RESEARCH

Metabolic Responses of Two Contrasting

Sorghums to Water-Deficit Stress
John J. Burke,* Paxton Payton, Junping Chen, Zhanguo Xin, Gloria Burow, and Chad Hayes

Abstract
Water-deficit stress responses in sorghum [Sorghum bicolor (L.) Moench] have been described
in the literature as preflowering drought tolerant (postflowering senescent) or postflowering
drought tolerant (preflowering drought sensitive). The underlying physiological mechanisms
associated with these drought traits remain
unclear. It was hypothesized that the preflowering drought sensitivity of stay-green lines could
be related to reported differences in osmotic
potential among stay-green and senescent lines
resulting in an inability of the cultivars to either
sense or respond the soil drying until the rate
of drying is too great for the stay-green lines to
compensate. The objective of this study was
to measure stress-induced changes in relative water content, abscisic acid (ABA), proline,
dhurrin, sucrose, and carbon assimilation during the onset of water-deficit stress in the preflowering drought-tolerant line SC121111E and
the postflowering drought-tolerant line BTx642
to determine if there were differential responses
to the onset of soil drying. In both greenhouse
and field studies, it was found that SC121111E
had lower relative water contents and accumulated higher levels of ABA and proline than the
BTx642. The SC121111E also showed increases
in carbon assimilation shortly after the cessation of irrigation that declined with prolonged
stress. These results provide new insights into
the differential responses of pre and postflowering drought-tolerant sorghum lines.

USDA-ARS, Cropping Systems Research Lab., 3810 4th Street, Lubbock, TX 79415. Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the USDA and does
not imply approval or recommendation of the product to the exclusion
of others that may be suitable. USDA is an equal opportunity provider
and employer. Received 25 April 2014. *Corresponding author (john.
burke@ars.usda.gov).
Abbreviations: YL, lethal leaf water potential; ABA, abscisic acid;
DW, dry weight; FW, fresh weight; OA, osmotic adjustments; RWC,
relative water content; RWCL, lethal relative water content; Tc, canopy
temperature; TW, turgid weight.

orghum is recognized for its overall tolerance to water deficit

and drought stress. To evaluate sorghums capacity to withstand drought stress, earlier research compared the relative water
requirements of sorghums and corn (Zea mays L.) (Cates, 1922;
Martin, 1930; Miller, 1916, 1923). These studies showed not only
that sorghum outproduced corn under water-limiting conditions,
but also demonstrated genetic diversity among sorghum lines for
water use. Indirectly, as farmers grasp the value of sorghums tolerance to water deficit and drought stresses, acreage of the crop
increased threefold from 1930 to 1958, from 8.83 to a maximum
of 26.89 million acres (USDA, 2014). There are two distinct
types of drought-stress responses identified and described in sorghum: (i) preflowering (panicle differentiation to flowering) and
(ii) postflowering (grain fill to physiological maturity of grain)
drought tolerance (Rosenow and Clark, 1982; Rosenow et al.,
1983). Plants under stress from panicle differentiation to flowering exhibit preflowering responses. This type of stress directly

Published in Crop Sci. 55:344353 (2015).

doi: 10.2135/cropsci2014.04.0322
Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA
All rights reserved. No part of this periodical may be reproduced or transmitted in any
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or any information storage and retrieval system, without permission in writing from
the publisher. Permission for printing and for reprinting the material contained herein
has been obtained by the publisher.

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crop science, vol. 55, january february 2015

affects panicle size, grain number, and grain yield. Symptoms include leaf rolling, uncharacteristic leaf erectness,
leaf bleaching, leaf tip and margin burn, delayed flowering, saddle effect, poor panicle exertion, panicle blasting
and floret abortion, and reduced panicle size. Meanwhile,
symptoms of postflowering drought stress include premature plant death, stalk collapse and lodging, increased
susceptibility to stalk rots, and significant reduction in
seed size, all of which result in decreased yield potential.
Genetic variation for pre or postflowering drought
tolerance has been detected in sorghum (Rosenow and
Clark, 1982; Rosenow et al., 1983). A number of studies have identified genetic diversity for traits related to
drought tolerance separately (Borrell et al., 1999; Burgess
et al., 2002; Burke et al., 2010; Garud et al., 2002; Harris
et al., 2007; Haussmann et al., 2002; Kassahun et al.,
2010; Mahalakshmi and Bidinger, 2002; McIntyre et al.,
1997; Rosenow et al., 1996; Sanchez et al., 2002; Tuinstra
et al., 1997, 1998; Walulu et al., 1994; Xu et al., 2000).
However, while germplasm sources of pre and postflowering drought tolerance have been characterized, sources
with combined tolerance to both pre and postflowering
drought stresses have not been identified.
Diversity in genotypic responses to drought or water
deficits was described earlier with studies focusing on
differential stomatal sensitivities, leaf water potentials,
and soil moisture extraction (Blum, 1974; Blum and
Ebercon, 1976; Henzel et al., 1975, 1976). In a study by
Henzel et al. (1975), selections were classified as sensitive
or insensitive to water deficit, with cultivars Shallu and
Alpha selected as two sensitive types and T.S.1598C and
M351 as two insensitive ones. They reported that stomatal sensitivity varied significantly among the entries
during progressive water deficit. The stomatal conductance of leaves of Alpha and Shallu decreased rapidly as
leaf water potential declined, whereas it declined more
slowly in I.S.1598C and M351 (Henzel et al., 1976). In
another study, Ackerson et al. (1980) examined the relationship between stomatal activity and leaf water potential
components for several sorghum genotypes as function of
growth stage under semiarid field conditions and started
classifying lines as senescent or nonsenescent types. They
found that the more senescenttype hybrids (RS610 and
ATX378 TX7000) initiated stomatal control at higher
leaf water potentials than did the nonsenescent genotypes.
Shackel et al. (1982) identified genotypic differences in
leaf osmotic potential among senescent and nonsenescent
(stay green) grain sorghum cultivars grown under irrigation and drought. The higher osmotic levels should result
in enhanced water uptake in stay-green lines under wellwatered and low levels of drought.
In this study, we investigated metabolic responses of
the stay-green line BTx642 and the senescent line SC1211
11E (Burke et al., 2010) during the onset of water-deficit
crop science, vol. 55, january february 2015

stress in the greenhouse and field. Focusing on the reported

differences in osmotic potential among stay-green and
senescent lines (Shackel and Hall, 1983), we examined
whether BTx642 and SC121111E responded differently
to the onset of water stress. Further we examined the relationship between elevated sucrose and higher cyanogenic
glucoside (dhurrin) levels (Burke et al., 2013) in stay-green
sorghums that could result in higher cellular osmotic levels
compared with senescent lines. To determine if the preflowering drought sensitivity of stay-green lines could be
related to differential inability of the cultivars to either
sense or respond the soil drying until the rate of drying is
too great for the stay-green lines to compensate, we evaluated two stress-response metabolites, ABA (Walker-Simmons, 1987) and proline (Bates et al., 1973) accumulation,
in response to the onset of water-deficit stress of senescent
and stay-green sorghums under greenhouse and field environments. Information from this study will provide insight
into the ability of preflowering drought-tolerant (senescent) and postflowering drought-tolerant (stay-green) sorghum to respond to the onset of water-deficit stress.

Materials and methods

Greenhouse Cultural Practices

BTx642 and SC121111E sorghum seeds were planted into

33-cm diameter pots containing Sunshine Mix #1 soil (Sun Gro
Horticulture Distributors Inc., Bellevue, WA). Three BTx642
seeds were planted at 0 and 180 degrees and three SC121111E
seeds were planted at 90 and 270 degrees orientation within
each pot. The pots were placed in a greenhouse set to provide
a 31 and 27C day and night cycle. A 16 and 8 h photoperiod
was maintained using 430 W high-pressure sodium lights (P.
L. Light Systems, Beamsville, ON, Canada). Nutrients were
maintained by daily application with Peters Excel fertilizer
(Scotts-Sierra Horticultural Products Company, Marysville,
OH) through an automated watering system. For each experiment, fifteen pots were prepared and divided into three five-pot
replicates. At the five-leaf stage, plants were thinned to one
plant at each of the four planting locations within the pot. With
two plants for each line per pot, a total of ten plants were evaluated per replicate at anthesis. On the day of treatment (Day 0),
all pots were fully watered early in the morning. Pot weights
were measured at 1300 h, 5 h after watering. Drought treatment was applied by withholding watering for the evaluation
period. Pots were weighed daily at a set time (1300 h) to obtain
a relative measure of water use by the plants. The experiment
was performed twice.

Field Cultural Practices

Field studies were performed during the 2011 and 2012 growing seasons using a surface drip irrigation system in Lubbock,
TX (Amarillo fine sandy loam; fine-loamy, mixed, superactive,
thermic Aridic Paleustalfs). The experimental plots were 5.5
m in length with 1 m between rows and received one preplant
furrow irrigation that filled the furrow to the peak of the beds
7 d before planting. BTx642 and SC121111E sorghum seeds

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were planted at 100 seeds per plot at a depth of 3 cm using

a John Deere MaxEmerge Planter modified for use in small
plot research (Kincaid Equipment Manufacturing, Haven, KS).
Surface drip irrigation (32 mm) was applied once a week after
planting until the plants reached boot stage when drought-stress
treatment started. The last surface irrigation was applied on the
day of initiation drought-stress treatment (Day 0) followed by
withholding watering. Plant water status was determined from
the relative water content of leaf samples from the third leaf
from the top of the canopy.

Dhurrin and Sucrose Determination on HighPerformance Liquid Chromotography

The dhurrin and sucrose concentrations in leaf tissues were
determined from three replicate samples of five punches collected for analysis according to the procedure described by
Burke et al. (2013). Five plants were sampled for each replication for a total sampling of 15 random plants. For leaf position
description, the youngest leaf is considered as Position 1 and
leaf positions increase basipetally. Dhurrin and sucrose from
five 1-cm punches per set from the third leaf were extracted
and samples equivalent to 50 to 100 g of fresh weight of leaf
tissue were separated on a 4.6 by 250-mm YMC Polyamine
II column (Waters, Milford, MA) with a mobile phase of 70%
acetonitrile in water (v/v) at a flow rate of 1.0 mL min1. The
amount of dhurrin and sucrose was analyzed using a VP Series
high-performance liquid chromotography system fitted with
a SIL-10AD auto-injector and an evaporative light scattering
detector-LT (Shimadzu, Columbia, MD). Presence of dhurrin
and sucrose in samples was identified by its retention time in
comparison with the corresponding dhurrin and sucrose standards. Quantification of dhurrin and sucrose concentrations
was calculated using peak area.

Photosynthesis Measurements
Leaf-level net carbon assimilation was measured on the most
recently matured leaf using a portable infrared gas analyzer
(Li-COR, Model LI-6400, Lincoln, NE). Leaf-to-air vapor
pressure deficit, air temperature, and relative humidity of the
cuvette were set to ambient environmental values for the growth
environment maintained constant for all measurements. The
reference CO2 concentration was set at 400 mol mol-1 and
photosynthetically active radiation was set to saturating conditions (1800 mol m-2 s-1 for the glasshouse study and 2000 mol
m-2 s-1 for the field study). For both studies, irradiance was supplied using a light-emitting diode (Licor LI-6400002). Once
the system was stable (approximately three minutes), data were
logged three times for each leaf during the course of 1 min and
then averaged for each plant to be used as a statistical unit. All
photosynthesis and transpiration measurements were performed
between 0900 and 1400 h in both glasshouse and field studies.

Stress Measurements
The relative water content (RWC) was determined for 1-cm 2
leaf punches taken from the third leaf from the top of five
plants per replicate. Leaf punches were immediately sealed in
an Eppendorf vial and placed on ice until returning to the laboratory. Three replications per treatment were evaluated. The
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fresh weights (FW) of the leaf punches were recorded immediately on returning to the laboratory and then the punches
were hydrated in water overnight at 4C in the dark. After
hydration, the samples were taken out of the water, dried of any
surface moisture with a paper towel, and immediately weighed
to obtain the turgid weight (TW). Samples were then oven
dried at 70C for 24 h and weighed (after cooling to room
temperature) to determine dry weight (DW). Relative water
content was calculated using the following equation: RWC (%)
= [(FW DW)/(TW DW)]100.
For ABA and proline analysis, a total of 15 1-cm leaf punches
per replicate were collected from five randomly selected plants.
Abscisic acid levels were determined according to the method
described by Walker-Simmons (1987) and proline accumulation was determined according to Bates et al. (1973).
Canopy temperatures were measured at 15-min intervals during the 2-wk period of soil drying using the Standard
SmartCrop sensors (Smartfield, Inc., Lubbock, TX).

Experimental Design
and Statistical Analyses
Experiments conducted in the polyhouse were arranged using
completely randomized design with five replications. Field
experiments were also set up using completely randomized
design with three replications. All experiments were repeated
once and field experiments were done for two growing seasons
(consecutive years). Statistical significance between genotypes
was analyzed with Students t test through the statistical applications of Microsoft Excel software.

Results

Relative Leaf Water Content

Greenhouse and field studies were conducted to evaluate the temporal responses of the senescent sorghum line
SC121111E and the stay-green line BTx642 to progressive soil moisture deficits. Figure 1 shows the gravimetric
change in soil water content for pots containing two plants
from each genotype for two independent experiments.
For both experiments, daily evapotranspirative water
loss was between 2000 and 2500 g. On the basis of this
response and the presence of both genotypes in a single
pot, it was assumed that water-deficit stress treatments
between the two experiments was similar and SC1211
11E and BTx642 were exposed to similar levels of plantavailable soil water during the course of the experiments.
Because of the similarity in water usage between the two
experiments based on a paired Students t test, data on the
relative water content, dhurrin levels, and ABA levels are
presented as an average of the two experiments.
The leaf RWC of BTx642 and SC121111E were evaluated to provide a measure of the stress responses of the
plants to a drying soil. Figure 2A shows RWC in BTx642
was higher than that of SC121111E during the course of
the water-deficitstress treatment and the rate of decline
in RWC differed between the two genotypes in the
greenhouse study. Following the cessation of irrigation,

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Figure 1. Accumulative water use indicated by average daily pot

weight changes during two greenhouse drought stress experiments (closed circles, Exp. 1; open circles, Exp. 2).

SC121111E showed a decrease in leaf RWC compared

with the well-watered condition in just 24 h, while the
leaf RWC in BTx642 remained unchanged. The difference in RWC between the lines became greater as the
plants used the available soil water. SC121111E declined
to a RWC of 78% with 4 d of drying, while the BTx642
exhibited a RWC of 86%. The leaf relative water contents
of field-grown sorghum were determined following the
cessation of the weekly irrigation (Fig. 2B). Significant
differences between two sorghum lines commenced at 5 d
after cessation of irrigation and thereafter. BTx642 exhibited higher RWC (91%) than SC121111E (85% RWC) at
5 d after the cessation of irrigation.

Leaf Dhurrin Levels

Previously, we reported a correlation between leaf dhurrin
concentration and the stay-green rating of sorghum lines
(Burke et al., 2013). To follow up on this, significant differences in leaf dhurrin concentration were detected between
BTx642 and SC121111E during the course of the greenhouse experiment (Fig. 3A). BTx642 leaves showed >10fold higher dhurrin levels compared with SC121111E,
approximately 25 g mg1 of leaf sample compared with
2 g mg1 for SC121111E. Dhurrin levels in SC121111E
slightly increased during the onset of the water-deficit
stress but remained unchanged in BTx642. Dhurrin levels
from field-grown BTx642 plants were in the same range
as those measured in the greenhouse studies with values
ranging from 15 to 25 g mg1 (Fig. 3B). Notably, SC1211
11E exhibited higher dhurrin levels in the field compared
with the greenhouse studies. Dhurrin levels in SC1211
11E were about 4 g mg1 in the field study across the 2
wk of measurement compared with 2 g mg1 in plants
in the greenhouse study. Field-grown BTx642 exhibited
significantly higher dhurrin levels (three to fourfold) than
SC121111E during the course of the experiment.
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Figure 2. (A) Changes in leaf relative water contents (RWC) of

BTx642 (closed circles) and SC121111E (open circles) plants
throughout greenhouse drought-stress experiments. Data showed
were means and standard deviations of six replications. (B) Graph
showing the average RWC of BTx642 (closed circles) and SC1211
11E (open circles) throughout field water-stress experiments performed in 2011 and 2012 in Lubbock, TX.

Leaf Abscisic Acid Levels

A twofold difference in ABA levels was observed immediately on the cessation of watering (Fig. 4A). The SC1211
11E had 0.5 pmol mg1 DW on Day 0, while the BTx642
had 0.22 pmol mg1 DW. The level of ABA increased rapidly in the SC121111E during the 4 d when water was
withheld, with levels doubling by Day 1 and reaching a
level of 6.1 pmol mg 1DW by Day 4. Line BTx642 showed
no change in ABA level at 1 d after the cessation of watering and then gradually increased to a maximum of 1.5 pmol
mg1 DW by Day 4. Similar to the greenhouse studies, a
twofold difference in ABA levels was observed immediately on the cessation of watering (Fig. 4B). The SC1211
11E had 0.89 pmol mg1 DW on Day 1, while the BTx642
had 0.49 pmol mg1 DW. The level of ABA remained relatively constant during the first 4 d after irrigation and then

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Figure 3. (A) Dhurrin levels in leaf tissues of BTx642 (closed circles)

and SC121111E (open circles) throughout greenhouse droughtstress experiments. BTx642 exhibited a dhurrin concentration of
approximately 25 g mg1 of leaf sample, while the SC121111E
had levels below 2 g mg1. (B) Graph showing the average dhurrin levels of BTx642 (closed circles) and SC121111E (open circles)
throughout field water-stress experiments performed in 2011 and
2012 in Lubbock, TX.
1

increased to 4.6 pmol mg DW by 12 d after irrigation.

The BTx642 showed no change in ABA level for 4 d after
the cessation of watering, and then gradually increased to
a maximum of 2.5 pmol mg1 DW by Day 12. The more
than twofold increase in ABA in the SC121111E compared with BTx642 in the field was similar to the fourfold
increase observed in the greenhouse study.

Leaf Proline Levels

Greenhouse-grown plants of the two sorghum lines exhibited similar low leaf proline levels throughout the 5-d study
(Fig. 5A). Similarly, field-grown plants from the two lines
displayed similar proline levels during the first 5 d postwatering (Fig. 5B). Subsequently, proline concentrations increased
from Day 8 to 12 in the SC121111E plants, ending with a
leaf concentration of 21 nmol mg1 DW. Proline levels form
348

Figure 4. (A) Changes in abscisic acid (ABA) levels of BTx642

(closed circles) and SC121111E (open circles) plants throughout
greenhouse drought-stress experiments. (B) Graph showing the
average ABA levels of BTx642 (closed circles) and SC121111E
(open circles) throughout field water-stress experiments performed in 2011 and 2012 in Lubbock, TX.

BTx642 increased only up to 11 pmol mg1 DW during

this same period. Noticeably, a twofold difference in leaf
proline levels during the 12-d period was observed between
the two lines (Fig. 5B), mirroring the twofold difference in
ABA during this same period (Fig. 4B).

Leaf Sucrose Levels

Leaf sucrose concentrations of greenhouse-grown plants
are shown in Fig. 6A. Similar sucrose levels were observed
between the lines for the first 4 d of the study. On the
fifth day, sucrose levels in the SC121111E line decreased
from 61 to 49 g mg1 DW, while the BTX642 remained
constant. Sucrose levels of both the field-grown BTx642
and SC1211 were similar during the first 4 d without irrigation (Fig. 6B). Values were approximately 36 g mg1
on the first day and dropped to 23 g mg1 DW by Day
4. On Day 5, sucrose began to increase in the leaves of

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crop science, vol. 55, january february 2015

Figure 5. (A) Changes in proline levels of BTx642 (closed circles)

and SC121111E (open circles) plants throughout greenhouse
drought-stress experiments. (B) Graph showing the average proline levels of BTx642 (closed circles) and SC121111E (open circles) throughout field water-stress experiments performed in 2011
and 2012 in Lubbock, TX.

Figure 6. (A) Changes in sucrose levels of BTx642 (closed circles)

and SC121111E (open circles) plants throughout greenhouse
drought-stress experiments. (B) Graph showing the average sucrose levels of BTx642 (closed circles) and SC121111E (open circles) throughout field water-stress experiments performed in 2011
and 2012 in Lubbock, TX.

SC121111E to 32 g mg1 DW while BTx642 increased

to 25 g mg1 DW. Sucrose continued to increase in both
lines during the next 7 d after cessation of irrigation with
the SC121111E values reaching 51 g mg1 by Day 12 and
the BTx642 reaching 33 g mg1 during the same period.

before ceasing irrigation (Fig. 7B). In 2011, the SC121111E

level was 24.9 mol CO2 m-2 s-1 1 d before irrigation, and
the BTx642 levels were 29.0 mol CO2 m-2 s-1. One day
following the irrigation, the SC121111E levels increased
85% to 46.1 mol CO2 m-2 s-1 and the BTx642 levels
increased 23% to 35.7 mol CO2 m-2 s-1. Five days after the
irrigation the SC121111E photosynthesis levels declined
by 20% to 36.9 mol CO2 m-2 s-1 and the BTx642 levels
declined by 10% to 32.2 mol CO2 m-2 s-1. In 2012, the
SC121111E level was 34.35 mol CO2 m-2 s-1 3 d before
irrigation, and the BTx642 levels were 30.94 mol CO2 m-2
s-1. One day following the irrigation, the SC121111E levels
increased 25% to 42.7 mol CO2 m-2 s-1 and the BTx642
levels increased 13% to 34.9 mol CO2 m-2 s-1. Four days
after the irrigation the SC121111E photosynthesis levels
declined by 43% to 24.45 mol CO2 m-2 s-1 and the BTx642
levels declined by 22% to 27.45 mol CO2 m-2 s-1.

Leaf-Level Gas Exchange

Leaf-level gas exchange measurements as indicated by
carbon assimilation showed higher gas exchange levels in
greenhouse-grown BTx642 compared with SC121111E
across all days evaluated (Fig. 7A). One day after cessation of
watering, BTx642 exhibited gas exchange rates of 20 mol
CO2 m-2 s-1, while the SC121111E plants averaged 16
mol CO2 m-2 s-1. Both lines showed proportional declines
in leaf gas exchange 2, 3, 4, and 6 d postwatering. Leaf-level
gas exchange measurements showed similar gas exchange
levels in field-grown BTx642 compared with SC121111E
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Figure 8. Graph showing daily canopy temperatures of BTx642

(closed circles) and SC121111E (open circles) throughout a field
water-stress experiment performed in 2011 in Lubbock, TX. The
temperature difference between the SC121111E and BTx642
(TcSC121111E TcBTx642, closed diamonds) shows that the SC1211
11E exhibited higher plant temperatures than the BTx642 during the first week of water stress. During the second week, the
SC121111E had daily alternating lower and higher canopy temperatures from mornings to evenings.

Figure 7. (A) Changes in carbon assimilation levels of BTx642

(closed circles) and SC121111E (open circles) throughout greenhouse drought-stress experiments. (B) Graph showing the carbon
assimilation levels during the 2011 field study of BTx642 (closed
circles) and SC121111E (open circles) and BTx642 (closed squares)
and SC121111E (open squares) during the 2012 field study. The
vertical line at Day 0 separates measurement before and after irrigation.

Canopy Temperatures
Canopy temperatures (Tc) measured from 3 to 12 days
following irrigation on 20 June 2011 are shown in Fig. 8.
The field-grown BTx642 and SC121111E exhibited a
gradual rise in maximum day temperature from Day 3
to Day 9 as the soil water was being depleted. The daily
swing between minimum and maximum temperatures
increased from approximately 16C for Days 3 to 8 to
approximately 22C between Days 9 and 12. It is interesting to note that ABA (Fig. 4), proline (Fig. 5), and sucrose
(Fig. 6) concentrations increased significantly during the
8 to 12 days following the ceasing of irrigation. Evaluation of the difference in canopy temperatures between the
SC121111E and the BTx642 (TcSC121111E TcBTx642) (Fig.
8) revealed elevated canopy temperature in the SC1211
11E compared with the BTx642 throughout the study.
350

During the first 7 d of soil drying, SC121111E exhibited a one to two C higher temperature before and after
midday. The two lines reached similar maximum temperatures at midday as exemplified by the drop in TcSC1211
11E Tc BTx642 to a zero differential value. From Day 8 to
12 a different pattern was observed. In the mornings the
BTx642 exhibited higher temperatures than the SC1211
11E, and in the afternoons the SC121111E had higher
temperatures than the BTx642. The magnitude of this
difference increased as stress progressed. The comparison
of SC121111E and BTx642 canopy temperatures across
all temperatures shows that for the majority of the study
the SC121111E plants were one or more degrees Celsius warmer than the BTx642 (Fig. 9). The differences
between the lines appeared to become greater at temperatures below 27C and above 30C.

Discussion

Greenhouse Studies
The present greenhouse study was initiated to better
understand the significance of differential stomatal sensitivities of sorghum genotypes in relation to metabolic
responses to onset of water stress. Henzel et al. (1975)
described a method that used monitoring leaf diffusive
resistance after water was withheld from pots of sorghum
lines against a check line M351 in the same pot. They
reported that the ratio of the linear regression coefficient
of leaf diffusive resistance on soil water potential provided
a measure of relative stomatal sensitivity. The higher ratio
indicated that stomata were relatively more sensitive to

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crop science, vol. 55, january february 2015

Figure 9. Graph showing the comparison of canopy temperatures

(Tc) between SC121111E and BTx642 during the water-stress
experiment described in Fig. 8. The comparison of SC121111E
and BTx642 canopy temperatures across all temperatures shows
that for the majority of the study the SC121111E plants were one
or more degrees Celsius warmer than the BTx642. The differences
between the lines appeared to become greater at temperatures
below 27C and above 30C.

decreasing soil moisture. In a followup study Henzel et

al. (1976) evaluated the four genotypes that covered the
range in stomatal sensitivity found earlier by Henzel et
al. (1975). Shallu and Alpha were selected as two sensitive
types and I.S.1598C and M351 as two insensitive ones.
Stomatal sensitivity varied significantly among the entries
during progressive water deficit. The stomatal conductance of leaves of Alpha and Shallu decreased rapidly as
leaf water potential declined, whereas it declined more
slowly in I.S.1598C and M351. Their results suggested
that stomatal sensitivity might be an important element
of intergenotypic variation in resistance to drought. Jones
and Turner (1978) reported that Shallus decreased osmotic
potential at full turgor in fully expanded sorghum leaves
at a moderate level of stress (predawn leaf water potential of -0.85 MPa) was fully accounted for by increases
in sugars, potassium, and chloride. The contributions of
total inorganic ions and sugars (glucose and sucrose) were
approximately equal ( Jones et al., 1980). Ackerson et al.
(1980) examined the relationship between stomatal activity and leaf water potential components of several sorghum
genotypes under field conditions. They reported that the
more senescenttype hybrids initiated stomatal control at
higher leaf water potentials than did the non-senescent
genotypes. Basnayake et al. (1996) conducted an experiment to quantify the effect of the rate of development
crop science, vol. 55, january february 2015

of water deficit on the expression of maximum osmotic

adjustment (OA) and desiccation tolerance in three-grain
sorghum lines TAM422, Tx2813, and QL27. They also
reported that desiccation tolerance was determined as
the lethal relative water content (RWCL) and lethal leaf
water potential (YL) of leaves (i.e., the value of these traits
just before tissue death). Both TAM422 and Tx2813 had
higher maximum OA, higher RWCL, and lower YL than
QL27. QL27, with a low expression of osmotic adjustment, and Tx2813 and TAM422, both with high expression of osmotic adjustment, were previously identified by
Basnayake et al. (1995). A number of studies reported similar findings on genotypic differences between sorghum
lines in response to soil drying (Blum, 1974; Blum and
Sullivan, 1986; Premachandra et al., 1995; Premachandra
et al., 1992; Shackel and Hall, 1983).
An apparent trend observed from studies in previous
literature suggested that lines exhibiting rapid changes
in leaf diffusive resistance were generally associated with
senescent sorghums (Tx7078, Tx7000) and lines exhibiting gradual changes were associated with stay-green
sorghums (QL27, M351). Kidambi et al. (1990) reported
that the hybrid SC35 SC599 maintained a very stable
carbon assimilation rate across a wide range of environments. The authors suggested that the higher carbon
assimilation rate under poor conditions of SC35 SC599
may be due to the presence of nonsenescent characteristics in both parents. Hybrids containing a senescent-type
parent (Tx378 SC599) were very sensitive to poor conditions but very responsive to good conditions.
Our present study investigated physiological responses
of the senescent sorghum SC121111E (Burke et al.,
2010) and the stay-green sorghum BTx642 (Burke et al.,
2013) to the onset of water-deficit stress. The results of
the greenhouse studies provided additional insights into
the capacity and competitive advantage of BTx642 in
extracting soil water. Shackel et al. (1982) showed that
the drought-tolerant line M351 had lower leaf osmotic
potentials under both wet and dry conditions compared
with the more drought-sensitive line dwarf Redlan.
We found higher relative water contents in the leaves of
BTx642 under irrigated conditions (Fig. 2, Day 0) compared with the SC121111E. This result is consistent with
BTx642 having high sucrose (Burke et al., 2010) and
dhurrin (Burke et al., 2013) (Fig. 3, Day 0) levels under
wet conditions. In the competitive extraction of water
experiment, the BTx642 plants extracted more soil water
than the SC121111E plants and maintained higher carbon
assimilation rates across all levels of soil water deficit (Fig.
5). The differential stress levels in the pot experiments
were not only supported by the RWC levels but also in
the rate of ABA accumulation in leaves (Fig. 4).

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Metabolic Response to Drought Stress

We chose to evaluate ABA accumulation as a measure
of the plants response to the onset of water-deficit stress
because ABAs main function is to regulate plant water
balance and osmotic stress tolerance (Zhu, 2002).
Because ABA leads to the expression of early
response transcriptional activators, we chose to evaluate
ABA responses as a surrogate for the plants sensing of
the onset of water-deficit stress. Figure 4 clearly shows
SC121111E induced ABA accumulation sooner and to
a greater extent than BTx642 experiencing the same soil
water levels, suggesting increased sensitivity to soil moisture deficit in SC121111E.
The field evaluation of the stress responses was conducted
in 2011 (81.7mm) and 2012 (169 mm). Additionally, both
seasons had above-average daily temperatures for significant periods and were considered severe drought episodes.
The measurement of relative water content following the
last irrigation exhibited a similar RWC differential between
the BTx642 and the SC121111E to that of the greenhouse
study (Fig. 2). Additionally, the differential dhurrin levels
were also observed in the field and the greenhouse studies (Fig. 3). No significant changes in dhurrin content were
observed in response to the onset of water-deficit stress in
the present study. This is consistent with previous findings
reported by Burke et al. (Burke et al., 2013).
It is interesting that although the ABA levels before
initiation of the water-deficit stress were low, the SC1211
11E had twice the ABA levels of the BTx642 (Fig. 4).
Possibly, the slightly higher ABA levels in the SC121111E
before the onset of the stress may account for the 1 to 2
C warmer canopy temperatures, a direct result of stomatal
closure, observed during the study (Fig. 8). The ABA levels
increased from Day 5 through Day 12 with the SC121111E
exhibiting a twofold higher level of ABA accumulation in
the leaves. Maximum canopy temperatures increased gradually, peaking 9 d after cessation of irrigation (Fig. 8). The
temperature differences between SC121111E and BTx642
showed an interesting transition beginning 8 d after irrigation and increasing in intensity through Day 12. In the
mornings the BTx642 was warmer than the SC121111E
and in the afternoon the SC121111E was warmer than the
BTx642 (Fig. 8, closed diamonds). One interpretation of this
data is that the SC121111E stomata are open earlier in the
day than the BTx642. In a study of sorghum stress responses
Garrity et al. (1984) reported that during the reproductive
period the stomata became nearly insensitive to leaf water
potential and remained open at low leaf water potentials.
The SC121111E carbon assimilation had a greater response
to the presence or absence of irrigation (Fig. 7). The photosynthetic changes may have been the result of more
responsive stomata in the SC121111E plants. It is not necessarily surprising that stomatal responses under more severe
stresses might be different between the two lines studied.
352

The SC121111E accumulated twice the levels of proline

(Fig. 5) compared with the BTx642 during the second week
of stress and exhibited higher sucrose levels than BTx642
(Fig. 6) during this time. It is possible that stomata were
kept open to maximize carbon uptake for photosynthesis,
possibly to provide additional photosynthate for the exponentially increasing synthesis of osmolytes. The BTx642
plants only added the osmolytes at half the rate during this
same period. This may have occurred in part because of
the high dhurrin levels present in the BTx642 leaves (Fig.
3). An unfortunate side effect of prolonged opening of the
stomata in the SC121111E is that more water would be
lost from the leaf and ultimately could result in leaf desiccation. It is interesting to speculate that the scenario described
above may explain why the lower leaves of senescent-type
sorghums desiccate during postflowering water stress and
stay-green types maintain green leaves during the same
period of stress. Clearly additional studies are needed to test
this hypothesis; however, this scenario is circumstantially
supported by reports in the literature.
Acknowledgments
The authors thank Jacob Sanchez, Halee Hughes, Marie Syapin,
Lan Liu-Gitz, and DeeDee Laumbach for their excellent technical support.

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