip1 and Bnpgip2 accumulated the transgenic breeding against rice R. solani.
when treated with JA, but neither responded to SA (Li et al. Our analysis of putative cis-elements in promoters of
2003). In Medicago truncatula, both Mtpgip1 and Mtpgip2 Ospgip genes in japonica rice cultivar further supplied
responded to JA, but neither could be induced by SA or possible clues for the differential expression patterns of
ABA (Song and Nam 2005). In Populus deltoides, both these seven genes. In this study, it showed that all the
Pdpgip2 and Pdpgip4 were induced by the treatments of Ospgip genes in japonica rice Zhonghua 11 could be
SA and JA (Cheng et al. 2008). In this study, the responses induced by pathogen infection or phytohormone treat-
of Ospgip genes to different treatments in both indica and ments, and their promoters contained at least one cis-acting
japonica rice cultivars were analyzed. Most of the Ospgip regulatory element associated with pathogen or phytohor-
genes could be induced by multiple treatments. The dif- mone response. Most of these results were in accordance
ferences of Ospgip genes response to treatments in indica with the different expression patterns of Ospgip genes. For
and japonica rice were also found. For example, under GA3 instance, Ospgip1, 4, Osfor1 and Ospgip7 with W-box
treatment, all the Ospgip genes in Minghui 63 were up- element were highly regulated after fungal infection.
regulated; however Ospgip6 and 7 in japonica rice Osfor1 containing ABRE, TCA and GCC-elements in its
Zhonghua 11 were not induced by GA3 treatment. Fol- promoter region was activated under ABA, SA and JA
lowing KT treatment, all the Ospgip genes in Minghui 63 treatments. However, a part of the putative cis-elements did
were up-regulated with the exception that Ospgip2 was not support the actual expression patterns for some genes.
down-regulated. In contrast, in Zhonghua 11, the expres- For example, cis-element ABRE had been found in Osp-
sion of Ospgip2 was up-regulated, Ospgip6 was down- gip6 promoter, but it did not response to ABA treatment.
regulated and Osfor1 and Ospgip7 did not respond to KT Cis-elements, GARE and TCA-element, were detected in
treatment. These results suggest that the expression of promoter of Ospgip7, while neither GA3 nor SA treatment
Ospgip genes may be regulated by different pathways in could induce the expression of this gene. The promoter
indica and japonica cultivars. With the rapid development region of Ospgip3 contained GCC-box, while JA treatment
of sequencing technology, enrichment of the genomic could not induce the expression of Ospgip3. These results
sequence of various indica and japonica rice cultivars might suggest that regulation of genes expression depended

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Plant Cell Rep (2012) 31:1173–1187 1185

on the existing of not only cis-elements in their promoter (Solanum palustre, NCBI ID: AAT77428); TaPGIP1-2
regions, but also transcription factors. In addition, we could (Triticum aestivum, NCBI ID: CAJ55695, CAJ55696);
not identify W-box in the promoters of Ospgip2, 3 and 6, VvPGIP (Vitis vinifera, NCBI ID: AAM74142); ZmPGIP1-
but transcripts of all the three genes were induced after 2 (Zea mays, NCBI ID: NP_001147517, NP_001150670).
fungal infection. This result was not surprising since there
might be some other new cis-elements involved in the Nucleotide accession numbers
pathogen response, which had not been identified yet, in
the promoters of these genes. Atpgip1-2 (Arabidopsis thaliana, NCBI ID: AF229249,
In this research on Ospgip genes, two new Ospgip genes AF229250); Bnpgip1-3, 5-17 (Brassica napus, NCBI ID:
were identified and it was the first time that expression EU142023-EU142038); Hapgip (Helianthus annuus, NCBI
patterns of all the Ospgip genes (except Ospgip3) in dif- ID: EU112834); Ospgip1-6 (Oryza sativa, NCBI ID:
ferent tissues/organs through the entire life cycle were AM180652–AM180655, NM_001066567, NM_001068
analyzed using online database. The responses of all the 720); Rspgip (Rhodotypos scandens, NCBI ID: AF196946);
Ospgips to pathogen infection and different hormone Sbpgip1-2 (Sorghum bicolor, NCBI ID: XM_002463003,
treatments were also studied by using real-time PCR. NM_001157198); Zmpgip1-2 (Zea mays, NCBI ID: NM_
Analysis of cis-elements in the promoter regions of Os- 001154045, NM_001157198).
pgips also supplied some possible clues for the various
responses to different treatments of Ospgip genes. All these
results were very useful for uncovering the biology func-
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