Accepted Manuscript

Title: Evidence for the involvement of hydraulic root or shoot

adjustments as mechanisms underlying water deficit tolerance
in two Sorghum bicolor genotypes

Author: Moira R. Sutka Milena E. Manzur Victoria A. Vitali

Sandra Micheletto Gabriela Amodeo

PII: S0176-1617(16)00004-3
DOI: http://dx.doi.org/doi:10.1016/j.jplph.2016.01.002
Reference: JPLPH 52281

To appear in:

Received date: 18-8-2015

Revised date: 6-1-2016
Accepted date: 6-1-2016

Please cite this article as: Sutka Moira R, Manzur Milena E, Vitali Victoria A, Micheletto
Sandra, Amodeo Gabriela.Evidence for the involvement of hydraulic root or shoot
adjustments as mechanisms underlying water deficit tolerance in two Sorghum bicolor
genotypes.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2016.01.002

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 

Evidence for the involvement of hydraulic root or shoot adjustments as mechanisms underlying water deficit
tolerance in two Sorghum bicolor genotypes

Moira R. Sutka1, Milena E. Manzur1, Victoria A. Vitali1, Sandra Micheletto2, Gabriela Amodeo1*

1
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.  
2
CERZOS-CONICET, Camino La Carrindanga Km 7, (8000) Bahía Blanca, Argentina.

*
Corresponding author. Email: [email protected]

1 
 
 

Abstract
Sorghum bicolor (L.) Moench is an ancient drought-tolerant crop with potential to sustain high yields even in those
environments where water is limiting. Understanding the performance of this species in early phenological stages could
be a useful tool for future yield improvement programs. The aim of this work was to study the response of Sorghum
seedlings under water deficit conditions in two genotypes (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 genotypes initially developed a conserved and indistinguishable response in terms of drought
tolerance parameters (growth rate, biomass reallocation, etc.), water regulation displayed different underlying
strategies. To avoid water loss, both genotypes adjusted their plant hydraulic resistance at different levels: 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.  
 
 
Keywords: hydraulic conductance, seedling physiology, Sorghum bicolor, water deficit

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

2 
 
 

Introduction 
Sorghum [Sorghum bicolor (L. ) Moench.] is an ancient drought-tolerant crop grown around the world. This species is
remarkable for its ability to grow in semiarid and arid regions, even under low or erratic precipitation conditions where
other cereal crops are not capable of sustaining high yields. Sorghum is grown for food, feed, fiber and fuel
(Undersander et al., 2003; Clark 2007; FAO 2012). The versatility of its final destination together with its capacity to
grow also in environments where water becomes scarce as a consequence of climate change (Morris et al., 2013) are
renewing the interest in this species and its use as a next-generation biofuel (Carpita and McCann 2008; Saballos 2008)
is demanding studies to be performed in new environmental conditions. However, variations in climatic conditions, as
an increase of air temperature and humidity, could promote the pre-harvest sprouting while grain maturation is taking
place (Steinbach et al., 1997). In countries that are highly considered as Sorghum producers (i. e. Argentina in Latin
America, ca. 60% of total production; FAO 2012), pre-harvest sprouting phenomena has been observed in some inbred
lines (Rodriguez et al., 2009). This phenomenon is related to the interruption of dormancy during seed development in
plant and, as a consequence, the induction of germination process while still on the parent plant. To avoid this effect,
the currently sowed genotypes are obtained from parental lines with 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 sprouting are not completely dissected in terms of their ability to adjust physiological traits when
they are exposed to water deficit scenarios. Therefore, our aim is to explore the early plant responses to low water
availability in two Sorghum parental lines employed in Argentina -RedLandB2 and IS9530- with opposite sprouting
behavior, being RedLandB2 susceptible to pre-harvest sprouting and IS9530 resistant to such phenomena (Rodriguez,
pers. comm.).  
Crop tolerance in response to drought has been addressed in terms of modifications on morphology, anatomy
and physiology traits (Shanker et al., 2014; Tardieu et al., 2014). There are several studies related to the intraspecific
variability in crop species, but most of them are focused on a particular physiological trait (Tardieu and Tuberosa
2010). For instance, while several works are mainly focused on the stress impact on the aerial part of the plant (e. g.
transpiration rate, leaf area, carbon allocation, photosynthetic efficiency; Djanaguiraman et al., 2014; Ogbaga et al.,
2014), others are focused on the impact of drought on the root system (e. g. root biomass, root elongation rate, root
architecture; Passioura1988a; Sharp et al., 1988; Rogers and Benfey 2015). In the present work our purpose is to study
the whole plant physiological response to water deficit, combining plant hydraulic properties and integrating different
aspects of the root system.  
One of the main challenges of current drought research is to elucidate the dynamics of plant hydraulic
regulation. At the whole plant level, the capacity for moving water is represented by hydraulic resistance. At shoot
level, drought-tolerant plants can modify transpiration rate adjusting the number and/or stomatal aperture as well as leaf
area. At root level, water uptake is a highly regulated process as it is strongly affected by low water availability in the
soil. The anatomy (suberization in exo and endodermis, number and diameter of xylem vessels) and surface area of
roots affect water uptake capacity by modifying root hydraulic resistances (Steudle 2000; Ranathunge and Schreiber,
2011; Lynch et al., 2014). In addition, numerous studies have demonstrated the importance of the cell-to-cell pathway,
3 
 
 

in particular the role of water channels (i.e. aquaporins) in root water permeability (Javot et al., 2003; Li et al., 2014). It
has been reported that water channel gene expression can be regulated by drought, salinity and osmotic stress in
Arabidopsis (Seki et al., 2002), rice (Kawasaki et al., 2001), wheat (Ayadi et al., 2011), barley (Katsuhara et al., 2003),
maize (Zhu et al., 2005) and Sorghum (Liu et al., 2014; Reddy et al., 2015). The aquaporins demand rethinking water
relations as a whole, considering the adjustment capacity of the cell-to-cell pathway and its contribution to the strategy
developed by each genotype in water deficit conditions. Understanding the physiological mechanisms associated with
drought tolerance in this species at early phenological stages could be a useful tool for future yield improvement
programs (Richards 2006). Thus, the aim of this work is to study the response of Sorghum seedlings under water deficit
conditions in the two genotypes RedLandB2 and IS9530. We characterize not only anatomical and physiological
parameters at the whole plant level but also analyze the water movement regulationat root and shoot level.

Materials and methods  

Plant material and culture conditions 
Experiments were performed using two Sorghum [Sorghum bicolor (L.) Moench] inbred lines, RedLandB2 and IS9530,
which are parental genotypes that originated the regularly sown lines in Argentina (Rodriguez, pers. comm.). Plants
were grown under controlled environmental conditions with a 16/8 h light/dark cycle (light intensity of 205 ± 10 µmol
m-2 s-1), a 60 ± 2.5% relative humidity in a 21ºC growth chamber. Seeds were sown in plastic containers (330 mL) filled
with sterilized sand and moistened with Hoagland solution: 1.25 mM KNO3, 0.75 mM MgSO4, 1.5 mMCa(NO3)2, 0.5
mM KH2PO4, 50 μMFeEDTA, 50 μM H3BO3, 12 μM MnSO4, 0.70 μM CuSO4, 1 μM ZnSO4, 0.24 μM Na2MoO4, and
100 μM Na2SiO3 (Javot et al., 2003). Ten days after germination seedlings were separated in two groups, one of them
was irrigated periodically and in the other one the irrigation was withdrawn during 15 days. Except for plant growth
rate experiments (Fig S2), seedlings in the three-four leaf stage were used at day 15th for all the measurements and
finally harvested for biomass determinations.  
 
Plant growth rate and leaf area 
Whole seedlings of both Sorghum genotypes were harvested every three days (3 seedlings per day per treatment) during
13 days. Immediately after each harvesting, plants were divided into root and aerial parts and photographed to measure
the increase in root and shoot length (cm) as a function of time (days). Root length is represented by the measurement
of the longest root of each seedling. Digital images were analyzed using ImageJ 1.48v software (http://rsb. info. nih.
gov/ij/). To determine fresh weight (FW) of plants, shoots and roots were weighed immediately after cut, and then
samples were dried at 60 ºC during 48 h (constant weight) to obtain the dry weight (DW). At the end of treatments, leaf
blades were photographed with a digital camera to determine leaf area. Images were processed with the software
mentioned above.  
 

4 
 
 

 
Relative water content (RWC) and soil water content (SWC) 
Relative water content (RWC) was determined according to Turner (1981). Briefly, leaves of both treated or control
seedlings were cut and immediately weighted (fresh weight, FW), followed by immersing them during 24h in distilled
water to determine turgid weight (TW) and finally dried at 60 ºC during 48 h to obtain dry weight (DW). Relative water
content was calculated as: RWC = (FW-DW)/(TW-DW)*100. Samples of soil were weighted to obtain fresh weight
(SFW) and then dried at 60 ºC during 48 h to obtain dry weight (SDW). Soil water content was calculated as: SWC =
[(SFW-SDW)/(SDW)]*100.  
 
Stomatal conductance (gs)  
Stomatal conductance was measured using a portable steady state diffusion leaf porometer (model SC-1, Decagon
devices, Pullman, WA, USA). Measurement was done at center of the last fully expanded leaf (abaxial face). All the
measurements were made between 10:30 and 11:30 AM. Data were analyzed from 3-7 plants from five independent
experiments per treatment.  
 
Predawn water potential (Ψh) 
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 4, Argentina) to determine the water
potential (Scholander et al., 1965). Measurements were done predawn in 17 seedlings obtained from six independents
experiments.  
 
Osmotic potential (Ψosm) 
Leaf blade osmotic potential was measured as previously described by Madhied et al., (2008). Briefly, leaf blades were
placed in small column with holes at the bottom and immediately frozen in liquid nitrogen. After thawing, the column
was placed inside in 1.5 ml centrifuge tube and centrifuged at 4000g for 4 min at 4ºC using a microcentrifuge. Sap
osmolarity of each sample was measured in a vapor pressure osmometer (Vapro 5520, Wescor, USA) and used to
calculate osmotic pressure according to Van’t Hoff’s equation.  
 
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

5 
 
 

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 channel inhibitors on the Lo, plants were incubated for 10 minutes with 50 µM HgCl2 (Carvajal et al., 1996; Sutka
et al., 2011; Zhang and Tyerman 1999) before determination of the exudate flow at the three different pressures.  
 
Stomatal density and open/close ratio 
Stomatal density was determined following techniques described by Foster (1950). Completely expanded leaf blades
were separated and the middle portion of the blade was diaphanized with 96% ethanol until all chlorophyll was
extracted. Then, blades were treated with 5% (w/v) NaOH, bleached with 50% (v/v) sodium hypochlorite and samples
were incubated overnight with chloral 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 microscope (Zeiss Axioskop 2, Japan) and a digital camera (Nikon E8700, Japan) was used to photograph the
samples (between 19 and 28 photographs per treatment). The number and state (open-close) of stomata were measured
at 400X in each microphotograph by means of free available software (ImageJ 1.48v software; http://rsb. info. nih.
gov/ij/).  
 
Xylem vessel number and diameter 
In order to characterize the anatomy along the roots, xylem vessels were quantified and measured. For this, roots of
control and treated plants (three plants per condition and one root per plant) were finely cut at different positions behind
the root apex (0.5, 1, 2 and 4 cm) using a razor blade, mounted and observed under a light microscope (Zeiss Axioskop
2, Japan). A digital camera (Nikon E8700, Japan) was used to photograph the samples. Numbers of xylem vessels were
counted from images and xylem diameter was measured using the software above mentioned.  

Statistical analyses 
Two-way analysis of variance (ANOVA) was used to analyze the effects of genotype and water deficit treatment on
morphological and physiological seedling responses. Post-hoc Tukey's tests were employed for mean comparisons (P ≤
0. 05). Variable normality and homogeneity of variances were previously verified in order to satisfy ANOVA's
assumptions. All statistical analyses were performed with the InfoStat 2014 package (Di Rienzo et al., 2013). Results
are presented as mean ± s.e. (standard error of the mean). Each sample size (n) is indicated in the figure legends.
Different upper-case letters indicate significant differences (P ≤ 0.05) between genotypes; lower-case letters indicate
significant differences between treatments within each genotype.

6 
 
 

Results 
The effect of water deficit on seedling growth is strictly conserved in both genotypes 
Seedlings of the two studied genotypes increased root biomass and reduced shoot biomass after 15 days of water deficit
as shown by the shoot:root biomass ratio (Fig. 1a). During the 15 days of treatment, soil water content diminished
between 80-87% compared to the well watered condition (control) for both genotypes (Fig. S1). The soil water content
was ca. 13% for control condition that is consistent with field capacity values in sandy soil conditions (Brady 1990). In
accordance with Sorghum tolerance to water limiting environments, Sorghum seedlings submitted to such condition
showed no significant differences in any of the parameters measured during the first seven days when compared to well
watered seedlings (Fig. S2). After this period, both genotypes -RedLandB2 and IS9530- started to reduce fresh weight
and shoot length (Figs. S2a, S2e.) and significant differences from well watered seedlings were observed. The
reduction profile of those parameters in roots was indistinguishable in the two genotypes (Figs. S2b, S2f). Both of 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 panels) while this
effect was not significant on root length of either genotype (Fig. 2, bottom panels).  

Genotypes showed contrasting hydraulics response to water deficit in shoots 

Relative water content (RWC) was studied in both genotypes of Sorghum as a meaningful index of plant water status.
RedLandB2 and IS9530 showed similar RWC under well watered condition (Fig. 3). However, after 15 days of WD,
IS9530 reduced RWC (30%) while RedLandB2 remained unchanged, indicating that RedLandB2 is able to maintain its
water status even in WD conditions. Water (Ψh) and osmotic potential (Ψosm) were also measured in both Sorghum
genotypes. As expected, WD reduced significantly 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 transpiration, 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 control condition (Figs. 5a, 5b). 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
decreased observed (Figs. 3, 4a). To further explore the stomata status under WD condition, density and open:close
7 
 
 

stomatal ratio were also analyzed. Under water deficit conditions the genotypes showed differences in terms of stomatal
density per leaf blade face. RedLanB2 showed a higher density at the adaxial face while IS9530 remained unchanged
(Fig. 5c). It was observed that RedLandB2 was the only genotype that slightly reduced the open:close stomatal ratio
(Fig. 5d). This last result is consistent with the stomatal conductance measurements (Fig. 5b).  
 
Genotypes showed different participation ofroot water radial pathway under water deficit 
Both genotypes equally presented a high Lo in control conditions and equally reduced it under WD condition (Fig. 6).
However, when we measured Lo in the presence of an aquaporin inhibitor (HgCl2) in well watered seedlings, only
IS9530 showed a significant reduction of Lo (42%; P < 0.05) while, in RedLandB2 Lo remained 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 experiments performed with seedlings grown in hydroponic
conditions, we assayed the effect of propionic acid, as another aquaporin inhibitor, in the root hydraulic conductance.
Only IS9530 showed a reduction of 48% compare to the control condition, similar to 42% obtained in the experiments
with HgCl2 (n=5-16 plants from 3 independent experiments, data not shown). The Lo reduction in IS9530 might reflect
the contribution of aquaporins -sensitive to mercurial compounds- to water movement and therefore the contribution 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 diameter of root xylem vessels in both Sorghum genotypes along 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 pattern in both genotypes and
treatments (Fig. S3a; P > 0.05). Only the diameter of xylem vessels was affected in RedLandB2 but not in IS9530.
Throughout, RedLandB2 showed a reduction of its vessel diameters under WD conditions, much more markedly next
to root tip (0.5 cm; Fig. S3b).  

Discussion  
The low shoot:root biomass ratio observed in well-watered conditions (Fig. 1a) may be due to the ontogenetic stage of
plants, since during the early growth more assimilates are allocated to roots in annual plants and, as development
continues, the dry matter allocation changes to aerial parts (Gregory et al., 1997). The initial plant response to drought
is the inhibition of shoot growth and the maintenance of root growth, as an adaptive response to maintain the water
uptake and reduce water loss by transpiration (Wu and Cosgrove, 2000; Sinclair et al., 2005). For some annual crops,
dry biomass accumulation follows a sigmoid pattern in root systems and also it is reported in shoots, although the both
phases of growth may not match exactly (Gregory, 2006). In general, a shortage of resources in the root environment -
as water deficit- causes changes in biomass assimilation patterns, favoring the root system growth (Brouwer, 1963).
Thus, it can be expected that there is a displacement in the biomass reallocation pattern between root and shoot under
water deficit conditions.
As expected for a tolerant crop, seedlings of both Sorghum genotypes showed a conserved response under
limiting water conditions (Muller et al., 2011). Ours experiments were done at an early stage (25 days old, 3-4
8 
 
 

expanded leaves seedlings), so it is not expected that the final state of the biomass accumulation pattern is achieved
(Fig S2). At that stage, there was a marked increase in root biomass with a significant impact on the total plant biomass,
as root:total biomass ratio indicated (0.68 to 0.85 and 0.70 to 0.85 for RedLandB2 and IS9530, respectively). The root
growth maintenance in water deficit conditions is a clear benefit to provide plant water supply as it has been previously
reported (Sharp and Davies, 1979; Malik et al., 1979; Meyer and Boyer 1981, van der Weel et al., 2000). Interestingly,
both genotypes maintain their biomass allocation response to water deficit, although they developed different strategies
to water movement regulation, as discussed below. These results are in accordance with those published by Fracasso et
al., (2016) where four genotypes of Sorghum, with different tolerance, were screened under drought stress conditions.
 
Both genotypes of Sorghum showed different strategies to deal with water deficit  
Under water deficit conditions, RedLandB2 maintained the relative water content of leaves by means of reducing the
water potential, stomatal conductance and the open:close stomata ratio (Figs. 3, 4 and 5). By contrast, IS9530 reduced
its relative water content (Fig. 3) and water potential (ca. 10 times more than Control) but maintained its stomatal
conductance and a high open:close stomata ratio (Figs. 4 and 5). A reduction in transpiration rate is considered a typical
response to droughts tress (Gholipoor et al., 2010). In particular for Sorghum, it was reported that the maintenance of
low stomatal conductance -under high VPD and low soil water potential conditions- could be advantageous under water
limited environments (Choudhary et al., 2013). However, in our approach, the low VPD (data not shown) and the soil
water content (Fig. S1) was the same for the two tested genotypes. Transpiration rate depends on both stomatal
conductance and root water absorption, both processes involved in maintenance of water homeostasis (Pittermann,
2010). Then, plants have to minimize water loss when the environmental conditions are compromising water uptake
(Hommel et al., 2014). Here, one of the differences found between genotypes lies in the physiological strategy to adjust
shoot-water demand, suggesting a refined mechanism in water movement regulation. Although, RedLandB2 was able
to increase the stomatal density in the leaf abaxial face (Fig. 5c), the strategy is to reduce the number of open stomata,
and thus decrease the stomatal conductance under water deficit (Fig. 5b, 5d). There is an agreement that the different
strategies developed for plants to deal with drought can be merged into two contrasting behaviors: anisohydric and
isohydric (Klein T, 2014; Martinez-Vilalta et al., 2014). Anisohydric plants sustain transpiration rate even in stressful
conditions, decreasing water potential (Ψh) and relative water content (RWC). By contrast, under the same scenario,
isohydric plants reduce transpiration, maintaining constant Ψhand RWC (Moshelion et al., 2015). In this framework, the
employed Sorghum genotypes could be associated with such distinct strategies, considering IS9530 as anisohydric and
RedLandB2 as isohydric.  
Focusing on water uptake by roots, its capacity to manage water under deficit conditions could be defined by
different cues that might determine the main pathway for water transport. It is well known that water transport across
roots occur through three different pathways: apoplastic, symplastic and transcellular (Steudle 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 to
experimentally dissect them. Water transport along the root can be characterized by measuring its hydraulic
conductance (Tyree 2003), which can be modified under stress. Our results demonstrate that both Sorghum genotypes
9 
 
 

studied here reduced their root hydraulic conductance when soil water availability was diminished (Fig. 6). Dissecting
water pathways allowed us to analyze its main components as discussed below.  
First, mercurial compounds are normally used to evaluate the contribution of cell-to-cell pathway to root water
transport, i.e. aquaporin activity (Carvajal et al., 1996; Sutka et al., 2011; Zhang and Tyerman, 1999). In Sorghum
genotypes, only IS9530 showed 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 modulating 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 mercurial 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 (Choudhary et al., 2013). Liu and coworkers
(2014) studied the effect of silicon application in the amelioration of water loss during osmotic stress. In their case,
application of HgCl2 decreased the transpiration rate of seedlings and by silicon application increased the transcription
levels of several root aquaporin genes. Thus, the use of mercurial compounds to reveal the aquaporin participation in
the root water movement in this specie is also effective. Moreover, Sorghum aquaporins have been identified, and their
expression under several abiotic stresses was also reported (Reddy et al., 2015). Thus, the drop in Lo observed in
IS9530, in the presence of mercurial compounds, could be strongly related to the role of aquaporins in the cellular
pathway.  
Second, xylem anatomy could provide us some clue of the contribution of each pathway when comparing
genotypes (for both control and water deficit conditions). The number of xylem vessels showed the same pattern in both
genotypes and conditions (Fig. S3a). However, the diameter of xylem vessels was reduced in RedLandB2 when it was
exposed to water limiting conditions (Fig. S3b), while IS9530 showed smaller xylem vessels than RedLandB2 in all
conditions. These results are in accordance with Cruz et al., (1992) where the diameter of root xylem vessels
diminished in water deficit conditions. These could explain the Lo drop (increase of root hydraulic resistance) observed
in RedLandB2 and it could be probably associated with an extra suberin deposition in the root exodermis (Passioura,
1982), as reported for other species with similar anatomical properties (Schreiber et al., 2005). However, in IS9530 the
response is different, since there are no changes in the number and diameter of the xylem vessels. This could indicate
that the cellular pathway could make a difference in terms of water balance.

Differential contribution of root to plant hydraulic resistance

The plant hydraulic adjustments can be explored analyzing the contribution of root and aerial components to hydraulic
resistance. In order to discuss this water management, different parameters have been considered: soil water content
(SWC), stomatal conductance (gs) and root hydraulic conductance (Lo). Plants of both genotypes cope with the same
intensity of water deficit (i. e. the same pattern of soil water loss, Fig S1, same VPD) regulating their water
management at different points. If the stomatal conductance (gs) or root hydraulic conductance (Lo) are reduced, then
10 
 
 

there is an increase in the total hydraulic resistance (Lambers et al., 2008). In RedLandB2, total hydraulic resistance is
given at aerial level, as stomatal conductance indicates, but not in IS9530 (Fig. 5), where the root seems to be more
important in water movement. In water deficit conditions, both genotypes reduced root hydraulic conductance (Fig. 6)
increasing the resistance to water movement. RedLandB2 also showed a reduction in stomatal conductance (reducing
the number of open stomata, Fig. 5b and 5d) having a resistance larger than IS9530. The response of RedLandB2 could
be associated with the capacity of this genotype to anticipate the imminent reduction in water availability. However, it
could be useful to know the related genes associated to the early response to drought stress, as those described by Pasini
et al., (2014). Therefore, RedLandB2 could use water in a conservative way, delaying the water deficit effect (Siqueira
et al., 2008), while IS9530 would be regulating at root level by modulating aquaporins activity. It seems likely that
plants of IS9530 could have a decoupling between roots and aerial part, which could be reflected on the interaction
between absorption and transpiration.

Conclusions
The differential response found between Sorghum genotypes implicates a distinctive plasticity on water management
when they grow under water limiting conditions at seedling stage. On one hand, RedLandB2 is able to respond to soil
water restriction earlier in stomatal conductance than IS9530, which do not close its stomata during the 15 days of
experiments. On the other hand, IS9530 modulates root water uptake by slightly increasing total hydraulic resistance.
We also discuss the importance of the cellular pathway for IS9530, associated with water channel activity.  
In summary, this work adds evidence in relation to water management of Sorghum species, highlighting the whole plant
responses to soil water deficit. We integrated the shoot and root morphological and physiological responses, including
the cellular pathway of water movement when seedlings were exposed to water deficit conditions. The different
strategies developed by both Sorghum genotypes could be an interesting attribute of this crop species, since they can
adjust the water management at different levels. This performed approach adds new information for Sorghum which
can be used to evaluate phenotypic plasticity for changing environments. Our result provides also new insight in terms
of anisohydric and isohydric strategies that can be developed by plants in relation to water management. However, an
exhaustive study to address the physiological/molecular mechanisms triggered in response to water deficit is still
necessary, including in this scenario the role of aquaporins. Besides, our approach make available new evidences
related to the performance of Sorghum seedlings of two genotypes with contrast sprouting behavior grown in water
limited conditions. This information could be use to improve crop yield in those environments where the soil water
content is low and the risk of sprouting phenomena is low too.
 

ACKNOWLEDGEMENTS 

We thank M. V. Rodriguez from Laboratorio de Biología de Semillas IFEVA (CONICET-FAUBA), for access to seed
of the genotypes used in the experiments. This research was supported by grants from UBACyT1417; PIP-CONICET
2012 and FONCyT (PICT11-2239 and PICT14-0744), all grants to GA. MRS and GA are members of the National
Research Council of Argentina (CONICET).  
 
 
 
 

11 
 
 

References

Ayadi M, Cavez D, Miled N, Chaumont F, Masmoudi K. Identification and characterization of two plasma membrane
aquaporins in durum wheat (Triticum turgidum L. subsp. durum) and their role in abiotic stress tolerance. Plant
PhysiolBiochem 2011;49:1029–39.
Brady, NC. The nature and properties of soils. 10ma ed. MacMillan Pub. Co. , New York, 1990.
Brouwer R. Some aspects of the equilibrium between overground and underground plant parts. Jaarboek van het
Institut voor Biologisch en Scheikundig onderzoek aan Landbouwgewassen, 1963;31-9.
Carpita NC, McCann MC. Maize and Sorghum: genetic resources for bioenergy grasses. Trends in Plant Sci
2008;13:415–20.
Carvajal M, Cooke DT, Clarkson DT. Responses of wheat plants to nutrient deprivation may involve the regulation of
water-channels function. Planta 1996;199:372-81.
Choudhary S, Sinclair TR, Vara Prasad PB. Hydraulic conductance of intact plants of two contrasting Sorghum lines,
SC15 and SC1205. Funct Plant Biol 2013;40:730–8.
Clark A. Managing cover crops profitably. 3rd ed. Sustainable Agriculture Research and Education (SARE) Handbook
Series Book 9. Sustainable Agriculture Research and Education (SARE) Program, College Park, MD, 2007.
Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M, Robledo CW. InfoStat versión. Grupo InfoStat,
FCA, Universidad Nacional de Córdoba, Argentina, 2013. URL http://www. infostat. com. ar
Djanaguiraman M, Vara Prasad PV, Murugan M, Perumal R, Reddy UK. Physiological differences among Sorghum
(Sorghum bicolor L. Moench) genotypes under high temperature stress. Environ Exp Bot 2014;100:43–54.
FAO Global Perspectives Unit (www. fao. org/economic/esa/esag/)
Foster AS. Techniques for the study of venation patterns on the leaves of angiosperms. Proceedings of the
7th International Congress of Botany, Stockholm, Sweden, 1950;586–7.

Fracasso A, Trindade L, Amaducci S. Drought tolerance strategies highlighted by two Sorghum bicolor races in a dry-
down experiment, J Plant Physiol 2016;190:1–14.
Gholipoor M, Prasad PVV, Mutava RN, Sinclair TR. Genetic variability of transpiration response to vapor pressure
deficit among Sorghum genotypes. Field Crops Res 2010;119:85–90.
Gholipoor M, Sinclair TR, Prasad PVV. Genotypic variation within Sorghum for transpiration response to drying soil.
Plant Soil 2012;357:35–40.
Gregory PJ. Plant roots: growth, activity and interactions with the soil. Oxford Blackwell, London, 2006.
Gregory PJ, Palta JA, Batts GR. Root system and root:mass ratio-carbon allocation under current and projected
atmospheric conditions in arable crops. Plant Soil 1997;187:221–8.
Hommel R, Siegwolf R, Saurer M, Farquhar GD, Kayler Z, Ferrio JP, Gessler A. Drought response of mesophyll
conductance in forest understory species – impacts on water-use efficiency and interactions with leaf water movement.
Physiol Plant 2014;152:98–114.
Javot H, Lauvergeat V, Santoni V, Martin-Laurent F, Güçlü J, Vinh J, Heyes J, Franck KI, Schäffner AR, Bouchez D,
Maurel C. Role of a single aquaporin isoform in root water uptake. Plant Cell 2003;15:509–22.
Katsuhara M, Koshio K, Shibasaka M, Hayashi Y, Hayakawa T, Kasamo K. Over-expression of a barley aquaporin
increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants. Plant Cell Physiol 2003;44:1378–83.
Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K, Galbraith D, Bohnert HJ. Gene expression
profiles during the initial phase of salt stress in rice. Plant Cell 2001;13:889–905.
12 
 
 

Klein T. The variability of stomatal sensitivity to leaf water potential across tree species indicates a continuum between
isohydric and anisohydric behaviours. Functional Ecology 2014;28:1313-20.
Lambers H, Chapin III FS, Pons TL. Plant Physiological Ecology, Springer, New York, USA, 2008.
Li G, Santoni V, Maurel C. Plant aquaporins: Roles in plant physiology. Biochim Biophys Acta 2014;1840:1574–82.
Liu P, Yin L, Deng X, Wang S, Tanaka K, Zhang S. Aquaporin-mediated increase in root hydraulic conductance is
involved in silicon-induced improved root water uptake under osmotic stress in Sorghum bicolor L. J Exp Bot
2014;65:4747–56.
Lynch JP, Chimungu JG, Brown KM. Root anatomical phenes associated with water acquisition from drying soil:
targets for crop improvement. J Exp Bot 2014;65:6155–66.
Mahdieh M, Mostajeran A, Horie T, Katsuhara M. Drought stress alters water relations and expression of PIP-type
aquaporin genes in Nicotiana tabacum plants. Plant Cell Physiol 2008;49:801–13.
Malik RS, Dhankar JS, Turner NC. Influence of soil water deficits on roots growth cotton seedlings Plant and Soil
1979;53:109-15.
Martínez-Vilalta J, Poyatos R, Aguadé D, Retana J, Mencuccini M. A new look at water transport regulation in plants.
New Phytol 2014;204:105-15.
Matsuo N, Ozawa K, Mochizuki T. Genotypic differences in root hydraulic conductance of rice (Oryza sativa L.) in
response to water regimes. Plant Soil 2009;316:25–34.

Meyer RF, Boyer JS. Osmoregulation, solute distribution, and growth in soybean seedlings having low water potentials.
Planta 1981;151:482-9.
Miyamoto N, SteudleE, Hirasawa T, Lafitte R. Hydraulic conductivity of rice roots. J Exp Bot 2001;52:1835–46.
Morris GP, Ramub P, Deshpande SP, Hash CT, Shah T, Upadhyaya HD, Riera-Lizarazu O, Brown PJ, Acharya CB,
Mitchell SE, Harriman J, Glaubitz JC, Buckler ES, Kresovich S. Population genomic and genome-wide association
studies of agroclimatic traits in . Sorghum. Proc Natl Acad Sci USA2013;110:453–8.
Moshelion M, Halperin O, Wallach R, Oren R, Way DA. Role of aquaporins in determining transpiration and
photosynthesis in water-stressed plants: crop water-use efficiency, growth and yield. Plant Cell Environ 2015;38:1785-
93.
Muller B, Pantin F, Génard M, Turc O, Freixes S, Piques M, GibonY. Water deficits uncouple growth from
photosynthesis, increase C content, and modify the relationships between C and growth in sink organs. J Exp Bot
2011;62:1715-29.
Ogbaga CC, Stepien P, Johnson GN. Sorghum (Sorghum bicolor) varieties adopt strongly contrasting strategies in
response to drought. Physiol Plant 2014;152:389–401.
Pasini L, Bergonti M, Fracasso A, Marocco A, Amaducci S. Microarray analysis of differentially expressed mRNAs
and miRNAs in young leaves of Sorghum under dry-down conditions. J. Plant Physiol 2014;171:537–48.

Passioura JB. Root signals control leaf expansion in wheat seedlings growing in drying soil. Aust J Plant Physiol
1988a;15:687–93.
Passioura JB. Water in the Soil-Plant-Atmosphere Continuum. Physiological Plant Ecology II, Encyclopedia of Plant
Physiology Volume 12 / B, 1982, pp 5–33.
Pittermann J. The evolution of water transport in plants: an integrated approach. Geo 2010;8:112–39.
Ranathunge K, Schreiber L. Water and solute permeabilities of Arabidopsis roots in relation to the amount and
composition of aliphatic suberin. J Exp Bot 2011;62:1961–74.

13 
 
 

Reddy PS, Rao TSRB, Sharma KK, Vadez V. Genome-wide identification and characterization of the aquaporin gene
family in Sorghum bicolor (L. ). Plant Gene 2015;1:18–28.
Richards, RA. Physiological traits used in the breeding of new cultivars for water-scarce environments. Agric Water
Manage 2006;8080:197–211.
Rodríguez MV, Mendiondo GM, Maskin L, Gudesblat GE, Iusem ND, Benech-Arnold RL. Expression of ABA
signalling genes and ABI5 protein levels in imbibed Sorghum bicolor caryopses with contrasting dormancy and at
different developmental stages. Ann Bot 2009;104:975–85.

Rogers ED, Benfey PN. Regulation of plant root system architecture: implications for crop advancement. Curr Opin
Biotechnol 2015;32:93-8.
Saballos A, Ejeta G, Sanchez E, Kang CH, Vermerris W. A genomewide analysis of the cinnamyl alcohol
dehydrogenase family in Sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the brown midrib Gene.
Genetics 2009;181:78395.
Schölander PF, Hammel HT, Bradstreet ED, Hemmingsen EA. Sap pressure in vascular plants: negative hydrostatic
pressure can be measured in plants. Science 1965;148:339–46.
Schreiber L, Franke R, Hartmann K, Ranathunge K, Steudle E. The chemical composition of suberin in apoplastic
barriers affects radial hydraulic conductivity differently in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea
mays L. cv. Helix). J Exp Bot 2005;56:1427–36.
Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, Satou M,
Akiyama K, Taji T, Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y, Shinozaki K. Monitoring the
expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA
microarray. Plant J 2002;31:279–92.
Shanker AK, Maheswari M, Yadav SK, Desai S, Bhanu D, Attal NB, Venkateswarlu B. Drought stress responses in
crops. Funct Integr Genomics 2014;14:11-22.
Sharp RE Davies WJ. Solute regulation and growth by roots and shoots of water-stressed maize plants. Planta
1979;147:43-9.
Sharp RE, Silk WK, Hsiao TC. Growth of the maize primary root at low water potential. I. Spatial distribution of
expansive growth. Plant Physiol 1988;87:50-7.
Sinclair TR, Holbrook NM, Zwieniecki MA. Daily transpiration rates of woody species on drying soil. Tree Physiol
2005;25:1469–72.
Siqueira M, Katul G, Porporato A. Onset of water stress, hysteresis in plant conductance, and hydraulic lift: Scaling soil
water dynamics from millimeters to meters. Water resources res 2008;44,14.
Steinbach HS, Benech-Arnold RL, Sánchez RA. Hormonal regulation of dormancy in developing Sorghum seeds. Plant
Physiol 1997;11:149–54.
Steudle E, Murrmann M, Peterson CA. Transport of water and solutes across maize roots modified by puncturing the
endodermis. Further evidence for the composite transport model of the root. Plant Physiol 1993;103:335–49.
Steudle E. Water uptake by roots: effects of water deficit. J Exp Bot 2000;51:1531–42.
Sutka M, Li G, Boudet J, Boursiac Y, Doumas P, Maurel C. Natural variation of root hydraulics in Arabidopsis grown
in normal and salt-stressed conditions. Plant Physiol 2011;155:1264–76.

Tardieu F, Parent B, Caldeira CF, Welcker C. Genetic and physiological controls of growth under water deficit. Plant
Physiol 2014;164:1628–35.

14 
 
 

Tardieu F, Tuberosa R. Dissection and modelling of abiotic stress tolerance in plants. Curr Opin Plant Biol
2010;13:206–12.
Turner NC. Techniques and experimental approach for the measurements of plant water status. Plant Soil 1981;58:339–
66.
Tyree MT. Hydraulic properties of roots. In “Ecological Studies” 2003;168:125–50. (Springer-Verlag Berlin
Heidelberg)
Undersander D J, Smith LH, Kaminski AR, Kelling KA, Doll JD. Sorghum forage. In: Alternative Field Crop Manual,
University of Wisconsin-Exension, Cooperative Extension, 2003.
van der Weele CM, Spollen WG, Sharp RE, Baskin TI. Growth of Arabidopsis thaliana seedlings under water deficit
studied by control of water potential in nutrient-agar media. J Exp Bot 2000;51:1555-62.
Wu Y, Cosgrove DJ. Adaptation of roots to low water potentials by changes in cell wall extensibility and cell wall
proteins. J Exp Bot 2000;51:1543–53.
Zhang WH, Tyerman SD. Inhibition of water channels by HgCl2 in intact wheat root cells. Plant Physiol 1999;120:849-
58.
Zhu C, Schraut D, Hartung W, Schäffner AR. Differential responses of maize MIP genes to salt stress and ABA. J Exp
Bot 2005;56:2971–81.

15 
 
 

Figure Legends

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 and IS9530 grown for 15 days under two experimental conditions:
control (C) and water deficit (WD). Each bar represents means ± s.e. of 18 plants (N=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, only for fresh shoot and dry root biomass.

16 
 
 

Fig. 2. Shoot and root length from 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 16 plants (N= 6
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.

17 
 
 

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). Different upper-case letters indicate significant differences (P< 0.05) between genotypes;
lower-case letters indicate significant differences between treatments within each genotype.

18 
 
 

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.

19 
 
 

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.

20 
 
 

Fig. 6. Properties of root water dynamics: the effect of drought and HgCl2 on root hydraulic conductance (Lo) of
seedlings from two Sorghum genotypes, RedLandB2 and IS9530. Seedlings were grown under control (C) and water
deficit (WD) conditions. In those experiments performed in the presence of 50 µM HgCl2, seedlings were pre-incubated
ten minutes before Lo measurements. Each bar represents means ± s. e. of 9-12 plants (N= 5 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.

21 
 
 

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

Control Drought Control Drought

Fresh weight Shoot 540.9±47.6Aa 267.0±33.5Ab 642.5±68.7Aa 263.2±37.1Ab

(mg)
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-2s-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 (µl 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

22