Journal of Plant Biochemistry and Biotechnology

https://doi.org/10.1007/s13562-023-00838-0

ORIGINAL ARTICLE

Expression of a truncated maize SPS increases source capacity in maize

Stephen M. G. Duff1 · Keith Kretzmer2

Received: 18 August 2022 / Accepted: 14 March 2023

© The Author(s) 2023

Abstract
In an attempt to increase source capacity, transgenic corn was generated by expressing a truncated maize sucrose phosphate
synthase (ZmSPSΔ482) under two leaf mesophyll cellspecific promoters (CAB and PPDK). The endogenous and truncated
SPS proteins from transgenic leaf extracts were distinguishable by protein immunoblot analysis. The expression of transgenic
SPS protein across events varied from very low to very high and included several cosuppressed events. SPS activity showed
a diurnal pattern in both transgenic and wild-type maize leaves. In greenhouse experiments, transgenic maize had higher
leaf sucrose and lower leaf starch, suggesting a shift in carbon partitioning from starch to sucrose. Conversely, cosuppressed
events had lower leaf sucrose and higher leaf starch. A field test was performed to compare sucrose and starch in positive
and negative isolines of hybrid maize CAB and PPDK ZmSPSΔ482 events. In the field, many positive isolines had higher
levels of both leaf sucrose and starch than the negative isolines. This suggests that in the field, with higher light intensity the
shift in carbon partitioning from starch to sucrose, observed under greenhouse conditions did not occur. This in turn suggests
that the environment affects the phenotype of the transgenics and that in the field, there was an overall increase in carbon
assimilation. Six events from each construct were tested in a pilot multi-density yield trial but overall, no effect on yield was
observed. Therefore, although the transgenic plants had more source capacity, this did not translate into higher seed yield.

Keywords sucrose phosphate synthase (SPS) · Yield · Source · Sink · Truncated protein · Diurnal

Abbreviations 1999). In maize, sucrose is primarily produced in mesophyll

SPS Sucrose phosphate synthase
SPP Sucrose phosphate phosphatase
PPDK Pyruvate, orthophosphate dikinase
CAB Chlorophyll a/b binding protein
Introduction
Starch and sucrose are primary stable end products of car-
bon assimilation in higher plants. Both fluctuate diurnally in
leaves and reach their highest measurable levels late in the
day. Starch is synthesized and stored in the chloroplast while
sucrose is synthesized in the cytosol from triose phosphates
transported from the chloroplast. While starch is considered
a storage form of assimilated carbon, sucrose is the main
transport carbohydrate in higher plants (Hofstra and Nelson
1969; Stitt et al. 1987; ap Rees 1987; Lunn and Furbank
* Stephen M. G. Duff
stephen.duff@bayer.com
1
Bayer Crop Science, Chesterfield, MO, USA
2
Wildwood, MO 63025, USA
cells (Lunn and Hatch 1997; Downton and Hawker 1973;
Usuda and Edwards, 1980; Furbank et al. 1985; Lunn and
Furbank 1999). It is then transported to non-photosynthetic
tissues, or sinks, for storage or immediate energy production.
In reproductive organs such as kernels, sucrose is hydro-
lyzed to hexose sugars which are then processed to provide
the precursors for starch, lipids, and protein production.
Increasing sucrose production in leaves may increase
source capacity available for sink tissues, and especially to
developing kernels during high sink demand and may be a
useful approach to increase grain yield.
Sucrose phosphate synthase (SPS, EC 2.4.2.14) is a key
enzyme for sucrose synthesis in mature photosynthetic tis-
sues (Stitt et al. 1987; Huber and Huber 1996 and references
therein). SPS catalyzes the rate-limiting step in sucrose
biosynthesis through the formation of sucrose-6-phospate
from fructose 6-phosphate and UDP-glucose (Winter and
Huber 2000). Sucrose 6-phosphate is further broken down
to sucrose by a closely associated enzyme, sucrose 6-phos-
phate phosphatase (SPP, Echeverria et al. 1997). SPS
activity is highly regulated in plants, both allosterically
by glucose 6-phosphate and phosphate, and by reversible

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phosphorylation (Lunn and Furbank 1999; Winter and
Huber 2000; Takahashi et al. 2000).
Several reports have shown that plants have multiple
forms of SPS. In potato, at least four SPS isozymes have
been resolved by electrophoresis, and their expression shown
to differ with development, tissue type and environmental
signals such as low temperature (Reimholz et al. 1997).
Three unique SPS genes were cloned from citrus fruit and
their expression shown to be tissue specific (Komatsu et al.
1996). In rice, two isoforms of SPS that differ in both their
tissue distribution and their kinetic properties have been
described (Ingram et al. 1997). More recently additional
SPS isoforms were identified (Lutfiyya et al. 2007, Castleden
et al. 2004). In maize two isoforms SPS1 and SPS6 appear
to be highly specific for leaves.
SPS isoforms appear to fall into several groups which
occur across phylogenetic groups which may have functional
significance and Lutfiyya et al. (2007) analyzed sequence
features across groups and concluded that, “the presence
of regulatory sites on the proteins appears to be consistent
across all members of the group leading to the speculation
that they may be diagnostic features and that it might be
possible to assign specific functions to entire groups of SPS
genes.”
There are numerous reports of SPS over-expression in
transgenic plants, especially in C ­ 3 species. For example,
there are reports of the over-expression of maize SPS in
tomato (Galtier et al. 1993; Galtier et al. 1993; Micallef et al.
1995; Worrell et al. 1991; Laporte et al. 1997), potato (Ishi-
maru et al. 2008), canola and sugar beets (Van Asseche et al.
1999), tobacco (Baxter et al. 2001, 2003), wheat (Sparks
et al. 2001) and Arabidopsis (Signora et al., 1998). A spin-
ach SPS has been over-expressed in cotton (Haigler et al.
2007), and an Arabidopsis SPS has been over-expressed
in tobacco (Ishimaru et al. 2008). In general, higher leaf
sucrose/starch ratios, and in some cases increased photo-
synthetic rates have been observed as well as higher tomato
and potato solids (Ishimaru et al. 2008) or cotton fiber thick-
ness (Haigler et al. 2007). Overexpression of an Arabidop-
sis transcription factor (AtbZIP53) increased expression of
some SPS transcripts and led to sucrose accumulation in
Fig. 1  Structure of truncated
and full length maize SPS
proteins. The figure shows
the position of the three main
regulatory phosphorylation sites
on SPS with Ser162 removed
in the truncated and partially
deregulated enzyme (Takahashi
2000, ZmSPSΔ49)
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Journal of Plant Biochemistry and Biotechnology
Arabidopsis leaves (Thalor et al 2012). Anur et al., (2020)
overexpressed SPS in transgenic sugarcane which like maize
is a C4 monocot, and showed that overexpression of SPS
increased sucrose, soluble invertase activity as well as glu-
cose and fructose.
Studies have suggested that the overall yield of maize
plants may be limited by their source capacity, that is, the
amount of leaf carbohydrate produced and/or exported by
phloem (Sarquis et al. 1988, Prioul and Schwebel-Dugue
1992). Other studies have suggested the contrary position,
that photosynthesis is not limiting yield in major crops
(Richards 2000), Maize is a ­C4 plant which means that quan-
tum yields of photosynthesis are much higher than they are
for most C
­ 3 crops at high temperatures, further complicating
the question of whether source capacity is a limiting factor
for grain yield in modern maize hybrids (Sage and Monson
1999). To directly test the hypothesis that source productiv-
ity limits yield, achieving higher sucrose synthesis and/or
transport must first be demonstrated.
Higher plant SPS is highly regulated allosterically and
by several different posttranslational modifications includ-
ing phosphorylation related to dark/light, 14-3-3 binding
and osmotic stress (Fig. 1, Huang and Huber 2001). Studies
of S158 variants of tobacco leaf SPS including wildtype,
S158A, S158T, S158E and S158; analogous to S161 in
maize SPS1 demonstrated the importance of light/dark regu-
latory phosphorylation at this residue.
Here we study the over-expression of truncated maize
SPS1 (Lutfiyya et al. 2007, ZmSPSΔ482) targeted to meso-
phyll cells using two mesophyll-specific promoters. Since
transformation with full-length SPS1 failed to produce a
phenotype, SPS1 gene was truncated to remove a phospho-
rylation site involved in light/dark regulation of enzyme
activity by a CDPK (Fig. 1, Huang and Huber 2001) which
we hoped would lower the chance that the transgenic plant
would deregulate it.
The mesophyll specific promoters, pyruvate, orthophos-
phate dikinase (PPDK) promoter and chlorophyll a/b bind-
ing (CAB) protein promoter had previously been shown to
target proteins mainly, if not exclusively, to mesophyll cells
in maize and show diurnal patterns of expression (Taniguchi
Journal of Plant Biochemistry and Biotechnology
et al. 2000; Bansal et al. 1992). We describe the cloning
and construction of the truncated gene, the quantification
of over-expressed SPS protein by immunoblot analysis, the
screening of R0 events for over-expression, initial green-
house testing to demonstrate gene efficacy by measuring car-
bohydrate levels (sucrose and starch) in leaves of F1 inbred
events, demonstration of increased SPS protein and enzyme
activity in selected transgenic events, demonstration of gene
efficacy in field hybrid events, and finally, the results of a
yield study with selected transgenic events.
Materials and methods
SPS1 gene isolation
7 unique maize SPS gene sequences were identified from
internal and public databases in which SPS1 was the major
SPS gene expressed in leaves (Lutfiyya et al. 2007). The
published sequence of the maize leaf SPS cDNA clone (Gen
Bank Acc. No. 401114) was used to design the following
synthetic oligonucleotide primers:
5′TAT​GTG​GTC​GAA​CTT​GCA​AGA3′,
5′CCGA​ ATT
​ CTC ​ ACA ​ TGC ​ CGC ​ TGG ​ AAG ​ TCT ​ TGG
​ AG​
ACT​TGC​TTC​AG3′,
5′TAT​GTG​GTC​GAA​CTT​GCA​AGA3′,
5′CCG ​ CAT ​ CAC ​ T TC ​ G GC ​ C CA ​ A AT ​ T GG ​ G GG ​ CAT​
TGA3′.
Three PCR products, spanning the entire length of the
SPS cDNA clone, were generated using first-strand DNA,
reverse transcribed from maize leaf mRNA. The most 5′
and 3′ PCRproducts were used to screen a maize scutellum
library. A nearly full-length cDNA, missing 15 nucleotides
at the 5’ end was isolated. The 15 nucleotides were added by
inserting a synthetic DNA fragment based on the published
DNA sequence. The full-length cDNA was expressed in E.
coli using both lacZ and tac promoters and in plant cells and
in leaf protoplasts using the e35S promoter.
Truncation of maize SPS1
Higher plant SPS enzymes are regulated by reversible phos-
phorylation on an N-terminal serine residue (Winter and
Huber 2000). In maize SPS1 this residue is Ser161 (Fig. 1).
To create a maize SPS which not be inactivated at the major
site of regulatory phosphorylation (Huang and Winter), a
truncated form of the gene was generated, missing the first
5′ 482 nucleotides of the SPS1 coding sequence (SPSΔ482),
which translated into a protein missing the first N-terminal
162 amino acids. Synthetic DNA oligonucleotide primers
were used in PCRreactions, to produce the truncated protein
in Fig. 1. The truncated SPS was then expressed in E. coli.
The specific activity of the truncated SPS under limiting
conditions (Toroser et al. 1999) in crude bacterial extracts
was 2.7-fold greater than that of the full-length SPS (data not
shown), suggesting that it had been deregulated.
Maize transformation with ZmSPSΔ482
Two transformation vectors were constructed to target
expression to maize leaf mesophyll cells. The vector con-
structs, herein called Zeke and Pat, employed the CAB and
PPDK promoters, respectively. Each construct contained the
truncated SPS Δ 482 gene engineered with an ATG start
codon at the 5’ end. The full length SPS1 gene, from which
the ZmSPSΔ482 gene was made, was 93% identical at the
DNA level and 100% identical at the protein level to the
maize SPS1 gene, which had previously been tested in plants
(Van Asseche et al. 1999).
The Zeke construct contained the gene of interest cassette
(CAB:Hsp70:ZmSPSΔ482:Nos3′) and a selectable marker
cassette (Lox-35 s:Hsp70:NptII:Nos3′-Lox). The Lox sites
allow for the eventual removal of the selectable marker,
NptII, for commercial events.
In the first step of vector construction, a “shuttle” vec-
tor was created. An HpaI fragment containing the CAB
promoter and the Hsp70 intron from pMON47112 were
joined with an HpaI fragment containing the SPSΔ482
gene and Nos3’. In the final step, a NotI fragment from
pMON17704 containing the gene of interest cassette
(CAB:Hsp70:ZmSPSΔ482:Nos3′), was joined with a NotI
linearized fragment of pMON36176, which contained the
NptII selectable marker flanked by Lox sites and the back-
bone necessary for Agrobacterium transformation.
The Pat construct contained the gene of interest cassette
(PPDK:Hsp70:ZmSPSΔ482:Nos3′) and a selectable marker
cassette (Lox-35 s:Hsp70:NptII:Nos3′-Lox). This vector was
created by ligating an HpaI fragment, which contained the
PPDK promoter and the 5’ portion of the Hsp70 intron, to
an HpaI fragment of the Zeke vector, which contained the
3’ end of the Hsp70 intron, the SPSΔ482 gene, the Nos3’
element, along with the Lox-flanked selectable marker
(NPTII) and the backbone necessary for Agrobacterium
transformation.
The Pat and Zeke vectors were screened for the correct
orientation of the promoter/intron fragments and were sub-
sequently used to stably transform an elite inbred maize
variety.
SPS event screening
Gene insert copy number was determined by NPTII TaqMan
assay at R0. Only events with 1 or 2 insert copies were
advanced.
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Selections of advanced events in which zygosity had
previously been determined (e.g., homozygous positive or
negative parents) were routinely screened by gene specific
PCR. DNA from leaf tips of individual plants was extracted
using a Sigma DNA isolation kit. After DNA extraction,
the presence of the SPSΔ482 gene was determined by PCR
using an Hsp70 intron forward primer (hsp 727–5′) and a
SPSΔ482 reverse primer (11,710-8528-3′) which yielded a
1.1 kb fragment. DNA integrity was confirmed by amplify-
ing the endogenous ADH gene.
Additional event selections were screened by an NPTII
TaqMan assay, primarily to determine segregation ratios
and zygosity. At several points during event selection, the
gene-specific PCR assays were confirmed by SPS protein-
immunoblot analysis.
SPS protein extraction
All greenhouse and field efficacy plants were screened for
expression by immunoblot (western) analysis. Typically, a
small leaf portion was removed from a V4 to V10 plant,
frozen on dry ice and stored at − 80 °C. The frozen leaf
sample was ground to a coarse frozen powder in an Eppen-
dorf tube on dry ice and extracted into 300 µL of PBST
containing 1% (v/v) Protease Inhibitor Cocktail for plant
cell and tissue extracts (Sigma Catalog No. P 9599). After
thawing on wet ice, tubes containing extract were vortexed
and centrifuged at 14,000 rpm for 4 min. An 80 µL portion
of the supernatant was transferred to a tube containing 20
µL of 6X SDSSample Buffer (Laemmli 1970) and boiled
for 10 min. A second 50 µL portion of supernatant was used
for protein concentration by Bradford analysis, using Sigma
bovine serum albumin as a protein standard.
Protein immunoblot analysis
Denatured extract samples (16 µg total protein) in SDS Sam-
ple Buffer were loaded onto 10% SDS-PAGE gels (Bio-Rad
Catalog No. 161-1119). The gels were electrophoresed in
Tris-Glycine-SDS Running Buffer (25 mM Tris, 192 mM
glycine, 0.1% SDS, pH 8.3) for 60 min at 100 V plus 30 min
at 150 V. Gel proteins were transferred to Immunoblot
PVDF membranes (Bio-Rad Catalog No. 162–0177) in
Transfer Buffer (25 mM Tris, 192 mM glycine, 20% MeOH)
for 100 min at 250 mAmps. Membranes were blocked over-
night in Tris Buffered Saline with Tween (TBST) + 5% milk
at 4 °C. Blocked membranes were washed 3X with TBST
and incubated for 90 min at room temperature in TBST + 5%
milk containing SPS primary antibodies from rabbit (Bru-
neau et al. 1991). After incubation with primary antibody,
membranes were washed 3X with TBST and incubated for
60 min at RT in TBST containing anti-rabbit secondary
antibody (1:10,000; Anti-Rabbit IgG-Alkaline Phosphatase,
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Journal of Plant Biochemistry and Biotechnology
Sigma). Membranes were washed 3X in TBST and incu-
bated in AP substrate (SigmaFast BCIP/NBT, Sigma Cata-
log No. B 5655) until protein bands were stained purple.
SPS activity assay
Leaf samples were crushed to a frozen powder with liquid
­N2. 1.2 mL of SPS Extraction Buffer (50 mM Tris–HCl,
pH 7.5, 400 mM sucrose, 10% (v/v) glycerol, 1 mM phe-
nylmethane sulfonyl fluoride 5 mM dithiothreiotol, 1 mM
trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane
(E64), 10 µg/mL leupeptin, and 1% (v/v) Plant Protease
Inhibitor Cocktail (Sigma Catalog No. P 9599) was added
to the sample. The buffer was ground into the mixture with
care taken to make sure that the mixture did not thaw. More
liquid ­N2 was added to completely homogenize the powder.
A portion of this homogenized mixture was then added to
a 1.5 mL microcentrifuge tube on dry ice. The tube was
removed from the dry ice and allowed to thaw. Immediately
upon thawing the tube was centrifuged at 14,000 rpm in a
microcentrifuge for 4 min at 4 °C.
Immediately prior to assay, a portion of the supernatant
was desalted on a G-25 Spin Column (Quick Spin Protein
Columns, Roche Diagnostics Corp.), which had been previ-
ously equilibrated with SPS Extraction Buffer.
To assay SPS activity, 65 µL of desalted extract was
added to 65 μL of assay buffer containing 50 mM HEPES
(pH 7.5), 10 mM ­MgCl2, 10 mM UDP-glucose, 10 mM fruc-
tose 6-phosphate, 36 mM glucose 6-phosphate and 10 mM
NaF. Substrate-negative controls were run with the same
buffer without UDP glucose, glucose 6-phosphate or fruc-
tose 6-phosphate. The mixture was vortexed and incubated
at 25 °C for 15–30 min. The reaction was terminated by the
addition of 65 µL of 30% (w/v) KOH and the remaining
fructose 6-phosphate was destroyed by boiling the sample
at 100 °C for 20 min. This treatment also converts sucrose-6
phosphate to sucrose which can then be detected by the
anthrone method (Yemm and Willis 1954). To detect the
product sucrose, 800 µL of 0.3% anthrone in 27N H ­ 2SO4 was
added to the sample, which was then vortexed, centrifuged
at 14,000 rpm for 2 min and then incubated at 37 °C for
20 min. The absorbance was read at 625 nm using a spec-
traphotometer. Activity was calculated by using a sucrose
standard curve. One unit of enzyme is the amount which can
produce 1 µM sucrose/min at 25 °C.
Greenhouse efficacy tests of inbred F1 events
R1 seed was generated by crossing R0 maize plants with
themselves. F1 seed was generated from R0 events by back-
crossing expressing (and co-suppressed) plants to wild-type
inbred plants. Since the R0 plant was hemizygous, F1 indi-
viduals of single copy R0 parents would be expected to be
Journal of Plant Biochemistry and Biotechnology
segregating 1:1. This process removed negative phenotypic
effects due to regeneration and created positive and negative
isolines to conduct efficacy experiments. Events in which F1
individuals had aberrant segregation were eliminated.
Three separate greenhouse experiments were conducted
in order to test for the effect of the expression of SPS. Each
experiment was similarly designed in a similar manner. The
greenhouse was programmed for 14 h of daylight at 30 °C
and 10 h dark at 24 °C, 60% humidity. To minimize tem-
perature and humidity gradients in the greenhouse wildtype
and transgenic plants were always planted next to each other.
Since the leaf levels of both starch and sucrose show a diur-
nal pattern (low levels early in the day accumulating to high
levels late in the day), leaves were harvested at several time
points. SPS expression in each individual plant was deter-
mined by protein immunoblot analysis blot at V4. Positive
and negative plants were then randomized within an event
block. Each block was bordered by a row of wild type plants
and the aisle or outside rows were bordered by 2 rows of
plants to eliminate border effects. Since comparisons are
always made against the negative isoline control we have
elected to combine the three experiment together for analy-
sis; however, they are described individually below.
Leaf carbohydrate analysis of PPDK:ZmSPSΔ482 (Pat)
events
A first experiment was conducted in a greenhouse with
events selected to have moderate to high expression of
SPSΔ482 (Table SI and S2). These included two moder-
ate expressing events (Pat87 and Pat94), 3 high expressing
events (Pat47, Pat63, and Pat81) and 1 co-suppressed event
(Pat20), along with a block of control plants. On the day of
leaf harvest, light intensity peaked at approximately 950 µE.
At V6, the upper most-recently expanded leaf was
removed from the plant, folded into an envelope and
immediately frozen on dry ice. Leaf samples were stored
at − 80 °C until extraction for carbohydrates. Leaves were
harvested at 10 a.m., 1 p.m., 6 p.m., and 11 p.m. A single
leaf was sampled from each of four separate plants within an
event block. Each plant was sampled only once.
Leaf carbohydrate analysis of CAB:ZmSPSΔ482 (Zeke)
events
A second experiment was conducted in a greenhouse with
Zeke (CAB:SPSΔ482) events which were selected in order
to test a range of expression of SPS, as measured at R0
(Table S1 and S2). These included a low expressing event
(Zeke122), 2 moderate expressing events (Zeke83 and
Zeke113), 2 high expressing events (Zeke9 and Zeke108)
and 1 co-suppressed event (Zeke10). At V6, leaves were
harvested for carbohydrate analysis at 4 time points: 10 a.m.,
1 p.m., 4 p.m., and 6 p.m. Daytime light intensities reached
approximately 600 µE. There were up to 10 replicate leaves
harvested for each treatment and each plant was sampled
only once. Leaves were stored at − 80 °C until extraction
for carbohydrates.
Leaf carbohydrate analysis of combined study
of PPDK:ZmSPSΔ482 (Pat) and CAB:ZmSPSΔ482 (Zeke)
events
A final greenhouse efficacy experiment was conducted in
mid spring with a combination of F1 PPDK:ZmSPSΔ482
(Pat) events and F1 CAB:SPSΔ482 (Zeke) events. As in the
two previous greenhouse experiments, events were chosen
so that a range of expression levels, based on R0 expres-
sion levels, could be compared. Gene expression was deter-
mined by PCRand events were blocked, randomizing posi-
tive and negative plants within each event block. There were
2 low expressing Pat events (Pat87 and Pat95), 2 moderate
expressing Pat events (Pat18 and Pat89), and 1 very high
expressing Pat event (Pat47). There were 3 low expressing
Zeke events (Zeke17, Zeke112, and Zeke113), 1 moderate
expressing Zeke event (Zeke64), and 1 high expressing Zeke
event (Zeke69). A very high expressing Zeke event, Zeke11
turned out to have no plants expressing SPS. There were no
plants for the co-suppressed event, Zeke58. Zeke113 was
subsequently determined to be segregating.
Because of the limited number of plants, the numbers
and time points for sampling was variable. Up to 10 plants
were sampled at 1:30 p.m., 4 p.m., and 6 p.m. Plants were at
V8 to V9 at the time of sampling. Greenhouse light inten-
sities approached 1100 𝜇 E during the middle of the day.
The upper most recently expanded leaf was harvested into a
paper envelope and frozen on dry ice and stored at − 80 °C
until extraction for carbohydrates.
Field efficacy test of pat and Zeke hybrid events
Several field tests were established to measure the efficacy
of SPS overexpression in hybrid maize leaves. One test
was a comparison of positive and negative isolines of Pat
and Zeke events. Positive and negative isoline plants were
planted side-by-side in 2-row plots. This test was replicated
in the field twice. A similarly designed test, replicated once,
compared positive and negative isoline plants of Zeke co-
suppressed events.
In total there were 2 co-suppressed Zeke events (Zeke 10
and Zeke 58), 6 expressing Pat events (Pat18, Pat 66, Pat85,
Pat87, Pat89, and Pat95), and 6 expressing Zeke events
(Zeke11, Zeke17, Zeke19, Zeke64, Zeke69, and Zeke112).
Zeke112 was represented twice in the Day 1 Harvest plots.
Leaves were harvested on 2 consecutive days designated Day
1 and Day 2. On Day 1, leaves were harvested at 9 a.m., 1
13

p.m., 3 p.m., and 5 p.m. On Day 2, leaves were harvested at 1
p.m., 6 p.m., and 7 p.m. Light intensities approached at least
1700 𝜇 E at midday on both Day 1 and Day 2. Plants were
approximately at V8 at harvest. After harvest, leaves were
frozen on dry ice and stored at − 80 °C until extraction for
carbohydrates. The results from Day 1 and Day 2 harvests
are similar but for the sake of brevity we will only show
analyzed data from the Day 2 harvest in this study.
Yield test and yield components test of pat and Zeke hybrid
events
A yield component field trial was performed to compare pos-
itive and negative hybrids of 6 expressing Pat and 6 express-
ing Zeke events. The tests were planted at 6 locations and at
two densities (25 and 37 plants/row).
Hybrid leaf carbohydrate analysis
Whole frozen leaves were ground to a powder by crushing
and grinding on stainless-steel trays kept on dry ice using
a rolling pin. Liquid N ­ 2 was used to keep the tissue brit-
tle. Large veins were discarded. For the greenhouse efficacy
experiments, frozen leaf powder was extracted for carbo-
hydrate analysis and powder was stored at – 80 °C until
extraction. For the field efficacy experiment, leaf powder
was lyophilized, and dry leaf powder was extracted for car-
bohydrate analysis.
Approximately 50 mg of frozen leaf powder, or approxi-
mately 20 mg of dried leaf powder, was transferred to a
tared, 1.4 mL Falcon tube and weighed.
Leaf powder was extracted into warm 80% (v/v) ethanol
with three successive extractions which extracted over 95%
of the soluble sugars. First, 800 µL of 80% ethanol was dis-
pensed to extraction tubes containing powder samples using
a multi-channel pipettor. The tubes were sealed with a Beck-
man cap mat and incubated at 55 °C for 15 min, shaking the
rack two- or three-times during incubation. The plates were
centrifuged at 1800 rpm and 500 µL of the supernatant was
transferred to a Nalgene 96-well plate with 2 mL wells. Then
500 µL of 80% ethanol was added to the extraction tubes
and the extraction steps were repeated, removing 500 µL
supernatant to the previous extracts. A final 300 µL of 80%
ethanol was added to all tubes, repeating the extraction steps,
and transferring a final 400 µL of supernatant to the Nalgene
plate, resulting in a final extraction volume of 1.4 mL.
The green ethanolic extract was clarified by adding
30 mg of activated charcoal to each well. The plate wells
were sealed with a cap mat and the plate was inverted
several times over 15 min, returning the plate to wet ice.
The plates were centrifuged at 1800 rpm and 800 µL of
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Journal of Plant Biochemistry and Biotechnology
clear supernatant was transferred to a 96-well strip tube
plate. The supernatant was dried in a SpeedVac and resus-
pended in 200 µL MQ-H2O and stored at − 20 °C for sugar
analysis.
For starch analysis, the extract pellet was washed 4
times with absolute ethanol to completely remove pig-
ments and oven dried at 65 °C for several days. Starch in
the pellet was gelatinized by adding 400 µL MQ-H2O to
the tubes, sealing with a cap mat, and boiling in a water
bath for 15 min using a “rig” designed to keep the tube
contents from boiling out of the tube. After boiling, the
tubes were cooled on wet ice.
The gelatinized starch was enzymatically digested to
glucose. To digest the starch, 500 μL of 100 mM acetate
buffer, pH 4.5, containing 5 U/500 µL amyloglucosidase
(Sigma Catalog No. A 7420) was added to the tubes using
a multichannel pipettor and the tubes were sealed with a
cap mat and incubated for 4 h. at 55 °C. The tubes were
inverted several times during incubation. After 4 h., the
tubes were boiled for 10 min and stored at − 20 °C until
assaying for glucose.
Extract sugars, i.e., glucose, fructose and sucrose, and
starch-glucose, were measured enzymatically in microtiter
plates by coupling substrate conversion with the reduction
of NADP and measuring absorbance increase at 340 nm.
Each assay plate contained appropriate standards and
extract sugar concentrations were derived from the linear
curve(s) of that plate.
For the glucose/fructose assay (Bernt and Bergmeyer
1974), 20 µL of aqueous resuspended extract was assayed
in a final well volume of 300 µL containing 100 mM
Triethanolamine buffer, pH 7.6, 10 mM M ­ gCl2, 1.1 mM
ATP, and 0.8 mM NADP. After reading the background
absorbance at 340 nm using a microtiter plate reader, 5
µL of an enzyme mix containing glucose 6-phosphate
dehydrogenase (0.2U/well) and hexokinase (0.4 U/well)
was added, and the OD was read at 340 nm after 10 min.
The OD change was due to glucose. Then.phosphoglucoi-
somerase (1U/well) was added and the OD change was
read after 15 min. The OD change was due to fructose.
For the sucrose assay (Bergmeyer and Bernt 1974),
10 µL of aqueous resuspended extracts were transferred
to microtiter plate wells containing 100 µL of 100 mM
acetate, pH 4.65 containing invertase (1U/well) to invert
sucrose to glucose (plus fructose). The resulting glucose
was measured by adding buffer, substrates, and enzymes
as described above.
For the assay of glucose in the starch digest, 10 µL of
digest was transferred to microtiter plate wells and assayed
as described above. Glucose, fructose, and sucrose were
expressed as µmol/gFW (or µmol/gDW), and starch was
expressed as glucose-equivalents, µmol/gFW (or µmol/
gDW).
Journal of Plant Biochemistry and Biotechnology

Results 2000). Total SPS activity was measured in leaf tissue col-
lected diurnally from two expressing PPDK:SPSΔ482 events
Variable levels of higher expression of SPSΔ482‑SPS and one co-suppressed PPDK:SPSΔ482 event and wild type
in PPDK:SPSΔ482 and CAB:SPSΔ482 maize events SPS. Activity in both over-expressing events was signifi-
cantly higher than wild-type and showed a similar diurnal
A total of 85 PPDK:SPSΔ482 (Pat) events and 105 pattern (Fig. 3.). Activity levels were approximately twofold
CAB:SPSΔ482 (Zeke) events were tested for expression at higher at 1 p.m. and 6 p.m. SPS activity of the co-suppressed
R0 by immunoblot analysis. Based on the intensity of the
101 kDa SPSΔ482 band, event expression levels were com-
paratively assigned as very low, low, moderate, high, very
high, or negative. Co-suppressed events were also identified
in which both the endogenous upper band and transgenic
lower band were reduced or absent. Figure 2 depicts protein
immunoblots for 12 of the events followed in this study.
They include events over the entire range of expression.
There were no apparent differences in levels of expression
between PPDK:ZmSPS1Δ482 and CAB:ZmSPS1Δ482
events. Events were advanced for multiple reasons includ-
ing seed return and expression. Tables S1 and S2 summa-
rize the expression of all Pat and Zeke events generated. In
2 Pat events and 6 Zeke events both the endogenous SPS
and SPSΔ482 band were reduced or missing, indicating
that these events were co-suppressed in these events. One
of these events (Zeke 10) is included in this study. No visual
phenotypes were observed in plants expressing SPSΔ482.

Higher activity of SPS in leaves of PPKK:SPSΔ482
and CAB:SPSΔ482 maize events
Endogenous SPS activity in leaves is known to vary diur-
nally (Kerr et al. 1985, and Kalt-Torres et al. 1987), due to
both circadian and light/dark regulation (Winter and Huber
Fig. 3  Total SPS activity in leaf extracts of wild type and selected
PPDK:ZmSPSΔ482 (Pat) R0 transgenic corn events: Leaf discs were
collected diurnally at 4 time points from 2 expressing events (Pat 81
and Pat 63) and 1 co-suppressed (Pat 20) event, and wild type. Lights
came on at 5 a.m. and were off at 9 p.m. (16 h. photoperiod). Each
value is the average of three leaf samples from an individual plant.
Activity is expressed as total sucrose produced/30 min/mg total pro-
tein

Fig. 2  SPS Protein Expression

from leaf extracts of plants
sampled at R0: The figure
includes all events selected in
the study for greenhouse, field
efficacy or yield evaluation.
a PPDK-ZmSPSΔevents; b
CAB:ZmSPSΔ482 events. See
Tables S1 and S2 for relative
expression levels of the entire
set of ZmΔ482-SPS proteins at
R0 based on protein immunob-
lot analysis

13

event was reduced by approximately 85 to 90% and did not
show a diurnal pattern although it did increase between
10 a.m. and 12 p.m.
Although the SPSΔ482 protein was not expected to
undergo diurnal deregulation, the diurnal expression pattern
of total SPS activity in the transgenic maize plants might be
due to the upregulation of the PPDK and CAB during the
day and/or a carryover of the diurnal expression of the native
SPS protein with the transgenic over the entire diurnal cycle.
SPS activity in leaves harvested at 1 p.m. of wild-type
and additional SPS events is shown in Table 1. Activity
was increased by approximately two- to three-fold. Since
advancements had already been made using expression from
protein immunoblots (Fig. 2), only one of these events (Pat
18) ended up advanced for further study.
The expression of truncated SPS modulates leaf
carbohydrate levels in inbred maize
In three separate experiments sucrose and starch levels were
measured in inbred greenhouse grown transgenic maize and
their isoline controls. The sucrose and starch data is sum-
marized in Table 2. Sucrose and starch generally followed a
diurnal pattern which peaked at 6 p.m.. In all events (except
the co-suppressed event) and at all time points, sucrose
trended higher in the transgenic plants compared to their iso-
line controls. In most events and at most timepoints (except
the co-suppressed events) starch trended lower in transgenic
maize compared to their isoline control. In 6 cases (event
x timepoint) sucrose was significantly higher in transgenic
plants and in 11 cases starch was significantly lower that the
isoline control. In no cases was starch significantly higher
than the isoline control.
In the co-suppressed event, Zeke 10, the opposite pattern
was observed. Sucrose trended or was significantly lower in
transgenic plants than their isoline control and starch trended
or was significantly higher in transgenic maize than the iso-
line control.
Table 1  SPS specific activity Event Activity (µmol/
in R0 transgenic maize leaves: min/mg protein
R0 plants were grown in the
GH and wild type plants.
Zeke11 12.5
Leaves were harvested at 1 p.m.
Zeke25 18.3
for activity assay. Green font
indicates the transgenic mean Pat18 14.9
is higher than the wild-type Pat33 12.4
control and red font indicates
Wild-type 5.5
the transgenic mean is less that
the control. Salmon shading
indicates a difference between
the transgenic and control with
a p < .05. Activity values are
mean values of 3 separate leaf
extracts from a single plant
13
Journal of Plant Biochemistry and Biotechnology
The expression of truncated SPS modulates leaf
carbohydrate levels in hybrid field grown maize
Figure 4 shows a comparison of sucrose and starch values
of leaves in positive and negative isolines of Pat and Zeke
hybrid events grown in the field.
In Zeke over-expressing events (Fig. 4a), a strong trend
toward higher starch is observed across most cases. Over-
all, 14 out of 18 cases (a case = an event x a time point)
trended positive with 9 cases being significantly positive. A
strong trend toward higher sucrose is also observed with 17
cases trending positive with 5 that were significantly posi-
tive. Interestingly, there were three cases (Zeke11 at 6 p.m.,
Zeke19, 1 p.m., Zeke112, 6 p.m.) in which both sucrose and
starch was significantly higher than the isoline control.
In Pat events (Fig. 4b), starch was mixed, and sucrose
trended positive in all Pat cases and was significantly posi-
tive in 3 cases.
The two co-suppressed events (Zeke10 and Zeke58)
tested in the field showed a different pattern (Fig. 4c). Starch
levels were trended or were significantly higher in transgenic
plants and sucrose levels were generally lower.
The expression of truncated SPS does not change
seed yield in field grown hybrid maize
Six Pat and six Zeke events were tested for yield. Positive
and negative hybrid isolines from each event were planted
at two densities (25 and 37 plants/row) at six field loca-
tions. None of the events had a significant difference in yield
between the positive and negative isoline (Table 3). In addi-
tion, the overall yield for all Pat and Zeke events was almost
identical in the positive and negative isolines.
Discussion
We have successfully over-expressed a truncated maize SPS
gene (ZmSPSΔ482) in maize. We have shown that leaves
from transgenic plants over-expressing SPS have increased
SPS activity.
Starting with a hypothesis that improving source capacity
could increase maize yield, we devised a method to suc-
cessfully over-express a gene (SPS) by targeting expression
to a specific cell type (mesophyll leaf cells of maize). We
then screened events based first on copy number and RNA
expression. Next, we tested the activity and protein levels
of the transgenic protein in the tissue and classified over-
all expression levels as co-suppressed, none, low, medium
and high. We then tested the metabolic efficacy of the gene
by determining the steady-state sucrose and starch levels in
the transgenic plants, first using inbred plants in the green-
house, and finally using hybrid plants in the field. Based
Journal of Plant Biochemistry and Biotechnology

Table 2  Comparison of diurnal sucrose starch

changes in sucrose and starch Event Expression Time Gene mean p value mean p value
measured in leaf extracts Pat18 over 130PM Neg 42.03 12.67
between positive and negative Pat18 over 130PM Pos 42.83 0.89 6.20 0.04
isoline plants of F1 inbred Pat18 over 4PM Neg 44.77 16.06
PPDK:ZmΔ482-SPS (Pat) and Pat18 over 4PM Pos 47.82 0.73 9.87 0.04
CAB:ZmΔ482-SPS (Zeke) Pat18 over 6PM Neg 38.33 15.83
Pat18 over 6PM Pos 58.01 0.07 10.16 0.07
events. Units are in µmol/
Pat47 over 130PM Neg 37.54 11.88
gram wet weight. Leaves were
Pat47 over 130PM Pos 43.36 0.25 7.52 0.04
collected from plants at the V6 Pat47 over 6PM Neg 43.96 20.54
stage Pat47 over 6PM Pos 55.96 0.19 16.06 0.18
Pat89 over 130PM Neg 40.81 10.06
Pat89 over 130PM Pos 43.37 0.73 10.50 0.85
Pat89 over 4PM Neg 34.03 22.58
Pat89 over 4PM Pos 46.48 0.13 13.53 0.00
Pat89 over 6PM Neg 49.88 19.38
Pat89 over 6PM Pos 59.02 0.31 18.66 0.74
Pat95 over 6PM Neg 38.04 21.80
Pat95 over 6PM Pos 50.89 0.02 20.44 0.65
Zeke10 co-sup 10AM Neg 21.98 7.72
Zeke10 co-sup 10AM Pos 18.61 0.67 10.49 0.47
Zeke10 co-sup 1PM Neg 37.59 19.48
Zeke10 co-sup 1PM Pos 30.86 0.05 35.20 0.01
Zeke10 co-sup 6PM Neg 26.50 19.66
Zeke10 co-sup 6PM Pos 23.10 0.59 35.86 0.12
Zeke108 over 10AM Neg 14.67 3.38
Zeke108 over 10AM Pos 18.48 0.07 2.97 0.65
Zeke108 over 1PM Neg 32.59 13.68
Zeke108 over 1PM Pos 37.56 0.30 10.66 0.04
Zeke108 over 4PM Neg 34.39 18.88
Zeke108 over 4PM Pos 42.13 0.03 17.01 0.06
Zeke108 over 6PM Neg 27.62 19.60
Zeke108 over 6PM Pos 36.94 0.00 12.78 0.08
Zeke112 over 6PM Neg 37.56 14.02
Zeke112 over 6PM Pos 40.66 0.38 17.65 0.10
Zeke113 over 6PM Neg 48.20 27.29
Zeke113 over 6PM Pos 50.72 0.71 18.33 0.01
Zeke122 over 10AM Neg 19.12 6.02
Zeke122 over 10AM Pos 19.97 0.68 6.07 0.89
Zeke122 over 1PM Neg 34.20 16.76
Zeke122 over 1PM Pos 40.16 0.05 16.09 0.68
Zeke122 over 4PM Neg 36.29 23.54
Zeke122 over 4PM Pos 41.19 0.05 23.12 0.87
Zeke122 over 6PM Neg 29.45 19.19
Zeke122 over 6PM Pos 33.09 0.24 22.68 0.06
Zeke17 over 6PM Neg 28.27 11.90
Zeke17 over 6PM Pos 35.78 0.46 22.02 0.46
Zeke64 over 6PM Neg 42.10 21.70
Zeke64 over 6PM Pos 44.26 0.76 15.89 0.04
Zeke69 over 130PM Neg 29.83 7.36
Zeke69 over 130PM Pos 44.27 0.03 6.62 0.60
Zeke69 over 4PM Neg 37.41 17.56
Zeke69 over 4PM Pos 49.22 0.29 14.01 0.03
Zeke69 over 6PM Neg 36.74 18.90
Zeke69 over 6PM Pos 46.35 0.01 14.84 0.04
Zeke9 over 10AM Neg 20.71 11.31
Zeke9 over 10AM Pos 24.57 0.11 5.93 0.02
Zeke9 over 1PM Neg 28.74 26.31
Zeke9 over 1PM Pos 33.09 0.04 15.15 0.07
Zeke9 over 4PM Neg 30.75 27.59
Zeke9 over 4PM Pos 36.91 0.09 18.64 0.05
Zeke9 over 6PM Neg 24.65 26.08
Zeke9 over 6PM Pos 27.06 0.65 24.23 0.72
Pat81 over 6PM Neg 26.13 11.63
Pat 81 over 6PM Pos 34.72 0.08 18.47 0.05
Pat81 over 11PM Neg 15.08 28.40
Pat81 over 11PM Pos 17.65 0.22 23.71 0.21

13
 Journal of Plant Biochemistry and Biotechnology

Fig. 4  Sucrose and starch in leaves of hybrid PPDK:ZmSPSΔ482 tistically higher sucrose or starch than negative line (p < 0.5); red,
(Pat) events and CAB:ZmSPSΔ482 (Zeke) events of the Hybrid Field positive isoline has statistically lower sucrose or starch than positive
Efficacy Test. Blue, no significant difference in sucrose or starch level isoline. Units are in µmol/gram dry weight. a Pat non-co-suppressed
between positive and negative isoline; green, positive isoline has sta- lines. b Zeke non-co-suppressed lines. c Co-suppressed lines

13
Journal of Plant Biochemistry and Biotechnology
Fig. 4  (continued)
on protein expression and inbred sucrose levels we picked
several events and tested them in a multi-location yield trial
to determine yield efficacy.
Since the SPS protein used in this study has many regu-
latory features (Lutfiyya et al. 2007), it is possible that a
higher plant SPS might be necessary to improve source
capacity in higher plants. Alternatively, isozymes from dif-
ferent phylogenetic groups (Lutfiyya et al. 2007) might have
different yield effects. More events will need to be tested to
determine the correlations between SPS expression level and
yield as well as sucrose/starch levels and yield. Also, it is
possible that higher levels of sucrose actually inhibit certain
processes and it may be necessary to stack SPS with a leaf
sucrose transporter to ensure that the additional sucrose is
transported out of the leaf to the sink tissues. Finally, other
SPS stacks with other genes in the same or different meta-
bolic pathways in the same or different tissues, including
sucrose phosphorylase (Narimatsu et al 2004), fructose 1
6-bisphosphate aldolase, sucrose phosphate phosphatase,
glutamine synthetase, and glucose dehydrogenase could be
considered to test various hypotheses.
Earlier attempts to over-express SPS in maize using an
e35S promoter had been unsuccessful. Leaf tissue expres-
sion experiments showed that the e35S promoter targets
expression to bundle sheath cells. In C4 plants having Kranz
anatomy such as maize, CO2 carboxylation and chloro-
plast starch accumulation are compartmentalized in bundle
sheath cells, while CO2 reduction by Rubisco and sucrose
biosynthesis are compartmentalized in mesophyll cells. In
this study, two different promoters were employed to tar-
get over-expression of SPS specifically to leaf mesophyll
cells, the PPDK promoter and the CAB promoter. These
two promoters also drive diurnal expression (Taniguchi et al.
2000; Bansal et al. 1992). The results of this study indicate
that targeting of SPS to leaf mesophyll cells is necessary for
expression in maize.
In this experiment the expression of transgenic SPS pro-
tein in maize leaves varied from very low to very high, based
on immunoblot intensity. We also identified individual trans-
genic events in which both endogenous and transgenic SPS
proteins were co-suppressed. The effect of transgenic SPS
expression was measured by comparing sucrose and starch
levels in leaves of positive and negative isoline plants grown
side by side in three separate greenhouse studies (Table 2).
The sucrose data from the greenhouse tests was strikingly
consistent. Every event overexpressing SPS had trended to
higher sucrose than the control at every time point. Many
of these were significant and more were almost significant.
The one exception which proves the rule is Zeke10 (co-
suppressed) which trended lower or was significantly lower
at every time point. The starch data, almost as strikingly,
had the opposite trend. Most events at most time points had
lower starch. Zeke10 (co-suppressed) showed exactly the
opposite trend to higher starch and lower sucrose at every
time point. The inverse correlation trends between sucrose
and starch across transgenic vs. isoline and across higher
13
 Journal of Plant Biochemistry and Biotechnology

Table 3  Yield Analysis of hybrid PPDK:ZmΔ482-SPS (Pat) and In field-grown hybrid maize, expression of SPS clearly
CAB:ZmΔ482-SPS (Zeke) events. Gene Positive is compared to neg- increases leaf sucrose levels, although the expression of
ative, across Densities, by Construct and by Event. Units are in kg/
hectare
PPDK-SPS had no clear effect on leaf starch levels (pos-
sibly a slight decrease). In these PPDK-SPS plants there
Denisty Event Pos Neg Diff p value may be a slight inverse trend between sucrose and starch
High Pat 18 11,283 11,745 − 462 0.34 level. CAB-SPS plants clearly have significant increases
High Pat 66 11,676 11,977 − 301 0.5 in starch and sucrose often simultaneously. In these events
High Pat 85 12,616 12,313 303 0.49 SPS is clearly enhancing source capacity in field-grown
High Pat 87 11,897 12,377 − 479 0.27 hybrid maize. The difference between PPDK-SPS and
High Pat 89 11,653 11,743 − 90 0.84 CAB-SPS efficacy further suggests that fine-tuning of the
High Pat 95 12,385 11,872 513 0.24 level and location of SPS expression will be necessary to
High Zeke 11 11,890 12,461 − 571 0.2 maximize source capacity. Overall, these results further
High Zeke 17 12,168 12,086 81 0.85 support the conclusion that SPS is a pinch point in leaf
High Zeke 19 12,315 12,228 87 0.84 metabolism.
High Zeke 64 12,566 12,423 143 0.75 Results from other transgenic experiments in maize in
High Zeke 69 12,266 12,380 − 114 0.8 which another sucrose synthesis pathway enzyme (fructose
High Zeke112 11,492 12,211 − 719 0.1 1,6-bisphosphate aldolase, FDA) did not show a consist-
Low Pat 18 11,474 11,545 − 71 0.87 ent sucrose phenotype (data not shown) also reinforce the
Low Pat 66 11,122 11,535 − 413 0.35 conclusion since only the rate-limiting enzyme would be
Low Pat 85 11,282 11,702 − 420 0.34 expected to have a large and consistent effect on sucrose.
Low Pat 87 11,268 11,145 123 0.78 Yield improvements in the beginning of the last century
Low Pat 89 10,937 11,466 − 529 0.23 have come about by increases in kernel number per hec-
Low Pat 95 11,986 11,217 769 0.08 tare (Egli, 2015, 2019) requiring higher plant populations
Low Zeke 11 11,143 11,253 − 110 0.8 and through a concomitant increase in both source and sink
Low Zeke 17 11,323 11,400 − 77 0.86 strength suggesting that increasing source capacity alone
Low Zeke 19 11,517 11,274 243 0.58 with no increase yield. Although the activity of SPS can
Low Zeke 64 11,004 10,875 129 0.77 satisfy demand for carbon assimilate (Prioul and Schwe-
Low Zeke 69 11,076 11,208 − 132 0.77 bel-Dugue 1992), cause and effect has not been established
Low Zeke112 11,029 11,005 24 0.96 with the possibility that sink and source work together to
Ave 11,640 11,727 − 86 determine yield (Egli 2015). Since there was no increase in
seed yield it might be expected that vegetative mass would
be higher in the transgenic plants; however, we could not
expression vs. co-suppression suggests there may be a limi- confirm this since we did not measure biomass.
tation either due to the light or in inbred maize compared Therefore, it is perhaps not surprising that we did not
to hybrid maize (Bellasio and Griffiths 2014). Although we see an effect on yield. In the multi-location study of six
did not measure photosynthesis directly, it is likely that pho- PPDK:SPSΔ482 events and six CAB:SPSΔ482events, there
tosynthesis was not inhibited but rather a reportioning of was no significant effect on yield (p > 0.05) in any event at
carbon between sucrose and starch similar to the relationship either planting density. Only one event, Pat 95, had a yield
between them in endosperm (Salerno 1986). This is signifi- increase of 625 kg/hectare over both densities which was
cant since there are several reports that sucrose accumulation nearly significant (p = 0.066). Since maize source and sink
inhibits photosynthesis [Stitt et al. 2010, McCormick et al. are thought to be tightly controlled (Seebauer et al. 2010)
2009, Inman-Bamber et al. 2011), and exogenous sucrose this result might be expected and suggests that to increase
supply strongly reduces the net C ­ O2 assimilation in sugar- maize yield both source and sink (and perhaps other factors)
cane (Lobo et al 2015). Overall, there appears to be room to will have to be considered.
increase source capacity in inbred maize.
Supplementary Information The online version contains supplemen-
It is noteworthy that the expression of SPS did not change
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 13562-0​ 23-0​ 0838-0.
the overall diurnal pattern of sucrose/starch leaf accumula-
tion nor was sucrose accumulation in SPS (over)expressors Acknowledgements The authors would like to acknowledge several
correlated with expression based on western. This suggests researchers who contributed to this project including Stan Dotson, Kim
Hahn, Christine Hull, Sarah Miller, Oscar Heredia, Kim Brunick, Gary
that SPS may be near limiting in maize leaves or alterna-
Lee, and Heather Conn. For leadership contributions they would like to
tively, higher levels of sucrose synthesis may be inhibitory. acknowledge Jill Deikman and Philip Miller. We like to acknowledge
In either case, improvement of source capacity will require Oscar Sparks for identifying the retrieving the raw yield data from the
fine-tuning of SPS expression. correct field experiments

13
Journal of Plant Biochemistry and Biotechnology
Funding The authors are former or current employees of Bayer Crop
Science, a manufacturer of seeds.
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.
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