Identification of a set of genes from genotypes of common bean tolerant and susceptible to water stress for a macroarray-based selection strategy

Abstract

Globally, drought is the main factor that reduces common bean yield. For this reason, breeding alternatives, such as molecular marker-assisted selection, that focus on various functional genes directly involved in the response to water stress, such as those encoding late embryogenesis abundant (LEA), early response to dehydration (ERD), and dehydrin proteins, have been implemented. The aim of this study was to identify differentially expressed genes of Phaseolus vulgaris in drought-tolerant cultivars Pinto Saltillo (PS) and Pinto Villa (PV), and drought-susceptible cultivars Bayo Madero (BM) and Canario 60 (C60) in vegetative and reproductive stages. Relative water content (RWC) in leaf tissue was measured. Twenty-eight P. vulgaris genes obtained from GenBank and from a subtractive suppressive library from the PS cultivar were analysed, and their expression profiles were examined by reverse transcription polymerase chain reaction (RT-PCR). Then, cDNA arrays were developed and hybridised to confirm expression which was finally validated by quantitative PCR (qPCR). The usefulness of the identified genes as selection criteria for the tolerance of different genotypes to drought was examined using cDNA arrays. Expression of 21 genes was induced by drought. The cDNA arrays confirmed that expression of 19 of these genes increased in the vegetative stage upon exposure to the drought, and a higher expression was observed in the reproductive stage compared with vegetative stage V4. Only five genes induced by the drought were found to have a lower expression in the susceptible cultivars compared with the tolerant ones. During recovery after the drought in the reproductive stage, 13 of the 21 induced genes remained transcriptionally active including LEA3 and dehydrin. The RWC during the drought in vegetative stage V4 decreased by about 55 % in all cultivars, but at the onset of flowering, it increased to 80 % in PV and PS. In contrast, in the susceptible cultivars, it remained at 55 %. Using qPCR validation, expression induction was confirmed in the drought-tolerant cultivars. Polyubiquitin2, LEA3, LEA4, and dehydrin were useful genes for selecting drought-tolerant genotypes under field conditions. Additional key words: dehydrin, expression patterns, LEA, qPCR, relative water content.

Full text

BIOLOGIA PLANTARUM 60 (3): 496-504, 2016 DOI: 10.1007/s10535-016-0587-5
496
Identification of a set of genes from genotypes of common bean tolerant
and susceptible to water stress for a macroarray-based selection strategy
G.M. GUTIERREZ-BENICIO1, J.G. RAMIREZ-PIMENTEL1, J.A. ACOSTA-GALLEGOS2,
C.L. AGUIRRE-MANCILLA1, J.C. RAYA-PEREZ1, A.P. RODRIGUEZ-VERA2,
and V. MONTERO-TAVERA2*
Tecnológico Nacional de México, Instituto Tecnológico de Roque. Celaya, 38110, México1
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Celaya, 38110, México2
Abstract
Globally, drought is the main factor that reduces common bean yield. For this reason, breeding alternatives, such as
molecular marker-assisted selection, that focus on various functional genes directly involved in the response to water
stress, such as those encoding late embryogenesis abundant (LEA), early response to dehydration (ERD), and dehydrin
proteins, have been implemented. The aim of this study was to identify differentially expressed genes of Phaseolus
vulgaris in drought-tolerant cultivars Pinto Saltillo (PS) and Pinto Villa (PV), and drought-susceptible cultivars Bayo
Madero (BM) and Canario 60 (C60) in vegetative and reproductive stages. Relative water content (RWC) in leaf tissue
was measured. Twenty-eight P. vulgaris genes obtained from GenBank and from a subtractive suppressive library from
the PS cultivar were analysed, and their expression profiles were examined by reverse transcription polymerase chain
reaction (RT-PCR). Then, cDNA arrays were developed and hybridised to confirm expression which was finally
validated by quantitative PCR (qPCR). The usefulness of the identified genes as selection criteria for the tolerance of
different genotypes to drought was examined using cDNA arrays. Expression of 21 genes was induced by drought. The
cDNA arrays confirmed that expression of 19 of these genes increased in the vegetative stage upon exposure to the
drought, and a higher expression was observed in the reproductive stage compared with vegetative stage V4. Only five
genes induced by the drought were found to have a lower expression in the susceptible cultivars compared with the
tolerant ones. During recovery after the drought in the reproductive stage, 13 of the 21 induced genes remained
transcriptionally active including LEA3 and dehydrin. The RWC during the drought in vegetative stage V4 decreased by
about 55 % in all cultivars, but at the onset of flowering, it increased to 80 % in PV and PS. In contrast, in the
susceptible cultivars, it remained at 55 %. Using qPCR validation, expression induction was confirmed in the drought-
tolerant cultivars. Polyubiquitin2, LEA3, LEA4, and dehydrin were useful genes for selecting drought-tolerant genotypes
under field conditions.
Additional key words: dehydrin, expression patterns, LEA, qPCR, relative water content.
Introduction
Common bean (Phaseolus vulgaris L.) is one of the
important crops because of its nutritional and
nutraceutical qualities (Guzman-Maldonado et al. 2002)
but a yield is severely affected by drought (Moreno-
Garcia et al. 2012). Drought is a multidimensional stress
that affects plants at various levels of organization
(Acosta et al. 1999). A water deficit during the
reproductive phase decreases bean yield more drastically
than a water deficit during the vegetative phase (Acosta-
Diaz et al. 2004, Aguilar-Benitez et al. 2012). Traditional
breeding has been applied to increase bean production
under drought stress (Ayele 1994, Kornegay et al. 1997)
leading to development of common bean cultivars
tolerant to drought, including Pinto Villa (PV) and Pinto
Saltillo (PS) obtained by Acosta et al. (1999) and
Sánchez-Valdez et al. (2004), respectively. However,

Submitted 24 May 2015, last revision 4 October 2015, accepted 5 November 2015.
Abbreviations: BCIP - 5-bromo-4-chloro-3-indolyl phosphate p-toluidine; CAB proteins - chlorophyll a and b binding proteins;
ERD - early response to dehydration; GMFL - Glycine max full-length; LEA - late embryogenesis abundant; MIPS - myo-inositol-1-
phosphate synthase; NCED - 9-cis-epoxycarotenoid dioxygenase; RT-qPCR - reverse transcription - quantitative polymerase chain
reaction; RWC - relative water content; ZEP - zeaxanthin epoxidase.
Acknowledgements: This research was supported by the INIFAP-SAGARPA 1225619346 project as well as CONACYT and
CONCYTEG fellowships to G.M. Gutierrez-Benicio.
* Corresponding author; fax: (+52) 4616115323; e-mail: montero.victor@inifap.gob.mx

MACROARRAY-BASED SELECTION STRATEGY
497
traditional breeding is a long process, and genotype-
environment interactions make rapid advances difficult
(Rosas et al. 2003). An alternative method is molecular
marker-assisted selection which allows identification of
genotypes that are tolerant to water deficit in the early
stages of plant development (Tanksley et al. 1989). These
molecular markers include expression markers which
provide information on the functions of various genes
under different stresses, environmental conditions, or
upon different cellular events. Overexpression of several
genes in response to water deficit has been observed in a
variety of plant species. In Arabidopsis thaliana and
P. vulgaris, 9-cis-epoxycarotenoid dioxygenase (NCED)
gene induction has been observed (Melhorn et al. 2008),
and the zeaxanthin epoxidase (ZEP) gene has been
identified in Nicotiana plumbaginifolia, Arabidopsis
thaliana, and Solanum lycopersicum (Bittner et al. 2001).
These genes are related to synthesis of abscisic acid, a
growth regulator that mediates a response to drought
stress. A group of induced genes encoding late
embryogenesis abundant (LEA) proteins, which protect
membranes from damage by dehydration, have been
studied during osmotic stress (Colmenero-Flores et al.
1999). Another pair of genes, RD29A and RD29B, have
mainly been studied in Arabidopsis thaliana and are
related to synthesis of transcription factors (Liu et al.
1998, Nakashima et al. 2000). In the bean cv. PV,
overexpression of 28 genes, associated with drought
tolerance, that encode LEA proteins, transcription factors,
photosynthesis-related proteins, and protein stabilization-
related proteins has been reported (Barrera-Figueroa et al.
2007). In vascular tissue of cv. PS, three genes that
encode proteins related to mobilization signals in phloem
and to the integrity of the vascular tissue under water
stress have been identified (Montero-Tavera et al. 2008).
The aim of this study was to identify differentially
expressed genes in drought-tolerant cvs. PS and PV and
drought-susceptible cvs. Bayo Madero (BM) and Canario
60 (C60) under water stress in vegetative and
reproductive stages.
Materials and methods
Bean (Phaseolus vulgaris L.) seeds from two drought-
tolerant cultivars PV and PS, and two susceptible
cultivars BM and C60 were germinated on moistened
filter paper in a growth chamber (ConvironTM) at 27 °C,
and after 5 d, the seedlings were transplanted into
Sunshine® substrate mix No. 3 (SunGro®, USA) and
transferred to a greenhouse (a 12-h photoperiod, an
irradiance of 570 mol m-2 s-1, day/night temperatures of
28/15 °C and an air humidity of 50 - 64 %). For each
bean genotype, 4 groups of 10 plants were established. A
control group was irrigated throughout the entire plant
life cycle. In the second group of plants, irrigation was
suspended for 5 d during vegetative stage V4 (White
1988), and in the third group of plants, irrigation was
suspended for 5 d at the onset of flowering (the
reproductive stage). In the fourth group of plants,
irrigation was suspended for 5 d twice during V4 stage
and the onset of flowering. On the fifth day of water
suspension, the plants showed wilting foliage and mostly
folded leaves. After that, the plants were irrigated and
allowed to recover for 10 d. Leaves were collected after
the water stress and after the recovery and stored at
-80 °C for further processing.
Relative water content (RWC) of leaves was
determined as described in Woo et al. (2008). Leaf tissue
samples (15 mm in diameter) were collected for each
treatment, and fresh mass (FM) was recorded. Then, the
samples were saturated with water at room temperature
for 18 h, and saturated mass (SM) was recorded. Finally,
the samples were dried in an oven at 80 °C for 24 h, and
dry mass (DM) was determined. To calculate the RWC,
the following formula was used:
RWC [%] = [(FM - DM)/(SM - DM)]  100.
The total RNA was extracted from leaf tissue as
described by Logemann et al. (1987). The RNA
concentration was standardised to 0.4 g dm-3, and
mixtures that included material from 10 plants for each of
the experimental conditions were examined. The quality
of RNA was assessed on a 1.5 % (m/v) agarose gel under
denaturing conditions. A cDNA was synthesised by real-
time polymerase chain reaction (PCR), and each reaction
mixture consisted of 0.001 cm3 of 3’ SMARTTM CDS
primer IIA, 0.002 cm3 of RNA (0.8 µg), 0.001 cm3 of
SMARTTM IIA primer, 0.001 cm3 of a deoxynucleotide
solution mix (0.01 M), and 0.006 cm3 of water. This
mixture was incubated at 65 °C for 5 min, and then
0.005 cm3 of a 5 reaction buffer (Invitrogen, Carlsbad,
USA), 0.002 cm3 of dithiotreitol (0.1 M) and 0.0005 cm3
of Super Script II enzyme were added. The sample was
incubated at 42 °C for 50 min, and the reaction was
stopped immediately by incubation at 70 °C for 15 min.
The cDNA amplification from the four bean cultivars and
each treatment was standardised to the 26S ribosomal
housekeeping gene (Ruiz-Nieto et al., 2015). To study
expression profiles associated with drought-related genes,
eight pairs of primers that had been previously
constructed in our laboratory and had been designed from
a suppressive subtractive library from cv. PS under
drought conditions were used in addition to 13 primer
pairs designed from a Gene bank of cv. PV (Barrera-
Figueroa et al., 2007, Table 1 Suppl). Each PCR mixture
contained 0.012 cm3 of water, 0.001 cm3 of a
deoxynucleotide solution mix (0.01 M), 0.002 cm3 of a
Taq buffer (Invitrogen), 0.0008 cm3 of MgCl2, 0.001 cm3
of each primer (1 μM), 0.0002 cm3 of Taq polymerase
(Invitrogen), and 0.002 cm3 of cDNA (500 ng). To
visualize the amplified genes, a 1.5 % (m/v) agarose gel
was used, and the bands were observed using 0.1 % (m/v)
ethidium bromide staining.
The gene fragments amplified by RT-PCR were cut

G.M. GUTIERREZ-BENICIO et al.
498
from the agarose gel, purified with a QIAquick kit
(Qiagen, Germany), ligated into the cloning vector
pGEM-T-Easy (Promega, Madison, USA), and
transformed into cells of competent E. coli strains DH-5α
and JM 107 by heat shock (Sambrook and Russell, 2006).
The transformed cells were plated on a Lourie-Brot agar
growth medium supplemented with 0.1 g dm-3 ampicillin,
50 g dm-3 5-bromo-4-chloro-3-indolyl β-D-galactopyra-
noside (X-Gal) and 23.8 g dm-3 isopropyl β-D-1-thio-
galactopyranoside (IPTG) (Sigma, St. Louis, USA) and
incubated at 37 °C for 18 h. The positive clones were
selected and propagated in a Lourie-Brot liquid medium.
The plasmid DNA was purified using the Quick
minipreps method (Zhovic and Yong 1990), and the
resulting DNA was denatured and used for analysis of
cDNA arrays.
Duplicates of 0.750 µg of the plasmid DNA
containing selected gene probes were printed onto
Hybond-N+ nylon membranes (Amersham Biosciences,
Uppsala, Sweden). A 26S ribosomal gene fragment was
used as positive control for hybridisation, and water and
the plasmid without an insert were used as negative
control. For all the treatments, the double stranded (ds)
cDNA was synthesised with an advantage cDNA
polymerase enzyme mix (Clontech, Palo Alto, USA) and
subsequently purified with a QIAquick kit. A total of
1 750 µg of each purified ds cDNA was used as probe. A
total of 28 probes were synthesised, corresponding to
each of the treatments. The probes were labelled with
biotin-11-dUTP by random priming (Feinberg and
Vogelstein 1983) with a biotin DecaLabel™ DNA
labelling kit (Fermentas, Lithuania). Hybridization was
detected with a biotin chromogenic detection kit
(Fermentas) using streptavidin coupled to alkaline
phosphatase and nitroblue tetrazolium - 5-bromo-4-chloro-
3-indolyl phosphate p-toluidine as chromogenic substrate.
Densitometric image analysis of cDNA array
membranes was performed with the TotalLabQuant
TL120 v. 2008 software to identify differentially
expressed genes. The spots in the membrane images were
normalised for brightness and contrast with respect to the
signal of the 26S ribosomal gene, and then the intensity
of the signal of each gene was measured. For
densitometric analysis, the signal intensity of the 26S
gene was normalised in each of the bean cultivars, and
the standard deviation was calculated. The threshold of
gene expression was estimated with respect to the control
for each of the identified genes and compared to the
control for each of the identified genes.
To confirm the expression profiles revealed by
RT-PCR and cDNA arrays, six of the genes identified as
differentially expressed were evaluated by quantitative
PCR (qPCR) using a Step One real-time PCR system
(Applied Biosystems). The genes examined encoded
Glycine max cDNA, clone: GMFL01-08-H07 (GMFL),
polyubiquitin2, α-carboxyl-transferase, sulphur-rich
proteins, an elongation factor, and LEA3, and a selection
criterion for evaluation was high transcriptions in cvs. PS
and PV. The reaction mixtures contained 0.0125 cm3 of
Master Mix (Thermo Scientific, USA) with a fluorophore
SYBR Green/ROX, 0.0015 cm3 of appropriate primers
(0.005 M), 0.01 cm3 of water, and 0.001 cm3 of a single-
stranded cDNA (0.05 g dm-3). To determine amplification
efficiency, calibration curves were constructed from five
serial dilutions (1:10) of the cDNA (a 0.4 µg cm-3 initial
concentration) with three replications. The experimental
conditions were standardised using a primer for the 26S
gene. The results were analysed with the comparative CT
method using a mathematical model proposed by Pfaffl
(2002) and the REST 2009 v. 2.0.13 software (Qiagen).
To determine functionality of the cDNA array,
36 different bean genotypes were subjected to drought at
the beginning of the flowering stage, and leaf tissue was
sampled after the drought and re-irrigation treatments. In
the field, 18 cultivars characterised as drought-tolerant
were planted. Six lines were crosses between the
genotypes Pinto Durango/PS, six were black bean
cultivars, and other six were red bean cultivars. Sampling
was performed when the water content of the soil was
23 %. In the greenhouse, an additional 18 cultivars with
unknown drought tolerance were planted. Lines, crosses,
and cultivars developed by INIFAP (Guanajuato, Mexico)
were included among these, and drought treatment was
maintained until the water content of the soil was 7 %.
Four drought response genes, LEA3, LEA4, DEHYDRIN,
and POLYUBIQUITIN2, were used as hybridization
probes. The probe and array were developed as described
above, and the total cDNA from each genotype was
printed in duplicate on nylon membranes. Subsequently,
densitometry analysis was performed, and the ratio of the
expressions during the drought treatment and in the
irrigated control (drought/irrigation) was calculated.
Values of the ratio greater than 1.0 were considered as
induction of gene expression.
Results
The analysis of RWC in plant leaves (Fig. 1) shows that
the control plants (under irrigation) of all the cultivars
maintained the RWC at approximately 80 %. In the plants
subjected to the drought stress during V4 stage, the RWC
decreased to approximately 55 % and returned back to
80 % after the recovery. When the second water stress
was applied in the reproductive stage, the RWC in the
tolerant cvs. PV and PS remained similar to that of the
control, whereas in the susceptible cvs. C60 and BM
decreased to 50 %. When the PV and PS plants received
the single stress during the reproductive stage, their RWC
decreased near to 60 %. These results demonstrate that
the plants subjected to the water stress during the
vegetative stage activated response mechanisms to reduce
damage during further water stress.
Twenty-one genes showed differential expressions

MACROARRAY-BASED SELECTION STRATEGY
499
during the drought treatment based on the RT-PCR
(Fig. 1 Suppl.). The activation of transcription was
mainly identified in the tolerant cvs. PV and PS in which
the induction of genes was identified after the drought in
vegetative stage V4 although some genes remained active
also after the recovery. In the reproductive stage, gene
expressions observed after the second stress were similar
to those observed in plants that received only the single
stress. Most of the genes induced in the reproductive
stage (except PS and BM) remained active after the
recovery. In the susceptible cvs. C60 and BM, a slight
induction of gene expression was observed.
The densitometric analysis of cDNA arrays revealed
differential expression between the tolerant and
susceptible cultivars (Tables 1 and 2). The tolerant cvs.
PV and PS showed the highest number of induced genes
under the water stress, whereas the susceptible cvs. C60
and BM showed repression of most identified genes. The
highest overall expressions were identified in PV
(1.42- to 22.7-fold) and PS (1.02- to 19.4-fold), whereas
in the susceptible cultivars, the expressions ranged
between 1.03- and 3.36-fold. The drought stress during
the reproductive stage induced a higher expression than
in V4 stage especially in the tolerant plants. A higher
Fig. 1. Leaf relative water content (RWC) in tolerant cvs. PS and PV and susceptible cvs. BM and C60. 1 - control in the vegetative
stage, 1E - first water stress at vegetative stage V4, r1E - recovery after first water stress, 2 - control in the reproductive stage,
2E - second water stress at reproductive stage, F - one stress in the reproductive stage. Means with the same letter are no
t
significantly different according to Tukey (P  0.05).
Table 1. Differential gene expression in response to drought in the tolerant cultivars as revealed by cDNA arrays. Ratio of the first
stress to the control (1E/C) and recovery (R1E/C); ratio of the second stress to the control (2E/C) and recovery (R2E/C); ratio of a
single stress at the beginning of flowering to the control (EF/C) and recovery (RF/C). Induced genes (dark grey), constitutive genes
(white), and repressed genes (light grey).
Encoded proteins Pinto Saltillo Pinto Villa
1E/C R1E/C 2E/C EF/C 1E/C R1E/C 2E/C R2E/C EF/C RF/C
Hypothetical protein 1.44 1.34 0.96 3.44 8.00 4.35 3.16 0.87 0.91 0.84
Clone GMFL 01-08-H07 2.68 0.10 4.31 6.99 8.60 1.10 3.40 0.94 1.39 1.95
Protein ERD 1.71 1.54 5.09 4.83 2.60 1.56 2.08 0.81 1.20 0.32
Sulphu
r
-rich proteins 3.17 1.21 4.39 5.51 4.77 0.48 5.21 0.30 1.78 1.46
Photosystem II proteins 0.54 0.63 2.02 2.83 4.56 2.26 6.50 3.33 2.95 2.64
Peptidase family M13 1.13 0.11 1.23 3.85 8.76 6.19 5.30 0.37 1.30 1.9
LEA3 0.92 1.03
11.2 17.6 4.83 2.68 13.1 1.8 3.41 0.62
Microsatellite SSR-IAC 105 1.33 5.47 1.75 2.32 2.97 1.51 0.97 0.37 0.48 1.82
Type 2 metallothionein 3.99 0.83 1.08 0.97 2.47 2.40 13.5 1.45 0.96 1.34
Polyubiquitin2 2.15 2.30 1.35 11.5 1.70 1.15 0.45 0.03 0.06 0.16
Elongation factor 1A 3.05 2.18 3.07 2.32 4.48 1.72 2.04 0.06 0.53 0.38
Myo-inositol-1-
p
hosphate synthase 1.51 1.18 1.97 1.46 1.42 0.60 2.76 0.45 0.11 0.68
Hypothetical protein CAB71113 1.25 0.51 0.16 1.55 3.54 1.87 0.93 0.25 1.93 0.54
Periplasmic-cytochrome C 5.82 0.49 4.23 6.22 0.43 0.31 1.10 0.77 1.68 0.37
Dehydrin 1.02 1.23 2.04 3.51 2.65 1.35 3.09 0.81 2.55 1.19
Transmembrane a.a. transporter protein 4.63 5.43 0.64 1.63 1.9 0.61 5.09 0.87 0.24 6.06
LEA4 1.13 0.16 16.6 10.2 2.01 0.95 5.10 1.28 1.12 0.28
Glycosylation enzyme 0.60 0.23 6.94 12.9 3.82 1.30 4.53 2.39 1.76 1.92
Clone 169 2.3 0.73 1.33 5.54 0.67 2.23 4.43 1.03 1.49 0.99
α-Carboxyltransferase 1.05 1.11 1.06 2.91 2.97 1.25 0.46 0.14 0.25 0.29
Clone 033 0.94 0.83 9.11 19.4 3.44 1.22 22.7 3.40 0.81 3.00

G.M. GUTIERREZ-BENICIO et al.
500
Table 2. Differential gene expression in response to drought in the susceptible cultivars as revealed by cDNA arrays. The ratio of the
first stress to the control (1E/C) and recovery (R1E/C); ratio of the second stress to the control (2E/C) and recovery (R2E/C); ratio of
a single stress at the beginning of flowering to the control (EF/C) and recovery (RF/C). Induced genes (dark grey), constitutive genes
(white), and repressed genes (light grey).
Encoded proteins Bayo Madero Canario 60
1E/C R1E/C 2E/C EF/C 1E/C R1E/C 2E/C R2E/C EF/C RF/C
Hypothetical protein 1.03 0.95 0.22 0.29 3.80 9.94 0.32 0.69 0.12 0.69
Clone GMFL 01-08-H07 0.84 0.63 0.36 0.14 0.63 0.19 0.41 0.53 0.19 0.53
Protein ERD 1.43 0.43 0.32 0.14 0.48 1.49 0.17 0.68 0.06 0.68
Sulphu
r
-rich proteins 0.0 0.41 0.3 0.31 0.62 0.37 1.37 0.76 0.04 0.76
Photosystem II proteins 0.94 0.88 0.21 0.32 0.52 0.41 0.47 0.66 0.10 0.66
Peptidase family M13 1.54 1.05 0.19 0.31 0.34 0.93 0.16 0.36 0.07 0.36
LEA3 1.00
1.43 0.68 0.80 0.37 0.43 0.33 0.18 0.21 0.18
Microsatellite SSR-IAC 105 0.96 1.24 0.66 0.80 0.47 0.44 0.18 0.73 0.27 0.73
Type 2 metallothionein 0.96 2.53 0.54 0.68 1.24 0.38 0.19 0.28 0.26 0.28
Polyubiquitin2 0.93
2.21 0.78 0.90 0.54 0.44 0.25 0.28 0.54 0.28
Elongation factor 1A 0.54 0.77 0.67 0.56 0.61 0.45 1.69 0.84 0.99 0.84
Myo-inositol-1-
p
hosphate synthase 1.38 1.86 1.25 0.40 1.36 3.36 0.31 0.61 0.11 0.61
Hypothetical protein CAB71113 1.34 6.62 0.13 0.38 1.03 0.46 0.40 0.65 0.06 0.87
Periplasmic cytochrome C 0.50 2.00 0.56 2.12 1.48 0.34 0.24 0.87 0.08 1.59
Dehydrin 0.59 0.41 0.53 0.85 0.36 0.80 0.17 0.68 0.13 0.65
Transmembrane a.a. transporter protein 0.33 0.78 0.11 0.04 0.06 0.25 0.21 0.14 0.08 0.14
LEA4 0.66 0.83 1.39 1.43 0.47 0.64 0.22 0.48 0.06 0.21
Glycosylation enzyme 2.27 1.08 0.25 0.42 0.48 0.66 0.12 0.51 0.13 0.14
Clone 169 1.25 0.67 0.07 0.29 0.47 0.51 0.11 0.56 0.06 0.26
α-Carboxyltransferase 4.83 0.89 0.46 1.32 0.39 0.82 0.32 0.76 0.12 0.91
Clone 033 1.06 0.19 1.07 0.43 0.53 1.89 0.40 0.82 0.28 1.10
Table 3. Differential expression (drought/irrigation ratio) as assessed by qPCR in different cultivars (PS, PV, BM, and C60) after the
first water stress (1E) and after the second water stress (2E). Means  SD; ** - highly significant differences (P < 0.01), * -
significant differences (P < 0.05). The arrows indicate a greater expression than the control.
Encoded proteins PS1E PV1E BM1E C601E
Clone GMFL 01-08-H07 ↑ 3.46  0.23 ** ↑ 3.44  0.25** ↑ 2.50  0.26* ↑ 13.80  0.19*
Polyubiquitin2 ↑ 10.70  0.35* ↑ 355.44  0.25** 0.24  0.44** 0.38  0.34*
α-Carboxyltransferase ↑ 46.84  0.28** ↑ 5.40  0.16* ↑ 3.56  0.51* 1.00  0.17
PS2E PV2E BM2E C602E
Sulphu
r
-rich proteins ↑ 119.00  0.11** ↑ 135.70  0.14** ↑ 24.12  0.12* ↑ 24.99  0.25*
Elongation factor 1A ↑ 128.45  0.19** ↑ 16.93  0.18** 0.22  0.17 0.23  0.27*
LEA3 ↑ 34.89  0.09* ↑ 100.54  0.18** ↑ 9.07  0.27* ↑ 17.60  0.11*
expression during the reproductive stage was observed in
cv. PV in the double water stress treatment compared to
the single stress-treated plants, whereas cv. PS showed an
opposite effect.
Regarding the tolerant cultivars, 17 genes were
induced in PS and 19 genes in PV during the first stress
(in V4); those genes were associated with the expression
of early response to dehydration (ERD) proteins, sulphur-
rich proteins, photosystem II proteins, LEA3, poly-
ubiquitin2, myo-inositol-1-phosphate synthase, dehydrin,
LEA4, and α-carboxyltransferase. During the second
stress (the reproductive stage), 16 genes were induced in
PV and 14 genes were induced in PS, whereas when the
single stress was applied in the reproductive stage, PS
had a higher number of induced genes than PV (20 and
7 genes, respectively); at this stage, the induced genes
encoded LEA3, dehydrin, LEA4, photosystem II proteins,
and ERD. In the susceptible ciltivars in the vegetative
stage, four genes were activated in BM and five in C60.
During the second stress, only two genes were induced in
C60. After the single stress during the reproductive stage,
the expressions of three genes were induced in BM,
whereas none were activated in C60. During the
recovery, several genes that were activated during the
first water stress remained active. In PS, there were nine
genes that remained active. In PV, 13 genes maintained
the same expression. In C60, two genes maintained the
same expression, but in BM, no genes remained active.

MACROARRAY-BASED SELECTION STRATEGY
501
Table 4. Drought-induced gene expression in bean genotypes grown under field conditions as evaluated by cDNA array. Soil
moisture was decreased to 23 %. The ratio of expression during drought treatment and irrigated control.
Genotypes
L
EA3
P
OLYUBIQUITIN2
D
EHYDRI
N
L
EA4 Mean
Pt. Dgo/Pt. Sal-2-3
Pt. Dgo/Pt. Sal-6-7
Pt. Dgo/Pt. Sal-6-6
Pt. Dgo/Pt. Sal-11-3
Pt. Dgo/Pt. Sal-4-4
Pt. Dgo/Pt. Sal-11-2
SCR13
SER 83
SER 118
SCR 11
SCR 18
INTA SEQUIA
SCN 7
SEN 56
SEN 26
SEN 70
SEN 44
ELS 15 55
0.499
0.916
0.989
1.165
4.284
0.266
0.958
0.987
1.595
3.778
1.957
2.909
0.840
0.647
0.726
0.412
0.918
0.554
0.453
0.840
1.726
2.142
1.604
1.130
0.902
5.228
1.116
1.993
1.461
1.623
1.136
0.656
0.247
0.416
0.481
1.286
0.385
0.406
0.912
0.810
0.906
0.702
0.447
1.375
1.278
1.531
0.927
1.116
0.873
0.809
0.619
0.522
1.963
1.128
0.698
1.000
0.943
0.993
0.835
0.578
0.513
1.155
0.986
0.986
0.882
0.991
1.253
0.839
0.797
0.670
2.427
0.791
0.508
0.790
1.142
1.277
1.907
0.669
0.705
2.186
1.243
2.072
1.306
1.659
1.025
0.737
0.597
0.505
1.447
0.939
Table 5. Drought-induced gene expression in bean genotypes grown under greenhouse conditions as evaluated by cDNA array. Soil
moisture was decreased to 7 %. The ratio of expression during drought treatment and irrigated control.
Genotypes
L
EA3
P
OLYUBIQUITIN2
D
EHYDRI
N
L
EA4 Mean
Pv/ps -11
SER 83
Rosa la bufa
G4523
SER 18
PV
F.J. Leon
SCR 6
F.J. Dalia
Bayo Madero
SCR 16
F.M. Dolores
Pinto Salinas
Pt. San Luis
Pv/Ps -8
Pv/Ps -281
Pt. San Rafael
Pt. Raramuri
0.354
0.210
0.813
0.824
1.075
0.562
0.755
0.572
0.250
0.742
0.377
0.355
0.716
0.500
1.030
0.812
0.719
1.343
1.111
1.200
1.134
0.841
0.987
0.930
2.347
0.871
0.837
1.117
1.032
1.361
1.065
0.983
0.491
1.035
1.283
0.834
0.651
0.880
0.770
0.907
0.794
1.216
1.143
0.778
0.266
0.919
0.422
0.738
0.685
0.892
0.736
0.524
0.896
0.943
0.656
0.797
0.427
1.061
1.744
0.593
1.193
1.021
0.237
0.943
0.791
0.889
0.701
0.684
0.730
0.489
0.432
0.567
0.693
0.771
0.786
0.908
1.150
0.825
1.359
0.810
0.397
0.930
0.655
0.835
0.791
0.764
0.746
0.715
0.832
0.921
From the genes activated during the reproductive stage
and examined during the recovery, PV was the only
cultivar in which six activated genes maintained the same
expression. The genes encoding photosystem II proteins,
LEA3, a glycosylation enzyme, polyubiquitin2, an ERD
protein, sulphur-rich proteins, elongation factor 1A, and
dehydrin maintained the same expression from V4 to the
reproductive stage and throughout the recovery (Table 2
Suppl.).
The qPCR results confirm that the six selected genes
were differently expressed among the tolerant and
susceptible cultivars in response to the drought (Table 3).
In cvs. BM and C60, four genes showed a lower
expression than in cvs. PV and PS; in contrast, GMFL
was expressed at a higher level in C60 than in BM, PV,
and PS. However, the expressions differed among the
three methods used, and higher levels were observed
using qPCR. For the susceptible cultivars, three genes,
for which no expression was observed by RT-PCR, were
detected by qPCR although the expressions of these
genes were low.
Of the 36 bean genotypes evaluated with cDNA array,
12 genotypes (greenhouse and field conditions) were
found to tolerate the drought. From the genotypes

G.M. GUTIERREZ-BENICIO et al.
502
established in the field (Table 4), red genotypes were
highlighted in selection, and five of the six evaluated
showed an induction of expression for three genes. The
lines obtained by crossing Pinto genotypes were also
selected as well as two black genotypes which showed
expression of two genes. Of the greenhouse grown
genotypes, two showed an increase in expression of two
genes (Table 5). In contrast, the genotype F.J. León
showed induction of three genes. The cDNA array used
in this research to select drought tolerant genotypes
allowed to identify bean cultivars with this feature. Most
drought-responsive genes were identified in bean
genotypes produced under field conditions with a history
of conventional breeding tolerance to water stress; on the
other hand, a low number of genes in response to drought
were identified in genotypes produced under greenhouse
conditions with no history of breeding to this stress; this
confirms the functionality of the cDNA array proposed
here to be incorporated into a breeding programme of
molecular marker-assisted selection.
Discussion
Previous investigations have reported changes in gene
expression in response to water stress, including changes
in expression of genes involved in production of enzymes
for synthesis of osmolytes and proteins with protective
functions such as LEA proteins, antioxidant enzymes, and
transcription factors (Moreno 2009). The genes expressed
under the water stress reported in this paper include those
associated with synthesis of various proteins such as
LEA3, LEA4, and dehydrin (LEA2). These proteins are
essential for a drought response because they provide a
hydrophilic microenvironment due to their random coil
structure; thus, they can substitute for water and help to
maintain the structures of various proteins as well as
membrane integrity (Battaglia et al. 2008). Therefore,
they have been widely studied in various organisms, and
transcripts have been identified in vascular and non-
vascular plants in response to dehydration (Salmi et al.
2005). Another group of proteins involved in water stress
are ERD proteins which protect the cell membrane
against damage by dehydration (Barrera-Figueroa et al.
2007, Alves et al. 2011). The ERD proteins, such as
ERD4, are rapidly induced by water deficit and have been
characterized mainly in maize, Arabidopsis, and rice (Rai
et al. 2012). We identified transcriptional induction under
the drought stress treatment at an early stage of
development. Additionally, within the set of induced
genes, we identified several genes with a possible role in
photosynthesis (Jaramillo-Giraldo et al. 2009). During
drought stress, there is an imbalance of ionic homeostasis
that results in an increased content of Ca2+ which acts
asmessenger in signalling pathways to activate
transcription of genes responsive to drought. Millar et al.
(1994) reported that an increasing content of cytosolic
Ca2+ induces transcription of genes encoding structural
photosystem II proteins and chlorophyll a and b binding
(CAB) proteins. In our study, we identified photosystem
II proteins, CABs, and a periplasmic cytochrome.
Transcription of genes related to photosynthetic processes
is critical because their joint action could ensure
photosynthetic efficiency in response to dehydration.
Four genes related to metabolism were identified. The
first of them was related to biosynthesis of sulphur-rich
proteins with possible involvement in synthesis of storage
proteins in seeds of common bean (Yin et al. 2011).
Further, albumins with sulphur-containing amino acids
have been identified in different cultivars of common
bean (Raya-Perez et al. 2014). The second gene
associated with metabolism encodes a protein related to
carboxyltransferase which takes part in fatty acid
synthesis, specifically with acetyl-CoA carboxylase
(Natsumi et al. 2013). Other induced genes belong to the
amino acid transporter superfamily and include amino
acid transporter family 1 (ATF1) and solute carrier 38
(SLC38) (Wipf et al. 2002). Another gene from this
group is elongation factor 1A which is involved in
protein synthesis and has been isolated and characterized.
In rice, the same genes (refa1, refa2, refa3, refa4) are
expressed in different tissues and stages of plant growth
(Shin-ichiro et al. 1998). These four genes were
identified after drought treatments and emerged as help to
cellular metabolism to prevent structural alterations and
lipid oxidation that can occur due to water deficit.
Furthermore, metallothionein2 is a typical class of plant
proteins that chelates heavy metals such as cadmium and
copper, and this type of protein has been observed in rice
and Arabidopsis (Zhou and Goldsbrough 1995, Zhou
et al. 2006). METALLOTHIONEIN genes have been
characterized in Jatropha curcas (Shalini et al. 2014),
and the data suggest that induction of these genes during
drought might contribute to detoxification mechanisms.
Myo-inositol-1-phosphate synthase (MIPS), which is
involved in biosynthesis of myo-inositol, was identified
and is an important second messenger in water stress
signal transduction (Irvine and Schell 2001). The MIPS
has been identified to show differential expressions in
Phaseolus vulgaris (Johnson and Wang 1996), and Abid
et al. (2009) reported the identification of 60 MIPS genes
in different organisms involved in tolerance to biotic and
abiotic stresses. Various proteins, both structural and
functional, are affected by water deficit, and therefore,
mechanisms, such as glycosylation and ubiquitination,
are required to counteract this effect. In a list of drought-
induced genes, the gene POLYUBIQUITIN2 encodes a
protein that marks proteins for degradation or recycling
(Welchman et al. 2005), and glycosylation enzymes play
a substantial role in maintaining cellular homeostasis by
modifying various molecules, primarily proteins (Wang
and Hou 2009). These genes might be involved in
recovery of cell damage due to water stress. In addition to
the set of the identified genes, hypothetical genes GMFL,

MACROARRAY-BASED SELECTION STRATEGY
503
microsatellite SSR-IAC 105 of Phaseolus vulgaris,
peptidase family M13, clone 169, and clone 033 were
identified. Although they do not seem to have a direct
relationship with the water deficit response, their
expressions were altered upon the drought treatment.
In this study, the identification of several genes
induced by water stress in widely recognized cultivars
that are tolerant to drought was crucial; moreover, the
induction of expression in early development during the
vegetative phase and the continuous expression of some
transcripts after the plants recovered were vital
information. Additionally, an understanding of the
expression of these genes during the reproductive stage,
which is sensitive to drought damage, might be helpful
for elucidating molecular mechanisms involved in the
response to drought. Thus, this set of identified genes is
of major agricultural and biotechnological importances.
In this paper, the utility of some genes that are directly
related to drought response, mainly LEA3, LEA4,
POLYUBIQUITIN2, and DEHYDRIN, have been verified,
and cDNA array was demonstrated as a feasible tool for
selection of different drought tolerant bean genotypes
under field conditions. Based on this work, these genes
should be considered as expression markers in
biotechnological applications to identify promising
genotypes.
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