(Ash Content) Pcp082

Physiological and Molecular Approaches to Improve Drought
Resistance in Soybean
Lakshmi P. Manavalan1, Satish K. Guttikonda1, Lam-Son Phan Tran1,2, and Henry T. Nguyen1,
Mini Review
1National Center for Soybean Biotechnology and Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA
Drought stress is a major constraint to the production and carbon isotope discrimination; EST, expressed sequence tag;
yield stability of soybean [Glycine max (L.) Merr.]. For ge, leaf epidermal conductance; HI, harvest index; LASH, leaf
developing high yielding varieties under drought ash; LG, linkage group; OA, osmotic adjustment; PI line, plant
conditions, the most widely employed criterion has introduction line; QTL, quantitative trait locus; RAPD,
traditionally been direct selection for yield stability over random amplied polymorphic DNA; RIL, recombinant
multiple locations. However, this approach is time inbred line; RFLP, restriction fragment length polymorphism;
consuming and labor intensive, because yield is a highly RWC, relative water content; SSR, simple sequence repeat;
quantitative trait with low heritability, and inuenced by STS, sequence-tagged site; TE, transpiration efciency; TF,
differences arising from soil heterogeneity and transcription factor; WUE, water use efciency.
environmental factors. The alternative strategy of indirect
selection using secondary traits has succeeded only in a
few crops, due to problems with repeatability and lack of
Introduction
phenotyping strategies, especially for root-related traits. Soybean is the worlds leading economic oilseed crop. Pro-
Considerable efforts have been directed towards cessed soybeans are also the largest source of vegetable oil
identifying traits associated with drought resistance in and protein feed. In addition to being a source of macronu-
soybean. With the availability of the whole genome trients and minerals, soybeans contain secondary metabo-
sequence, physical maps, genetics and functional genomics lites such as isoavones (Sakai and Kogiso 2008), saponins,
tools, integrated approaches using molecular breeding phytic acid, oligosaccharides, goitrogens (Liener 1994) and
and genetic engineering offer new opportunities for phytoestrogens (Ososki and Kennelly 2003). Global produc-
improving drought resistance in soybean. Genetic tion of soybean in 2007 was around 219.8 million metric
engineering for drought resistance with candidate genes tonnes (mmt). The country with the greatest output was
has been reported in the major food crops, and efforts for the USA, producing 70.4 mmt, followed by Brazil (61 mmt),
developing drought-resistant soybean lines are in progress. Argentina (47 mmt) and China (14.3 mmt) (Soystats 2008).
The objective of this review is to consolidate the current While soybean has long been important in Japan for the pro-
knowledge of physiology, molecular breeding and func- duction of traditional foods such as tofu, miso, shoyu and
tional genomics which may be inuential in integrating vegetable oil, the consumption of soybean-based products is
breeding and genetic engineering approaches for drought increasing worldwide because of the reported benecial
resistance in soybean. effects including lowering of cholesterol, prevention of
Keywords: Drought resistance Genetic engineering cancer, diabetes and obesity, and protection against bowel
Marker-assisted selection Physiological traits Soybean and kidney diseases (Friedman and Brandon 2001). Soybean
[Glycine max (L.) Merr]. is also viewed as an attractive crop for the production of
biodiesel (Pimentel and Patzek 2008). It also has the ability
Abbreviations: AFLP, amplied fragment length to x atmospheric nitrogen (Burris and Roberts 1993) and
polymorphism; BAC, bacterial articial chromosome; CID, therefore requires minimal input of nitrogen fertilizer which
2Present address: Signaling Pathway Research Unit, Gene Discovery Research Group, RIKEN Plant Science Center, 1-7-22 Suehiro,
Tsurumi, Yokohama, 230-0045 Japan.

Corresponding authors: Lam-Son Phan Tran, E-mail: [email protected]; Fax, +81-45-503-9591; Henry T. Nguyen,
E-mail, [email protected]; Fax: +1-573-882-1469.
Plant Cell Physiol. 50(7): 12601276 (2009) doi:10.1093/pcp/pcp082, available online at www.pcp.oxfordjournals.org
The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
1260 Plant Cell Physiol. 50(7): 12601276 (2009) doi:10.1093/pcp/pcp082 The Author 2009.
Approaches to improve soybean drought resistance
often accounts for the single largest energy input in phenology, photoperiod sensitivity, developmental plastic-
agriculture. ity, leaf area maintenance, heat tolerance, osmotic adjust-
Plants are subjected to a range of abiotic and biotic ment, early vigor, rooting depth and rooting density.
stresses that affect their growth and development. In par- Additionally, transpiration efciency and leaf reectance are
ticular, it is predicted that water decit will continue to be a the other two traits related to WUE (Purcell and Specht
major abiotic factor affecting global crop yields (Sharma and 2004).
Lavanya 2002). One-third of the worlds population resides Signicant progress in breeding for drought resistance
in water-stressed regions, and with elevated CO2 levels in the cannot be made unless the stress is imposed year after year
atmosphere and climatic changes predicted in the future, to allow direct selection for yield. For this reason, a eld with
drought could become more frequent and severe. The resil- poor moisture-holding capacity, soil uniformity and a rea-
ience of legume crops against present-day weather extremes, sonable drought each year is important, but is unfortunately
such as drought, excess water, heat, cool weather during very difcult to achieve (Pathan et al. 2007). In order to
grain lling, and early frost, is considered to predict their enable high throughput and reliable gravimetric measure-
adaptation to future climate change (Cutforth et al. 2007). ments of water relation traits and water use in soybean, it is
In soybean, drought reduces yield by about 40% (Specht also important to include modern facilities such as a rainout
et al. 1999). Depending on hybrid characteristics, soybeans shelter in a breeding program. By using a rainout shelter with
use about 450700 mm of water during the growing season a movable roof, researchers at the National Center for Plant
(Dogan et al. 2007). The most critical period for water stress Gene Research (NCPGR) in Wuhan, China, have succeeded
in soybean has been reported to be during the owering in dissecting the genetics of complex traits such as drought
stage and the period following owering (Meckel et al. resistance (Pennisi 2008). The plant-wise drought treatment
1984). strategy developed at the NCPGR provides a useful method
Plants use various mechanisms to cope with drought for independent evaluation of the individual components of
stress. These may be classied into three groups: drought drought resistance (Yue et al. 2006). In addition, current
escape, drought avoidance and drought tolerance (Turner advances, such as automated plant phenotyping platforms
et al. 2001). Drought escape allows the plant to complete its to study the plant responses to soil water decit under con-
life cycle during the period of sufcient water supply before trolled conditions (Granier et al. 2006), an automated rotat-
the onset of drought. Normally the life cycle is shorter and ing lysimeter system for evapotranspiration and plant growth
plants set some seeds instead of complete crop failure. An measurements (Lazarovitch et al. 2006), non-destructive
example of drought escape is the Early Soybean Planting measurement of plant water status over time using portable
System, now used widely in the southern USA. In this system, unilateral nuclear magnetic resonance (Capitani et al. 2009),
short season cultivars are planted during MarchApril in and other precision equipments to quantify plant water
regions where later maturing cultivars have previously been use should be exploited. Screening of genotypes in such
grown. The early maturing cultivars start owering in late controlled facilities has the advantage of quantifying the
April to early May and set pods in late May, thus completing morpho-physiological traits that inuence drought adapta-
the reproductive stage before the period of possible drought tion, which in turn will complement yield selection criteria.
in JulyAugust (Heatherly and Elmore 2004). The second In this review, we rst concentrate on the major physio-
mechanism, drought avoidance, involves strategies which logical traits related to the mechanisms of drought resis-
help the plant maintain high water status during periods of tance in soybean (Fig. 1). Although the emphasis of this
stress, either by efcient water absorption from roots or by article is on soybean, the basic concepts are applicable to
reducing evapotranspiration from aerial parts. The third drought resistance improvement in other crops as well.
mechanism, drought tolerance, allows the plant to maintain Recent advances in marker-assisted selection, functional
turgor and continue metabolism even at low water poten- genomics and genetic engineering approaches to achieve
tial, e.g. by protoplasmic tolerance or synthesis of osmopro- drought resistance in soybean will also be discussed.
tectants, osmolytes or compatible solutes (Nguyen et al.
1997). In soybean, a widely accepted equation for grain yield
(Y) under water-limited conditions is a function of three
Physiological and biochemical responses
components, namely the amount of water transpired (T), associated with drought resistance in
water-use efciency (WUE) and harvest index (HI); soybean
Y = T WUE HI (Turner et al. 2001). Maintenance of opti-
mum transpiration, leading to increased WUE, is one of the Root-related traits
strategies to improve yield in soybean. Nine secondary traits Root morphology and plasticity. Soybean plants often
have been reported to be associated with the likelihood of undergo substantial water decits, even though water is
increasing or maintaining T during drought. These traits are readily available only slightly deeper in the soil. Plants can
Plant Cell Physiol. 50(7): 12601276 (2009) doi:10.1093/pcp/pcp082 The Author 2009. 1261
L. P. Manavalan et al.
Drought resistance
Drought avoidance Drought tolerance
High relative water content High harvest index

(less water use)
Leaf pubescence
Low epidermal
conductance Partial closure of stomata
High transpiration Less ureide accu-

efficiency mulation in petiole
Early vigor/ground cover Leaf osmotic adjustment
ABA, ROS scavenging

Fibrous and moderate
lateral roots
Deeper taproot early

in the season
Fig. 1 Traits associated with drought resistance in soybean. ROS, reactive oxygen species.
adapt to drought by developing a longer taproot which Because root growth and water deployment are critical to
helps reach the lower soil layers where water is available maintaining function in different environmental conditions,
(Taylor et al. 1978). In addition, an extensive brous root plasticity for root traits is a critical factor to acquire resources.
system can be useful for foraging subsoil surface moisture Water stress tends to increase biomass partitioning to roots,
and nutrients such as phosphorus. increasing the root : shoot ratio. Non-irrigated soybeans
One of the major factors inuencing soybean rooting showed an increase in root length when compared with
depth is taproot elongation rate. As taproots are the rst irrigated plants, especially in the subsoil (Huck et al. 1983).
formed roots in soybean, identication of genotypes Signicant correlations exist in soybean between drought
with rapidly elongating taproots under non-stress condi- resistance and various root traits such as dry weight, total
tions may allow the determination of deeper rooting ability. length, and volume and number of lateral roots (Read and
Genotypic variation in the taproot elongation rate in soy- Bartlett 1972, Liu et al. 2005a). These authors proposed using
bean cultivars has been well documented in greenhouse these traits as indicators for drought resistance. Soybean
studies. Kaspar et al. (1984) evaluated 105 diverse soybean plant growth and development are classied into vegetative
genotypes from different maturity groups. Taproot elonga- (V) and reproductive (R) stages. The V stage comprises
tion within a maturity group varied by as much as 1.3 cm d1, stages VE to Vn, where VE is vegetative cotyledon emergence
and contrasting cultivars were selected for eld study. The and Vn are numbered according to how many fully devel-
results of these studies indicated that the group of cultivars oped trifoliate leaves are present. The R stage comprises ow-
with faster elongation rates was also more able to deplete ering in stages R1 and R2, pod formation in stages R3 and R4,
soil water 120 cm below the surface. Limited research has and seed lling and maturation development in stages R5R8.
been reported regarding genetic variability in producing lateral In soybean, when drought was imposed during the later
roots. Under stress conditions, the number of lateral roots per stages of vegetative and/or the early stages of reproductive
unit of taproot length signicantly increased, but no promotion growth (R1R2), a large increase in root growth was observed
of taproot length or diameter was observed (Read and Bartlett in non-irrigated plants, especially in the deeper soil layers.
1972). Root growth was less affected when drought was imposed at
the R4 stage and ceased at the R5 stage (Hoogenboom et al. marker selection or genetic engineering to develop soybean
1987). In another study, it was demonstrated that soybean with better root-related traits.
plants which experienced drought before owering had Nitrogen xation under drought. Nitrogen (N2) xation in
higher seed yields than those stressed after owering, legumes is very sensitive to soil drying. In drought condi-
because they had already developed a large root system tions, soybean not only suffers losses of CO2 accumulation
before owering (Hirasawa et al. 1994). This shows that if a and reduced leaf area development, but its symbiotic N2
plant develops a large root system during its early vegetative xation is also especially vulnerable (Sinclair and Serraj 1995).
growth, it would be in an excellent position to maintain In dry soils, this results in a reduced supply of N2 for protein
turgor under drought conditions. production, which is the critical seed product of the plant,
All of these early studies were based on popular cultivars/ and consequently lower crop yields (Purcell and King 1996).
genotypes, and less is known about the rooting ability and/ Several factors have been related to the inhibition of N2 xa-
or plasticity of root growth in exotic plant introduction (PIs) tion under drought, including reduced oxygen availability,
lines. The line PI416937 has an exceptionally branched reduction in carbon ux to nodules, decline in nodule sucrose
brous root system (Carter and Rufty 1993, Hudak and Pat- synthase activity and an increase in ureides and free amino
erson 1996) which has shown drought resistance in the eld acids (Durand et al. 1987, Gonzalez et al. 1995, Arrese-Igor
by limiting transpiration during the high evaporative et al. 1999, King and Purcell 2005). In particular, a close rela-
demand period (Fletcher et al. 2007). The brous root trait tionship was found between leaf and nodule water potential
of PI416937 also conferred increased nitrogen xation (Durand et al. 1987). Nitrogenase activity showed a 70%
through increased root surface area. PI416937 possesses the decrease during the rst 4 d of drought, while photosynthe-
genetic capability to continue root growth in compacted sis only declined by 5%. This implies that water stress exerts
soils (Busscher et al. 2000). However, no correlation was an inuence on nitrogenase activity that is independent of
demonstrated between the rooting ability and drought rate of photosynthesis. It was also found that water decit
resistance of this PI line, although a positive correlation directly affected nodule activity through increased resis-
between rooting ability and seed protein concentration was tance to oxygen diffusion to the bacteroid. The increase in
found (Pantalone et al. 1996). oxygen diffusion resistance, decrease in nitrogenase-linked
The vast diversity of PI lines and cultivars worldwide pro- respiration and enzyme activity, accumulation of respiratory
vides valuable resources for identication of germplasm substrates and oxidized lipids, and the up-regulation of anti-
which can be used in breeding for root traits. However, the oxidant genes together demonstrate that the respiratory
laborious procedures involved in separation of roots and the activity of bacteroids is impaired in drought conditions, and
lack of rapid screening procedures are the major constraints that oxidative damage occurs in nodules prior to any detect-
for genetic improvement of root traits. Since a deep taproot able effect on sucrose synthesis or leghemoglobin (Naya
system associated with a moderate number of lateral roots et al. 2007). Soil drying also leads to the accumulation of
to extract soil water is the most promising genetic mecha- ureides in soybean leaves and is thought to be an inhibitor of
nism for improving soybean drought resistance, research in nodulation (Sinclair and Serraj 1995).
this direction is imperative. Substantial genetic variation was found in the sensitivity
Overall, root traits show strong potential for improve- of N2 xation in response to soil drying (Sall and Sinclair
ment of drought resistance through breeding. However, it 1991). When comparing soybean germplasm with or with-
would be extremely difcult to perform selection based on out the capacity to maintain tissue turgidity, and therefore
measuring the root phenotype. Instead, molecular tagging leaf and nodule function, under drought conditions at the
will facilitate breeding for root-related traits. The candidate reproductive stage, the germplasm able to maintain tissue
gene approach is another alternative methodology, which turgity showed the least reduction in grain yield (Patterson
involves choosing a candidate gene for root traits from and Hudak 1996). Screening for petiole ureide levels is an
public data, obtaining primer sequences to amplify the gene, effective initial approach to identify soybean lines whose N2
uncovering polymorphisms, developing a convenient proce- xation is more tolerant of soil drying. By screening a large
dure for large-scale genotyping, identifying a population for collection (> 3,000) of soybean PI lines in this way, eight lines
association studies, carrying out an association study of the were identied that exhibited N2 xation drought tolerance
candidate gene with trait phenotype and nally verifying the (Sinclair et al. 2000). In a further study, Sinclair et al. (2003)
uncovered associations. This approach was successful in found that ureide catabolism independent of manganese
nding candidate genes associated with root number in rice was active in six of the eight PI lines. The variety Jackson,
under low moisture conditions (Vinod et al. 2006). which is more tolerant of N2 xation in drying soils, has been
Understanding the physiological mechanisms and genetic used as a parent for developing high yielding lines for drought
regulation of root adaptation to drought will help to identify conditions. Two high yielding progeny lines for non-irrigated
specic genes and metabolic pathways for either gene-based conditions that exploit the N2 xation drought tolerance
trait of Jackson are considered a potential genetic resource has clearly established that decreases in stomatal conduc-
for improved symbiotic N2 xation and increased yield under tance are associated with severe plant water decits in soy-
rainfed conditions (Sinclair et al. 2007). bean (Bennett et al. 1987, Liu et al. 2005b). Further research
It is unclear whether N2 xation under drought is only is needed to establish the physiological and biochemical basis
regulated at the whole-plant level (e.g. by a systemic nitrogen for stomatal closure during intermittent drought conditions
feedback mechanism) or whether it can also occur at a local as well as mild soil water decits, particularly under eld con-
nodule level. There is evidence that signaling for biological ditions. Control of stomatal conductance under drought is a
N2 xation regulation can be provided by an N2 feedback promising physiological trait for developing drought resis-
mechanism involving shoot N2 status (Serraj et al. 2001). tance in soybean, considering the non-destructive nature of
King and Purcell (2005) suggested that a combination of the measurement and availability of precise instruments for
ureide and aspartic acid levels in nodules, plus the transport the measurement of conductance. However, a better under-
of several amino acids from the leaves, might be involved in standing of the quantitative inheritance of this trait is vital if
such a feedback inhibition mechanism in soybean. A recent it is to be used for this purpose.
study using a partially droughted split- root system indicated Epidermal conductance. A second shoot-related physio-
that N2 xation activity under drought stress is mainly con- logical trait which may help identify cultivars with increased
trolled at the local level rather than by a systemic N2 signal drought tolerance and prolonged crop survival during severe
(Marino et al. 2007). More research should be directed water stress is low leaf epidermal conductance (ge). Total leaf
toward understanding the molecular genetics of the factors conductance to water vapor is the sum of the stomatal and
limiting and regulating the response of N2 xation under cuticular diffusive pathways acting in parallel (van Gardingen
drought. and Grace 1992). When the stomata are open, cuticular con-
ductance is generally a negligible fraction of total conduc-
Shoot-related traits tance. However, in water-stressed or dark-adapted leaves
Stomatal conductance. One of the shoot-related physiologi- in which the stomata tend to be closed, the cuticular com-
cal traits that may affect drought tolerance is the decline in ponent of leaf epidermal conductance may exceed the
whole-plant water use during a soil water decit event. As a stomatal conductance (Boyer et al. 1997). Under severe
soil water decit develops, plants undergo a transition from water decit when stomatal closure is maximal, ge (the sum
the water-saturated phase, in which whole-plant water use of cuticular conductance and any residual stomatal conduc-
is not dependent on the soil water content, to a second tance) determines the rate of water loss from leaf tissues and
phase where water use is directly related to the availability of therefore the rate of progression toward critically low (inju-
soil water (Sinclair and Ludlow 1986). This transition is asso- rious) leaf water content. Species adapted to arid environ-
ciated with a reduction in the average stomatal conduc- ments tend to have low ge (Riederer and Schreiber 2001),
tance, and can occur at different soil water contents in and crop species or varieties with low ge are often those that
different species (Earl 2003). Stomatal conductance is a key survive the longest under severe soil water decits (Sinclair
variable inuencing leaf gas and water vapor exchange. The and Ludlow 1986, Jovanovi et al. 1996). Signicant variation
effects of drought stress on leaf growth, stomatal conduc- in epidermal conductance was found among 74 soybean
tance and plant water relations have been well addressed in accessions. Expression of genotypic differences in epidermal
soybean (Muchow 1985, Bennett et al. 1987, Ray and Sinclair conductance among the selected accessions was consistent
1998). It was shown that drought stress decreases relative across environments (Paje et al. 1988). Interestingly, these
leaf expansion rate, stomatal conductance and leaf turgor, differences in epidermal conductance were not associated
whereas it increases the content of ABA in the leaf and xylem with differences in stomatal density. Selection for lower epi-
(Liu et al. 2003). In this study, decreased stomatal conduc- dermal conductance could allow improved survival of leaves
tance coincided with an increase in xylem ABA and occurred under intermittent water stress, so that sufcient leaf area
before any signicant change in leaf turgor was detected, remains available for growth after the stress is relieved (Paje
indicating that chemical signals (root-originated ABA) con- et al. 1988). Recently, a signicant negative correlation
trol stomatal behavior at moderate soil water decit. Relative (r = 0.74) was reported between ge and WUE under drought,
stomatal conductance decreased exponentially with increas- which again supports the hypothesis that lower epidermal
ing relative xylem ABA (r2 = 0.98) (Liu et al. 2003). Genotypic conductance is a desirable trait for drought resistance
differences in the ability to keep stomata open despite inter- (Hufstetler et al. 2007). Perennial wild-type soybean exhib-
nal water stress have been reported in soybean (Vignes et al. ited lower values of ge compared with various soybean culti-
1986). When compared with soybean, other legumes such as vars and G. soja. Among the soybean genotypes, tropically
green gram (Vigna radiata), black gram (Vigna mungo) and adapted genotypes exhibited the lowest epidermal conduc-
cowpea (Vigna unguiculata) exhibited better stomatal con- tance and more negative osmotic potential when compared
trol over water loss (Vignes et al. 1986). Previous research with genotypes adapted to temperate regions, suggesting
that the adaptability of tropical genotypes might have WUE. The close association between genotypic and geno-
resulted from selection for survival under drier environ- typic mean yield (r = 0.71) provides evidence that soybean
ments (James et al. 2008a). Broad-sense heritability for WUE can potentially be improved simply by selecting for
epidermal conductance was signicantly different from zero genotypic mean yield (Specht et al. 2001). In a study using 24
in 10 hybrid populations and ranged from 60 to 93% (James soybean genotypes, WUE ranged from 2.7 g dry matter kg1
et al. 2008b). The magnitude of the heritability estimates water to 3.4 g dry matter kg1 water (Hufstetler et al. 2007).
suggested that the trait was highly heritable. However, to It has been proposed that the aperture of stomata could be
validate the usefulness of these traits in breeding for drought regulated in such a way that a partial closure of stomata at a
resistance, more research is needed to characterize the inter- certain level of soil water decit might lead to an increase in
relationship between ge, conductance, maintenance of leaf WUE (Liu et al. 2005b). While promising results were
turgor and plant survival. observed, many uncertainties still remain regarding the
Leaf pubescence density. Leaf pubescence is a common implementation of irrigation techniques in different crop
feature of xerophytic plants, as well as some crop plants species under varying environmental conditions. Therefore,
including soybean. Generally, leaf pubescence density a deep understanding of the physiological basis for stomatal
increases reectance from the leaf, resulting in lower leaf regulation and improved WUE in drought-stressed plants is
temperatures under high irradiance. Leaf pubescence den- required (Liu et al. 2005a).
sity is an important adaptive trait for soybean under water- Another relevant trait related to WUE is transpiration
stressed conditions. Densely pubescent lines had increased efciency (TE), which is the assimilation or dry matter accu-
vegetative vigor, greater root density and deeper root exten- mulation per unit of transpiration (Fischer 1981). TE is under
sion (Garay and Wilhelm 1983). Increased leaf pubescence genetic control and excludes amount of water lost by soil
density may also increase leaf boundary layer resistance by evaporation, and hence should be considered as a potential
up to 50%. Reduced leaf temperature, restricted transpira- trait.
tion water loss and enhanced photosynthesis due to lower Osmotic adjustment. Osmotic adjustment (OA) is dened
radiation penetration into the canopy were also reported to as the active accumulation of solutes that occurs in plant
be associated with the dense pubescence trait (Specht and tissues in response to an increasing water decit. OA is con-
Williams 1985). Moreover, extra-dense pubescence can sig- sidered a useful measure because it provides a means for
nicantly reduce the incidence of soybean mosaic virus maintaining cell turgor when tissue water potential declines.
infection. However, increased pubescence density controlled OA has been shown to maintain stomatal conductance and
by the Pd1 and Pd2 alleles produces both positive and nega- photosynthesis at lower water potentials, delayed leaf senes-
tive agronomic effects (Pfeiffer et al. 2003). In particular, cence and death, reduced ower abortion, improved root
these alleles have been shown to reduce seed yield, delay growth and increased water extraction from the soil as water
maturity and increase plant height, with a concomitant decit develops (Turner et al. 2001). The capacity for OA in
increase in lodging (Specht et al. 1985). Thus, the negative soybean ranges from 0.3 to 1.0 MPa, which is not as wide as
linkage between the leaf pubescence density and seed yield the ranges observed in chickpea (Cicer arietinum) and pigeon
should be considered before introgressing this trait into elite pea (Cajanus cajan) (0.11.3 MPa) (Cortes and Sinclair 1986,
germplasm. Turner et al. 2001). Among six common soybean cultivars
Water use efciency. The WUE of a plant is generally tested, leaf OA ranged from 0.3 to 0.5 MPa under prolonged
dened as the amount of biomass accumulated per unit of drought control conditions (S. Guttikonda, unpublished). In
water used. Genetic variation in WUE has been reported in a recent study, the leaf osmotic potential of cultivated and
recent years for many eld crops, including soybean (Mian wild soybean accessions was measured in order to assess the
et al. 1996). High WUE can contribute to crop productivity capacity for OA. Leaf osmotic potential at 70% relative water
under drought. The positive association between WUE and content (RWC) ranged from 2.3 to 3.5 MPa in soybean
total biomass yield in a drought environment suggests that cultivars and 4.3 MPa in the wild species G. latifolia and
improvement of the WUE of a crop plant should result in G. canescens (James et al. 2008a). There was a highly signica-
superior yield performance if a high HI can be maintained tion correlation (r = 0.98) between rate of decline in RWC
(Wright 1996). Soybean cultivar Young exhibited consider- and relative OA among seven different genotypes tested,
ably higher WUE (4.4 g DW l1) than did PI416937 (3.7 g suggesting that genotypes with higher osmotic potential
DW l1) in greenhouse conditions (Mian et al. 1996). In showed a slower rate of decline in RWC, indicating the abil-
another greenhouse study, soybean cultivar Jackson accu- ity of these genotypes to maintain turgor for longer periods
mulated more biomass and total nitrogen than PI416937 (James et al. 2008b).
with similar transpirational losses (Purcell et al. 1997). Most of the published studies on OA in crops have used
Signicant genetic differences were observed for , which is only leaf measurements, and few examined the potential
an empirical seed yield based on estimation of genotypic benets of OA upon crop yield (Serraj and Sinclair 2002).
One study in wheat (Morgan 1995) claimed putative bene- water decit (Meckel et al. 1984). When soybean plants were
ts of OA on crop yield. In chickpea, however, the relation- subjected to continuous water stress from the beginning of
ship between OA and yield under water-limited conditions growth stage R6 (early in seed lling) until maturity, the
was inconsistent (Leport et al. 1999, Moinuddin and Khanna- plants lost nitrogen and chlorophyll from their leaves more
Chopra 2004, Turner et al. 2007). Turner et al. (2007) studied rapidly than non-stressed plants (de Souza et al. 1997). Stress
advanced breeding lines of chickpea over different conti- also shortened the seed-lling period (R7 occurred up to 7 d
nents and demonstrated genotypic differences in OA, earlier), resulting in smaller seeds (maximum reduction of
but the phenotypic expression of OA was not stable and 32%) and lower yields (up to 44%). Water stress can induce
varied from year to year. It was also found that the difference senescence which cannot be stopped by reapplying water,
in OA has no effect on yield under terminal drought when and even short periods of water stress during seed lling
grown under rainfed conditions. These inconsistent results might decrease yield by up to 23% (Brevedan and Egli 2003).
could be due to differences in growth habitat, stress level By applying large quantities of nitrogen fertilizer to supplant
and phenological characteristics of the tested cultivars. An the dependency on nitrogen xation during water decit,
exception where OA may have an important benet during Purcell and King (1996) reported an 18% increase in soybean
drought is when it is directed to roots, where turgor mainte- yield. However, while inherently delayed senescence may be
nance might lead to an increased rooting depth and would advantageous for yield under optimal growing conditions, it
thus increase the amount of water available to the crop. may be of no consequence under post-anthesis stress
Emphasis should be given to studying the role of OA in root because then the over-riding stress factor will impose accel-
tips to improve crop growth under water decit conditions erated senescence or leaf killing.
(Serraj and Sinclair 2002). Another problem with selecting Interestingly, water stress at the ower induction, ower-
for OA is the limited number of genotypes that could be ing, pod formation and pod-lling stages of growth did not
screened using current water relation traits. Use of multiple signicantly affect the oil or protein content of soybean
psychrometers (Santamaria et al. 1990) and complex rela- seeds (Dornbos and Mullen 1992). Furthermore, drought
tionships between RWC and OA has limited screening to 50 had little effect on the fatty acid composition of the oil, but
genotypes per study. As suggested by Turner et al (2001), the high air temperature reduced the proportion of polyunsatu-
development and identication of molecular markers, as rated components. A recent study also showed that irriga-
in rice and wheat, would enable selection for OA in early tion generally had no signicant inuence on unsaturated
generation populations of soybean. fatty acid accumulation in seed oil in soybean genotypes
with altered fatty acid proles (Lee et al. 2008).
Soybean seed and grain lling during drought stress In conclusion, the key to a complete understanding of the
Yield as measured by the weight of seeds is reduced most by relationship between water stress and soybean seed growth
stress occurring during early formation and pod-lling stages. lies in determining the effect of stress on the supply of assim-
Water stress during early reproductive growth (owering and ilate to individual seeds.
pod set) reduces yield, usually as a result of fewer pods and
seeds per unit area. It has been shown that root-originated
Marker-assisted selection for drought resistance
xylem sap ABA can move to reproductive structures causing
kernel/pod abortion, presumably via inhibition of cell divi- In environments where water is limited, genetic improve-
sion in young ovaries (Liu et al. 2003). Stress during seed ll- ment of a crop for drought resistance is an economically
ing, and after pod and seed numbers are xed, could affect attractive option (Blum 2002). However, despite the large
the seed growth rate by reducing photosynthesis and, there- resources committed to soybean breeding, progress in
fore, the supply of assimilate available to the seed, ultimately improving drought resistance has been slow for a number of
affecting seed size. At the pro-embryo developmental stage, reasons. (i) Identifying lines with the highest yield potential
young ovules are undergoing active division, which coincides under optimum moisture conditions is an important selec-
with rapid pod expansion and is specically sensitive to tion criterion in soybean. Conversely, evaluating lines from
water decit during seed lling (Westgate and Peterson low yielding environments under drought conditions is
1993). In a rainout shelter study, the drought tolerance based often not considered, because small yield differences among
on seed yield varied among Brazilian soybean cultivars. Cul- lines do not separate high yielding genotypes from low yield-
tivars with high drought tolerance maintained a high crop ing genotypes. (ii) Historically, the emphasis in soybean
growth rate during the early reproductive stage, associated breeding was upon resistance to biotic stress rather than
with high seed yield (Oya et al. 2004). The length of the seed- abiotic stress such as drought, due to the complexity of trait
lling period is another important determinant of yield in all evaluation. This unfortunately resulted in a narrow genetic
grain crops, including soybean. Seed ll duration is under base for initiating drought resistance breeding programs. (iii)
genetic control (Smith and Nelson 1987) and is sensitive to Drought resistance is governed by several complex factors
including genotype, environment and the interaction of the and WUE depends on the maintenance of a constant con-
two (Carter et al. 1999). In the future, marker-assisted selec- centration of minerals in the transpiration stream. While
tion based on genotype will greatly increase breeding environmental conditions could cause variation in the min-
efciency. eral concentration, comparison of different genotypes under
Identication of quantitative trait loci (QTLs) related to the same environmental conditions would nonetheless
drought resistance is one of the promising approaches using provide repeatable responses. A molecular investigation may
marker-assisted selection (Pathan et al. 2007). In soybean, a provide some insight into the genetic relationships between
large number of QTLs have been identied for traits related WUE and leaf ash (LASH) (Mian et al. 1996).
to agronomic, physiological, seed composition, and both To identify QTLs associated with WUE (total plant dry
biotic and abiotic stress parameters (www.soybase.ncgr.org). weight/amount of water used in liters) and LASH in 36-day-
However, to date, only a few QTLs have been associated with old, greenhouse-grown plants, Mian et al. (1996) constructed
drought resistance traits (Table 1). Reported QTLs also a restriction fragment length polymorphism (RFLP) map
explain <10% of the phenotypic variation for those traits. from a soybean [G. max (L.) Merr] population of 120 F4-
Most studies for QTL identication have included single and derived lines from a cross of Young PI416937. Signicant
small size populations. The QTL markers identied by a (P < 0.01) phenotypic differences were detected among the
single population may not be automatically applicable to lines for both traits. A total of four and six independent RFLP
unrelated populations without marker validation and/or markers were associated with WUE and LASH, respectively,
ne mapping (Nicholas 2006). and, if combined, each group of markers would explain
As discussed above, WUE is one of the important traits 38 and 53% of the variability in the respective traits. One
associated with drought resistance in soybean. Since water marker locus (cr497-1), on USDA Linkage Group (LG) J,
consumption and its efcient use by crops are related to explained 13.2% of the variation in WUE, indicating the
yield, carbon isotope discrimination (CID) (, a measure of presence of a major QTL. The authors also found that LASH
the 13C/12C ratio in plant tissues compared with the air) may was negatively correlated with WUE (r = 0.40), and two
provide a useful indirect measure of genetic variation in TE, QTLs were associated with both WUE and LASH. For each of
i.e. the ratio of net assimilation to water transpired, in C3 these QTLs, the allele for increased WUE was associated with
species (Araus et al. 1998). The expensive equipment and reduced LASH.
high cost of analyses restrict the use of in breeding pro- To test the consistency of WUE QTLs across populations,
grams. However, ash content and K concentration have been Mian et al. (1998) screened another F2-derived soybean pop-
proposed as cheap and easily determined surrogates of ulation from the cross of S100 PI416937. They discovered
(Tsialtas et al. 2002). Due to its inherent analytical simplicity, that one QTL linked to the RFLP marker A063E for WUE, was
estimation of ash content in vegetative organs became an also found in the Young P1416937 population, but the
attractive alternative to for a preliminary screening phenotypic effect was <10%. These two loci, however, have
of large, genetically diverse populations (Masle et al. 1992). not yet been conrmed in any other genetic background or
Ash concentration (leaf and stem) of crested wheatgrass environmental conditions. To determine the genetic basis of
(Agropyron desertorum) was negatively associated with WUE and CID, 236 recombinant inbred lines (RILs) have been
(Mayland et al. 1993). The relationship between ash content developed from a cross between Minsoy and Noir 1. Only
Table 1 Quantitative trait loci (QTLs) for drought resistance in soybean

Mapping populations Reported traits Marker linked to QTL, linkage group (LG) and Reference
(QTL number) contribution (R2)
Hutcheson PI471938, 140 F4 population Yield (3), wilting (3) Satt226, LG-D2; Sat_375, LG-F1; Sat_074, LG-F2 Monteros et al. (2006)
Jackson KS4895, 81 RILs Leaf wilting (1) Sat_044, LG-K, R2 = 17 Bhatnagar et al. (2005)
Minsoy Noir 1,236 RILs Yield (1) Satt205-satt489, LG-C2, R2 = 7 Specht et al. (2001)
S-100 Tokyo, 116 F2 population Water use efciency (2) A489H, LG-L, R2 = 14A0631, unlinked, R2 = 8 Mian et al. (1998)
Young PI416937, 120 F4 population Water use efciency (5) B031-1, LG-G, R2 = 8.5 Mian et al. (1996)
A089-1, LG-H, R2 = 8.7
cr497-1, LG-J, R2 = 13.2
K375-1, LG-J, R2 = 7.5
A063-1, LG-C1, R2 = 5
LG, linkage group; R2, percentage of trait variance explained by a QTL; RIL, recombinant inbred line.
one QTL was identied for CID on LG-C2, with a phenotypic tagged site (STS) markers, developed from ESTs, which are
contribution of <10% and with no effect on (Specht et al. associated with drought responses, disease resistance, seed
2001). development and composition traits (Wu et al. 2008). In
So far QTLs in soybean under water decit conditions addition, a six-dimensional pool array has been recently
have been reported only for WUE and LASH. More studies developed from 49,152 BstyI soybean clones (6 genome
are needed to identify QTLs that inuence root architecture equivalents), comprising 208 BAC pools (Wu et al. 2008).
and shoot turgor maintenance. Mapping for new QTL(s)/ The integrated genetic and physical map will be useful for
gene(s) and determination of gene action under drought comparative genetic analysis, map-based cloning of QTLs of
will probably provide key resources to improve tolerance to desired traits, and genomic sequencing. In addition, yeast
drought stress in soybean. High-density genetic maps and articial chromosomes have also been created for the pur-
conrmed QTLs/genes, which are screened across the vari- pose of chromosome walking and in situ hybridization
ous environments and across genetic backgrounds, are the (Santra et al. 2003). Recently, the entire soybean genomic
most important criteria for developing drought-resistant sequence has been released with 66,153 protein-coding loci
soybeans through marker-assisted selection (Vuong et al. (http://www.phytozome.net/soybean#C).
2007). EST sequencing projects in soybean have resulted in a
wealth of DNA sequence information. A spotted cDNA
Functional genomics resources and tools for microarray is available containing 36,000 elements con-
drought research in soybean structed from soybean cDNAs, which were derived from a
variety of EST libraries representing a wide source of tissues
Plant functional genomics has emerged as a new and rapidly and organs, developmental stages and stress-related or
evolving scientic discipline to study the functions of genes. pathogen-infected plants (Vodkin et al. 2004). The ESTs iso-
In the last decade, considerable progress has been made in lated from the subtracted library of drought-stressed soybean
developing genomic resources for soybean, including the root tips and submitted to GenBank will greatly contribute
sequencing of the entire soybean genome of approximately to the stress-specic unigenes for further functional genom-
975 Mb (http://www.phytozome.net/soybean#C). For the ics work aimed at better understanding of the drought stress
genetic and genomic analyses of the soybean genome, pre- response of the soybean root system (Valliyodan and Nguyen
cise genetic and physical maps are important. Various types 2008). This result will provide an important resource for
of physical maps have so far been reported using RFLP, revealing the transcriptional regulatory networks which will
random amplied polymorphic DNA (RAPD), simple illuminate our understanding of the drought stress responses
sequence repeat (SSR) and amplied fragment length poly- of soybean at different developmental stages.
morphism (AFLP) markers (Shinozaki 2007). A high-density Full-length cDNAs are important tools for functional
genetic linkage map of soybean using expressed sequence analysis of genes, and provide a valuable resource for the
tag (EST)-derived microsatellite markers was generated improvement of soybean productivity and drought resis-
using a hybrid between the Japanese cultivar Misuzudaizu tance by biotechnology. Umezawa et al (2008) obtained
and the Chinese line Moshidou Gong 503 by Japanese 6570 new full-length sequences of soybean cDNAs derived
researchers (Hisano et al. 2007, Xia et al. 2007). The possibil- from tissues treated with different abiotic stresses, in order
ity of segmental duplications in the previously suggested to analyze gene functions and structures. About 88% of the
regions of the soybean genome was conrmed by inspecting soybean cDNA clones contained complete coding sequences
the co-linearity between the genomes of soybean and Lotus in addition to 5- and 3-untranslated regions.
japonicus. This comparison, combined with information Although the gene expression proling data created by
relating to regulatory networks deduced by transcriptional microarray analysis have been commonly used as valuable
proling, may provide clues toward understanding the evo- information in functional genomics or in the selection of
lutionary process of the paleopolyploid soybean genome candidate genes for genetic engineering of soybean plants,
(Hisano et al. 2007). Microsatellite markers have been dis- the integration of transcriptomics, proteomics and metabo-
covered from bacterial articial chromosome (BAC) libraries lomics represents the frontier of integrated functional
to create detailed genetic and physical maps of the soybean genomics. It provides an unprecedented opportunity to gain
genome, which together cover the soybean genome by a whole picture of the physiological and biochemical
>35-fold (Shoemaker et al. 2008). The constructed Williams responses to drought stress. Taking advantage of available
82 BstyI library contains 92,160 clones with an average insert proteomic technology, a proteomic analysis of drought-
size of 150 kb and covers approximately 12 genome equiva- treated soybean roots, leaves and seeds has revealed region-
lents (http://www.soybeangenomics.org/genome_mapping. specic regulation of metabolism in the growth zone of
php). A genetic map was then integrated into this physical water-stressed soybean roots (M. Yamaguchi, unpublished).
map by anchoring approximately 1,000 SSR and sequence While transcriptomic data and proteomic analyses do
not tell the whole story of what might be happening in a genomic lesions, and deletion mutants have been developed
cell, metabolic proling can give an instantaneous snapshot with -rays (Bhatia et al. 1999), X-rays and fast neutrons
of the physiology and biochemical changes in the cell (Men et al. 2002).
(Morgenthal et al. 2007). It has long been known that in Insertional mutagenesis is another method by which
addition to ABA, a variety of osmolytes, including certain gene expression can be modied in plants, creating mutants
sugars (such as rafnose family oligosaccharides, sucrose, with either gain or loss of function for drought studies.
trehalose and sorbitol), sugar alcohols (such as mannitol), T-DNA-tagged populations in rice (An et al. 2003) and maize
amino acids (such as proline) and amines (such as glycine (Cowperthwaite et al. 2002) proved to be very useful
betaine and polyamines), accumulate in plants in response resources for functional genomics, including drought func-
to drought stress (Umezawa et al. 2006, Seki et al. 2007). tional genomics. However, the development of T-DNA
However, the degree to which the metabolome is altered in mutants takes a lot of time for soybean transformation (57
response to drought stress has remained a mystery. The months), and considerable labor to generate an adequate
recent development of highly sensitive metabolite proling number of insertions (Parrott and Clemente 2004). To over-
technologies, such as gas chromatography or liquid chroma- come this problem, it has been suggested to couple T-DNA
tography coupled with mass spectrometry [GC-time-of-ight regions with transposon-based elements (Brutnell 2001).
(TOF)/MS or UPLC-Q-TOF-MS] makes such comprehensive Isolation of loss-of-function mutants may be hindered by
analysis possible. Recently, Urano et al (2009) used GC-TOF/ the lack of an obvious phenotype due to gene redundancy,
MS to detail the changes that occur in the metabolome of especially in a paleopolyploid such as soybean (Mathieu
Arabidopsis in response to drought stress, and to assess the et al. 2009). To circumvent this problem, a gain-of-function
extent to which the dehydrated metabolome is recongured approach can be adopted to generate mutants by trans-
by inactivation of the NCED3 gene which is involved in the forming plants with multiple enhancer sequences (Jeong
dehydration-inducible biosynthesis of ABA. Although the et al. 2006). By using the Ds-transposon system, Mathieu
role of a number of accumulated metabolites remained et al. (2009) have created activation-tagging, gene and
unknown, this study has revealed new molecular mecha- enhancer trap elements in soybean to establish a transpo-
nisms of dynamic metabolic networks in response to drought son-based mutagenesis repository which currently houses
stress. Additionally, genetic engineering using genes encod- around 900 soybean events, with anking sequence data
ing components of drought stress-related metabolic path- derived from 200 of these events (http://digbio.missouri.
ways has shown the potential to enhance drought resistance edu/gmgenedb/index.php).
in important crops, including rice, wheat and soybean The availability of these functional genomics tools and
(de Ronde et al. 2004a, de Ronde et al. 2004b, Umezawa et al. resources, coupled with the forthcoming completely anno-
2006, Seki et al. 2007). Thus, metabolomics, in addition to tated genome sequence, represents an exciting era in soy-
transcriptomics and proteomics, provides a major tool for bean genetics. With the availability of mutant populations
characterizing the drought stress response in soybean in the and major achievements in marker-assisted selection and
post-genomics era. soybean transformation, it is now possible to study and
Both gain-of-function and loss-of-function approaches characterize the genes related to drought resistance, leading
can be used to elucidate gene function, as well as provide ultimately to better soybean productivity.
novel tools and mutant resources for improving drought
resistance in plants (Timpte et al. 1994, Klein et al. 2004).
Antisense- and RNA interference (RNAi)-mediated tran-
Genetic engineering for drought resistance
scriptional or post-transcriptional gene silencing has been
used to study soybean gene function (Buhr et al. 2002, Nunes In response to stress, plants activate a number of defense
et al. 2005, Subramanian et al. 2005). Alternatively, fast neu- mechanisms that function to increase tolerance to adverse
trons, X-rays, -rays and chemical mutagenesis are effective conditions. A large array of genes is activated and conse-
methods to generate mutants in plants for drought research, quently a number of proteins are produced to contribute to
including legumes such as Medicago trunculata, L. japonicus the metabolic pathways that lead synergistically to the
and soybean. LegumeBase (http://www.legumebase.agr. enhancement of stress tolerance (Shinozaki and Yamaguchi-
miyazaki-u.ac.jp) provides a list of mutant resources in these Shinozaki 2000, Seki et al. 2003, Valliyodan and Nguyen 2006).
legumes. Chemical mutagenesis can be applied to soybean The response to drought stress, which involves a number of
followed by screening for mutations in a target of interest, biochemicalmolecular mechanisms, is complex, as sche-
using a strategy known as Targeting Induced Local Lesions IN matically represented in Fig. 2. Although the role of the vast
Genomes (TILLING). Recently, TILLING has been applied to majority of these genes in plant adaptation to environmental
four mutagenized soybean populations in two backgrounds stresses remains to be elucidated, the function of some of
(Cooper et al. 2008). Radiation has been also used to induce regulatory genes, which encode regulatory transcription
Drought
Disruption of cellular an ionic

homeostasis, and membrane functions
Signal sensing, perception

and transduction
Transcriptional control
through transcription factors Ion channels
(aquaporins, proton
Osmoprotectants
transporters)
Gene activation
Molecular
Antioxidants
Stress tolerance/resistance chaperones
through reactivation of cellular
homeostasis
Plant survival/recovery
Fig. 2 Molecular mechanisms associated with drought tolerance.
factors (TFs), and that of functional genes, which encode ZmNF-YB2 gene produced an 50% increase in yield relative
metabolic components, have been described (Yamaguchi- to controls when water was withheld from the planted eld
Shinozaki and Shinozaki 2006, Tran et al. 2007a,b). area during the late vegetative stage (Nelson et al. 2007).
The application of this emerging understanding to the A number of important successes have also been reported
genetic engineering of food crops has already led to exam- in creating drought tolerance in economically important
ples of improved drought tolerance and increased yield plants, by genetic engineering of functional genes. For exam-
under drought (Hu et al. 2006, Nakashima et al. 2007, Nelson ple, trehalose, a non-reducing disaccharide that functions as
et al. 2007, Hu et al. 2008). Introduction of the SNAC1 and a compatible solute to protect biological structures under
ZmNF-YB2 TFs into rice and maize, respectively, enhanced stress, has been used to increase drought tolerance in rice
the drought tolerance of transgenic plants as demonstrated and tomato (Garg et al. 2002, Cortina and Marcia 2005).
by eld studies. Transgenic rice overexpressing the SNAC1 Transgenic rice plants were developed with a trehalose-
gene had 2234% higher seed setting in the eld than the 6-phosphate synthase/phosphatase gene using an ABA-
negative control under severe drought stress conditions at inducible promoter. Under drought stress, these transgenic
the reproductive stage. SNAC1 transgenic rice plants are rice plants showed increased amounts of trehalose, leading
drought tolerant not only due to the increased expression of to higher levels of tolerance not only to drought, but also to
genes encoding proteins functioning in the production of salt and low temperature stresses, without any negative
osmolytes, detoxication and redox homeostasis, and in effect on plant growth or grain yield (Garg et al. 2002). Trans-
protection of macromolecules, but also because of the genic canola was generated using a farnesyltransferase
increased stomatal closure in transgenic leaves which pre- (ERA1) antisense construct driven by a drought-inducible
vents water loss from the plant (Hu et al. 2006). Similarly, rd29A promoter. The developed transgenic canola plants
SNAC2/OsNAC6 transgenic rice plants have improved showed increased ABA sensitivity, a signicant decrease in
drought tolerance because of the enhanced expression of a stomatal conductance and decreased transpiration when
large number of genes encoding proteins with predicted tested under drought conditions (Wang et al. 2005). Recently,
stress tolerance functions such as detoxication, redox transgenic tobacco was engineered to overexpress an isopen-
homeostasis and proteolytic degradation (Nakashima et al. tenyl transferase gene (IPT) from Agrobacterium tumefaciens
2007, Hu et al. 2008). Transgenic maize overexpressing the driven by the SARK promoter, which was isolated from bean
(Phaseolus vulgaris) and shown to be induced by both technologies, provide unprecedented opportunities to
drought stress and maturation. The transgenic plants showed understand global patterns of gene expression and their
improved drought tolerance by maintaining high water con- association with the development of specic phenotypes, as
tent and photosynthetic activity (Rivero et al. 2007). well as promising tools for the genetic improvement of
Although a considerable body of literature now supports plants cultivated in adverse environments by molecular
the development of stress-resistant transgenic crops by breeding or transgenic approaches.
genetic engineering as a promising approach, only one study
has been reported to date in soybean. Transgenic soybean
Concluding remarks
plants overexpressing the Arabidopsis 1-pyrroline-5-
carboxylate synthase gene, P5CR, showed greater tolerance Increased demand for soybean products over the past
to drought stress due to an increased free proline level and decade has continued to raise global soybean production
RWC and a reduced level of reactive oxygen species, particu- steadily upward. Indeed, rising demand has prompted pro-
larly hydrogen peroxide (de Ronde et al. 2004a, de Ronde ducers to shift acres from wheat to soybean. Although there
et al. 2004b, Kocsy et al. 2005). Since this report, much effort are numerous cultivated and wild soybean accessions in the
has been directed toward isolating drought stress-related soybean germplasm collections of the world, only a few
genes in all physiological and biochemical aspects of drought germplasm have been screened for drought tolerance. Com-
stress, TFs and their respective promoters, which will in turn bining genes from both wild and cultivated species shows
provide novel tools and resources for the development of promise to obtain genotypes with higher levels of tolerance.
engineered soybean with improved drought resistance. The During our survey of the literature, we found that there have
identied soybean candidate genes are usually tested for been only limited efforts to study soybean genotypic varia-
their ability to enhance drought tolerance in Arabidopsis tion for important physiological traits related to drought
before pursuing their engineering into soybean. Chen et al. tolerance, such as cell membrane thermostability, osmotic
(2007) isolated an AtDREB homologous gene GmDREB2 from adjustment, canopy temperature depression and metabolic
soybean. Constitutive overexpression of GmDREB2 increased traits such as antioxidants and ABA. Considering the impor-
the survival rate of transgenic plants without growth retar- tance of soybean to the world economy, the identication
dation under water decit and salinity stress conditions of suitable traits and genotypes associated with drought tol-
(Chen et al. 2007). In addition, a few basic-leucine zipper erance in soybean is absolutely necessary. More concen-
genes encoding bZIP TFs were cloned from soybean, and trated efforts are needed to screen for resistant germplasm,
found to be induced by drought and high salt treatments. By discover new candidate genes and combine these genes for
overexpressing some of these genes in Arabidopsis, the higher levels of drought tolerance in soybean. While com-
researchers found that transgenic plants have increased bining genes/traits, preference should be given to pyramid a
freezing and salt tolerance, but no signicant difference in set of genes/traits contributing to a whole regulatory path-
drought tolerance (Liao et al. 2008a, Liao et al. 2008b). In two way. With the recent availability of the entire soybean
other recent studies it was found that overexpression of a genome sequence and genome-wide expression proling
GmERF TF family member, the GmERF089 gene, and a chilling- data, it is possible to identify key genes regulating drought
inducible GMCHI gene promoted enhanced tolerance to tolerance. However, the identication and use of traits
drought stress in tobacco and Arabidopis, respectively related to drought tolerance and the development of suit-
(Zhang et al. 2008, Cheng et al. 2009). Recently, 31 GmNAC able screening techniques are the prime criteria for cultivar
genes, including the six genes previously identied by Meng development. Integration of genomics, proteomics and
et al. (2007), were identied and cloned from soybean. systems biology platforms will undoubtedly lead to the dis-
Systematic expression analysis of these 31 GmNAC genes covery of novel candidate genes and pathways involved in
demonstrated that nine genes are dehydration inducible drought resistance.
(Tran et al. 2009). These nine GmNAC genes and their respec- Transgenic approaches have been shown to be powerful
tive promoters are promising tools for genetic engineering tools to help understand and manipulate the responses of
to improve drought resistance of soybean, as the NAC family plants to stress, but this can be achieved only when studied
was reported to be a major group of TFs that play a role in by precise physiological and biochemical investigation of
root development and stress tolerance in plants (Tran et al. transgenic plants under stress conditions. Moreover, to
2004, Hu et al. 2006, Nakashima et al. 2007, Hu et al. 2008). ensure that the responses of the transgenic plants to water
To develop soybean plants with enhanced tolerance to stress treatments are comparable with those in eld condi-
drought stress, an understanding of the physiological and tions, the plants should be subjected to the same or compa-
biochemical responses and gene regulatory networks is rable drought regime that crops experience in the eld.
essential. Recent advances in soybean research, ranging from Greater emphasis should be placed on the severity and dura-
breeding programs to genome sequencing and genomics tion of the stress, the method of imposing the stress and the
parameters which will be measured. To facilitate accurate Brutnell, T.P. (2001) Transposon tagging in maize. Funct. Integr.
evaluation in the eld of the genotypes for drought resis- Genomics 2: 412.
tance, screening should be performed under managed Buhr, T., Sato, S., Ebrahim, F., Xing, A., Zhou, Y., Mathiesen, M., et al.
drought environments over multiple locations and years. An (2002) Ribozyme termination of RNA transcripts down regulate
seed fatty acid genes in transgenic soybean. Plant J. 30: 155163.
interdisciplinary approach combining the knowledge of
Burris, R.H. and Roberts, G.P. (1993) Biological nitrogen xation. Annu.
plant breeders, crop physiologists and molecular biologists Rev. Nutr. 13: 317335.
will be apposite to evaluate the complex response of plants Busscher, W.J., Lipiec, J., Bauer, P.J. and Carter, T.E. (2000) Improved
to develop better drought-resistant soybeans in future. root penetration of soil hard layers by selected genotypes. Comm.
Soil. Sci. Plant Anal. 31: 1920.
Funding Capitani, D., Brilli, F., Mannina, L., Proietti, N. and Loreto, F. (2009) In
situ investigation of leaf weater status by portable unilateral NMR.
United States Department of Agriculture (USDA)-Coopera- Plant Physiol. 149: 16481647.
tive State Research, Education, and Extension Service Carter, T.E., Jr., D Souza, P.I. and Purcell, L.C. (1999) Recent advances
(CSREES) and the Missouri Soybean Merchandising Council in breeding for drought and aluminum resistance in soybean.
(MSMC) (to the soybean genomics drought research pro- In Proceedings at the World Soybean Research Conference VI
gram at the University of Missouri). Chicago, IL. pp. 106125. Superior Printing, Champagne, IL.
Carter, T.E. and Rufty, T.W. (1993) A soybean plant introduction
Acknowledgements exhibiting drought and aluminum tolerance. In Adaptation of
Vegetables and other Food Crops to Temperature and Water Stress.
We would like to thank Donavan Aldrich and Theresa Edited by Kuo, G. pp. 335346. Asian Vegetable Research and
Musket for critically reading the manuscript. Development Center, Publ, Tainan, Taiwan.
Chen, M., Wang, Q.Y., Cheng, X.G., Xu, Z.S., Li, L.C., Ye, X.G., et al. (2007)
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Physiological and Molecular Approaches to Improve Drought

Tsurumi, Yokohama, 230-0045 Japan.

Drought avoidance Drought tolerance

High relative water content High harvest index

High transpiration Less ureide accu-

Early vigor/ground cover Leaf osmotic adjustment

ABA, ROS scavenging

Deeper taproot early

Table 1 Quantitative trait loci (QTLs) for drought resistance in soybean

Disruption of cellular an ionic

Signal sensing, perception

Fig. 2 Molecular mechanisms associated with drought tolerance.

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