South African Journal of Botany 123 (2019) 200–207

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Photosynthetic responses and tolerance to root-zone hypoxia stress of

five bean cultivars (Phaseolus vulgaris L.)
N.F. Velasco ⁎, G.A. Ligarreto, H.R. Díaz, L.P.M. Fonseca
Facultad de Ciencias Agrarias, Departamento de Agronomía, Universidad Nacional de Colombia, Bogotá, Colombia

a r t i c l e i n f o a b s t r a c t

Article history: Common bean (Phaseolus vulgaris L.) is very sensitive to waterlogging, which is considered a limiting factor for its
Received 31 May 2018 production. The objective of this study was to evaluate photosynthetic responses and tolerance to root-zone hyp-
Received in revised form 6 February 2019 oxia stress of five bean cultivars (Cerinza, Bachue, Bianca, Bacatá and NUA35). Bean seedlings of the five cultivars
Accepted 8 February 2019
were grown in hydroponics and subjected for 14 days to two treatments (i) high oxygen availability in the root
Available online xxxx
zone or (2) low oxygen availability or hypoxia stress. Results showed that all cultivars under hypoxia stress ex-
Edited by R Baraldi hibited reductions in photosynthesis rate (62%–82%), stomatal conductance (73%–97%), transpiration rate (51%–
92%), actual quantum yield, non-photochemical quenching, electron transport rate and in the content of chloro-
Keywords: phylls and carotenoids. An increase in leaf electrolyte leakage was also observed, which suggests a loss in the sta-
Photosynthesis bility of cell membranes because of hypoxia stress. The cultivars Bachue, Bianca, Cerinza and NUA35 showed an
Stomatal conductance increase in proline content (300%–750%) compared to plants grown with high oxygen. The cultivar with the
Proline highest water use efficiency and the lowest loss of dry mass was Cerinza, followed by NUA35. These cultivars,
Abiotic stress Cerinza and NUA35, showed a higher tolerance to hypoxia (0.36 and 0.38) according to the waterlogging toler-
Hydroponics
ance coefficient compared with the other cultivars grown with low oxygen availability. The performance of these
cultivars could be attributed to the greater reduction in stomatal conductance, maintaining low rates of transpi-
ration but without a high reduction in the CO2 intake. In this way, the tolerance in these cultivars could be asso-
ciated with the greater water use efficiency during the conditions of hypoxia, compared with the other cultivars.
© 2019 Published by Elsevier B.V. on behalf of SAAB.

1. Introduction
Common bean (Phaseolus vulgaris L.) is an important crop legume
species worldwide (Rahmani et al., 2011); its center of origin is in Me-
soamerica and South America, from Mexico to Argentina (Chacón
et al., 2005). Common bean is considered one of the most important
sources of protein and nutrients for more than 300 million people in re-
gions of Eastern Africa and Latin America (Reyna et al., 2003). The an-
nual global bean production is approximately 12 million metric tons,
with 5.5 million metric tons alone in Latin America Caribbean (LAC)
(Collaku and Harrison, 2002). However, the yield of the bean crop is af-
fected by water stress including both, shortage and excess of water due
to its sensitivity (Singer et al., 1996). In some regions of the tropics, the
presence of climatic events such as El Niño and La Niña has intensified
due to climate change and an increase in precipitations is expected
(FAOUN, 2015; Horchani et al., 2010). The increase in the frequency
and intensity of rainfall causes temporary waterlogging of the soils
⁎ Corresponding author.
E-mail addresses: nflorezv@unal.edu.co (N.F. Velasco), galigarretom@unal.edu.co
(G.A. Ligarreto), hrestrepod@unal.edu.co (H.R. Díaz), lpmorenof@unal.edu.co
(L.P.M. Fonseca).
and, therefore, a deficiency of oxygen in them, which affects the yield
of crops such as beans (Duran and Pinto, 2011; Lakitan et al., 1992;
Sairam et al., 2008).
Flooding causes modifications in the physicochemical properties of
the soil such as pH, redox potential and oxygen content (Conn, 1968).
For this reason, plants that grow under waterlogging conditions show
stress due to oxygen deficiency (hypoxia) or to its absence (anoxia)
(Bertholdsson, 2013; Striker, 2012). The damage and death that occurs
in plants under waterlogging conditions has been attributed mainly to
this deficiency of oxygen in the soil (Kozlowski, 1984a; Setter et al.,
2003). The first effect of waterlogging on plants is the reduction in the
production of ATP, generated by the change from aerobic to anaerobic
metabolism (Sairam et al., 2008). The decrease in ATP during
waterlogging causes a drop in the speed of all metabolic processes in
the plant, and therefore in growth (Bailey-Serres et al., 2012). The effect
of waterlogging conditions on plants includes the decrease in stomatal
conductance (gs), rate of photosynthesis (Pn), hydraulic conductance
and transpiration (E) (Kumar et al., 2013; Rao and Li, 2003). Also, in dif-
ferent plants such as millet (Pennisetum glaucum L.) and apple (Malus
domestica L.) it has been observed that waterlogging decreases water
use efficiency (WUE) (Jie et al., 2001; Zegada-Lizarazu and Iijima,
2005). The decrease in photosynthesis has been associated in part

https://doi.org/10.1016/j.sajb.2019.02.010
0254-6299/© 2019 Published by Elsevier B.V. on behalf of SAAB.
 N.F. Velasco et al. / South African Journal of Botany 123 (2019) 200–207
with the drop in gs, although the damage in the photosynthetic appara-
tus due to the over-reduction caused by an imbalance between the two
phases of photosynthesis has also been reported (Bota et al., 2004). The
over-reduction of the electron transport chain causes the generation of
reactive oxygen species (ROS) (El-Enany et al., 2013). An increase in
ROS produced via plasma membrane NADPH oxidase has also been
found under waterlogging conditions (Shao et al., 2013). ROS are highly
reactive molecules that cause damage in chlorophylls, proteins, nucleic
acids and lipids (Blokhina et al., 2003). An increase in the electrolyte
leakage (EL) has been found because of the lipid peroxidation in cell
membranes (Sairam et al., 2009). Damage to the integrity of the mem-
branes affects the efficiency of photosystem II, decreasing the rate of
photosynthesis and the production of assimilates (Blokhina et al.,
2003; Maxwell, 2000; Rao and Li, 2003). Therefore, the carbohydrate re-
serves and the efficiency in their use in the plant are affected (Li et al.,
2008; Van Nguyen et al., 2017). Also, a degradation of chlorophylls, pro-
teins and RNA, and a decrease in the concentration of nutrients has been
observed during waterlogging (Florez-Velasco et al., 2015; Li et al.,
2008). A decrease in chlorophyll a and b content has been observed in
several plants such as lulo (Solanum quitoense Lam.) and mung bean
(Vigna radiata L.) under waterlogging conditions (Florez-Velasco et al.,
2015; Kumar et al., 2013). The decrease in the production of ATP, as
well as the reduction in the rate of photosynthesis and the nutritional
imbalance reduce plant growth (Celik and Turhan, 2011; Florez-
Velasco et al., 2015; Kumar et al., 2013).
Plants present several response mechanisms to cope with
waterlogging stress (Elzenga and Veen, 2010; Sairam et al., 2008;
Voesenek and Bailey-Serres, 2015). In many plants, waterlogging stress
increases the synthesis of molecules such as proline that protect from
damage caused by oxidative stress as a tolerance mechanism (Ou
et al., 2011; Parvin and Karmoker, 2013; Singh and Singh, 1981). In dif-
ferent plants, features such as chlorophyll content and biomass produc-
tion have been used as indirect selection indices for tolerance to
waterlogging, since they have high heritability (Elzenga and Veen,
2010; Li et al., 2008). However, the tolerance to waterlogging is complex
because it is influenced by other factors such as temperature, soil type,
availability of nutrients and microorganisms present in the soil (Li
et al., 2008; Soltani et al., 2018). Since the main limitation in water-
logged soils is oxygen deficiency, one way to evaluate its effect on plants
is to use a hydroponic system with a decrease in the concentration of O2
in the nutrient solution, which allows the evaluation of many plants in a
limited space and under controlled conditions (Blokhina et al., 2003).
The responses of plants such as wheat (Triticum aestivum L.) and barley
(Hordeum vulgare L.) to waterlogging can be reproduced in anaerobic
nutrient solutions bubbled with N2 gas (Bertholdsson, 2013; Striker,
2008; Trought and Drew, 1980). However, N2 did not simulate the re-
sponses to soil anoxia because it often contains some O2, but it may be
useful to generate a hypoxia condition (Wiengweera and Greenway,
2004).
In common bean, it has been reported that genotypes respond differ-
ently to soil waterlogging (Aydogan and Turhan, 2015; Chacón et al.,
2005). Waterlogging tolerance in bean had been considered a complex
character controlled by several genes, which hinders introgression in
genetic improvement programs. However, Middle American genotypes
have greater hypocotyl length and greater adventitious root formation
than Andean beans, which allow them to better adapt to excess water
Table 1
Cultivars selected for evaluation in this assay.
Cultivar Institutions and years when delivered
NUA35 CIAT, 2009
Bacatá Universidad Nacional de Colombia, 2013
Bianca Universidad Nacional de Colombia, 2013
Bachue ICA, 1994
Cerinza ICA, 1991
201
in the soil (Soltani et al., 2018). For this reason, it is necessary to obtain
materials that are tolerant to hypoxia or waterlogging, not only by de-
termining tolerant genotypes, but also by identifying traits associated
with tolerance to this stress that are easy to evaluate. In Colombia,
there is a wide genetic variability in beans, which includes numerous
market classes and cultivars that have originated from traditional ge-
netic improvement of characteristics of resistance to diseases, earliness,
seed yield and seed color, shape and size (Mayor-Duran et al., 2016). So
far, there is no information available about the tolerance or susceptibil-
ity of these genotypes to waterlogging, or hypoxic conditions. The ob-
jective of this study was to evaluate the effect of hypoxic stress on the
physiological variables of gaseous exchange, water use efficiency, chlo-
rophyll fluorescence parameters, photosynthetic pigments, membrane
stability, dry mass and the degree of tolerance in five genotypes of com-
mon bean subjected to hypoxia under controlled conditions.
2. Materials and methods
2.1. Plant material and growth conditions
This study was carried out in the Agricultural Sciences Faculty of the
Universidad Nacional de Colombia. We evaluated the cultivars ICA
Cerinza (Delgado et al., 2013) and ICA Bachue, two commonly grown
cultivars in Colombia released by Instituto Colombiano Agropecuario
(ICA) in 1991 and 1994 respectively, cultivars Bacatá and Bianca, two
new cultivars developed at Facultad de Ciencias Agrarias, Universidad
Nacional de Colombia, and NUA35 (Delgado et al., 2013) of the Andean
nutrition program of the International Center of Tropical Agriculture
(CIAT). All these cultivars belong to the Andean gene pool Nueva Gra-
nada race (Table 1). These cultivars were chosen for their economic im-
portance or their potential to be cultivated in the country. Ten seeds per
each cultivar were surface-sterilized in 5% sodium hypochlorite for
15 min. and rinsed with distilled water. Seeds were germinated in a ger-
mination camera (25 °C and 60% RH) using quartz sand as a substrate.
The plants were grown under controlled environmental conditions for
14 days in a growth room with photoperiod of 12:12 light: dark and
photosynthetic active radiation (PAR) of 200 μmol m−2 s−1. The tem-
perature was set at 25 ± 3 °C and the relative humidity was maintained
between 65% and 75%. One-week-old seedlings were placed in plugged
holes in polystyrene foam sheets that were floating in a plastic tank. The
tank contained a nutritive solution that consisted of N 220 mg L−1,
P 44.5 mg L−1, K 210 mg L−1, Ca 144 mg L−1, Mg 57 mg L−1,
S 5 mg L−1, Fe 5.6 mg L−1, Mn 0.564 mg L−1, Cu 0.06 mg L−1, Zn
0.132 mg L−1, B 0.53 mg L−1, Mo 0.006 mg L−1, Co 0.0018 mg L−1.
The pH of the solution was fixed at 6.0, and the solution was changed
every 5 days (Nutriponics®, Walco, Colombia).
2.2. Hypoxia treatments
Four-week-old plants were subjected to two treatments of oxygen
availability in the root zone for 14 days: (i) high oxygen availability in
which the solution was aerated to maintain an adequate oxygen content
(ii) low oxygen availability or hypoxia in which the nutritive solution
was constantly bubbled 70% (v/v) nitrogen for the time indicated for
14 days. The oxygen concentration in the nutritive solution was moni-
tored with the Winkler test (Pomeroy and Kirschman, 1945). The values
Gene pools Weight of 100 seeds (g) Types
Andean 47 = large Pinto beans
Andean 60 = large Red beans
Andean 58 = large White beans
Andean 57 = large Red beans
Andean 52 = large Red beans
202 N.F. Velasco et al. / South African Journal of Botany 123 (2019) 200–207

Table 2 2.3. Gas exchange and water use efficiency

Analysis of variance (ANOVA) results for physiological parameters.

Source variation The photosynthetic rate (Pn) and the stomatal conductance (gs)
Abbr. Cultivar Hypoxia Cultivar × Hypoxia
were registered using a photosynthesis measurement system (LI-
6200, Lincoln, Nebraska, USA.). The measurements were taken in the
Total plant dry weight TDW *** *** **
second young fully expanded leaf from 9:00 to 11:30 h with a CO2 con-
Net photosynthesis Pn ** *** **
Stomatal conductance gs ** *** ** centration of 500 μL L−1 and a photosynthetic photon flux density
Transpiration E *** *** *** (PPFD) of 200 μmol m−2 s−1. The transpiration rate (E) was registered
Water use efficiency WUE ** ** ** using a portable steady-state porometer (LI-1600, Lincoln, Nebraska,
Chlorophyll a Chl a ns *** ns USA.). The water use efficiency (WUE) was calculated with Pn and E
Chlorophyll b Chl b ns * ns
Total chlorophyll Total Chl ns *** ns
data (Pn/E). Two measurements were carried out per plant.
Carotenoids Car ns *** ns
Electrolytes leakage EL ns *** ns 2.4. Chlorophyll fluorescence parameters
Proline *** *** ***
Actual quantum yield ФPSII ns * ns
To determine the state of the photosynthetic apparatus, the chloro-
Electron transport rate ETR ns * ns
Photochemical quenching qP ns *** * phyll fluorescence was measured in a dark-adapted leaf for 30 min.
Nophotochemical quenching NPQ ns * ns Using a MINI-PAM modulated fluorometer (Walz®, GmbH Effeltrich,
ANOVA: *, **, and *** significantly different at the 0.05, 0.01 and 0.001 probability levels,
Germany). Measurements were carried out on the same leaves that
respectively. NS, not significant at a = 0.05. were used to measure Pn. The Chl molecules were excited for 0.80 s
with 1500 μmol m−2 s−1 of actinic light. The parameters photosynthetic
electron transport rate (ETR), actual quantum yield (ФPSII), photochem-
of dissolved oxygen were 10 mg O2 L−1 for aerated solution and 1.5 mg ical quenching (qP), and non-photochemical quenching (NPQ) were
O2 L−1 (83 μm O2 30% of air saturation) for hypoxic solution. In this re- registered.
search a complete randomized factorial design was used, in which the
first factor was the cultivars and the second the oxygen availability in 2.5. Photosynthetic pigments and membrane permeability
the nutritive solution. Four replications were made by treatment and
the experimental unit was one plant. For both treatments, the variables Pigments were extracted in accordance with Sims and Gamon
were measured at the end of the experiment (14 days). (2002). For the extraction, 0.03 g of tissue from the second fully

12 0.8
A B
a ab
10 ab
0.8 a a
ab a a
Pn (Pmol m–2 s–1)

8 a
gs (mol m–2 s–1)

6 0.4

c
4 cd
cd 0.2 b
cd c
2 d c
d
d
0 0.0
Bacata Bachue Bianca Cerinza NUA35 Bacata Bachue Bianca Cerinza NUA35

10 6
a
C A-Oxygen
D
5
WUE (Pmol CO2/mmol H2O)

8 Hypoxia
a ab a
ab
b
E (mmol m–2 s–1)

4
6 a

3
4 c
2 bc bc
bc bc
d bc bc bc
2 d
1
e e d
0 0
Bacata Bachue Bianca Cerinza NUA35 Bacata Bachue Bianca Cerinza NUA35

Fig. 1. Gas exchange and water use efficiency in five common beans cultivars under adequate oxygen availability in the root zone (A-Oxygen) and hypoxia (Hypoxia). (A) Photosynthesis
(Pn); (B) Stomatal conductance (gs); (C) Transpiration (E) and (D) water use efficiency (WUE). The data shown are the means of four replicates, with the standard error indicated by the
vertical bars. Means denoted by the same letter do not significantly differ at P b .05 according to the Tukey test.
 N.F. Velasco et al. / South African Journal of Botany 123 (2019) 200–207 203

Table 3
Actual quantum yield (ФPSII), electron transport rate (ETR) and non-photochemical
quenching (NPQ) in five common beans cultivars under adequate oxygen availability in
WTC ¼ Total dry weight of plants subjected to hypoxia ðTDW−HÞ
the root zone (A-Oxygen) and hypoxia (Hypoxia).
 =Total dry weight of plants with oxygen availability
Treatments ФPSII ETR NPQ  ðTDW−AÞ
−2 −1
(μmol m s )

A-Oxygen 0.09 ± 0.00 a 69.34 ± 5.88 a 1.37 ± 0.06 a

Hypoxia 0.07 ± 0.00 b 49.12 ± 5.70 b 1.03 ± 0.06 b
Significance * * ***
CV (%) 47.01 47 23.65
Means denoted by the same letter do not significantly differ at P b .05 according to the
Tukey test.
expanded leaf of four plants per treatment was homogenized in 80% ac-
etone. The absorbance was determined at an optical density of 663 and
647 nm for chlorophyll and of 470 nm for carotenoids (Wellburn, 1994).
Permeability of the membranes was measured by the amount of elec-
trolyte leakage (EL). A piece of leaf tissue (5 cm2) was placed in a tube
with 20 mL of deionized water at 30 °C for 2 h and the electrical conduc-
tivity (EC1) was determined. Subsequently, the samples were boiled,
cooled at 25 °C and total electrical conductivity (EC2) was measured.
EL was expressed as: EC1/EC2 × 100 (Hamilton and Heckathorn, 2001).
2.6. Proline content
The proline content was determined in four plants from 0.5 g of leaf
tissue according to Bates et al. (1973). For the proline (Pro) determina-
tion, a 1:1:1 solution of proline, ninhydrin and glacial acetic acid was in-
cubated at 100 °C for 1 h. The reaction was arrested in an ice bath, the
chromophore was extracted with toluene and its absorbance was mea-
sured at 520 nm with a spectrophotometer (Evolution™ 300 UV–Vis
ThermoFíscher Scientific, Waltham, MA, USA). The proline concentra-
tion was determined using a standard curve and was expressed as
μmol proline g−1 fresh weight (FW).
2.7. Growth and waterlogging tolerance coefficient
Four plants per treatment were harvested at the end of the hypoxia
period. Then, the plant material was dried at 65 °C until the weight be-
came constant. The waterlogging tolerance coefficient (WTC) was used
to determine the hypoxia tolerance in the cultivars and, therefore, their
potential tolerance to waterlogging (Liu et al., 2010). The WTC was cal-
culated using the formula:
0.30
A-Oxygen
0.25 a
Hypoxia
ab
0.20
2.8. Data analysis
Two-way ANOVA was carried out to determine the effect of the
treatments in the analyzed variables. A fixed-effect model was used. A
mean comparison test was performed with a Tukey multiple range
test (p b .05).
3. Results
Table 2 shows the results of ANOVA. The five bean cultivars analyzed
in this assay exposed different photosynthetic performance with statis-
tical differences. Hypoxia affected all photosynthetic parameters evalu-
ated. Significant interaction between cultivars and hypoxia in the
parameters TDW, Pn, gs, E, WUE, proline and qP was found.
3.1. Gas exchange and water use efficiency
The variables of Pn, gs and E showed a significant reduction in plants
under hypoxia compared to those under adequate oxygen level in the
root zone for all cultivars (Fig. 1A, B and C). Bianca was the cultivar
with the highest reduction in Pn (86%), whereas NUA35 showed the
least reduction (62%) (Fig. 1A). Photosynthetic rate was reduced for cul-
tivars Bachue (82%), Cerinza (77%) and Bacatá (64%) under hypoxia. The
gs was reduced for cultivars Cerinza (97%) and NUA35 (93%), while it
was reduced by an average of 80% for Bacatá and Bachue under hypoxia.
The cultivar with the lowest reduction in gs was Bianca (73%) (Fig. 1B).
Transpiration showed the same behavior as gs under hypoxia (Fig. 1C).
The cultivars with the greatest reduction in E were Cerinza (94%) and
NUA35 (92%). Cultivars Bacatá and Bachue showed a reduction in E of
73% on average, while Bianca showed the lowest reduction (51%).
WUE increased under hypoxia for cultivars Cerinza (4.85 μmol CO2/
mol H2O) and NUA35 (3.05 μmol CO2/mol H2O), while for cultivar
Bianca the WUE showed a decrease (0.33 μmol CO2/mol H2O)
(Fig. 1D) compared to those under adequate oxygen level.
3.2. Chlorophyll fluorescence parameters
Hypoxia caused a reduction in ФPSII (22%), NPQ (25%) and ETR (29%)
in all cultivars (Table 3). Bianca was the cultivar that showed a higher
2.0
a
1.8 A-Oxygen
a
1.6 Hypoxia
Proline (Pmol g–1 FW)

ab ab
ab ab 1.4
ab ab
qP

0.15 ab 1.2
b 1.0 bc
0.10 c 0.8
0.6 cd
0.05 cd cd cd
0.4 d d
0.2
0.00
Bacatá Bachue Bianca Cerinza NUA35 0.0
Bacata Bachue Bianca Cerinza NUA35
Fig. 2. Photochemical quenching (qP) in five common beans cultivars under adequate
oxygen availability in the root zone (A-Oxygen) and hypoxia (Hypoxia). The data shown Fig. 3. Proline content in five common beans cultivars under adequate oxygen availability
are the means of four replicates, with the standard error indicated by the vertical bars. in the root zone (A-Oxygen) and hypoxia (Hypoxia). The data shown are the means of four
Means denoted by the same letter do not significantly differ at P b .05 according to the replicates, with the standard error indicated by the vertical bars. Means denoted by the
Tukey test. same letter do not significantly differ at P b .05 according to the Tukey test.
204 N.F. Velasco et al. / South African Journal of Botany 123 (2019) 200–207

8 Table 5
a Waterlogging tolerance coefficient (WTC) of five common beans cultivars under hypoxia
a A-Oxygen
in the root zone.
Hypoxia
6 Cerinza NUA35 Bacatá Bianca Bachue Significance CV (%)
b WTC 0.38 0.36 0.31 0.20 0.17 * 25.87
b
TDW (g)

bc CV: Coefficient of Variation.

4

c 3.6. Relationship between photosynthetic parameters and WTC

d
2
e e e Stomatal conductance declined with increasing WTC in a similar
way across all cultivars, allowing stomatal response to WTC to be de-
scribed with a single function that accounted for 78% of the variation
0
in gs. Cultivars Cerinza and NUA35, with lower gs, had the highest
Bacata Bachue Bianca Cerinza NUA35
WTC values (Fig. 5A). The WUE was positively correlated with WTC
(Fig. 5B), which means that cultivars with larger WUE tended to exhibit
Fig. 4. Total dry weight (TDW) in five common beans cultivars under adequate oxygen
availability in the root zone (A-Oxygen) and hypoxia (Hypoxia). The data shown are the
larger values of WTC. The proline content and WTC did not show any
means of four replicates, with the standard error indicated by the vertical bars. Means correlation; cultivars Cerinza and NUA35 showed intermediate proline
denoted by the same letter do not significantly differ at P b .05 according to the Tukey test. contents and high values of WTC (Fig. 5C).

reduction in qP (79%) under hypoxia compared to the plants with ade-
quate oxygen level (Fig. 2). There were no significant differences in Fv/
Fm between plants under hypoxia and those under adequate oxygen
level (data no shown).
3.3. Photosynthetic pigments and leaf membrane permeability
The content of pigments decreased in all cultivars under hypoxia
conditions for Chl a (24%), Chl b (13%), Total Chl (33%) and Car (26%).
Hypoxia caused an increase in EL (~38%) in all bean cultivars showing
a change in leaf membrane permeability (Table 4).
3.4. Proline content
There was an increase in the proline content under hypoxia in culti-
vars Bachue, Bianca, Cerinza and NUA35, being higher in cultivars
Bianca (366%) and Bachue (500%) and lower in NUA35 (206%)
(Fig. 3). The Bacatá cultivar did not increase the proline content under
hypoxic conditions.
3.5. Growth and waterlogging tolerance coefficient
The hypoxia treatment generated a reduction in TDW in all cultivars
(~ 70%) as previously described in bean (Soltani et al., 2018). Cultivar
NUA35 presented the lowest reduction in dry weight (53%) followed
by Cerinza (61.5%), while the greatest reduction in dry mass was
shown by cultivar Bachue (83%) compared to the plants with adequate
oxygen level (Fig. 4). Waterlogging tolerance coefficient was higher in
Cerinza (0.38), followed by NUA35 (0.36), while Bachue had the lowest
value (0.17). (See Table 5.)
4. Discussion
The lack of oxygen has been proposed as the main problem associ-
ated with waterlogging stress (Jackson and Drew, 1984; Kozlowski,
1984b). Hydroponic systems with N2-flushed nutrient solution may be
useful to generate low oxygen conditions in the root medium such as
those presented in the initial stages of waterlogging or when the soil
has a low gas-filled porosity (Drew, 1992; Wiengweera and
Greenway, 2004). Likewise, cultivar screening is a successful tool to
identify tolerance to root low oxygen stress that could be used in breed-
ing programs (Mustroph, 2018). In this study, the effect of stress due to
hypoxia in the root zone for 14 days on the photosynthetic traits of five
common bean cultivars was evaluated. Here, it was observed that culti-
vars NUA35, Bacata and Cerinza had the lowest decrease in Pn, while
Cerinza and NUA35 had the lowest decrease in E and gs under hypoxia.
These results suggest that cultivars Cerinza and NUA35 have a better
regulation of water loss through a greater reduction in stomatal conduc-
tance, maintaining low transpiration rates without a greater reduction
in the CO2 intake, compared with the other cultivars (Kumar et al.,
2013). However, other factors such as the decrease in the chlorophyll
content and the senescence of the leaf that were observed under hyp-
oxia in all cultivars also contributed to the decrease of photosynthesis
(Florez-Velasco et al., 2015; Gregersen et al., 2014). A reduction in Pn,
gs and E has been observed under hypoxia in plants such as wheat
(Triticum aestivum L.), cowpea (Vigna unguiculata L.), soybean (Glycine
max L.) and chickpea (Cicer arietinum L.) (Jung et al., 2008; Meena
et al., 2015; Shao et al., 2013; Takele and McDavid, 1994). The stomatal
closure and the maintenance of high water potentials have also been
observed as common effects of waterlogging in several species of herba-
ceous plants (Bradford, 1983). Nevertheless, a significant correlation be-
tween the stomatal conductance and the water potential under hypoxia
has not been found in some plants, which suggests that the water po-
tential is more determined by the osmotic potential and the pressure
potential under this stress (Ashraf and Arfan, 2005). An increase in

Table 4
Chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Total Chl), carotenoids (Car) y electrolyte leakage (EL) in five cultivars of common bean under adequate oxygen availability in
the root zone (A-Oxygen) and hypoxia (Hypoxia).

Treatments Chl a Chl b Total Chl Car EL (%)

(mg g−1 FW) (mg g−1 FW) (mg g−1 FW) (mg g−1 FW)

A-Oxygen 3.92 ± 0.15 a 1.92 ± 0.06 a 7.05 ± 0.18 a 0.72 ± 0.02 a 19.53 ± 0.94 b
Hypoxia 2.96 ± 0.16 b 1.68 ± 0.07 b 4.74 ± 0.18 b 0.53 ± 0.03 b 27.06 ± 1.06 a
Significance *** * *** *** ***
CV (%) 18.01 15.26 16.12 19.02 18.19

Means denoted by the same letter do not significantly differ at P b .05 according to the Tukey test.
 N.F. Velasco et al. / South African Journal of Botany 123 (2019) 200–207
0.20
0.18 A Cerinza
2=0.78
r r
WUE (µmol CO2/mmol H2O)
0.16 Bianca
0.14
gs (molm–2 s–1)
0.12
0.10 Bacsta
0.08 Bachue
NUA35
0.06
0.04
0.02
Cerinza
0.00
0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
WTC
Fig. 5. Stomatal conductance (gs) (A); Water use efficiency (WUE) (B); and Proline (C) versus Waterlogging tolerance coefficient (WTC) in five common bean cultivars under hypoxia in
the root zone. Vertical and horizontal bars represent ± standard error of means of four replicates. The circle shows the cultivars with higher tolerance.
WUE under hypoxia was observed for two of the cultivars, Cerinza and
NUA35. It has been found that genotypes susceptible to waterlogging
present higher reductions in WUE, and therefore, they gain less carbon
per unit of water lost (Meena et al., 2015).
Chlorophyll fluorescence is an indicator of the stability and function-
ality of PSII (Maxwell, 2000; Shao et al., 2013). Under hypoxia stress, the
ROS levels increase significantly and can damage the PSII (Shulaev and
Oliver, 2006). In all cultivars evaluated, hypoxia reduced the values of
ФPSII, ETR and NPQ. A smaller reduction in qP was also observed in cul-
tivars NUA35 and Cerinza. However, no differences were observed in
Fv/Fm between the plants under hypoxia and those under adequate ox-
ygen level, which suggests that the alterations in the PSII were minimal
under hypoxia. The maintenance of PSII functionality may be related to
the presence of protective molecules such as proline, whose content in-
creased in four of the cultivars. The damaging effects of singlet oxygen
and hydroxyl radicals on Photosystem II (PSII) can be reduced by pro-
line in isolated thylakoid membranes (PSII) (Alia et al., 1997).
The reduction in the content of photosynthetic pigments in plants
under hypoxia has been reported in several crops (Florez-Velasco
et al., 2015; Rao and Li, 2003). In this study, hypoxia caused the reduc-
tion in the contents of Chl a, Chl b, total Chl and Car in all cultivars.
The reduction in the chlorophyll content may be due to the increase in
ROS under hypoxia, because the chloroplasts are the main targets of
ROS-linked damage under stress (Khanna-Chopra, 2012). The reduction
in chlorophyll content can also be related to the reduction in the absorp-
tion of nutrients under hypoxia conditions, which is mainly limited by
the shortage of ATP required to fuel the H+ pumps needed to drive
energy-dependent ion transport (Greenway et al., 2012). In plants like
barley, it has been found that one of the main non-stomatal limitations
of photosynthesis is the decrease of photosynthetic pigments in the leaf
(Gregersen et al., 2014). Therefore, leaf chlorosis has been used as an
index of selection of plants susceptible to hypoxia stress in different
crops such as wheat, soybean and barley (Boru et al., 2003; Cai et al.,
1995; Cao et al., 1995; Ikeda et al., 1954; Reyna et al., 2003).
The stability of the cell membrane in plants strongly influences cel-
lular metabolism, and it is modified by the increase in ROS under stress
conditions (McKersie and Leshem, 1994). Here, it was observed that
hypoxia increased the electrolyte leakage in all cultivars suggesting an
increase in the permeability of cell membranes. This decrease in the sta-
bility of the membranes is due to lipid peroxidation caused by ROS,
whose levels increase significantly under hypoxia (Blokhina et al.,
2003). Similar results have been reported for pigeon pea (Cajanus
cajan L.) under oxygen deficiency (Bansal and Srivastava, 2015;
McKersie and Leshem, 1994; Meena et al., 2015). Under waterlogging
conditions, an increase in ROS has also been found due to the production
of O2− by the NADPH oxidase enzyme (Sairam et al., 2011).
205
6 2.0
B 1.8
C
5 2=0.80 r2=0.25
Bachue
1.6
Proline (µmolg–1 FW)
4
1.4 Bianca
Cerinza
3 1.2
2 1.0
NUA35
Bachue NUA35
0.8
1 Bacsta
0.6
Bacsta
0 Bianca 0.4
0.2
0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
WTC WTC
Proline is a nonessential amino acid that, besides being an osmolyte
and antioxidant, has a protective function of macromolecules and cellu-
lar structures (Kavikishor et al., 2005; Paleg et al., 1981; Stewart and Lee,
1974). In this research, it was found that hypoxia caused an increase in
the proline content in the leaves of Bachue, Bianca, Ceriza and NUA35
cultivars. Proline accumulation observed in bean cultivars under hyp-
oxia can avoid the damage caused by the increase in ROS (Aloni and
Rosenshtein, 1982; Paleg et al., 1981). Our results suggest that proline
is one of the defense mechanisms that are induced in four of the culti-
vars evaluated. An increase in proline content has been reported in
waterlogging-tolerant genotypes of Maize (Zea mays L.), jute
(Corchorus capsularis L.), tomato (Solanum lycopersicum L.) and in plants
of several species of the genus Capsicum (Horchani et al., 2010; Ou et al.,
2011; Singh et al., 1985). Proline accumulation has been observed in to-
mato hypoxia-tolerant cultivars and it has been suggested that this mol-
ecule can be used as a source of energy, nitrogen or carbon to reinitiate
growth after the stress period has ended (Aloni and Rosenshtein, 1982).
Tolerance to waterlogging in physiological studies is defined as the
survival or maintenance of growth rates under waterlogging in different
developmental stages (Setter et al., 2003). In this research, cultivars
Cerinza and NUA35 showed a higher WTC, in addition to showing the
lowest reduction in biomass. The lowest reduction in biomass is a trait
that has been associated with tolerance to hypoxia, due to its high her-
itability (Li et al., 2008).
Cultivars Cerinza and NUA35 also showed an increase in WUE under
hypoxia, a trait that has been associated with an efficient stomatal reg-
ulation that could be linked to tolerance under stress (Ou et al., 2011).
Likewise, cultivars Cerinza and NUA35 showed an increase in the pro-
line content higher than cultivar Bacatá, but lower than cultivars Bachue
and Bianca, which presented the lowest WTC. In plants under hypoxia, a
higher WUE generates both a greater potential to develop defense
mechanisms to cope with the stress and a greater potential to growth
after the stress finished; therefore, it generates higher performance
(Ou et al., 2011). Although proline production can be a mechanism to
contend with stress by hypoxia, the production of high amounts of
this amino acid decreases the availability of nitrogen and carbon for
the synthesis of other molecules during stress, and, therefore, can gen-
erate a decrease in growth (Aloni and Rosenshtein, 1982; Kavikishor
et al., 2005). In this study, no correlation was found between the proline
content and WTC, but it was found between the water use efficiency and
WTC.
5. Conclusions
Taken together, these results indicate that genotypes Cerinza and
NUA35 are more tolerant to hypoxic stress than genotypes Bacata,
206 N.F. Velasco et al. / South African Journal of Botany 123 (2019) 200–207
Bianca and Bachue. The tolerance of these genotypes can be conferred
by a higher WUE, a lower reduction in qP and a lower reduction in dry
mass production. Similarly, proline production can be a defense mecha-
nism that decreases the damage caused by hypoxic stress in cellular ho-
meostasis as reported in other plants. The reduction in dry mass
production could be considered as a good trait associated with tolerance
and could be used in breeding programs. The tolerance expression of
these cultivars could be associated with their wide adaptation due to
their broad geographic distribution in the Andean region including
Colombia. Tolerance can also be related to the presence of genes associ-
ated with hypoxia tolerance, so more research is needed to determine if
these genotypes can be new sources of tolerance. Although the number
of genotypes and the number of plants per genotypes evaluated in this
study were reduced, the traits found associated with hypoxia tolerance
could be common to other bean cultivars of the Andean gene pool. In
order to validate the results obtained here, it is important to carry out
other investigations, expanding both the number of genotypes and the
plants evaluated for each one.
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