Plant Science 272 (2018) 14–21

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Plant Science
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Review article

Review: The promise and limits for enhancing sulfur-containing amino acid T
content of soybean seed
⁎
Hari B. Krishnana, , Joseph M. Jezb
a
Plant Genetics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, University of Missouri, Columbia, MO 65211, USA
b
Department of Biology,Washington University in St. Louis, St. Louis, MO 63130, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Soybeans are an excellent source of protein in monogastric diets and rations with ∼75% of soybeans produced
Soybean worldwide used primarily for animal feed. Even though soybeans are protein-rich and have a well-balanced
Sulfur assimilation amino acid profile, the nutritive quality of this important crop could be further improved by elevating the
Essential amino acids concentrations of certain amino acids. The levels of the sulfur-containing amino acids cysteine and methionine in
Nutritive value
soybean seed proteins are inadequate for optimal growth and development of monogastric animals, which ne-
Seed storage proteins
cessitates dietary supplementation. Subsequently, concerted efforts have been made to increase the concentra-
tions of cysteine and methionine in soybean seeds by both classical breeding and genetic engineering; however,
these efforts have met with only limited success. In this review, we discuss the strengths and weakness of
different approaches in elevating the sulfur amino acid content of soybeans. Manipulation of enzymes involved
in the sulfur assimilatory pathway appears to be a viable avenue for improving sulfur amino acid content. This
approach requires a through biochemical characterization of sulfur assimilatory enzymes in soybean seeds. We
highlight recent studies targeting key sulfur assimilatory enzymes and the manipulation of sulfur metabolism in
transgenic soybeans to improve the nutritive value of soybean proteins.

1. Introduction
Soybean is a remarkable crop that in recent times has drastically
changed the landscape of agriculture in the United States of America.
Although soybeans have been cultivated in Asian countries for cen-
turies, they were introduced into the US as a forage crop in the early
20th century, but have steadily gained in importance and now ranks as
the second most important crop in US. The history of soybean in-
troduction to US has been well documented [1]. Currently, the US is the
leading global producer of soybean and accounts for ∼34% of world-
wide soybean production. In 2016, US farmers planted 83.7 million
acres of soybeans worth $38 billion post-harvest (https://www.ers.
usda.gov/topics/crops/soybeans-oil-crops/related-data-statistics/).
The two most important reserve components of soybean are the
protein and oil. Current commercial soybean varieties accumulate up to
36% protein and 18% oil in their seeds [2], which are processed to yield
both meal and oil (http://ncsoy.org/media-resources/uses-of-
soybeans/). Soybean is the most dominant oil seed crop and accounts
for ∼90% of US oilseed production [3]. Soybean oil is mainly used as
edible vegetable oil throughout the world and a portion of it is pro-
cessed for numerous industrial applications. In recent times, the use of
soybean for biodiesel production has been promoted by federal and
state subsidies. The other processed byproduct, soybean meal, serves as
the world's largest source of animal protein feed. Soybean meal is
considered as “gold standard” to which other protein sources are
compared [4].
Today, soybean meal is in most animal feed because of its high
protein content, balanced amino acid profile, ready availability and
relatively low cost; however, the nutritional value of this crop could be
improved by enhancing the content of certain amino acids. In parti-
cular, the sulfur-containing amino acids cysteine and methionine are
not found at adequate levels in soybean seed for optimal growth and
development of monogastric animals. The United Soybean Board’s
Better Bean Initiative (BBI) and poultry industry has identified the
improvement of sulfur amino acid content (Methionine + Cysteine) of
soybean from the current level of 1.4% to 2.1% as one of the primary
meal targets (http://soybeaninnovationlab.illinois.edu/files/
PoultrySoybeanUse.pdf). In this review, we highlight recent studies
targeting key sulfur assimilatory enzymes and the manipulation of
sulfur metabolism in transgenic soybeans to improve the nutritive value
of soybean proteins.

⁎
Corresponding author at: USDA-ARS, Plant Genetics Research Unit, 108 Curtis Hall, University of Missouri, Columbia, MO 65211, USA.
E-mail address: Hari.Krishnan@ARS.USDA.GOV (H.B. Krishnan).

https://doi.org/10.1016/j.plantsci.2018.03.030
Received 4 January 2018; Received in revised form 27 March 2018; Accepted 29 March 2018
Available online 04 April 2018
0168-9452/ Published by Elsevier B.V.
H.B. Krishnan, J.M. Jez
2. Importance of sulfur-containing amino acids in human diet and
animal feed
Sulfur-containing amino acids, cysteine and methionine, play a vital
role in human health and nutrition [5]. Methionine is an essential
amino acid and cysteine is considered to be a ‘conditionally’ essential
amino acid because animals can convert methionine to cysteine.
Mammals cannot synthesize either amino acid de novo and are de-
pendent on dietary sources to fulfill their sulfur amino acid require-
ments. Methionine plays key role in multitude of cellular functions and
has a central role in the initiation of mRNA translation. It serves as the
precursor of S-adenosylmethionine (SAM), which in turn regulates the
levels of other important metabolites like ethylene, biotin, and poly-
amines. Additionally, SAM is the main methyl group donor that reg-
ulates plant growth and development. Cysteine is an important struc-
tural and functional component of proteins and enzymes, but also
required for cellular components containing reduced sulfur, including
methionine, glutathione, homoglutathione, iron-sulfur clusters, vitamin
cofactors like biotin and thiamin, and multiple secondary metabolites
[6]. Recent studies suggest a role for these amino acids in promoting
human health, including cancer prevention, proper function of the
immune system, and development of cardiovascular disease [7]. As
such, these two sulfur-containing amino acids are indispensable for
human and animal nutrition.
It now known that not only the quantity but also the quality of
protein is critical for optimum growth of animals. This observation is
especially relevant with regard to sulfur-containing amino acids.
Deficiency of the sulfur amino acids limits animal growth, results in
lowered resistance to disease, and can retard mental and physical de-
velopment in children [7]. Diets devoid of animal products are deficient
in sulfur amino acids. Nutritional sulfur amino acid deficiency is
widespread in countries where the intake of animal products is very
limited and where diets are primarily based on pulses and cereals. The
dietary requirement of sulfur amino acids for humans ranges between 6
and 13 mg per kg body weight or 910–1120 mg per day [7,8]; levels
that a Western diet, which contains about 3.6 g per day sulfur amino
acids, fulfills [5].
For animals, soybean meal is a preferred protein source in poultry
and livestock feed rations. An ideal animal diet should include all nu-
trients required for maintenance, growth, reproduction, and production
of products such as meat, eggs, and milk (https://www.nrcs.usda.gov/
Internet/FSE_DOCUMENTS/stelprdb1046729.pdf). The high protein
content of soybean is ideally suited for formulation of animal rations;
however, one limiting aspect of soybean protein is the apparent defi-
ciency in cysteine and methionine. The sulfur amino acid content of
soybean is about 1.3 g per 100 g per g of protein, which does not ade-
quately meet the recommended 3.5 g of sulfur-containing amino acids
per 100 g protein [9]. Consequently, synthetic sulfur amino acids are
added during the formulation of diets to maintain the optimal growth
and development of poultry and livestock.
3. Supplementing with synthetic amino acids in mixed corn- and
soybean-based animal rations
Livestock rations are typically corn and soybean meal based. Poultry
and swine are the major consumers of soymeal and account for 22–55%
of total soymeal used in livestock rations [10]. Corn is deficient in ly-
sine, but rich in methionine, while soybeans contain relatively high
amounts of lysine but low amounts of sulfur-containing amino acids. By
balancing soybean and corn in livestock rations, adequate amounts of
cysteine and methionine can be provided; however, the amount of
methionine available in the soybean-corn based feeds is not optimal to
fully meet methionine requirements of poultry and swine [10]. This is
especially true with regard to poultry, where methionine deficiency
results in retarded growth and poor feed conversion, as well as poor
feather growth and increased feather pecking, which could lead to
15
Plant Science 272 (2018) 14–21
cannibalistic behavior [10]. In the case of pig rations, the most limiting
amino acid is lysine followed by methionine, threonine, and tryptophan
[11]. A review of the literature indicates that nursery and growing pigs
require 10.4–11.2 mg of digestible sulfur amino acids per g of body
weight gain [11]. Methionine deficiency can be overcome by inclusion
of excessive protein; however, this approach leads to excretion of excess
nitrogen, which contributes to environmental degradation and a sub-
stantial increase in feed cost.
Alternatively, adding synthetic amino acids to feeds provides a cost-
effective solution for meeting the essential amino acid requirements of
livestock. Inclusion of synthetic amino acids ensures high levels of feed
efficiency and protects the environment by lowering nitrogen excretion.
Additional benefits offered by amino acid supplementation include re-
duction in crude protein content, improved energy utilization, higher
availability of amino acids compared with protein bound amino acids,
and prevention of digestive disorders [10]. Although the inclusion of
synthetic amino acids has advantages, it is also more expensive and
organic producers oppose this practice. One potential way to avoid the
addition of synthetic molecules to feeds is to develop soybean cultivars
with elevated amounts of sulfur-containing amino acids. Population
growth mostly in developing countries has elevated the demand for
livestock products and prompted the feed industry to look for alter-
native and cost-effective sources of protein for animal feeds. Soybeans
with improved sulfur amino acid content will enhance the nutritive
value of soybean not only in animal feed but also as a protein source for
humans. In countries where the use of animal protein is limited, the use
of nutritionally improved soybean and their derived products such as
soy milk should aid in alleviating malnutrition.
4. Can traditional plant breeding improve sulfur-containing
amino acid content in soybean?
Better understanding of animal nutrition has highlighted the im-
portance of amino acid profile to animal performance. It is believed that
the value of soybean meal is less a function of its protein content and
more a function of its amino acid profile [12]. Increasing the con-
centration of essential amino acids, especially methionine and cysteine,
remains a goal in soybean breeding because this crop is the pre-
dominant protein source in monastic rations [13]. An estimated 10%
increase in methionine content in soybean seed will result in economic
benefit of $5 per ton [14].
There is an inverse relationship between soybean seed yield and
seed protein content. Early studies have examined the relationship
between protein and methionine content of soybean seeds [15,16]. No
correlation was found indicating methionine is not likely to decrease as
a result of selection for higher protein soybean lines. Variation in the
concentration of sulfur amino acid levels in soybean seeds has been
reported and could be influenced by environmental factors, nitrogen
source, and the availability of reduced forms of sulfur [17–20]. Quan-
titative trait loci (QTL) associated with methionine and cysteine con-
centrations in soybean seed have been identified and are found on
chromosomes 1, 3, 4, 5, 6, 7, 9, 10, 13, 15, 17, 18 and 20 (https://
soybase.org) [19,20]. Several recombinant inbred lines (RILs) were
found to contain cysteine and methionine levels above the United Na-
tions Food and Agriculture Organization (FAO) standards [19]. Using a
linkage map derived from soybean RIL mapping populations, new QTLs
for cysteine and methionine concentration were reported on chromo-
somes 2 and 20 [21]. Population-based mapping approaches such as
genome-wide association (GWA) scans revealed the presence of strong
candidate alleles for sulfur amino acid content on chromosomes 1 and 8
[22]. Even though several QTLs and candidate alleles associated with
amino acid content have been reported, they have not resulted in the
commercial development of soybean cultivars with improved sulfur
amino acid content.
Further investigations of the genetic basis for soybean seed protein
quality both by family-based and population-based mapping
H.B. Krishnan, J.M. Jez
approaches should aid plant breeders to increase the sulfur amino acid
content of soybean to the FAO standard; however, improvement of
current methods and the development of new approaches to rapidly
measure methionine and cysteine content of soybean seed is required to
achieve this goal. Currently, methods used to quantify these amino
acids in soybean are time-consuming and not cost-effective. HPLC is
routinely used to measure sulfur amino acids, but this slow and ex-
pensive procedure limits its use in plant-breeding efforts that require
screening large numbers of samples. Researchers have employed near-
infrared (NIR) spectroscopy for measuring amino acid in soybean seeds
[19,23–25], but this procedure has limitations in precisely quantifying
levels of sulfur amino acids. Moreover, better and practical field eva-
luation methods are also required to monitor soil sulfur and plant sulfur
status [26]. Development of rapid and cost-effective methods to quan-
tify cysteine and methionine will be critical for high-throughput
screening and will greatly aid soybean breeders in their endeavors to
develop soybean cultivars with enhanced sulfur amino acid content.
5. Why expression of heterologous methionine-rich proteins has
not improved the sulfur amino acid content of soybean seeds
Proteins rich in sulfur amino acids have been successfully in-
troduced into soybeans for the purpose of improving the protein quality
of soybean. Generation of transgenic soybeans expressing heterologous
seed proteins rich in sulfur-containing amino acids and modification of
endogenous seed protein expression have been described in the litera-
ture [27–36]; however, the overall improvements in sulfur amino acid
content in these transgenic soybeans have been modest, at the best.
Several reasons can explain the lack of success with this approach, but
one major reason appears to be associated with insufficient accumula-
tion of heterologous proteins in soybean. The accumulation of hetero-
logous seed proteins in transgenic soybeans is typically about 1% of
total seed weight [30,33]. In most cases, the accumulation of hetero-
logous proteins, many only detected by immunological methods, is
insignificant when compared to that of endogenous soybean seed sto-
rage proteins.
An examination of soybean seeds by transmission electron micro-
scopy (TEM) reveals that cotyledonary cells are primarily composed of
protein storage vacuoles and lipid bodies (Fig. 1). Protein storage va-
cuoles occupy a major portion of the cell volume. TEM of transgenic
soybeans expressing methionine-rich zein proteins reveals that they
accumulate as protein bodies of 0.5–1 μm in size (Fig. 1). Moreover, the
number of protein bodies seen in these cells is insufficient to alter the
sulfur amino acid content of the seed. If one wants to improve the sulfur
amino acid content of soybean seed, then it will be necessary to dras-
tically improve the accumulation of heterologous sulfur amino acid-rich
proteins.
Heterologous seed proteins are routinely expressed under the con-
trol of seed-specific promoters [30,33]; however, these promoters have
not been well characterized and may not contain distant cis-acting
elements required for maximum transgene expression. Improving
transgene expression for biotechnological application will require the
use of well characterized ‘full-length’ promoters [37]. The use of syn-
thetic promoters and inducible promoters should also be considered for
efficient expression of heterologous seed proteins in soybean. Ad-
ditionally, a large number of independently transformed plants need to
be systematically analyzed because random integration of transgene
and copy number will also impact transgene expression levels [37]. Any
strategy employed for maximum transgene expression should take into
consideration the targeting, folding and stability of the introduced
protein in a new environment.
6. Overcoming limits to the accumulation of sulfur-rich proteins
in soybean by targeting sulfur metabolism
Earlier studies demonstrate that heterologous expression of sulfur-
16
Plant Science 272 (2018) 14–21
Fig. 1. Transmission electron micrograph of transgenic soybean seed expressing
18 kDa δ-zein. The colorized version of the thin section of soybean cotyledon
reveals the presence of large protein storage vacuoles (PSV; magenta), lipid
bodies (LB; white) and prominent amyloplasts (A; green). The methionine-rich
18 kDa δ-zein accumulates in spherical protein bodies (PB; red). Note the
number of PBs and their relative size in comparison to that of PSV. CW, cell
wall.
rich proteins in legumes resulted in reduced accumulation of en-
dogenous of sulfur-rich seed proteins [38,39]. Based on this observa-
tion, we can speculate that there is limitation in the availability of
cysteine and methionine in legume seeds. Consequently, in the absence
of sulfur amino acid availability, the synthesis of heterologous protein
is promoted at the expense of endogenous sulfur-rich proteins. In-
creasing the capacity of soybean seeds to synthesize cysteine and me-
thionine could alleviate this situation. Clearly, efforts to improve the
sulfur amino acid content of soybeans show promise, but obstacles re-
main. Altering endogenous seed protein expression or expressing
transgenic proteins aimed to introduce a sulfur amino acid-rich protein
“sink” in transgenic soybeans, but fall short of increasing nutritional
value because of an inadequate supply of cysteine and methionine in
developing soybean seeds. An alternative approach to enhance the cy-
steine and methionine content of soybean involves metabolic en-
gineering of the sulfur assimilatory and methionine biosynthesis path-
ways (Fig. 2). This approach requires a thorough understanding of the
biochemical pathways for sulfur assimilation and cysteine and me-
thionine synthesis in soybean. The sulfur assimilatory and methionine
biosynthesis pathways have been well studied in microbes and model
plants such as Arabidopsis, but only limited studies have been carried
out on crops such as soybean.
The importance of sulfur nutrition and the effect of sulfur deficiency
on the agronomic performance of soybean have been previously re-
viewed [26]. Plants acquire sulfur in the form of sulfate from soil using
a family of sulfate transporters (SULTR), which are encoded by multi-
gene families, are responsible for the absorption of sulfate and its ac-
cumulation within cells [40–42]. Although SULTR genes have been well
characterized in Arabidopsis, their role in uptake of sulfur in soybean
has only been recently examined [43]. Twenty-eight putative SULTR
genes, classified into 4 groups, have been identified in the soybean
genome. Transgenic tobacco plants overexpressing a soybean sulfate
transporter gene (GmSULTR1;2b) accumulated higher levels of organic
matter, had enhanced biomass, and increased seed weight compared to
H.B. Krishnan, J.M. Jez
Fig. 2. Overview of plant sulfur metabolism from uptake to storage proteins.
control plants [43]. This observation indicates that generation of
transgenic soybean plants overexpressing sulfate transporter genes may
improve sulfur utilization efficiency through sulfur uptake.
Once in the plant, sulfate is converted to sulfide through a series of
enzymatic steps catalyzed by ATP sulfurylase, adenosine 5′-phospho-
sulfate reductase (APS reductase), and sulfite reductase in the sulfur
assimilation pathway (Fig. 2) [44–47]. To date, ATP sulfurylase and
APS reductase from soybean have been biochemically characterized
[48–50]. In soybean, both are encoded by large gene families with
isoforms having cytosolic and plastidal localization (Table 1). Expres-
sion of both ATP sulfurylase and APS reductase is elevated in early
development of leaves and seeds with deprivation of sulfur, nitrogen,
and phosphorus leading to increased transcript levels [48,49]. Devel-
opmentally, the timing of ATP sulfurylase and APS reductase expression
coincides with sulfur assimilation to support amino acid synthesis be-
fore accumulation of seed storage proteins [18]. Although studies in-
dicate that ATP sulfurylase is the committed step for sulfur assimilation,
as it catalyzes the thermodynamically unfavorable adenylation of sul-
fate to the high-energy molecule adenosine 5′-phosphosulfate, this step
of the pathway has not yet been targeted for engineering of sulfur
amino acid content in soybean. Overexpression of APS reductase in
maize can increase the flux through assimilative reduction pathway;
however, the resulting plants accumulated toxic intermediates resulting
in stunted growth [51]. To overcome this problem, an unregulated
bacterial APS reductase was expressed in maize with the help of me-
sophyll-specific PepC or bundle sheath cell-specific RbcS promoters. The
resulting transgenic maize accumulated high levels of methionine
without any apparent yield loss [52]. In addition, the use of transgenic
maize with increased seed methionine also promoted significant weight
gain compared with nontransgenic kernels in a feeding trail with chicks
[52]. The ability to improve the seed methionine content by over-
expression of an unregulated bacterial APS reductase in maize kernels
suggests that this enzyme in soybean could also a useful engineering
target.
For amino acid synthesis, sulfide is incorporated into cysteine
(Fig. 2). First, serine acetyltransferase (SAT) generates O-acetylserine
from serine and acetyl-CoA; this is followed by the reaction of sulfide
and O-acetylserine catalyzed by O-acetylserine sulfhydrylase (OASS;
also known as cysteine synthase and O-acetylserine-thiol-lyase). As with
the enzymes from the early steps of the sulfur assimilation pathway,
SAT and OASS are members of large gene families in soybean (Table 1)
[44,45,47,53,54]. Two SAT isoforms (one cytosolic and the other
17
Plant Science 272 (2018) 14–21
plastidal) have been biochemically characterized and the x-ray crystal
structure of the cytosolic form determined [54–56]. Both isoforms are
regulated by feedback inhibition through cysteine; however, the plas-
tidal isoform becomes insensitive to this control when phosphorylated
by a cyclin-dependent protein kinase [55]. OASS, like other members of
the β-substituted alanine synthase (BSAS) enzyme family, uses a pyr-
idoxal-dependent reaction to form cysteine [57–59]. To date, multiple
soybean OASS and related BSAS isoforms have been biochemically
characterized (Table 1) [53,58,60,61]. Interestingly, SAT and OASS
form a macromolecular complex that responds to changes in sulfur state
[62–69]. Formation of the protein complex structurally stabilizes and
activates of SAT, while inactiving OASS to promote O-acetylserine
synthesis under sulfur-sufficient conditions. Excess uncomplexed OASS
then catalyzes cysteine formation.
Expression of either SAT or OASS in a variety of plants, such as
maize, rice, lupin, potato, tobacco, and poplar, leads to a variety of
sulfur-linked outcomes, including increased cysteine and/or methio-
nine content [70–76]. To date, efforts to enhance cysteine biosynthesis
in soybean have focused on overexpression of OASS [77]. Transgenic
soybean expressing a cytosolic isoform of OASS had a 58–74% increase
in the protein-bound cysteine compared to wild-type plants and pro-
moted accumulation of cysteine-rich proteins, such as Bowman-Birk
protease inhibitor. Importantly, the resulting transgenic soybean seeds
contained a total of 4.5% sulfur-containing amino acids, which is suf-
ficient to meet the sulfur amino acid requirement of monogastric ani-
mals. However, overexpression of OASS in the transgenic soybean
plants resulted in growth reduction and impaired nodulation [78].
These negative impacts will preclude its utilization as a nutritionally
improved feed source since any reduction in the seed yield will not be
acceptable for soybean farmers.
Likewise, manipulation of the first committed step of methionine
biosynthesis (i.e., the formation of cystathionine from cysteine and O-
phosphoserine by cystathionine γ-synthase; Fig. 2) in soybean also
shows promise. Transgenic soybean plants overexpressing a methio-
nine-insensitive form of Arabidopsis cystathionine γ-synthase under
control of a seed-specific promoter contained up to 7-fold more soluble
methionine in developing seed and 2.3-fold greater total methionine in
the mature dry seed [79]. Interestingly, these transgenic soybean plants
also had a higher protein content suggesting a positive correlation be-
tween methionine content and protein synthesis [79]. No work has
investigated the contribution of later enzymes (cystathionine β-lyase,
and methionine synthase) to this pathway in soybean.
7. What is next?
Initial efforts toward improving sulfur-containing amino acid con-
tent in soybean are promising, but these are the first steps. Both tra-
ditional breeding and genetic engineering approaches remain to be
fully explored for this goal. As noted, family-based and population-
based mapping approaches are now available but limited by the lack of
rapid analytical methods. Likewise, the genetic and biochemical un-
derstanding of sulfur metabolism from assimilation to cysteine and
methionine biosynthesis in soybean has progressed, although gaps in
our understanding sulfate uptake, transport, and metabolism remain.
Other aspects of sulfur metabolism in soybean that have not re-
ceived much attention relates to the acquisition of sulfur from the soil
and characterization of enzymes in methionine biosynthesis. The up-
take of sulfur by plants is tightly regulated in response to the plants
mineral need and its availability in the soil. In plants, the sulfate
transporter family has been grouped into five subgroups, each group
with different functional role [40]. The soybean genome contains 28
putative sulfate transporter genes (https://phytozome.jgi.doe.gov/pz/
portal.html) and very little is known about the functions of these
transporters, how they are regulated, and their expression patterns. A
detailed characterization of sulfate transporters will be crucial for de-
signing strategies aimed at enhancing sulfate uptake and increasing the
H.B. Krishnan, J.M. Jez Plant Science 272 (2018) 14–21

Table 1
Summary of sulfur metabolism enzyme isoforms in soybean.
Gene Identification Number Isoform Protein Length/MW Predicted Localization cDNA Expression/Enzyme Activity

Glyma10g38760 GmATPS1 465 aa/51 kDa Plastidic Yes/Yesa

Glyma20g28980 GmATPS2 467 aa/51 kDa Plastidic ND/ND
Glyma13g06940 GmATPS3 488 aa/54 kDa Plastidic ND/ND
Glyma19g05020 GmATPS4 553 aa/62 kDa Plastidic ND/ND
Glyma09g00670 GmAPSR1 470 aa/52 kDa Plastidic Yes/Yesb
Glyma15g11540 GmAPSR2 472 aa/52 kDa Plastidic ND/ND
Glyma07g39130 GmAPSR3 466 aa/52 kDa Plastidic ND/ND
Glyma11g09890 GmSIR1 687 aa/77 kDa Plastidic ND/ND
Glyma12g02200 GmSIR2 688 aa/77 kDa Plastidic ND/ND
Glyma16g03080 GmSERAT1;1 286 aa/30 kDa Cytosolic Yes/Yesc
Glyma07g06480 GmSERAT1;2 286 aa/30 kDa Cytosolic Yes/ND
Glyma18g08910 GmSERAT2;1 391 aa/43 kDa Cytosolic/Plastidic Yes/Yesd
Glyma08g43940 GmSERAT2;2 387 aa/42 kDa – Yes/ND
Glyma02g46870 GmSERAT2;3 356 aa/39 kDa – Yes/ND
Glyma14g010840 GmSERAT2;4 351 aa/38 kDa – Yes/ND
Glyma16g22630 GmSERAT3;1 391 aa/44 kDa Secretory Yes/ND
Glyma02g04770 GmSERAT3;2 385 aa/42 kDa Cytosolic ND/ND
Glyma11g00810 GmOASS1 325 aa/34 kDa Cytosolic Yes/Yese,f
GmBSAS1;1
Glyma19g43150 GmOASS3 325 aa/34 kDa Cytosolic Yes/Yesf
GmBSAS1;2
Glyma03g40490 GmOASS2 325 aa/34 kDa Cytosolic Yes/Yesf,g
GmBSAS1;3
Glyma20g28630 GmBSAS1;4 315 aa/33 kDa Cytosolic Yes/ND
Glyma10g39320 GmBSAS1;5 286 aa/30 kDa Cytosolic ND/ND
Glyma02g15640 GmOASS4 394 aa/42 kDa Plastidic Yes/Yesf
GmBSAS2;1
Glyma07g32790 GmOASS5 389 aa/41 kDa Plastidic Yes/Yesf,g
GmBSAS2;2
Glyma09g39390 GmCAS 373 aa/40 kDa – Yes/Yesf
GmBSAS3;1
Glyma18g46920 GmBSAS3;2 372 aa/40 kDa – Yes/Yesg
Glyma15g41600 GmBSAS4;1 321 aa/34 kDa Cytosolic Yes/ND
Glyma10g30140 GmDES 324 aa/35 kDa Cytosolic Yes/Yesf
GmBSAS4;2
Glyma20g37280 GmBSAS4;3 323 aa/35 kDa Cytosolic Yes/ND
Glyma10g30130 GmBSAS4;4 323 aa/34 kDa Cytosolic Yes/Yesg
Glyma20g37290 GmBSAS4;5 295 aa/32 kDa Cytosolic ND/ND
Glyma03g00900 GmBSAS5;1 320 aa/35 kDa Plastidic ND/ND

Enzyme abbreviations are: ATPS, ATP sulfurylase; APSR, adenosine 5′-phosphosulfate reductase; SIR, sulfite reductase; SERAT, serine acetyltransferase; OASS, O-
acetylserine sulfhydrylase; BSAS, β-substituted alanine synthase; CAS, β-cyanoalanine synthase; DES cysteine desulfhydrase. Molecular weight was calculated based
on the complete amino acid sequence using Protparam (http://www.expasy.ch/tools/protparam.html). Subcellular localization was analyzed using TargetP (http://
www.cbs.dtu.dk/services/TargetP/). When a clear localization is not predicted, a “–” is shown. Confirmation of either cDNA expression or enzyme activity is noted,
as follows: Yes or ND = “not determined.”
a
Phartiyal et al. [48].
b
Phartiyal et al. [49].
c
Chronis and Krishnan [54].
d
Liu et al. [55].
e
Chronis and Krishnan [53].
f
Yi and Jez [58].
g
Zhang et al. et al. [60].

availability of sulfate in the developing soybean seeds.
Even though significant progress has been made in the biochemical
characterization of enzymes involved in soybean cysteine biosynthesis,
little is known about the enzymes involved in soybean methionine
biosynthesis [80]. For example, the methionine biosynthesis enzymes
cystathionine γ-synthase, cystathionine β-lyase, and methionine syn-
thase have not been well characterized. Attempts have been made to
increase the methionine content of legume seeds by heterologous ex-
pression of a mutated form of Arabidopsis cystathionine γ-synthase, but
its expression in transgenic soybean and Azuki bean increased the levels
of free amino acids without any effect on total methionine content of
the seed [81]. In contrast, ectopic expression of a gene encoding soy-
bean methionine synthase in tobacco resulted in an increase in me-
thionine content but was accompanied by growth abnormalities such as
stunting and delayed flowering [82]. Previous studies have also re-
ported growth abnormalities in plants overexpressing sulfur assim-
ilatory enzymes [83]. It is critical that development of soybean cultivars
with improved sulfur amino acid content is accomplished without any
negative effect on growth and yield. Undoubtedly, a greater under-
standing of biochemical pathways involved in sulfur uptake and as-
similation will facilitate progress in the development of superior soy-
bean varieties with improved protein quality.
RNA interference (RNAi) has been successfully used for enhancing
the nutritional quality of seed proteins [83]. For example, a high-lysine
maize was created by knocking out the expression of lysine-poor 22 kDa
zein. Similarly, the methionine content of maize seeds can be altered by
selective knockdown of different classes of zeins [84]. The β- and δ-
zeins are the major sink for methionine and β- and γ-zeins for cysteine
[85]. In contrast to maize, down regulation of abundant seed storage
proteins by RNAi has a minimal effect on the sulfur amino acid content
of seed proteins [39]. Unlike maize, currently we do not know if soy-
beans have also evolved specialized proteins for cysteine and methio-
nine accumulation and storage. Transgenic soybean plants over-
expressing OASS accumulate elevated levels of cysteine and Bowman-

18
H.B. Krishnan, J.M. Jez
Birk protease inhibitor. This protein could be assigned as a specialized
protein for cysteine accumulation and storage in soybean.
Genome editing tools such as Zinc finger nucleases (ZFNs),
Transcription Activator-Like Effector Nucleases (TALENs), and
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/
CRISPR-associated) (Cas) technologies have gained importance to se-
lectively alter genomic DNA sequences in vivo [86–88]. Unlike ZFNs
and TALENs, the CRISPR/Cas9 system offers several advantages in
terms of simplicity, accessibility, cost, and versatility [86]. Moreover,
recent work demonstrates it's utility as a simple and inexpensive
method for genome editing in soybean [89]. This technique could be
exploited to modify biosynthetic and catabolic fluxes by editing key
enzymes involved in the sulfur assimilatory reductive pathway. The
CRISPR-Cas9 technology will surely facilitate the development of nu-
tritionally-improved crops, including soybeans.
Manipulating expression of key enzymes in the sulfur assimilatory
pathway can increase the sulfur-containing amino acid content of
soybean. This approach could be linked with efforts to provide sulfur-
rich “sinks” in soybean seed. We envision the combination of expressing
multiple sulfur metabolic enzymes and sulfur-rich seed storage proteins
– this two-pronged “push and pull” approach remains to be explored as
a strategy for further increasing sulfur content for nutritional purposes
of livestock and humans. There is also the possibility of altering the
types of “sink” proteins. The predominant seed proteins of soybean are
the salt-soluble globulins, 7S β-conglycinin and 11S glycinin, which
account for about 70% of total seed proteins and are poor in sulfur
containing amino acids [90,91]. The overabundance of these two types
of protein tend to lower the overall methionine and cysteine content in
soybean seeds. Soybean accumulates only a few proteins that are en-
riched in the sulfur amino acids. For example, Bowman-Birk protease
inhibitor is a cysteine-rich protein and the soybean 2S albumin is a
methionine-rich protein. However, these proteins are only minor
components of soybean seed. One potential approach to increase the
accumulation of these sulfur rich proteins is by altering the promoter
sequences by “promoter bashing” [92]. By introducing point mutations
or deletions in the promoter region, it is possible to identify regions that
enhance or repress the transcription of the target genes. Use of tailored
promoters may be vital for enhanced expression of low abundant soy-
bean sulfur-rich proteins. The success of this approach requires detailed
knowledge of proteins that bind the modified promoter sequences to
enhance transcription of target genes.
Metabolic engineering of soybean seed composition can also be fa-
cilitated by the application of synthetic biology. For example, it was
recently demonstrated that a metabolic engineering strategy led to dual
production of eicosapentaenoic acid and the astaxanthin, critical
aquafeed ingredients, in soybean [93]. This impressive achievement
was made possible due to optimal genetic designs, exploitation of
synthetic biology and novel metabolic engineering strategies [93]. A
similar approach could be utilized to improve the sulfur amino acid
content of soybean by expressing multiple genes involved in the
synthesis of methionine. Efforts are underway to express enzymes in-
volved in Escherichia coli methionine biosynthetic pathway in soybean
to improve the methionine content of soybean (Tom Clemente, personal
communication). Four enzymes, homoserine O-succinyltransferase en-
coded by metA, cystathionine γ-synthase encoded by metB, cystathio-
nine β-lyase encoded by metC, and either cobalamin-dependent me-
thionine synthase encoded by metH or cobalamin-independent
methionine synthase encoded by metE are involved in conversion of
homoserine to methionine in E. coli [94]. By using Golden Gate as-
sembly these codon-optimized E. coli genes were cloned in a binary
vector for soybean transformation. If this approach is successful it will
greatly enhance the nutritional value of soybean meal for animal feed.
Artificially-modified genes have been employed to improve the
amino acid content of seed proteins [95]. For example, modified Brazil
nut 2S albumin, β-phaseolin, and maize α- and γ-zeins have been ex-
pressed in transgenic plants, but this approach was not successful due to
19
Plant Science 272 (2018) 14–21
the instability of these modified proteins [83]. To overcome this in-
stability problem, a new strategy was employed where seed-specific
endogenous genes with low expression levels in targeted species were
selected and fused with desired amino acid-rich motifs [96]. With this
approach, two rice endogenous genes were fused with lysine- and
threonine-coding motifs and expressed in transgenic rice seeds. The
expression of the synthetic fusion protein significantly elevated the
lysine, threonine, total amino acids, and crude protein content when
compared with wild type control seeds. Additionally, these transgenic
rice plants revealed no significant differences in plant growth and grain
yield when compared with wild type plants [96]. A similar strategy
could also be exploited to increase the sulfur amino acid content of
soybean seed proteins by over-expressing synthetic fusion proteins.
In addition, nearly all efforts to enhance the sulfur amino acid
content of soybean focus on the seed, which is a consequence of agri-
cultural and livestock feed demands, but other side-products of soybean
processing could benefit from improved cysteine and methionine con-
tent. For example, okara, the residue from soy milk production, is
∼25% protein and has multiple uses, including livestock feeds and pet
foods in Asia [97,98], that could benefit from altered amino acid pro-
files. With the advancement in proteomics and the availability of the
soybean genome sequence, it should be possible to identify other pro-
teins that are rich in the sulfur-containing amino acids that could also
be used to enhance sulfur nutrient levels and to improve soybean pro-
tein quality.
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