ARTICLE IN PRESS

Journal of Plant Physiology 163 (2006) 577—584

www.elsevier.de/jplph

Identification of genes induced upon water-deficit

stress in a drought-tolerant rice cultivar
Mayra Rodrı́guez, Eduardo Canales, Carlos J. Borroto, Elva Carmona,
Junior López, Merardo Pujol, Orlando Borrás-Hidalgo

Laboratory of Plant Functional Genomics, Head of the Plant Functional Genomic Department, Plant Division, Center
for Genetic Engineering and Biotechnology (C.I.G.B.), P.O. Box 6162, La Habana 10600, Cuba

Received 4 May 2005; accepted 5 July 2005

KEYWORDS Summary
cDNA-AFLP;
Among the abiotic stresses, the availability of water is the most important factor that
Drought stress;
limits the productive potential of higher plants. The identification of novel genes,
Gene expression;
determination of their expression patterns, and the understanding of their functions
Oryza sativa L.
in stress adaptation is essential to improve stress tolerance. Amplified fragment
length polymorphism analysis of cDNA was used to identify rice genes differentially
expressed in a tolerant rice variety upon water-deficit stress. In total, 103 transcript-
derived fragments corresponding to differentially induced genes were identified. The
results of the sequence comparison in BLAST database revealed that several
differentially expressed TDFs were significantly homologous to stress regulated
genes/proteins isolated from rice or other plant species. Most of the transcripts
identified here were genes related to metabolism, energy, protein biosynthesis, cell
defence, signal transduction, and transport. New genes involved in the response to
water-deficit stress in a tolerant rice variety are reported here.
& 2005 Elsevier GmbH. All rights reserved.

Abbreviation: AFLP; amplified fragment length polymorphism;
CDK; cyclin-dependent kinase; CDPKs; calcium-dependent pro-
tein kinases; GRP; glycine-rich protein; HSF; heat-shock tran-
scription factors; MAPKs; mitogen-activated protein kinases;
PTGRP; proline–threonine–glycine-rich protein; SRKs; S-locus
family of receptor protein kinases; TDFs; transcript-derived
fragments
Corresponding author. Tel.: +53 7 271 6022x3151;
fax: +53 7 33 1779.
E-mail address: orlando.borras@cigb.edu.cu
(O. Borrás-Hidalgo).
Introduction
Rice (Oryza sativa L. subsp. Indica) is the staple
food for more than half of the world’s population.
With limited water resources, future increases in
rice production will largely rely on rainfed produc-
tion. Upland rice, which relies strictly on rainfall as
a source of water, is often exposed to drought
stress and has developed drought-resistant traits
(Yadav et al., 1997). Abiotic stress is the primary

0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2005.07.005
 ARTICLE IN PRESS
578
cause of crop loss worldwide, reducing average
yields for most major crop plants by more than 50%.
In Cuba, 1 580 996 ha of soils are affected by
drought and desertification being 14% of total soil
(Naciones Unidas, 2000).
Among the abiotic factors that have shaped and
continue shaping plant evolution, water availability
is the most important (Bray et al., 2000). The
complex plant response to abiotic stress involves
many genes and biochemical–molecular mechan-
isms. Changes in gene expression are induced by a
complex of signal transduction events that have not
been clearly delineated. Various genes respond to
drought stress in several species, and functions of
their gene products have been predicted from
sequence homology with known proteins. Many
drought-inducible genes are also induced by salt
stress and low temperature, which suggests the
existence of similar mechanisms of stress responses
(Zhu, 2001).
Genes induced during drought-stress conditions
are thought to function not only in protecting cells
from water deficit by the production of important
metabolic proteins but also in the regulation of
genes for signal transduction in the drought-stress
response (Shinozaki et al., 2003). Thus, these gene
products are classified into three major groups. (1)
those that encode products that directly protect
plant cells against stresses such as heat stress
proteins or chaperones, LEA proteins, osmoprotec-
tants, antifreeze proteins, detoxification enzymes
and free-radical scavengers (Bray et al., 2000); (2)
those that are involved in signalling cascades and in
transcriptional control, such as mitogen-activated
protein kinases (MAPKs), calcium-dependent pro-
tein kinases (CDPKs) (Ludwig et al., 2004) and SOS
kinase (Zhu, 2001), phospholipases (Frank et al.,
2000), and transcriptional factors (Shinozaki and
Yamaguchi-Shinozaki, 2000); (3) those that are
involved in water and ion uptake and transport
such as aquaporins and ion transporters (Blumwald,
2000).
Stress-inducible genes have been used to im-
prove the stress tolerance of plants by gene
transfer. It is important to analyze the functions
of stress-inducible genes not only to understand the
molecular mechanisms of stress tolerance and the
responses of higher plants, but also to improve the
stress tolerance of crops by gene manipulation
(Seki et al., 2003).
Expression profiling has become an important
tool to investigate how an organism responds to
environmental changes. Sometimes these tran-
scriptional changes are successful adaptations
leading to tolerance while in other instances the
plant ultimately fails to adapt to the new environ-
M. Rodrı́guez et al.
ment and is labeled as sensitive to that condition.
Expression profiling can define both tolerant and
sensitive responses. These profiles of plant re-
sponse to environmental extremes are expected to
lead to regulators that will be useful in biotechno-
logical approaches to improve stress tolerance as
well as to new tools for studying regulatory genetic
circuitry (Hazen et al., 2003). cDNA-amplified
fragment length polymorphism (AFLP) is an effi-
cient, sensitive and reproducible technology that
offers several advantages over other methods of
gene expression analysis (Bachem et al., 1996).
The aim of this work is to identify genes that are
activated and associated with tolerance in rice
upon water-deficit stress. This is pursued by
isolation of differentially expressed transcription-
derived fragments that are induced upon water-
deficit stress in a tolerant rice variety, based on a
cDNA-AFLP approach.
Materials and methods
Plant materials and water-deficit treatment
Seeds from two varieties of rice (seed provided
by the Rice Researches Institute, Cuba) with
contrasting drought tolerance (O. sativa L. subsp.
Indica cv. ‘‘LC8866’’, tolerant and O. sativa L.
subsp. Indica cv. ‘‘IACuba-27’’, sensitive) were
germinated in 6-in pots containing normal soil
under controlled conditions in growth chambers at
23 1C. Seedlings were watered every day. Thirty-
day old seedlings from both varieties were drought
stressed by withholding water for a period of 3
weeks. Phenotypic analysis was developed at 1, 2
and 3 weeks after drought conditions.
RNA isolation and cDNA-AFLP
Leaf samples from ‘‘LC8866’’ variety were
harvested at 1, 2 and 3 weeks after drought
conditions. Also, leaf samples from unstressed
‘‘LC8866’’ variety were harvested at 1, 2 and 3
weeks. The samples were immediately frozen in
liquid nitrogen and stored at 70 1C for further RNA
extraction. Leaf samples from ‘‘LC8866’’ variety at
1, 2 and 3 weeks of drought conditions were pooled
before RNA extraction. A leaf samples pool from
unstressed ‘‘LC8866’’ variety at 1, 2 and 3 weeks
were used as control. Total RNA (10 mg) was
extracted using a commercially available kit (SV
total RNA Isolation System, Promega, Madison, WI)
according to the manufacturer’s instructions. The
integrity of the RNA was checked on a denaturing
 ARTICLE IN PRESS
Identification of rice stress tolerant genes
agarose gel. Finally, double-stranded cDNA was
synthesized (cDNA Synthesis System, Promega,
Madison, WI). cDNA prepared from ‘‘LC8866’’
variety under drought condition and unstressed
‘‘LC8866’’ variety were used in cDNA-AFLP analysis.
The cDNA-AFLP procedure described by Bachem et
al. (1996) was utilized with a few modifications.
The EcoRI/MseI enzyme combination was employed
and a total of 9 primer pairs (9 with EcoRI+1/MseI+1
combination and 9 with EcoRI+2/MseI+2 combina-
tions) were used to generate PCR amplification
products. The amplification products were sepa-
rated on a 6% polyacrylamide gel containing urea
(8.0 M) at 110 W until the bromophenol blue
reached the bottom of the gel. The cDNA bands
were stained with silver nitrate, according to the
protocol DNA Sequencing System kit (Promega,
Madison, USA).
Sequence determination of differentially
expressed TDFs
Differentially expressed (those that are ex-
pressed in the stressed tolerant variety but not in
unstressed tolerant variety) transcript-derived
fragment (TDFs) were marked, cut out and incu-
bated in 150 mL TE (10 mM Tris, pH 7.5, and 1 mM
EDTA, pH 8.0) overnight at 37 1C. Extracted target
bands were used as template for re-amplification
using PCR. The sequences were determined with an
automated sequencer (Perkin Elmer ABI PRISM Dye
Terminator Cycle sequencing kit and ABI Model 377
DNA sequencer) using an EcoRI+TA and MseI+GT
primer pair. Nucleotide sequences as well as
translated sequences were compared with nucleo-
tide and protein sequences of the GenBank non-
redundant databases and sequences of the
expressed sequence tag databases by using the
BLAST sequence alignment program (Altschul et al.,
1997).
RNA blot analysis
In a separate experiment, total RNA from
‘‘LC8866’’ variety (tolerant) and ‘‘IACuba-27’’
variety (sensitive) rice varieties at 0, 1, 2 and 3
weeks under drought conditions were size-fractio-
nated on a 1.2% agarose/0.4 M formaldehyde RNA
gel and transferred to Hybond N+ Nylon membrane
(Amersham-Pharmacia, Buckinghamshire, UK).
Probes were generated from PCR-amplified frag-
ments of selected TDFs with higher E value using
the ReadyPrime random primed DNA labeling kit
(Amersham-Pharmacia, Buckinghamshire, UK) with
[32P] (ICN Biomedicals, Irvine, CA, USA). Blots were
579
hybridized and washed according to standard
procedures (Sambrook et al., 1989).
Results
The IACuba-27 and LC8866 rice varieties are used
in the production and as parental into rice breeding
program in Cuba. The IACuba-27 is a short-cycle
variety with high yield and resistance to main
disease, but very sensitive to water-stress condi-
tion. This variety was obtained after mutagenesis
process of O. sativa L. subsp. Indica cv. ‘‘J104’’. On
the other hand, ‘‘LC8866’’ variety was introduced
from Vietnam and the origin is unknown. This
variety showed high tolerance to water-stress
condition in Cuban soils (Alvarez et al., 1999).
Phenotypic changes in the sensitive variety were
observed 2 weeks upon water-deficit stress. Plants
from sensitive variety were significantly affected
after water-deficit stress, while tolerant variety
remained green. However, stress symptoms were
observed after 3 weeks upon water-deficit stress in
both varieties. These symptoms were higher in the
sensitive variety (Fig. 1).
Rice genes whose expression was regulated by
drought stress were identified by a cDNA-AFLP
technique. cDNA-AFLP analysis can reveal altered
expression of any gene provided that it carries the
restriction sites that have been chosen for analysis.
Highly reproducible banding patterns were ob-
tained with the EcoRI+TA and Mse I+GT primer pair.
Using this primer pair, a total of 103 bands were
obtained to be differentially expressed in tolerant
variety (LC8866) upon water-deficit stress, com-
pared to unstressed ‘‘LC8866’’ variety. The origin of
these 103 TDFs was from leaf samples pool of
stressed tolerant variety at 1, 2 and 3 weeks under
drought conditions. In addition, all differentially
expressed TDFs were up-regulated and sequenced.
Following sequencing, the TDFs were organized
into several categories according to their putative
function. The majority of the TDFs were grouped
into defense/stress, signal transduction, transpor-
ters, metabolism, protein synthesis, energy and
unknown function categories (Fig. 2). The genes of
known function were sorted into the 6 primary
functional categories. The largest set of genes
(20%) was assigned to the defense/stress category,
while genes involved in transporter and protein
synthesis constituted the smallest groups, compris-
ing 12%, respectively. Genes with unknown function
constituted 4% of the TDFs collection.
The results of the sequence comparison in BLAST
database revealed that several differentially
 ARTICLE IN PRESS
580
Figure 1. Behavior of tolerant (LC8866) and sensitive (IACuba-27) rice varieties after 0 (A), 1 (B), 2 (C) and 3 (D) weeks
upon water-deficit stress.
Figure 2. Classification of identified TDFs according to
the putative function. All clones with scores below 10 5
for the E value were considered as significant and
included. Genes were grouped using the same functional
classification used for Arabidopsis thaliana MIPS (http://
www.mips.biochem.mpg.de).
expressed TDFs were significantly homologous to
stress regulated genes/proteins isolated from rice
or other plant species. TDFs showing homology to
rust resistance-like protein, glycine-rich protein
(GRP), calcium-dependent protein kinase, S-recep-
M. Rodrı́guez et al.
tor kinase, cyclin-dependent kinase (CDK) C,
carbamoyl phosphate synthase and ethylene-re-
sponsive protein kinase were found (Table 1).
Differential gene expression patterns were com-
pared between drought-stress tolerant variety and
drought-stress sensitive variety by RNA blot analy-
sis. RNA from sensitive variety for RNA-blot analysis
was only isolated at 0, 1 and 2 weeks after stress
condition, because the quality of the leaves was
not good after 3 weeks under stress. The analysis
was performed on a selection of most interesting
differentially expressed TDFs according to E value.
The RNA blot analysis indicated that expression
levels of the transcripts in the tolerant variety were
very high compared to the sensitive variety for
which transcript levels were either very low or
undetectable (Fig. 3). It is possible that lack of
hybridization reflects differences in sequence
between the two cultivars. For that reason, we
hybridized some of the probes to genomic DNA from
the sensitive cultivar and verified that the genes
were present and sequences conserved between
the two cultivars (data non shown).
The ‘‘GBAS12’’ transcript which exhibited se-
quence homology with rust resistance-like protein
was up-regulated in the first week under drought
 ARTICLE IN PRESS
Identification of rice stress tolerant genes 581

Table 1. Homology of transcription-derived fragment (TDF) sequences.

AFLP fragment Accession number Sequence homology/match E value

GBAS02 Q9LXP8 Hypothetical 26.4 kDa protein 7e 12

GBAS08 Q9HXK5 2-isopropylmalate synthase 7e 07
GBAS12 AAM0301 Rust resistance-like protein RP1-3 [Zea mays] 3e 31
GBAS14 BM929246 Rice root nitrate-induced subtracted library [Oryza sativa] 1e 05
GBAS19 P22198 Serine/threonine protein phosphatase [Zea mays] 4e 22
GBAS23 S20846 Glycine-rich protein [Zea mays] 2e 29
GBAS30 AAK38343.1 Seven transmembrane protein Mlo7 [Zea mays] 3e 12
GBAS36 BAA13232.1 Calcium-dependent protein kinase [Zea mays] 5e 34
GBAS37 T02053 S-receptor kinase (EC 2.7.1.-) KIK1 precursor [Zea mays] 1e 52
GBAS39 CAC85725.1 Putative carbamoyl phosphate synthase small subunit [Nicotiana tabacum] 3e 34
GBAS57 BG451891 Drought Medicago truncatula cDNA, mRNAsequence 4e 18
GBAS60 AB049715 Pisum sativum ssa-4 mRNA for putative senescence-associated protein 4e 07
GBAS77 BE363768 Water-stressed 1 (WS1) Sorghum bicolor cDNA, mRNA sequence. 5e 19
GBAS80 CAC51391.1 Cyclin dependent kinase C [Lycopersicon esculentum] 2e 76
GBAS81 AF096250.1 Ethylene-responsive protein kinase TCTR1 [Lycopersicon esculentum] 2e 23

Figure 3. RNA blot analysis for transcription-derived fragments isolated from tolerant rice variety upon water-deficit
stress. RNA blots were prepared using total RNA (10 mg each lane) isolated from tolerant variety (LC8866) or sensitive
variety (IACuba-27), after 0, 1, 2 and 3 weeks under stress, respectively. Ribosomal RNAs were stained with ethidium
bromide (EtdBr).

stress in tolerant variety and afterwards it appears
to be reduced. TDF that had sequence homology
with GRP (GBAS23) was activated after the second
week of drought treatment, while it was reduced at
the third week in tolerant variety. The ‘‘GBAS37’’
homologous to a precursor of S-receptor kinase and
‘‘GBAS80’’ with homology to CDK C showed the
same behavior in tolerant variety, respectively.
Meanwhile, ‘‘GBAS37’’ transcript was also up-
regulated in sensitive variety; however, the ex-
pression was not high. The ‘‘GBAS36’’ and
‘‘GBAS39’’ transcripts that showed homology to
CDPK and a carbamoyl phosphate synthase were
induced in the first week and remained expressed
 ARTICLE IN PRESS
582
during all time points evaluated in tolerant variety.
Finally, the homolog of the ethylene-responsive
protein kinase (GBAS81) was induced during 1 and 2
weeks upon water-deficit stress in tolerant variety
and repressed after 3 weeks, respectively. Never-
theless, this transcript was only expressed 2
weeks upon water-deficit stress in sensitive variety
(Fig. 3).
Discussion
When plants are subjected to drought stress, a
number of physiological responses have been
observed. Osmotic adjustment, as a process of
active accumulation of compatible osmolytes in
plant cells exposed to water deficit, may enable (1)
a continuation of leaf elongation, though at
reduced rates; (2) stomatal and photosynthetic
adjustments; (3) maintained root development and
soil moisture extraction; (4) delayed leaf senes-
cence; (5) better dry matter accumulation and
yield production for crops in stressful environments
(Boyer, 1982).
cDNA-AFLP analysis was used to identify differ-
entially expressed genes in tolerant variety upon
water-deficit stress. Genes induced by drought
stress has been allocated to at least 10 different
functional categories: metabolism; energy; tran-
scription; protein destination; transport; cell com-
munication/signal transduction; cell rescue,
defence, death and ageing; cellular organiza-
tion; cell growth, cell division and DNA syn-
thesis and cellular transport (Bray, 2002). Most
of the transcripts identified here were genes
related to metabolism, energy, protein biosynth-
esis, cell defence, signal transduction, and trans-
port (Fig. 2).
We found that ‘‘GBAS12’’ exhibited sequence
homology with rust resistance-like protein reveal-
ing that stress response is a complex regulatory
network of signals that allows plants to respond
optimally to their changing environment. However,
no report about the relation of this protein with
drought stress in plants has been found.
In addition, the ‘‘GBAS36’’ transcript showed
homology to CDPK. It has been reported that cold,
drought and salinity induce transient Ca2+ influx
into the cell cytoplasm. Channels responsible for
this Ca2+ influx represent one type of sensor for
these stress signals. CDPKs are implicated as
important sensors of Ca2+ influx in plants in
response to these stresses. CDPKs are encoded by
multigene families, and expression levels of these
genes are spatially and temporally controlled
M. Rodrı́guez et al.
throughout development. In addition, a subset of
CDPK genes responds to external stimuli. Biochem-
ical evidence supports the idea that CDPKs are
involved in signal transduction during stress condi-
tions. Furthermore, loss-of-function and gain-of-
function studies revealed that signaling pathways
leading to cold, salt, drought or pathogen resis-
tance are mediated by specific CDPK isoforms
(Ludwig et al., 2004).
On the other hand, ‘‘GBAS23’’ had sequence
homology with GRP. Similar GRP from normalized
library of drought-stressed rice seedlings was
reported in O. sativa (Reddy et al., 2002). GRPs
containing more than 60% glycine have been found
in different tissues from many eukaryotic species.
Some of these proteins are components of the cell
walls of many higher plants. In most cases, it has
been shown that they are accumulated in the
vascular tissues and that their synthesis is part of
the plant’s defence mechanism. Other distinct
types of GRPs are characterized by having struc-
tures and functions similar to animal cytokeratins
or by a domain with typical RNA-binding motifs
(Mousavi and Hotta, 2005). Report on wild tomato
species (Lycopersicon chilense) revealed that a
proline-, threonine-, and GRP is down-regulated by
drought stress. The level of the mRNA in leaves and
stems of 8-d drought-stressed plants decreased 5-
to 10-fold compared with that in regularly watered
plants. Proline–threonine–glycine-rich protein
(PTGRP) was the first drought-regulated protein
that has been precisely localized in the cell wall
(Harrak et al., 1999). In our research, the RNA-blot
analysis showed that ‘‘GBAS23’’ appeared to be up-
regulated at the second week of drought stress;
however, at the third week a very weak hybridiza-
tion signal was observed. This fact is pointing out
that its expression was suppressed after 2 weeks.
Currently, the role of GRPs in abotic stress in plants
is still unknown.
According to the sequence analysis, ‘‘GBAS37’’
seems to be related to the S-locus family of
receptor protein kinases (SRKs) because its homol-
ogy to a precursor of S-receptor kinase. A similar
finding was recently reported by Bassett et al.
(2005) who isolated a gene induced by water-deficit
treatment from peach.
The carbamoyl phosphate synthase has been
reported in mammalian as a part of carbamoyl-
phosphate synthetase–aspartate carbamoyltrans-
ferase-dihydroorotase, a multienzymatic protein
required for the de novo synthesis of pyrimidine
nucleotides and cell growth carbamoylphosphate is
a common intermediate in the metabolic pathways
leading to the biosynthesis of arginine and pyrimi-
dines (Hewagama et al., 1999). Studies carried out
 ARTICLE IN PRESS
Identification of rice stress tolerant genes
by Graves et al. (2000) indicated its direct link
between activation of the MAPK cascade and de
novo biosynthesis of pyrimidine nucleotides. MAPKs
play a key role in plant responses to stress and
pathogens. In the last few years, MAPK cascades
have been proposed as major pathways for signal
transduction into intracellular response. MAPKs
identified from different plant species are induced
in response to wounding, pathogen attack, drought,
temperature and osmotic stress (Kumar et al.,
2003). It should be noted that ‘‘GBAS39’’ had a very
strong hybridization signal during the water stress
treatment and it showed homology to carbamoyl
phosphate synthase. We could suggest that carba-
moyl phosphate synthase play an important role
during drought stress in rice.
Additionally, ‘‘GBAS80’’ transcript showed
homology to CDK. CDKs control cell cycle progres-
sion through timely coordinated phosphorylation
events (Vanstraelen et al., 2004). In human cells, as
well as in Arabidopsis, phosphorylation of heat-
shock transcription factors (HSFs) through various
kinases may integrate growth signals. As yet, it is
unknown whether CDKs are involved in HSF phos-
phorylation in animals or whether MAPKs play a role
in HSF regulation in plants (Schöffl et al., 1998).
Stress gene induction occurs primarily at the
level of transcription, and regulating the temporal
and spatial expression patterns of specific stress
genes is an important part of the plant stress
response (Karam et al., 2002). Induction of gene
expression does not necessarily imply that a gene
will play an adaptive role. Depending upon the
conditions to which the plant was subjected, some
of genes that are expressed may indicate that the
plant has been subjected to a secondary stress.
It is uncertainly whether these genes are induced
directly by cellular water deficit or by resulting
secondary stress. Techniques such as silencing or
over-expression of certain signaling components
may confirm their role in particular pathways. In
conclusion, further characterization and functional
analysis of the genes that are identified in this
study can lead to a more comprehensive under-
standing of stress tolerance to drought in rice.
Acknowledgments
We are grateful to Rice Researches Institute,
Cuba for providing the seeds for the experiments.
Also, we are grateful to MACROGENE for sequencing
service. The work was supported by the Cuban
State Council.
583
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