Journal of Plant Physiology 192 (2016) 13–20

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Journal of Plant Physiology

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

Physiology

Evidence for the involvement of hydraulic root or shoot adjustments

as mechanisms underlying water deficit tolerance in two Sorghum
bicolor genotypes
Moira R. Sutka a , Milena E. Manzur a , Victoria A. Vitali a , Sandra Micheletto b ,
Gabriela Amodeo a,∗
a
Departamento de Biodiversidad y Biología Experimental e Instituto de Biodiversidad y Biología Experimental y Aplicada (IBBEA, CONICET-UBA), Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, Ciudad Universitaria, Pabellón II, (C1428EGA) Buenos Aires,
Argentina
b
CERZOS-CONICET, Camino La Carrindanga Km 7, (8000) Bahía Blanca, Argentina

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

Article history: Sorghum bicolor (L.) Moench is an ancient drought-tolerant crop with potential to sustain high yields
Received 18 August 2015 even in those environments where water is limiting. Understanding the performance of this species in
Received in revised form 6 January 2016 early phenological stages could be a useful tool for future yield improvement programs. The aim of this
Accepted 6 January 2016
work was to study the response of Sorghum seedlings under water deficit conditions in two genotypes
Available online 8 January 2016
(RedLandB2 and IS9530) that are currently employed in Argentina. Morphological and physiological
traits were studied to present an integrated analysis of the shoot and root responses. Although both
Keywords:
genotypes initially developed a conserved and indistinguishable response in terms of drought tolerance
Hydraulic conductance
Seedling physiology
parameters (growth rate, biomass reallocation, etc.), water regulation displayed different underlying
Sorghum bicolor strategies. To avoid water loss, both genotypes adjusted their plant hydraulic resistance at different levels:
Water deficit RedLandB2 regulated shoot resistance through stomata (isohydric strategy), while IS9530 controlled root
resistance (anisohydric strategy). Moreover, only in IS9530 was root hydraulic conductance restricted in
the presence of HgCl2 , in agreement with water movement through cell-to-cell pathways and aquaporins
activity. The different responses between genotypes suggest a distinct strategy at the seedling stage
and add new information that should be considered when evaluating Sorghum phenotypic plasticity in
changing environments.
© 2016 Elsevier GmbH. All rights reserved.

1. Introduction ing studies to be performed in new environmental conditions.

However, variations in climatic conditions, as an increase of air
Sorghum [Sorghum bicolor (L.) Moench.] is an ancient drought- temperature and humidity, could promote the pre-harvest sprout-
tolerant crop grown around the world. This species is remarkable ing while grain maturation is taking place (Steinbach et al., 1997).
for its ability to grow in semiarid and arid regions, even under low In countries that are highly considered as Sorghum producers (i.
or erratic precipitation conditions where other cereal crops are not e. Argentina in Latin America, ca. 60% of total production; FAO,
capable of sustaining high yields. Sorghum is grown for food, feed, 2012), pre-harvest sprouting phenomena has been observed in
fiber and fuel (Undersander et al., 2003; Clark, 2007; FAO, 2012). some inbred lines (Rodríguez et al., 2009). This phenomenon is
The versatility of its final destination together with its capacity related to the interruption of dormancy during seed development
to grow also in environments where water becomes scarce as a in plant and, as a consequence, the induction of germination
consequence of climate change (Morris et al., 2013) are renewing process while still on the parent plant. To avoid this effect, the
the interest in this species and its use as a next-generation biofuel currently sowed genotypes are obtained from parental lines with
(Carpita and McCann, 2008; Saballos et al., 2009) is demand- a different sprouting behavior. Although Sorghum is a drought-
tolerant species (Gholipoor et al., 2012) those genotypes that
had been selected for their performance to deal with pre-harvest
∗ Corresponding author.
sprouting are not completely dissected in terms of their ability to
E-mail address: amodeo@bg.fcen.uba.ar (G. Amodeo).
adjust physiological traits when they are exposed to water deficit

http://dx.doi.org/10.1016/j.jplph.2016.01.002
0176-1617/© 2016 Elsevier GmbH. All rights reserved.
14 M.R. Sutka et al. / Journal of Plant Physiology 192 (2016) 13–20

scenarios. Therefore, our aim is to explore the early plant responses 1.5 mMCa(NO3 )2 , 0.5 mM KH2 PO4 , 50 ␮MFeEDTA, 50 ␮M H3 BO3 ,
to low water availability in two Sorghum parental lines employed 12 ␮M MnSO4 , 0.70 ␮M CuSO4 , 1 ␮M ZnSO4 , 0.24 ␮M Na2 MoO4 ,
in Argentina -RedLandB2 and IS9530- with opposite sprouting and 100 ␮M Na2 SiO3 (Javot et al., 2003). Ten days after germi-
behavior, being RedLandB2 susceptible to pre-harvest sprouting nation seedlings were separated in two groups, one of them was
and IS9530 resistant to such phenomena (Rodriguez, pers. comm.). irrigated periodically and in the other one the irrigation was with-
Crop tolerance in response to drought has been addressed in drawn during 15 days. Except for plant growth rate experiments
terms of modifications on morphology, anatomy and physiology (Fig. S2), seedlings in the three-four leaf stage were used at day
traits (Shanker et al., 2014; Tardieu et al., 2014). There are sev- 15th for all the measurements and finally harvested for biomass
eral studies related to the intraspecific variability in crop species, determinations.
but most of them are focused on a particular physiological trait
(Tardieu and Tuberosa, 2010). For instance, while several works are 2.2. Plant growth rate and leaf area
mainly focused on the stress impact on the aerial part of the plant
(e.g. transpiration rate, leaf area, carbon allocation, photosynthetic Whole seedlings of both Sorghum genotypes were harvested
efficiency; Djanaguiraman et al., 2014; Ogbaga et al., 2014), others every three days (3 seedlings per day per treatment) during 13
are focused on the impact of drought on the root system (e.g. root days. Immediately after each harvesting, plants were divided into
biomass, root elongation rate, root architecture; Passioura, 1988; root and aerial parts and photographed to measure the increase in
Sharp et al., 1988; Rogers and Benfey, 2015). In the present work root and shoot length (cm) as a function of time (days). Root length
our purpose is to study the whole plant physiological response to is represented by the measurement of the longest root of each
water deficit, combining plant hydraulic properties and integrating seedling. Digital images were analyzed using ImageJ 1.48 v soft-
different aspects of the root system. ware (http://rsb.info.nih.gov/ij/). To determine fresh weight (FW)
One of the main challenges of current drought research is of plants, shoots and roots were weighed immediately after cut, and
to elucidate the dynamics of plant hydraulic regulation. At the then samples were dried at 60 ◦ C during 48 h (constant weight) to
whole plant level, the capacity for moving water is represented obtain the dry weight (DW). At the end of treatments, leaf blades
by hydraulic resistance. At shoot level, drought-tolerant plants were photographed with a digital camera to determine leaf area.
can modify transpiration rate adjusting the number and/or sto- Images were processed with the software mentioned above.
matal aperture as well as leaf area. At root level, water uptake is
a highly regulated process as it is strongly affected by low water 2.3. Relative water content (RWC) and soil water content (SWC)
availability in the soil. The anatomy (suberization in exo and endo-
dermis, number and diameter of xylem vessels) and surface area Relative water content (RWC) was determined according to
of roots affect water uptake capacity by modifying root hydraulic Turner (1981). Briefly, leaves of both treated or control seedlings
resistances (Steudle, 2000; Ranathunge and Schreiber, 2011; Lynch were cut and immediately weighted (fresh weight, FW), followed
et al., 2014). In addition, numerous studies have demonstrated the by immersing them during 24 h in distilled water to determine
importance of the cell-to-cell pathway, in particular the role of turgid weight (TW) and finally dried at 60 ◦ C during 48 h to
water channels (i.e. aquaporins) in root water permeability (Javot obtain dry weight (DW). Relative water content was calculated as:
et al., 2003; Li et al., 2014). It has been reported that water channel RWC = (FW-DW)/(TW-DW)*100. Samples of soil were weighted to
gene expression can be regulated by drought, salinity and osmotic obtain fresh weight (SFW) and then dried at 60 ◦ C during 48 h to
stress in Arabidopsis (Seki et al., 2002), rice (Kawasaki et al., 2001), obtain dry weight (SDW). Soil water content was calculated as:
wheat (Ayadi et al., 2011), barley (Katsuhara et al., 2003), maize SWC = [(SFW-SDW)/(SDW)]*100.
(Zhu et al., 2005) and Sorghum (Liu et al., 2014; Reddy et al., 2015).
The aquaporins demand rethinking water relations as a whole, con- 2.4. Stomatal conductance (gs )
sidering the adjustment capacity of the cell-to-cell pathway and its
contribution to the strategy developed by each genotype in water Stomatal conductance was measured using a portable steady
deficit conditions. Understanding the physiological mechanisms state diffusion leaf porometer (model SC-1, Decagon devices, Pull-
associated with drought tolerance in this species at early pheno- man, WA, USA). Measurement was done at center of the last fully
logical stages could be a useful tool for future yield improvement expanded leaf (abaxial face). All the measurements were made
programs (Richards, 2006). Thus, the aim of this work is to study between 10:30 and 11:30 AM. Data were analyzed from 3–7 plants
the response of Sorghum seedlings under water deficit conditions from five independent experiments per treatment.
in the two genotypes RedLandB2 and IS9530. We characterize not
only anatomical and physiological parameters at the whole plant 2.5. Predawn water potential ( h )
level but also analyze the water movement regulationat root and
shoot level. Prior to measurement, whole aerial part (stem, leaf blade and
leaf sheath) were placed in a plastic box covered with Parafilm® and
introduced in a Scholander pressure chamber (Biocontrol Model
2. Materials and methods 4, Argentina) to determine the water potential (Schölander et al.,
1965). Measurements were done predawn in 17 seedlings obtained
2.1. Plant material and culture conditions from six independents experiments.

Experiments were performed using two Sorghum [Sorghum 2.6. Osmotic potential ( osm )
bicolor (L.) Moench] inbred lines, RedLandB2 and IS9530, which
are parental genotypes that originated the regularly sown lines Leaf blade osmotic potential was measured as previously
in Argentina (Rodriguez, pers. comm.). Plants were grown under described by Mahdieh et al. (2008). Briefly, leaf blades were placed
controlled environmental conditions with a 16/8 h light/dark in small column with holes at the bottom and immediately frozen
cycle (light intensity of 205 ± 10 ␮mol m−2 s−1 ), a 60 ± 2.5% rel- in liquid nitrogen. After thawing, the column was placed inside in
ative humidity in a 21 ◦ C growth chamber. Seeds were sown in 1.5 ml centrifuge tube and centrifuged at 4000 × g for 4 min at 4 ◦ C
plastic containers (330 mL) filled with sterilized sand and moist- using a microcentrifuge. Sap osmolarity of each sample was mea-
ened with Hoagland solution: 1.25 mM KNO3 , 0.75 mM MgSO4 , sured in a vapor pressure osmometer (Vapro 5520, Wescor, USA)
 M.R. Sutka et al. / Journal of Plant Physiology 192 (2016) 13–20 15

and used to calculate osmotic pressure according to Van’t Hoff’s

equation.

2.7. Measurement of root hydraulic conductance (Lo )

Root hydraulic conductance measurements were carried out

as described by Miyamoto et al. (2001), with some modifications.
Before starting the measurements, shoots were cut off using razor
blades and roots were washed with tap water. The root system
of freshly detopped Sorghum seedlings was immersed in a 50 mL
container filled with hydroponic culture medium and inserted
into a pressure chamber (Biocontrol Model 4, Argentina). Root-
shoot junction was carefully threaded through the metal lid of the
chamber and sealed using low-viscosity dental paste (A + Silicone,
Densell). The flux of exudates root (Jv ) induced by pressure (P) at
0. 2, 0.3 and 0.4 MPa was determined as described by Matsuo et al.
(2009) with some modifications. At each pressure, exuded sap was
collected at the surface of the cut root-shoot junction for 3 min
on a small piece of tissue paper that was previously pre-weighed.
The tissue paper was then weighed on an analytical balance with
a sensitivity of 0.1 mg. This process was repeated three times for
each pressure. Root hydraulic conductance was obtained from the
linear Jv vs. (P) relationship. To measure the effect of water chan-
nel inhibitors on the Lo , plants were incubated for 10 min with
50 ␮M HgCl2 (Carvajal et al., 1996; Sutka et al., 2011; Zhang and
Tyerman, 1999) before determination of the exudate flow at the
three different pressures.

2.8. Stomatal density and open/close ratio

Stomatal density was determined following techniques

described by Foster (1950). Completely expanded leaf blades were Fig. 1. Shoot:Root biomass ratio (a) fresh (b) and dry (c) biomass of shoot (white
bars) and root (grey bars) for seedlings of two genotypes of Sorghum: RedLandB2
separated and the middle portion of the blade was diaphanized
and IS9530 grown for 15 days under two experimental conditions: control (C) and
with 96% ethanol until all chlorophyll was extracted. Then, blades water deficit (WD). Each bar represents Means ± S.E. of 18 plants (N = 7 independent
were treated with 5% (w/v) NaOH, bleached with 50% (v/v) sodium experiments). Different upper-case letters indicate significant differences (P < 0.05)
hypochlorite and samples were incubated overnight with chloral between genotypes; lower-case letters indicate significant differences between
treatments within each genotype, only for fresh shoot and dry root biomass.
hydrate (Foster, 1950). After that, blades were mounted in gelatin
in two positions: abaxial and adaxial. Two replicates per treatment
and genotype were made. The slides were observed under a light with the InfoStat 2014 package (Di Rienzo et al., 2013). Results are
microscope (Zeiss Axioskop 2, Japan) and a digital camera (Nikon presented as Mean ± S.E. (standard error of the mean). Each sample
E8700, Japan) was used to photograph the samples (between 19 size (n) is indicated in the figure legends. Different upper-case let-
and 28 photographs per treatment). The number and state (open- ters indicate significant differences (P ≤ 0.05) between genotypes;
close) of stomata were measured at 400X in each microphotograph lower-case letters indicate significant differences between treat-
by means of free available software (ImageJ 1.48 v software; http:// ments within each genotype.
rsb.info.nih.gov/ij/).
3. Results
2.9. Xylem vessel number and diameter
3.1. The effect of water deficit on seedling growth is strictly
In order to characterize the anatomy along the roots, xylem ves- conserved in both genotypes
sels were quantified and measured. For this, roots of control and
treated plants (three plants per condition and one root per plant) Seedlings of the two studied genotypes increased root biomass
were finely cut at different positions behind the root apex (0.5, 1, and reduced shoot biomass after 15 days of water deficit as shown
2 and 4 cm) using a razor blade, mounted and observed under a by the shoot:root biomass ratio (Fig. 1a). During the 15 days of
light microscope (Zeiss Axioskop 2, Japan). A digital camera (Nikon treatment, soil water content diminished between 80 and 87% com-
E8700, Japan) was used to photograph the samples. Numbers of pared to the well watered condition (control) for both genotypes
xylem vessels were counted from images and xylem diameter was (Fig. S1). The soil water content was ca. 13% for control condition
measured using the software above mentioned. that is consistent with field capacity values in sandy soil conditions
(Brady, 1990). In accordance with Sorghum tolerance to water limit-
2.10. Statistical analyses ing environments, Sorghum seedlings submitted to such condition
showed no significant differences in any of the parameters mea-
Two-way analysis of variance (ANOVA) was used to analyze the sured during the first seven days when compared to well watered
effects of genotype and water deficit treatment on morphological seedlings (Fig. S2). After this period, both genotypes -RedLandB2
and physiological seedling responses. Post-hoc Tukey’s tests were and IS9530- started to reduce fresh weight and shoot length (Figs.
employed for mean comparisons (P ≤ 0.05). Variable normality and S2a and e.) and significant differences from well watered seedlings
homogeneity of variances were previously verified in order to sat- were observed. The reduction profile of those parameters in roots
isfy ANOVA’s assumptions. All statistical analyses were performed was indistinguishable in the two genotypes (Figs. S2b and f). Both of
16 M.R. Sutka et al. / Journal of Plant Physiology 192 (2016) 13–20

Table 1
Different traits for two Sorghum genotypes: RedLandB2 and IS9530 grown under control or water deficit condition.

Variable RedLandB2 IS9530

Control Water deficit Control Water deficit

Fresh weight (mg) Shoot 540.9 ± 47.6Aa 267.0 ± 33.5Ab 642.5 ± 68.7Aa 263.2 ± 37.1Ab
Root 469.7 ± 38.6Aa 410.8 ± 39.3Aa 551.2 ± 53.3Aa 458.5 ± 50.0Aa
DW (mg) Shoot 480.3 ± 3.7Aa 380.0 ± 3.5Ab 580.2 ± 4.6Aa 420.1 ± 4.6Ab
Root 1020 ± 22.4Aa 2070.2 ± 36.5Ab 1380.7 ± 20.9Aa 2370.3 ± 39.4Ab
Length (cm) Shoot 24.4 ± 1.4Aa 20.1 ± 0.7Ab 26.9 ± 1.0Aa 21.9 ± 0.7Ab
Root 9.6 ± 0.8Aa 10.5 ± 0.7Aa 9.5 ± 0.5Aa 13.3 ± 1.9Aa
Total biomass (DW, mg) 150.3 ± 25.0Aa 245.2 ± 39.4Ab 197.0 ± 21.2Aa 279.3 ± 43.7Ab
Shoot–Root ratio biomass 0.63 ± 0.1Aa 0.22 ± 0.03Ab 0.49 ± 0.1Aa 0.25 ± 0.1Ab
Leaf area (cm2 ) 21. 98 ± 1.1Aa 11.19 ± 1.2Ab 24.38 ± 1.96Aa 11.01 ± 1.5Ab
Water content (mg) 90.9 ± 0.4Aa 84.78 ± 1.9Ab 90.73 ± 0.5Aa 82.07 ± 2.6Ab
Relative water content 96.9 ± 1.3Aa 81.74 ± 7.4Aa 95.82 ± 1.3Aa 70.16 ± 7.5Ab
Osmotic potential (MPa) −0.79 ± 0.03Aa −0.8 ± 0.05Aa −0.79 ± 0.03Aa −0.95 ± 0.2Aa
gs (mmol m−2 s−1 ) 7.14 ± 1.7Aa 2.55 ± 0.7Aa 10.24 ± 1.96Ba 86.87 ± 1.8Ba
Waterpotential (MPa) −0.13 ± 0.02Aa −0.58 ± 0.1 Ab −0.12 ± 0.02Aa −1.23 ± 0.2Bb
Lo (␮l s−1 MPa−1 ) 3.6E-4 ± 4.3E-5Aa 1.1E-4 ± 3.1E-5Ab 6.3E-4 ± 3.3E-5Ba 1.3E-4 ± 4.9E-5Ab

DW: dry weight, gs : stomatal conductance, Lo : hydraulic conductance. Values are Mean ± S.E. of five replicates. Different bold upper-case letters indicate significant differences
(P < 0.05) between genotype; bold lower-case letters indicate significant differences between treatments within each genotype.

them also increased their root dry weight under water deficit (WD)
(Fig. S2d). Therefore, the analysis of the responses of Sorghum to
WD was explored after 15 days of water shortage. In both Sorghum
genotypes, total fresh biomass diminished under WD conditions
due to a reduction of the aerial fresh weight of the plant while root
fresh weight remained without changes (Fig. 1b). While the shoot
dry biomass decreased in both genotypes (Fig. 1c, Table 1), total dry
biomass showed an increase as a consequence of a rise in dry root
biomass. These results demonstrate that both Sorghum seedlings
were able to cut back water supply to the aerial part and sustain the
water status in roots. Seedlings of both Sorghum genotypes showed
a marked decrease only on shoot length (P < 0.05, Fig. 2, upper pan-
els) while this effect was not significant on root length of either
genotype (Fig. 2, bottom panels). Fig. 3. Relative water content of seedlings of two genotypes of Sorghum: RedLandB2
and IS9530, grown for 15 days under two conditions: control (C) and water deficit
(WD). Each bar represents Mean ± S.E. of 18 plants (N = 7 independent experiments).
3.2. Genotypes showed contrasting hydraulics response to water
Different upper-case letters indicate significant differences (P < 0.05) between geno-
deficit in shoots types; lower-case letters indicate significant differences between treatments within
each genotype.
Relative water content (RWC) was studied in both genotypes
of Sorghum as a meaningful index of plant water status. Red-
condition (Fig. 3). However, after 15 days of WD, IS9530 reduced
LandB2 and IS9530 showed similar RWC under well watered
RWC (30%) while RedLandB2 remained unchanged, indicating that
RedLandB2 is able to maintain its water status even in WD condi-
tions. Water (h ) and osmotic potential (osm ) were also measured
in both Sorghum genotypes. As expected, WD reduced signifi-
cantly the water potential in RedLandB2 and IS9530. RedLandB2
showed a four-fold reduction compared to control condition while
IS9530 showed a ten-fold reduction compared to control condition
(Fig. 4a). Interestingly, neither of the genotypes showed differences
in osmotic potential values, for both control and WD conditions
(Fig. 4b), indicating that solute accumulation is not responsible for
the observed reduction in water potential.
In order to study how water deficit affects plant transpira-
tion, we measured two related variables in the aerial part: leaf
area and stomatal conductance. Both RedLandB2 and IS9530 had
similar values of leaf area and stomatal conductance under con-
trol condition (Fig. 5a and b). However, both genotypes reduced
their leaf area under WD conditions, only RedLandB2 decreased
the stomatal conductance, while IS9530 kept conductance values
similar to the control condition (Fig. 5b), which is consistent with
the mentioned reduction in RWC and the higher water potential
Fig. 2. Shoot and root length from seedlings of two genotypes of Sorghum: Red- decreased observed (Figs. 3 and 4a). To further explore the stomata
LandB2 and IS9530, grown for 15 days under two conditions: control (C) and status under WD condition, density and open:close stomatal ratio
water deficit (WD). Each bar represents Mean ± S.E. of 16 plants (N = 6 independent
were also analyzed. Under water deficit conditions the genotypes
experiments). Different upper-case letters indicate significant differences (P < 0.05)
between genotypes; lower-case letters indicate significant differences between showed differences in terms of stomatal density per leaf blade face.
treatments within each genotype. RedLanB2 showed a higher density at the adaxial face while IS9530
 M.R. Sutka et al. / Journal of Plant Physiology 192 (2016) 13–20 17

Fig. 4. Water potential (a) and osmotic potential (b) of seedlings of two Sorghum genotypes, RedLandB2 and IS9530, grown for 15 days under two conditions: control (C) and
water deficit (WD). Each bar represents Mean ± S.E. of 13–24 plants (N = 5–7 independent experiments). Different upper-case letters indicate significant differences (P < 0.05)
between genotypes; lower-case letters indicate significant differences between treatments within each genotype.

Fig. 5. Leaf area (a), stomatal conductance (b), stomatal density (c) and open/close ratio (d) of seedlings from two Sorghum genotypes: RedLandB2 and IS9530, grown for
15 days under two conditions: control (C) and water deficit (WD). Each bar represents Mean ± S.E. of 13–24 plants (N = 5–7 independent experiments). Different upper-case
letters indicate significant differences (P < 0.05) between genotypes; lower-case letters indicate significant differences between treatments within each genotype.

remained unchanged (Fig. 5c). It was observed that RedLandB2 independent experiments, data not shown). The Lo reduction in
was the only genotype that slightly reduced the open:close stoma- IS9530 might reflect the contribution of aquaporins -sensitive to
tal ratio (Fig. 5d). This last result is consistent with the stomatal mercurial compounds- to water movement and therefore the con-
conductance measurements (Fig. 5b). tribution of the root cell-to-cell pathway to water management.
It was also important to verify if the root anatomy (in terms
of vascular system) was affected. We analyzed the number and
3.3. Genotypes showed different participation of root water
diameter of root xylem vessels in both Sorghum genotypes along
radial pathway under water deficit
the length of the root (from 0.5 to 4.0 cm). The number of vessels
increased from the root apex to the base following a similar pat-
Both genotypes equally presented a high Lo in control condi-
tern in both genotypes and treatments (Fig. S3a; P > 0.05). Only the
tions and equally reduced it under WD condition (Fig. 6). However,
diameter of xylem vessels was affected in RedLandB2 but not in
when we measured Lo in the presence of an aquaporin inhibitor
IS9530. Throughout, RedLandB2 showed a reduction of its vessel
(HgCl2 ) in well watered seedlings, only IS9530 showed a signifi-
diameters under WD conditions, much more markedly next to root
cant reduction of Lo (42%; P < 0.05) while, in RedLandB2 Lo remained
tip (0.5 cm; Fig. S3b).
indistinguishable from control conditions. Mercury chloride is the
most common aquaporin inhibitor in plants, although it must been
used with care due to its possible toxicity. In a complementary set of 4. Discussion
experiments performed with seedlings grown in hydroponic condi-
tions, we assayed the effect of propionic acid, as another aquaporin The low shoot:root biomass ratio observed in well-watered con-
inhibitor, in the root hydraulic conductance. Only IS9530 showed ditions (Fig. 1a) may be due to the ontogenetic stage of plants, since
a reduction of 48% compare to the control condition, similar to 42% during the early growth more assimilates are allocated to roots
obtained in the experiments with HgCl2 (n = 5–16 plants from 3 in annual plants and, as development continues, the dry matter
18 M.R. Sutka et al. / Journal of Plant Physiology 192 (2016) 13–20

allocation changes to aerial parts (Gregory et al., 1997). The initial nance of low stomatal conductance -under high VPD and low soil
plant response to drought is the inhibition of shoot growth and water potential conditions- could be advantageous under water
the maintenance of root growth, as an adaptive response to main- limited environments (Choudhary et al., 2013). However, in our
tain the water uptake and reduce water loss by transpiration (Wu approach, the low VPD (data not shown) and the soil water content
and Cosgrove, 2000; Sinclair et al., 2005). For some annual crops, (Fig. S1) was the same for the two tested genotypes. Transpiration
dry biomass accumulation follows a sigmoid pattern in root sys- rate depends on both stomatal conductance and root water absorp-
tems and also it is reported in shoots, although the both phases of tion, both processes involved in maintenance of water homeostasis
growth may not match exactly (Gregory, 2006). In general, a short- (Pittermann, 2010). Then, plants have to minimize water loss when
age of resources in the root environment -as water deficit- causes the environmental conditions are compromising water uptake
changes in biomass assimilation patterns, favoring the root sys- (Hommel et al., 2014). Here, one of the differences found between
tem growth (Brouwer, 1963). Thus, it can be expected that there is genotypes lies in the physiological strategy to adjust shoot-water
a displacement in the biomass reallocation pattern between root demand, suggesting a refined mechanism in water movement reg-
and shoot under water deficit conditions. ulation. Although, RedLandB2 was able to increase the stomatal
As expected for a tolerant crop, seedlings of both Sorghum density in the leaf abaxial face (Fig. 5c), the strategy is to reduce
genotypes showed a conserved response under limiting water con- the number of open stomata, and thus decrease the stomatal con-
ditions (Muller et al., 2011). Ours experiments were done at an ductance under water deficit (Fig. 5b and d). There is an agreement
early stage (25 days old, 3–4 expanded leaves seedlings), so it that the different strategies developed for plants to deal with
is not expected that the final state of the biomass accumulation drought can be merged into two contrasting behaviors: anisohydric
pattern is achieved (Fig. S2). At that stage, there was a marked and isohydric (Klein, 2014; Martínez-Vilalta et al., 2014). Anisohy-
increase in root biomass with a significant impact on the total dric plants sustain transpiration rate even in stressful conditions,
plant biomass, as root:total biomass ratio indicated (0.68–0.85 decreasing water potential (h ) and relative water content (RWC).
and 0.70–0.85 for RedLandB2 and IS9530, respectively). The root By contrast, under the same scenario, isohydric plants reduce tran-
growth maintenance in water deficit conditions is a clear bene- spiration, maintaining constant h and RWC (Moshelion et al.,
fit to provide plant water supply as it has been previously reported 2015). In this framework, the employed Sorghum genotypes could
(Sharp and Davies, 1979; Malik et al., 1979; Meyer and Boyer, 1981; be associated with such distinct strategies, considering IS9530 as
van der Weele et al., 2000). Interestingly, both genotypes maintain anisohydric and RedLandB2 as isohydric.
their biomass allocation response to water deficit, although they Focusing on water uptake by roots, its capacity to manage water
developed different strategies to water movement regulation, as under deficit conditions could be defined by different cues that
discussed below. These results are in accordance with those pub- might determine the main pathway for water transport. It is well
lished by Fracasso et al. (2016) where four genotypes of Sorghum, known that water transport across roots occur through three dif-
with different tolerance, were screened under drought stress con- ferent pathways: apoplastic, symplastic and transcellular (Steudle
ditions. et al., 1993; Steudle, 2000). The latter two pathways are normally
referred as cell-to-cell pathway, due to the fact that it is impossible
4.1. Both genotypes of Sorghum showed different strategies to to experimentally dissect them. Water transport along the root can
deal with water deficit be characterized by measuring its hydraulic conductance (Tyree,
2003), which can be modified under stress. Our results demon-
Under water deficit conditions, RedLandB2 maintained the rel- strate that both Sorghum genotypes studied here reduced their root
ative water content of leaves by means of reducing the water hydraulic conductance when soil water availability was diminished
potential, stomatal conductance and the open:close stomata ratio (Fig. 6). Dissecting water pathways allowed us to analyze its main
(Figs. 3, 4 and 5). By contrast, IS9530 reduced its relative water con- components as discussed below.
tent (Fig. 3) and water potential (ca. 10 times more than Control) First, mercurial compounds are normally used to evaluate the
but maintained its stomatal conductance and a high open:close contribution of cell-to-cell pathway to root water transport, i.e.
stomata ratio (Figs. 4 and 5). A reduction in transpiration rate is aquaporin activity (Carvajal et al., 1996; Sutka et al., 2011; Zhang
considered a typical response to drought stress (Gholipoor et al., and Tyerman, 1999). In Sorghum genotypes, only IS9530 showed
2010). In particular for Sorghum, it was reported that the mainte- a hydraulic conductance that was significantly inhibited by HgCl2
(Fig. 6) indicating that the cell-to-cell pathway might contribute
in adjusting the root water transport capacity in this genotype. In
contrast, RedLandB2 maintained Lo unchanged even when roots
were incubated in HgCl2 (Fig. 6). These results are consistent with
the idea that RedLandB2 seedling deals with water deficit mod-
ulating the water status of the aerial part. Recently, the use of
mercurial compounds revealed the participation of aquaporins in
the root hydraulic conductance in Sorghum plants (Choudhary et al.,
2013; Liu et al., 2014). Two Sorghum lines (SC15 and SC1205), with
dissimilar leaf and root conductance (Kleaf , Lo ), showed different
transpiration rate when they were exposed to HgCl2 . The authors
interpreted this response as a consequence of the impact of mercu-
rial compound on roots, and it is consistent with the fact that SC15
has a larger number of water channels in its roots than the other line
Fig. 6. Properties of root water dynamics: the effect of water deficit and HgCl2 on
root hydraulic conductance (Lo ) of seedlings from two Sorghum genotypes, Red- (Choudhary et al., 2013). Liu et al. (2014) studied the effect of silicon
LandB2 and IS9530. Seedlings were grown under control (C) and water deficit (WD) application in the amelioration of water loss during osmotic stress.
conditions, and incubated with HgCl2 (Hg). In those experiments performed in In their case, application of HgCl2 decreased the transpiration rate
the presence of 50 ␮M HgCl2 , seedlings were pre-incubated ten minutes before Lo of seedlings and by silicon application increased the transcription
measurements. Each bar represents Means ± S.E. of 9–12 plants (N = 5 independent
levels of several root aquaporin genes. Thus, the use of mercurial
experiments). Different upper-case letters indicate significant differences (P < 0.05)
between genotypes; lower-case letters indicate significant differences between compounds to reveal the aquaporin participation in the root water
treatments within each genotype. movement in this specie is also effective. Moreover, Sorghum aqua-
 M.R. Sutka et al. / Journal of Plant Physiology 192 (2016) 13–20 19

porins have been identified, and their expression under several resistance. We also discuss the importance of the cellular pathway
abiotic stresses was also reported (Reddy et al., 2015). Thus, the for IS9530, associated with water channel activity.
drop in Lo observed in IS9530, in the presence of mercurial com- In summary, this work adds evidence in relation to water
pounds, could be strongly related to the role of aquaporins in the management of Sorghum species, highlighting the whole plant
cellular pathway. responses to soil water deficit. We integrated the shoot and root
Second, xylem anatomy could provide us some clue of the con- morphological and physiological responses, including the cellu-
tribution of each pathway when comparing genotypes (for both lar pathway of water movement when seedlings were exposed
control and water deficit conditions). The number of xylem ves- to water deficit conditions. The different strategies developed by
sels showed the same pattern in both genotypes and conditions both Sorghum genotypes could be an interesting attribute of this
(Fig. S3a). However, the diameter of xylem vessels was reduced crop species, since they can adjust the water management at dif-
in RedLandB2 when it was exposed to water limiting conditions ferent levels. This performed approach adds new information for
(Fig. S3b), while IS9530 showed smaller xylem vessels than Red- Sorghum which can be used to evaluate phenotypic plasticity for
LandB2 in all conditions. These results are in accordance with Cruz changing environments. Our result provides also new insight in
et al. (1992) where the diameter of root xylem vessels diminished in terms of anisohydric and isohydric strategies that can be devel-
water deficit conditions. These could explain the Lo drop (increase oped by plants in relation to water management. However, an
of root hydraulic resistance) observed in RedLandB2 and it could exhaustive study to address the physiological/molecular mech-
be probably associated with an extra suberin deposition in the root anisms triggered in response to water deficit is still necessary,
exodermis (Passioura, 1982), as reported for other species with including in this scenario the role of aquaporins. Besides, our
similar anatomical properties (Schreiber et al., 2005). However, in approach make available new evidences related to the performance
IS9530 the response is different, since there are no changes in the of Sorghum seedlings of two genotypes with contrast sprouting
number and diameter of the xylem vessels. This could indicate that behavior grown in water limited conditions. This information could
the cellular pathway could make a difference in terms of water be use to improve crop yield in those environments where the soil
balance. water content is low and the risk of sprouting phenomena is low
too.

4.2. Differential contribution of root to plant hydraulic resistance

Acknowledgements

The plant hydraulic adjustments can be explored analyzing the

We thank M. V. Rodriguez from Laboratorio de Biología de Semil-
contribution of root and aerial components to hydraulic resistance.
las IFEVA (CONICET-FAUBA), for access to seed of the genotypes
In order to discuss this water management, different parameters
used in the experiments. This research was supported by grants
have been considered: soil water content (SWC), stomatal con-
from UBACyT1417; PIP-CONICET 2012 and FONCyT (PICT11-2239
ductance (gs ) and root hydraulic conductance (Lo ). Plants of both
and PICT14-0744), all grants to GA. MRS and GA are members of
genotypes cope with the same intensity of water deficit (i.e. the
the National Research Council of Argentina (CONICET).
same pattern of soil water loss, Fig. S1, same VPD) regulating their
water management at different points. If the stomatal conduc-
tance (gs ) or root hydraulic conductance (Lo ) are reduced, then Appendix A. Supplementary data
there is an increase in the total hydraulic resistance (Lambers
et al., 2008). In RedLandB2, total hydraulic resistance is given at Supplementary data associated with this article can be found,
aerial level, as stomatal conductance indicates, but not in IS9530 in the online version, at http://dx.doi.org/10.1016/j.jplph.2016.01.
(Fig. 5), where the root seems to be more important in water move- 002.
ment. In water deficit conditions, both genotypes reduced root
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