ORIGINAL PAPER

Over-expression of OSRIP18 increases drought and salttolerance in transgenic rice plants

Shu-Ye Jiang • Ritu Bhalla • Rengasamy Ramamoorthy •

Hong-Fen Luan • Prasanna Nori Venkatesh •

Minne Cai • Srinivasan Ramachandran

Received: 6 February 2011 / Accepted: 5 October 2011 / Published online: 26 October 2011

� Springer Science+Business Media B.V. 2011

Abstract Both drought and high salinity stresses are

major abiotic factors that limit the yield of agricultural

crops. Transgenic techniques have been regarded as

effective ways to improve crops in their tolerance to

these abiotic stresses. Functional characterization of

genes is the prerequisite to identify candidates for such

improvement. Here, we have investigated the biolog-

ical functions of an Oryza sativa Ribosome-inactivat-

ing protein gene 18 (OSRIP18) by ectopically

expressing this gene under the control of CaMV 35S

promoter in the rice genome. We have generated 11

independent transgenic rice plants and all of them

showed significantly increased tolerance to drought

and high salinity stresses. Global gene expression

changes by Microarray analysis showed that more than

100 probe sets were detected with up-regulated

expression abundance while signals from only three

probe sets were down-regulated after over-expression

of OSRIP18. Most of them were not regulated by

drought or high salinity stresses. Our data suggested

that the increased tolerance to these abiotic stresses

in transgenic plants might be due to up-regulation

of some stress-dependent/independent genes and

OSRIP18 may be potentially useful in further improv-

ing plant tolerance to various abiotic stresses by

over-expression.

Keywords Drought stress �Ectopic over-expression �High salinity stress � Ribosome-inactivating protein �Rice

Abbreviations

Mg Megnaporthe grisea

PEG Polyethylene glycol

qRT-PCR Quantitative real-time RT-PCR

RACE Rapid amplification of cDNA ends

RIP Ribosome-inactivating proteins

WT Wild type

Xoo Xanthomonas oryzae pv oryzae

Introduction

Crop plants are frequently exposed to various envi-

ronmental stresses such as heat, oxidative stress, and

Shu-Ye Jiang and Ritu Bhalla contributed equally to this work.

Electronic supplementary material The online version ofthis article (doi:10.1007/s11248-011-9568-9) containssupplementary material, which is available to authorized users.

S.-Y. Jiang � R. Bhalla � R. Ramamoorthy �H.-F. Luan � P. N. Venkatesh � M. Cai �S. Ramachandran (&)

Rice Functional Genomics Group, Temasek Life Sciences

Laboratory, 1 Research Link, The National University

of Singapore, Singapore 117604, Singapore

e-mail: sri@tll.org.sg

Present Address:R. Bhalla

Republic Polytechnic, 9 Woodlands Ave 9, Singapore

738964, Singapore

123

Transgenic Res (2012) 21:785–795

DOI 10.1007/s11248-011-9568-9

heavy metal toxicity and so on. Among them, drought

and high salinity are the major abiotic stresses

(Mahajan and Tuteja 2005). These stresses may

decrease average yields for most major crops by more

than 50% (Bray et al. 2000). Therefore, it is important

to develop stress tolerant crops to minimize these

losses to cope with the increasing food requirements.

Scientists and crop breeders have developed several

strategies to improve plant tolerance to various abiotic

stresses including traditional and biotechnology-based

breeding methods. Transgenic techniques have been

frequently employed to improve crop tolerance to

various abiotic stresses. An important step for trans-

genic improvement is to identify and characterize

candidate genes with the potential to further increase

the abiotic tolerance. Based on the tolerance mecha-

nisms to abiotic stresses, osmotic regulation should be

regarded as major elements to prevent plants from

stress damages. A large number of transgenic crops

have been produced with higher tolerance to various

abiotic stresses by increasing osmoprotectants such as

glycine-betaine, proline, sugar alcohols (mannitol,

trehalose, myo-inositol and sorbitol), and polyamines

as well as by regulating K?/Na? homeostasis (for

review, see Jiang and Ramachandran 2010). On the

other hand, a detoxification strategy, which could

effectively eliminate reactive oxygen species (ROS)

produced by abiotic stress, also can be used for

improving stress tolerance (Mittler 2002). In addition

to these, studies have shown that transcription factors

play very important roles in stress tolerance mecha-

nisms. Up to now, at least 10 types of transcription

factors have been involved in abiotic stress regula-

tions. Over-expression or loss-of-function of some

genes encoding some of transcription factors can

significantly increase tolerance to abiotic stresses

(Yamaguchi-Shinozaki and Shinozaki 2006). Apart

from these, other signaling transduction genes also

play important roles in stress responsive pathways and

also can be used for transgenic improvement. These

candidates may involve in receptor-coupled phospho-

relay, phosphoionositol-induced Ca2? changes, mito-

gen activated protein kinase (MAPK) cascade, and

transcriptional activation of stress responsive genes

(for review, see Jiang and Ramachandran 2010).

Ribosome-inactivating proteins (RIPs) are N-gly-

cosidases that specifically cleave nucleotide N–C

glycosidic bonds. The ability of N-glycosidase to

depurinate the sarcin/ricin (S/R) loop of the large

rRNA of prokaryotic and eukaryotic ribosomes is a

common enzymatic activity for all RIPs. Besides this,

other enzymatic activities of RIPs have also been

reported including nuclease and superoxide dismutase

(SOD) (for review, see Stirpe and Battelli 2006). On

the other hand, expression of many RIP genes was

regulated by various biotic stresses including various

viruses (Iglesias et al. 2005; Girbes et al. 1996), Fungi

(Vivanco et al. 1999; Wei et al. 2005; Xu et al. 2007)

and insects (Dowd et al. 1998; Gatehouse et al. 1990;

Kumar et al. 1993; Zhou et al. 2000). Thus, transgenic

techniques have been employed to improve plants in

their tolerance to biotic stresses by over-expressing

RIP genes. Currently, transgenic tobacco, tomato,

potato and rice plants have been generated by over-

expressing various foreign RIP genes and increased

tolerance to various biotic stresses including viruses

and fungal pathogens has been reported (Lodge et al.

1993; Jach et al. 1995; Maddaloni et al. 1997;

Zoubenko et al. 1997; Desmyter et al. 2003; Yuan

et al. 2002). However, limited protection against

infection by viruses or fungal pathogens was also

observed in transgenic plants expressing some RIP

genes (Kim et al. 1999; Bieri et al. 2000; Schaefer et al.

2005).

Besides biotic stresses, evidence showed that the

expression of many RIP genes were also regulated by

various abiotic stresses including drought/polyethyl-

ene glycol (PEG) (Bass et al. 2004; Wei et al. 2005;

Jiang et al. 2008), salinity (Rippmann et al. 1997;

Jiang et al. 2008), H2O2 (Iglesias et al. 2005) and heat

or osmotic stress (Stirpe et al. 1996). However,

transgenic analyses were focusing on biotic stress

resistance and no data showed the effect of RIP genes

on abiotic stresses. On the other hand, all RIP genes

used for transgenic analysis were foreign genes and no

data showed the biological effects by over-expressing

an endogenous RIP gene. Furthermore, although more

than 160 RIPs or their genes were purified or

identified, reported data were mainly focusing on the

analyses of enzymatic activities and/or expression

under various stresses (Girbes et al. 2004; Jiang et al.

2008). Little is known on phenotypic effects following

the ectopic expression of the genes. In this study, we

analyzed their biological functions by over-expressing

an endogenous tapetum-specific RIP gene in rice. Our

data showed that over-expression of this gene could

significantly increase the tolerance to both drought and

high salinity stresses in rice plants.

786 Transgenic Res (2012) 21:785–795

123

Materials and methods

Plant materials and growth conditions

under normal and stress treatments

We used rice (Oryza sativa) variety Nipponbare

(japonica rice) for all the experiments. Rice seeds

were germinated in water and were planted in green-

house under natural light and temperature conditions.

For drought treatment, 2-week old seedlings were

treated with 30% PEG to simulate water stress and

whole plants were collected at various time intervals

(0, 0.5, 1, 2, and 3 h), and frozen with liquid nitrogen

prior to total RNA preparation. For high salinity stress,

the similar staged seedlings were treated with 200 mM

NaCl solution. Samples for RNA extractions were

collected at 0, 2, 4, 8 and 16 h time intervals.

Isolation and ectopic expression analysis

of OSRIP18 as well as characterization

of transgenic plants

The full-length cDNA was isolated by RACE using

the Clontech RACE system following the manufac-

turer’s protocol. Primer sequences used for RACE

were designed based on the annotated cDNA

sequences listed in Supplemental Table S1. After

sequencing and verification, the amplified fragment

was cloned into pGEM-T Easy vector (Promega). The

full-length cDNA was subcloned into pCAMBIA1300

Ti-derived binary vector (CAMBIA, Canberra, Aus-

tralia; http://www.cambia.org.au) under the control of

Cauliflower mosaic virus 35S promoter (Fig. 1a). The

construct was transformed into the Nipponbare gen-

ome by the Agrobacterium-mediated method (Hiei

et al. 1994). Transgenic plants from T0 to T4 gener-

ations were used for phenotype characterization under

normal and stressed conditions to check if the phe-

notypes are stable or not from generation to genera-

tion. We got homozygous plants by segregation

analysis, which were used for all specific experiments.

All phenotype investigations including abiotic stress

treatments and seeding rate measurement were carried

out by three biological replicates.

Southern and northern blot analysis

Genomic DNA samples were prepared using leaves of

seedlings by the SDS method (Dellaporta et al. 1983).

After restriction enzyme digestions, 6 lg of DNA

was separated using 0.7% agarose gels and trans-

ferred onto nylon membranes for southern blot. For

northern blot, 30 lg of total RNAs from each tissue

were fractionated on 0.8% agarose gel containing

formaldehyde. Probes were synthesized by PCRs with

EcoR I Hind III

MIII UT3 UT64 UT66 UT71 WT UT3 UT64 UT66 UT71 WT MVIII

YL ML YP MP YR MR

A

C

B

Fig. 1 Molecular characterization of transgenic plants harbor-

ing the CAMV 35S-OSRIP18 construct. a A schematic

representation of the transgene expression cassette. NOSnopaline synthase gene terminator. b T-DNA copy number

analysis in the transgenic plants UT3, UT64, UT66 and UT71

shown by southern blot. DNA samples from WT and transgenic

plants were restricted by EcoRI and HandIII and then

transferred into nylon membrane for hybridization using

hygromycin probe. c Expression analysis of transgenic plants

shown by northern blot analysis. Total RNA samples from

various tissues were transferred into nylon membrane for

hybridization using OSRIP18 as a probe. YL young leaf, MLmature leaf, YP young panicle, MP mature panicle, YR young

root, MR mature root

Transgenic Res (2012) 21:785–795 787

123

DIG Probe Synthesis Kit (Roche), using the primers

listed in Supplementary Table S1. Southern and

northern hybridizations were carried out according

to the manufacturer’s instructions (Boehringer

Mannheim).

Quantitative real-time RT-PCR analysis

We used qRT-PCR analysis to revise the microarray

results. All steps including total RNA preparation,

cDNA synthesis, primer designing and cDNA real-

time PCR analysis were carried out according to Jiang

et al. (2007). All primer sequences were listed in the

Supplementary Table S1. Two biological duplicates

were carried out and technical triplicates for quanti-

tative assays for each of the duplicate were performed

for all qRT-PCR analysis.

Microarray hybridization and data analysis

Two-week-old WT and transgenic UT64 seedlings

grown under normal conditions and under drought and

salinity stresses (treated with 30% PEG for 1 h or

200 mM NaCl for 2 h, respectively) were used as

starting materials. Total RNA samples were prepared

using RNeasy Plant mini kit (Qiagen). The concen-

tration of total RNA was determined using NanoDrop

ND-1000 spectrophotometer. Only those RNA sam-

ples with an A260/A280 ratio of 1.9-2.1 were used for

microarray analysis. We used Affymetrix GeneChip

Rice Genome Arrays (Cat# 900599) for the analysis.

This array contained probe sets designed from

approximately 48,564 Japonica and 1,260 Indica

sequences and is estimated to represent around

46,000 rice genes. One-cycle target labeling, hybrid-

ization to arrays, washing, staining, and scanning were

carried out according to the manufacturer’s instruc-

tions (Affymetrix). Hybridization data were analyzed

using Affymetrix GeneChip Operating Software

(GCOS 1.4). We identified differentially expressed

genes using empirical criterion of more than twofold

change and significant student’s t test of P \ 0.05

based on two biological replicates. A set of differen-

tially expressed genes was selected for qRT-PCR

analyses to confirm their expression patterns using

gene-specific primers as listed in Supplementary

Table S1.

Results

Identification and characterization of OSRIP18

We have previously carried out a genome-wide survey

of the RIP domain family members in the rice genome

and have identified 31 RIP genes in the genome (Jiang

et al. 2008). Among them, OSRIP18 was expressed

only in young panicles and further investigation

showed that the gene was detected only in tapetum

layer (Jiang et al. 2008). The gene encodes a type 1

RIP with the locus name LOC_Os07g37090 from the

Michigan State University (MSU) rice genome anno-

tation database (previous TIGR rice genome annota-

tion database, now moved to MSU, http://rice.

plantbiology.msu.edu/). We have isolated this gene

by RACE (Rapid Amplification of cDNA Ends)

(Frohman et al. 1988) according to the annotated

coding sequence. The amplified products were

sequenced and the results showed that the gene con-

tains no intron with 56 bp 50-UTR and 84 bp 30-UTR

and its sequence has been attached in the Supple-

mentary Table S2. The gene encodes a protein with

298 amino acids and is a homolog of the previously

reported gene RA39 with accession number

AB053261 (Ding et al. 2002).

Molecular characterization of transgenic plants

ectopically expressing OSRIP18

To investigate the biological functions of the OSRIP18

gene, it was ectopically expressed under the control of

35S promoter. Totally, 33 transgenic plants were

generated with this construct. These plants were

integrated 1–4 copies of the construct by southern

blot analysis. Among them, 11 independent transgenic

plants contained single copy insertion of the T-DNA

and they were used for further investigation. The copy

number analysis by southern blot hybridization was

shown in Fig. 1b. To investigate if the integrated gene

was expressed or not, total RNA samples were isolated

from the transgenic line UT64 and were transferred

into nylon membrane for northern blot hybridization.

The blotting data showed that the gene was ectopically

expressed with high level in all six tissues tested

including young and mature leaves, young and mature

panicles, young and mature roots (Fig. 1c). Northern

blot analysis for other two transgenic lines UT66 and

UT71 showed that the integrated OSRIP18 were also

788 Transgenic Res (2012) 21:785–795

123

expressed in all tested tissues with differential tran-

script abundance (Supplementary Figure S1).

Ectopic expression of OSRIP18 increased

plant tolerance to water and high salinity

stress in Oryza sativa

Since this gene is tapetum-specifc, upon over-expres-

sion we expected the difference in male gametophyte

development and subsequently abnormal fertility. On

the contrary, our data showed that all 11 independent

transgenic plants with single copy of T-DNA insertion

were fertile and the seeding rates had no co-relation

with ectopic expression this gene. Some of the data

were shown in Fig. 2a. Although OSRIP18 showed no

significant difference in their expression levels under

stress treatments during seedling stage, a few other

RIPs were shown to be involved in various biotic and

abiotic stresses (Jiang et al. 2008). Therefore, these

transgenic plants were exposed to various stresses to

investigate their responses to these stresses by com-

pared with wild type (WT) plants. Our results showed

that transgenic plants exhibited no significant differ-

ences in response to both biotic stresses (Megnaporthe

grisea) and Xoo (Xanthomonas oryzae pv oryzae) as

well as to cold stress when compared with WT plants.

Besides these stresses, we also investigated the effect

of PEG on the transgenic plants. After treatment for

2 h under 30% PEG solution, transgenic plants still

showed normal leaf phenotype while WT plants

exhibited withered and curved leaves (Fig. 2b). The

result suggested that ectopic expression of this gene in

rice increased its tolerance to 30% PEG stress. We also

surveyed the effect of 200 mM NaCl treatment on the

transgenic rice plants. After 8 h of treatment, similar

results were observed (data not shown), suggesting a

Fig. 2 Phenotypic characterization of transgenic plants over-

expressing OSRIP18. a The seeding rates of transgenic lines

compared with WT. The seeding rate was determined using the

ratio of normal seeds among total florets in the panicles. The

symbol ‘‘*’’ indicates the highest seeding rate among all 11

transgenic lines, ‘‘**’’ indicates the lowest rate and ‘‘***’’

indicates the average seeding rate of these transgenic lines.

b Higher tolerance of transgenic plants (right) to 30% PEG

treatment for 2 h compared with WT plants (left). c Improved

tolerance of transgenic plants (right) to natural drought

conditions compared with WT plants. The similar stages of

WT and transgenic plants were subjected to natural drought

conditions. The photo was taken after no watering for 25 days.

Three biological replicates have been carried for all experiments

Transgenic Res (2012) 21:785–795 789

123

role for this gene in drought/high salinity stresses.

Interestingly, the increased tolerance to drought and

high salinity was also observed during reproductive

stage (Fig. 2c). Since we have carried out the northern

blot analysis for three lines UT64, UT66 and UT71

(Fig. 2b and Supplementary Figure S1), we further

investigated the effect of expression abundance on the

tolerance to both abiotic stresses. Our data show that

the over-expressed OSRIP18 gene exhibits different

expression abundance among different transgenic

lines. However, these lines showed the similar toler-

ance to both PEG and high salinity stresses. This may

be due to that the transcript abundance is high enough

to enhance the tolerance even in the lines with the

lower expression level. In fact, these lines with two or

more copies of the T-DNA insertions, which lines may

be with higher level of expression abundance, also

showed similar tolerance to both abiotic stresses. On

the other hand, our data also showed that the observed

phenotypes were stable in all tested generations. We

have not observed any significant difference in their

tolerance to abiotic stresses between homozygous and

heterozygous plants. However, homozygous plants

were still used for all specific experiments for better

operation.

Subsequently, we selected two independent trans-

genic plants UT-64 and UT-66 for further investiga-

tions since most of 11 independent transgenic plants

showed similar response to both PEG and NaCl

treatment during our preliminary investigation.

Firstly, 2-week old transgenic plants were submitted

to 30% PEG for 4 h and the treated plants were washed

thoroughly with water to get rid of the PEG and then

transferred to normal growth conditions. After

2 weeks, we found that transgenic plants grew better

and stronger (Fig. 3a). We investigated their survival

ratios after 2 weeks of growth. For WT plants, only

around 50% of plants can be survival while more than

90% of transgenic plants survived (Fig. 3b, left panel).

These data showed that their tolerance to drought

stress for both transgenic plants has been significantly

increased. Secondly, both UT-64 and UT-66 were

planted under 200 mM NaCl-containing soil for

4 days before transferred to normal growth conditions.

We observed that 94% of UT-64 and 89% of UT-66

plants survived in this high salinity stress compared to

WT plants (with a survival ratio of 57%; Fig. 3b, right

panel). These data confirmed that transgenic plants

showed significantly higher tolerance to high salinity

stress. Now we have got the fourth generation of

transgenic plants and they still showed better tolerance

to both PEG and NaCl treatments.

Global gene expression changes regulated

by over-expression of OSRIP18

To explore the possible roles of OSRIP18 in stress

signaling pathways, microarray analyses were carried

out. We have submitted four total RNA samples for

such analyses including these samples from WT

plants, 30% PEG-treated WT plants, 200 mM NaCl-

treated WT plants and transgenic plants. A sum-

mary of Microarray data analysis was presented in

* * ** *

0

20

40

60

80

100

WT UT-64 UT-66 WT UT-64 UT-66PEG High salinity

Surv

ival

rat

io (

%)

A

B

Fig. 3 Tolerance test to both PEG and high salinity stresses

shown by survival rates. a Higher resistance of transgenic plants

(down) compared with WT plants (up) to 30% PEG treatment

for 4 h followed by growing in normal conditions for 2 weeks.

b Bar charts showing the survival ratios of WT and transgenic

plants under drought and salt stresses. WT and transgenic lines

were treated with 30% PEG for 4 h and then transferred into

normal growth conditions. For high salinity stress, both WT and

transgenic lines were treated with 200 mM NaCl for 4 days

followed by normal growth conditions. After 2 weeks of growth

under normal conditions, the numbers of plants survived were

scored for calculation and the t test was performed on these

scores for statistical analysis. Three replicates were carried out

and around 100 two-week-old WT or transgenic plants in

replicates were used for this experiment. Both marks ‘‘*’’ and

‘‘**’’ indicate significant difference by t test at P value \0.05

and 0.01, respectively. Three biological replicates have been

carried for all experiments

790 Transgenic Res (2012) 21:785–795

123

Supplementary Table S3. Differentially expressed

probes/genes were identified according to the descrip-

tion in Methods. We have identified total of 735 and

723 probes with up-regulated expression signal under

the 30% PEG and 200 mM NaCl treatments, respec-

tively, when compared with un-treated WT plants

(Fig. 4a). Among them, expression abundance from

330 probes was up-regulated by both PEG and NaCl

treatments. However, only 129 probes were detected

with significantly increased expression signal in the

transgenic plants when compared with un-treated WT

plants (Fig. 4a).

Among 129 up-regulated genes (probe sets) after

ectopic expression of OSRIP18, 124 were not regu-

lated by PEG or high salinity stress (Supplementary

Table S4). The remaining five genes were up-

regulated by PEG or high salinity stress (Supplemen-

tary Table S4). These genes encoded various proteins

including transcription factors, protein kinases, auxin-

responsive proteins and son on. Some of these genes

might be candidates for drought/salinity stress

response although most of them were not regulated

by PEG/high salinity stress. For example, we have

identified three probe sets (Os.26569.1.S1_at, Os.4830.

1.S1_at and Os.52268.1.S1_at) (Supplementary Table

S4). Their corresponding genes encoded methyltrans-

ferase family proteins and were up-regulated by

OSRIP18. Previous reports showed that some members

of this family were involved in drought stress (Nar-

asimha Chary et al. 2002; Vincent et al. 2005). Other

examples are some F-box domain encoding genes with

probe sets Os.19525.1.S1_at, Os.20614.4.S1_x_at,

Os.23289.1.S1_at and Os.50659.1.S1_at (Supplemen-

tary Table S4). Previous reports showed that F-box

proteins played roles in drought tolerance through

abscisic acid signaling pathway (Zhang et al. 2008;

Koops et al. 2011). Thus, our data suggest that increased

tolerance may be also due to the up-regulation of these

genes following ectopic expression of this gene.

On the other hand, we have detected 475 and 452

probes with down-regulated expression abundance

under the PEG and NaCl stresses, respectively

(Fig. 4b). Among them, the expression abundance of

95 probes was down-regulated by both stresses.

However, only three probe sets were down-regulated

in the transgenic plants over-expressing OSRIP18

(Supplementary Table S4). They were all from genes

encoding membrane-related proteins. Since the gene

OSRIP18 encoded a protein which exhibited the RNA

N-glycosidase activity, biological synthesis of some

proteins may be inhibited by ectopic expression of this

gene. As a result, transcription of these genes may be

induced as a compensation of inhibited protein

synthesis. This may provide an explanation why the

transcript abundance was up-regulated for 129 probes

and only three probes showed reduced expression

abundance.

To confirm the results of GeneChip analysis, a set

of six genes up-regulated by OSRIP18 over-expres-

sion or PEG/high salinity stress were selected for

quantitative real-time RT-PCR (qRT-PCR) analyses.

These genes were listed in Fig. 5a and they encoded

expressed unknown proteins, hypothetical proteins,

potassium transporter and b-glucosidase precursor.

The results showed similar expression patterns signals

validating the microarray results (Fig. 5b). For exam-

ple, both microarray and qRT-PCR analyses showed

that four genes with cDNA accession No. AK071513,

Fig. 4 Summary of differentially expressed genes between WT

and transgenic plants under both PEG and high salinity stresses.

a Venn diagram showing the classification of genes induced by

30% PEG, 200 mM NaCl and/or over-expression of OSRIP18based on microarray analysis. b Venn diagram showing the

classification of genes suppressed by 30% PEG, 200 mM NaCl

and/or over-expression of OSRIP18 based on microarray

analysis

Transgenic Res (2012) 21:785–795 791

123

AK064111, AK070962 and AK108619 were all up-

regulated only by ectopic expression of OSRIP18; the

remaining two genes were also up-regulated by PEG

stress (AK241580) or by both PEG and NaCl

(AK102508).

Discussion

OSRIP18 might play a role in increasing tolerance

to both drought and high salinity

Based on our previous report, the expression of

OSRIP18 was tapetum-specific in WT plants (Jiang

et al. 2008). Its expression can not be induced by both

PEG and high salinity stresses during seedling stage

(Jiang et al. 2008). However, the up-regulated

expression by these stresses can be observed during

panicle development (Jiang et al. 2008). These results

suggested that the gene might play a role in increasing

tolerance to drought and high salinity during panicle

development but not in seedling stage. After over-

expression, active expression can be detected in all

tested developmental stages and as a result, the

transgenic plants exhibited higher tolerance to both

stresses in these stages.

The role of OSRIP18 in rice plants during panicle

development and its potential application

on improving crop tolerance to abiotic stresses

by over-expression

During the stage of reproductive development, plants

are more sensitive to various environmental stresses

Probe set cDNA accession No

Detected Signal Descriptions

WTCK WTPEG WTNaCl UT64

Os.10266.1.S1_at AK071513 16.75 20.75 26.05 1072.00** Expressed protein

Os.17158.1.S1_at AK241580 77.80 234.30* 87.80 203.90* Potassium transporter

Os.20851.1.A1_x_at AK064111 36.05 18.10 46.15 3379.75** Hypothetical protein

Os.32889.1.S1_at AK102508 80.75 209.40* 178.20* 1390.70** Expressed protein

Os.8442.1.S1_at AK070962 258.80 217.50 245.60 889.85** Beta-glucosidase precursor

OsAffx.22999.1.S1_at AK108619 672.50 331.80 454.70 12834.90** Expressed protein

0

100

200300

400

500

600700

800

900

AK064111

mR

NA

rel

ativ

e am

ount

0

1

2

3

4

5

6

AK070962

mR

NA

rel

ativ

e am

ount

050

100150200250300350400450500

AK071513

mR

NA

rel

ativ

e am

ount

0

0.5

1

1.5

2

2.5

3

3.5

4

AK102508

mR

NA

rel

ativ

e am

ount

05

101520253035404550

AK108619

mR

NA

rel

ativ

e am

ount

0

1

2

3

4

5

6

1 2 3 4

1 2 3 4

1 2 3 4

1 2 3 4 1 2 3 4

1 2 3 4

AK241580

mR

NA

rel

ativ

e am

ount

A

B

Fig. 5 Six up-regulated genes (probe sets) revealed by

microarray analysis and their qRT-PCR expression profiles.

a Average expression signals of 6 up-regulated probe sets, their

corresponding full-length cDNA and descriptions of their

encoded proteins. b qRT-PCR analysis of up-regulated genes

in WT and transgenic plants. WT control without treatment (1);

WT plants treated with 30% PEG for 1 h (2) and 200 mM NaCl

for 2 h (3) and transgenic plants without treatment (4). Both

marks ‘‘*’’ and ‘‘**’’ in a and b indicate significant difference by

t test at P value\0.05 and 0.01, respectively

792 Transgenic Res (2012) 21:785–795

123

including drought and high salinity (Reddy and Goss

1971; Saini 1997; Lauchli and Grattan 2007; Barnabas

et al. 2008). Some stress-related genes or quantitative

trait loci (QTL) have been identified during this stage

(Lanceras et al. 2004; Hu et al. 2006). The OSRIP18

gene exhibited panicle-specific expression by qRT-

PCR analysis and tapetum-specific expression by

analyzing the promoter-GUS transgenic plants (Jiang

et al. 2008). The transgenic plants over-expressing

OSRIP18 showed no obvious phenotype difference

during vegetative or reproductive development stages

under normal growth conditions. In addition, this gene

was induced by PEG and high salinity treatments

during panicle development (Jiang et al. 2008) and

over-expression plants showed increased tolerance to

both stresses (Figs. 2, 3). All these data suggest that

the gene may play a role as a member of natural

defense system against various environmental condi-

tions including drought and high salinity stresses.

Previous reports showed that many RIP genes were

up-regulated by both biotic and abiotic stresses and

over-expression of some RIP genes could improve

plants against virus infection (see ‘‘Introduction’’).

However, no data shows that RIP genes can be used for

improving plant tolerance to abiotic stresses. Our data

has clearly showed that OSRIP18 could be used for

further improving rice or other crop plants in their

tolerance to both drought and high salinity stresses and

demonstrated that rice plants have the genetic

potential to survive under higher salt or drought

stresses by over-expressing internal genes.

On the other hand, some ectopically expressed RIPs

have been demonstrated to be toxic to ribosomes of

some plants or their corresponding transgenic plants

exhibited low fertility in some species (Maddaloni

et al. 1997), which may retard its breeding application.

In several studies, inducible promoters were employed

when a RIP gene was over-expressed to improve the

tolerance to biotic stresses (Logemann et al. 1992;

Maddaloni et al. 1997). In this study, we have

analyzed the fertility of 11 independent transgenic

lines and no evidence showed that over-expression of

OSRIP18 in rice plants contributed to the abnormal

fertility even the endogenic OSRIP18 exhibited tape-

tum-specific expression. In fact, some reports also

showed indistinguishable phenotype in their fertility

after over-expression of a RIP gene (Lodge et al. 1993;

Bieri et al. 2000; Yuan et al. 2002; Desmyter et al.

2003).

The possible mechanisms behind the improved

abiotic tolerance by over-expressing OSRIP18

in rice plants

Ectopic expression of OSRIP18 increased plant toler-

ance to drought and high salt stresses. This may be due

to the re-organization of protein metabolism through

inhibiting protein synthesis by OSRIP18. Accumula-

tion of reactive oxygen species (ROS) is a result of

various environmental stresses (Foyer and Noctor

2005). The stress-induced ROS accumulation can be

reduced by a variety of enzymatic scavengers includ-

ing SOD. Therefore, plant stress tolerance may be

improved by higher levels of SOD in plants (Mittler

2002; Guo et al. 2003). RIPs have shown other enzyme

activities including SOD activity in addition to

N-glycosidase as described in the introduction. Thus,

the increased tolerance to drought and high salt by

over-expressing OSRIP18 may also be due to the

potential SOD activity of OSRIP18 although no data

showed the SOD activity of this protein.

Microarray analyses revealed 129 up- and 3 down-

regulated genes following over-expression of this gene

(Fig. 4 and Supplementary Table S4). Among the up-

regulated genes, most of them were not induced by

PEG/NaCl stress. However, they may still function in

stress response (See Results). On the other hand, 5

genes up-regulated by not only ectopic expression of

OSRIP18 but also PEG/NaCl stresses were of interest.

Two of these genes encoded expressed or hypothetical

protein and the other 3 genes with probe sets

Os.17158.1.S1_at, Os.46339.1.S1_at and Os.6024.1.

S1_at encoded potassium transporter 5, phosphatase

2c and calmodulin, respectively. Expression patterns

of potassium transporter gene with probe set

Os.17158.1.S1_at were also confirmed by qRT-PCR

analysis (Fig. 5). Potassium transporter, phosphatase

2c and calmodulin were shown to be involved in

salinity/drought stress signal pathways (Rubio et al.

1995; Meskiene et al. 1998; Perruc et al. 2004). All

these data suggested that the increased tolerance to

PEG/NaCl in transgenic plants might also due to up-

regulation of some stress-dependant/independent

genes.

Transgenic Res (2012) 21:785–795 793

123

References

Barnabas B, Jager K, Feher A (2008) The effect of drought and

heat stress on reproductive processes in cereals. Plant Cell

Environ 31:11–38

Bass HW, Krawetz JE, Obrian GR, Zinselmeier C, Habben JE,

Boston RS (2004) Maize ribosome-inactivating proteins

(RIPs) with distinct expression patterns have similar

requirements for proenzyme activation. J Exp Bot

55:2219–2233

Bieri S, Potrykus I, Futterer J (2000) Expression of active barley

seed ribosome-inactivating protein in transgenic wheat.

Theor Appl Genet 100:755–763

Bray EA, Bailey-Serres J, Weretilnyk E (2000) Responses to

abiotic stresses. In: Buchannan BB, Gruissem W, Jones RL

(eds) Biochemistry and molecular biology of plants.

American Society of Plant Biologists, Rockville,

pp 1158–1249

Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA mini-

preparation: version 2. Plant Mol Biol Rep 1:19–22

Desmyter S, Vandenbussche F, Hao Q, Proost P, Peumans WJ,

Van Damme EJ (2003) Type-1 ribosome-inactivating

protein from iris bulbs: a useful agronomic tool to engineer

virus resistance? Plant Mol Biol 51:567–576

Ding ZJ, Wu XH, Wang T (2002) The rice tapetum-specific

gene RA39 encodes a type I ribosome-inactivating protein.

Sex Plant Reprod 15:205–212

Dowd PF, Mehta AD, Boston RS (1998) Relative toxicity of the

maize endosperm ribosome-inactivating protein to insects.

J Agri Food Chem 46:3775–3779

Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant

signaling: a metabolic interface between stress perception

and physiological responses. Plant Cell 17:1866–1875

Frohman KB, Dush M, Marlin G (1988) Rapid amplification of

fulllength cDNAs from rare transcripts: amplification using

a single-specific oligonucleotide primers. Proc Natl Acad

Sci USA 85:8998–9002

Gatehouse A, Barbieri L, Stirpe F, Croy RRD (1990) Effects of

ribosome inactivating proteins on insect development—

differences between Lepidoptera and Coleoptera. Entomol

Exp Appl 54:43–51

Girbes T, de Torre C, Iglesias R, Ferreras JM, Mendez E (1996)

RIP for viruses. Nature 379:777–778

Girbes T, Ferreras JM, Arias FJ, Stripe F (2004) Description,

distribution, activity and phylogenetic relationship of

ribosome-inactivating proteins in plants, fungi and bacte-

ria. MiniRev Med Chem 4:467–482

Guo X, Ren Z, Zhao Y, Zhang H (2003) Over-expression of

SOD2 increases salt tolerance of Arabidopsis. Plant

Physiol 133:1873–1881

Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient

transformation of rice (Oryza sativa L) mediated by

Agrobacterium and sequence analysis of the boundaries of

the T-DNA. Plant J 6:271–282

Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L (2006)

Overexpressing a NAM, ATAF, and CUC (NAC) tran-

scription factor enhances drought resistance and salt tol-

erance in rice. Proc Natl Acad Sci USA 103:12987–12992

Iglesias R, Perez Y, de Torre C, Ferreras JM, Antolin P, Jimenez

P, Rojo MA, Mendez E, Girbes T (2005) Molecular

characterization and systemic induction of single-chain

ribosome-inactivating proteins (RIPs) in sugar beet (Betavulgaris) leaves. J Exp Bot 56:1675–1684

Jach G, Gornhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R,

Schell J, Maas C (1995) Enhanced quantitative resistance

against fungal disease by combinatorial expression of dif-

ferent barley antifungal proteins in transgenic tobacco.

Plant J 8:97–109

Jiang SY, Ramachandran S (2010) Improving crop tolerance to

abiotic stresses by plant biotechnology, chapter 15. In:

Kumar A, Roy S (eds) Applications of plant biotechnology:

in vitro propagation, plant transformations and secondary

metabolite production. I.K. International Publishing

House, New Delhi, pp 286–309

Jiang SY, Bachmann D, La H, Ma Z, Venkatesh PN, Rama-

moorthy R, Ramachandran S (2007) Ds insertion muta-

genesis as an efficient tool to produce diverse variations for

rice breeding. Plant Mol Biol 65:385–402

Jiang SY, Ramamoorthy R, Bhalla R, Luan HF, Venkatesh PN,

Cai M, Ramachandran S (2008) Genome-wide survey of

the RIP domain family in Oryza sativa and their expression

profiles under various abiotic and biotic stresses. Plant Mol

Biol 67:603–614

Kim JK, Duan X, Wu R, Seok SJ, Boston RS, Jang IC, Eun MY,

Nahm BH (1999) Molecular and genetic analysis of

transgenic rice plants expressing the ribosome inactivating

protein b-32 gene and the herbicide resistance bar gene.

Mol Breed 5:85–94

Koops P, Pelser S, Ignatz M, Klose C, Marrocco-Selden K,

Kretsch T (2011) EDL3 is an F-box protein involved in the

regulation of abscisic acid signalling in Arabidopsis tha-liana. J Exp Bot. doi:10.1093/jxb/err236

Kumar MA, Timm DE, Neet KE, Owen WG, Peumans WJ, Rao

AG (1993) Characterization of the lectin from the bulbs of

Eranthis hyemalis (winter aconite) as an inhibitor of pro-

tein synthesis. J Biol Chem 268:25176–25183

Lanceras JC, Pantuwan G, Jongdee B, Toojinda T (2004)

Quantitative trait loci associated with drought tolerance at

reproductive stage in rice. Plant Physiol 135:384–399

Lauchli A, Grattan SR (2007) Plant growth and development

under salinity stress. In: Jenks MA, Hasegawa P, Jain SM

(eds) Advances in molecular breeding toward drought and

salt tolerant crops. Springer, Netherlands, pp 1–32

Lodge JK, Kaniewski WK, Tumer NE (1993) Broad-spectrum

virus resistance in transgenic plants expressing pokeweed

antiviral protein. Proc Natl Acad Sci USA 90:7089–7093

Logemann J, Jach G, Tommerup H, Mundy J, Schell J (1992)

Expression of a barley ribosome-inactivating protein leads

to increased fungal protection in transgenic tobacco plants.

Nat Biotechnol 10:305–308

Maddaloni M, Forlani F, Balmas V, Donini G, Stasse L, Corazza

L, Motto M (1997) Tolerance to the fungal pathogen Rhi-zoctonia solani AG4 of transgenic tobacco expressing the

maize ribosome-inactivating protein b-32. Transgenic Res

6:393–402

Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses:

an overview. Arch Biochem Biophys 444:139–158

Meskiene I, Bogre L, Glaser W, Balog J, Brandstotter M,

Zwerger K, Ammerer G, Hirt H (1998) MP2C, a plant

protein phosphatase 2C, functions as a negative regulator

794 Transgenic Res (2012) 21:785–795

123

of mitogen-activated protein kinase pathways in yeast and

plants. Proc Natl Acad Sci USA 95:1938–1943

Mittler R (2002) Oxidative stress, antioxidants and stress tol-

erance. Trends Plant Sci 7:405–410

Narasimha Chary S, Bultema RL, Packard CE, Crowell DN

(2002) Prenylcysteine alpha-carboxyl methyltransferase

expression and function in Arabidopsis thaliana. Plant J

32:735–747

Perruc E, Charpenteau M, Ramirez BC, Jauneau A, Galaud JP,

Ranjeva R, Ranty B (2004) A novel calmodulin-binding

protein functions as a negative regulator of osmotic stress

tolerance in Arabidopsis thaliana seedlings. Plant J

38:410–420

Reddy PR, Goss JA (1971) Effect of salinity on pollen I. Pollen

viability as altered by increasing osmotic pressure with

NaCl, MgCl2, and CaCl2. Am J Bot 58:721–725

Rippmann JF, Michalowski CB, Nelson DE, Bohnert HJ (1997)

Induction of a ribosome-inactivating protein upon envi-

ronmental stress. Plant Mol Biol 35:701–709

Rubio F, Gassmann W, Schroeder JI (1995) Sodium-driven

potassium uptake by the plant potassium transporter HKT1

and mutations conferring salt tolerance. Science 270:

1660–1663

Saini HS (1997) Effects of water stress on male gametophyte

development in plants. Sex Plant Reprod 10:67–73

Schaefer SC, Gasic K, Cammue B, Broekaert W, van Damme

EJ, Peumans WJ, Korban SS (2005) Enhanced resistance to

early blight in transgenic tomato lines expressing heterol-

ogous plant defense genes. Planta 222:858–866

Stirpe F, Battelli MG (2006) Ribosome-inactivating proteins:

progress and problems. Cell Mol Life Sci 63:1850–1866

Stirpe F, Barbieri L, Gorini P, Valbonesi P, Bolognesi A, Polito

L (1996) Activities associated with the presence of ribo-

some-inactivating proteins increase in senescent and

stressed leaves. FEBS Lett 382:309–312

Vincent D, Lapierre C, Pollet B, Cornic G, Negroni L, Zivy M

(2005) Water deficits affect caffeate O-methyltransferase,

lignification, and related enzymes in maize leaves. A pro-

teomic investigation. Plant Physiol 137:949–960

Vivanco JM, Savary BJ, Flores HE (1999) Characterization of

two novel type I ribosome-inactivating proteins from the

storage roots of the Andean crop Mirabilis expansa. Plant

Physiol 119:1447–1456

Wei Q, Huang MX, Xu Y, Zhang XS, Chen F (2005) Expression

of a ribosome inactivating protein (curcin 2) in Jatrophacurcas is induced by stress. J Biosci 30:351–357

Xu J, Wang H, Fan J (2007) Expression of a ribosome-inacti-

vating protein gene in bitter melon is induced by Spha-erotheca fuliginea and abiotic stimuli. Biotechnol Lett

29:1605–1610

Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional

regulatory networks in cellular responses and tolerance to

dehydration and cold stresses. Ann Rev Plant Biol 57:

781–803

Yuan H, Ming X, Wang L, Hu P, An C, Chen Z (2002)

Expression of a gene encoding trichosanthin in transgenic

rice plants enhances resistance to fungus blast disease.

Plant Cell Rep 20:992–998

Zhang Y, Xu W, Li Z, Deng XW, Wu W, Xue Y (2008) F-box

protein DOR functions as a novel inhibitory factor for

abscisic acid-induced stomatal closure under drought stress

in Arabidopsis. Plant Physiol 148:2121–2133

Zhou X, Li XD, Yuan JZ, Tang ZH, Liu WY (2000) Toxicity of

cinnamomin—a new type II ribosome-inactivating protein

to bollworm and mosquito. Insect Biochem Mol Biol 30:

259–264

Zoubenko O, Uckun F, Hur Y, Chet I, Tumer N (1997) Plant

resistance to fungal infection induced by nontoxic poke-

weed antiviral protein mutants. Nat Biotechnol 15:

992–996

Transgenic Res (2012) 21:785–795 795

123