THE ROLE AND REGULATION OF
SUCROSE-PHOSPHATE SYNTHASE FROM
SUGARCANE (Saccharum species)
Bambang Sugiharto1,2 and Widhi Dyah Sawitri 1
1Center
for Development of Advanced Science and Technology and 2Department
of Biology, University of Jember
Jl. Kalimantan No 37, Kampus Tegalboto, Jember 68121, Indonesia.
Email : [email protected]
Photosynthetic carbon assimilation and sucrose synthesis in plants
Exported to
sink tissues
Sucrose-phosphate synthase (SPS) activities determine sucrose
accumulation in leafs of Saccharum species
Lines
Ni9
NiF8
NCo310
PEPC NADP
-ME
1.68
1.47
1.14
1.08
1.11
0.98
SPS
Sucrose
contents
37.74
38.70
25.77
77.50
84.44
55.90
Molokai
Babakan
Bois R
1.67
1.75
1.29
0.8
0.72
1.16
31.53
28.45
23.50
59.20
52.70
51.10
28NG
IN84
1.45
1.99
0.92
0.90
24.51
21.94
52.70
55.82
SES186
JW94
2.10
1.71
0.90
1.24
20.51
15.69
46.00
39.10
The enzyme activities are expressed as unit/mg prot and
sucrose contents in µg / g FW
Ni9 NiF8 NCo
Mol Bbkn
Bois 28NG IN84 SES JW94
Levels of LSU-Rubisco protein
in leafs of Saccharum species
PEPC : phosphoenolpyruvate carboxylase
NADP-ME: NADP malic enzyme
SPS : sucrose-phosphate synthase
Rubisco : ribulose-1,5-biphosphate
carboxylase/oxygenase
Sucrose contents in the leaf
of Saccharum species were
mainly fluctuated according
to the SPS activities
Sucrose contents
(μg Suc/g FW)
Induction of SPS activity during drought stress in sugarcane
Dry season is the best
condition for harvesting
sugarcane
SPS activity
(μg Suc/min/prot)
ABA induced SPS levels
1 Day
2 Days
SPS levels
Water stress (days)
Change in sucrose contents (upper), SPS activities
(middle) and SPS levels (lower) during 10 days
water stress treatments
Change in protein levels of SPS, GS1, GS2 and
LSU-Rubisco proteins after treatment of ABA
hormone at concentration 0, 10 and 100 µM.
The proteins levels were detected by Western
Blot analysis with a spesific antibody
Cloning and expression analysis of genes for sucrosephosphate synthase from sugarcane
Relative levels of mRNAs
SoSPS1
10
8
6
4
2
0
0
12
SoSPS2
24
48
Time after illumination (h)
Comparison of the amino acid sequences deduced
from cDNAs for SPS from sugarcane. Box sequences
I, II, III are functionally important regions. Dotted
Ser residues are the regulatory phosphorylation sites.
Detection of
transcripts of
SoSPS1 and
SoSPS2 in
different organ
of sugarcane
Sugiharto et al (1997) [Plant Cell Physiology 38(8):961-965]
SPS activity in leaf
(µg suc/min/µg prot)
Overexpression of SoSPS1-cDNA increased SPS levels and sucrose
accumulation in leaf of transgenic tomato
0.19
0.20
0.15
0.10
0.09
0.10
2.1
2.2
0.05
0.08
0.07
0.18
0.10
0.09
0.11
0.12
0.10
0.12
0.12
0.11
5.2
5.3
5.4
1.17
1.14
1.13
5.2
5.3
5.4
0.05
Sucrose content
In leaf (mg/g FW)
0.00
k
2.3
2.4
2.5
1.50
1.00
3.1
1.28
0.74
0.87
0.97
0.86
0.82
2.3
2.4
1.00
3.2
3.3
4.1
1.15
1.18
1.16
3.2
3.3
4.1
5.1
1.03
5.5
1.28
0.50
0.00
Western Blot
analysis with
antibody
against SPS1
k
2.1
K1
2.2
2.1
2.2
2.5
3.1
3.1
3.3
4.1
5.1
5.4
5.5
5.5
SPS activity in leaf
(µg/µg prot/min)
Overexpression of SoSPS1 cDNA increased SPS activity and
sucrose content in leaves of transgenic sugarcane
SoSPS1
Actin
Semi-quantitatif RT-PCR analysis of SoSPS1
(upper) and Actin (lower) expression in leaf
of transgenic sugarcane
SoSPS1
levels
Sucrose content in leaf
(mg/g fresh weight)
Transgenic sugarcane
WT 1 2
3
4
5
SPS activity in leaf of Wild type
and transgenic sugarcane
WT
E2
E6
E6a E8
E9
Post-translational regulation of sucrose-phosphate synthase in plant
Sucrose
biosynthesis
SPS regulation
metabolites
G6P Pi
SPS
Active
(light)
(dark)
Less active
sugarcane
SPS
Allosteric
regulation
G6P dependent (allosterically regulated)
0.5
[Sucrose] (mM)
Plant cell
Sugarcane SPS
0.4
0.3
0.2
20 mM of F6P
10 mM of F6P
5 mM of F6P
0.1
0
0 mM
2 mM
4 mM
[G6P]
6 mM
8 mM
• Activity of plant SPS is regulated by diurnal
cycles. Active under light and inactive during
dark condition.
• This allosteric regulation involving
metabolites G6P
• Increasing G6P levels up to around 5 mM
increased the SPS activity.
Expression and purification of recombinant SPS1 in Escherichia coli
Partial purification :
Full-length cDNA
encoding SoSPS1
(Sugiharto, et al. 1997.
Plant Cell Physiol.)
cDNA SoSPS1
pTrcHis-SPS
pTrc99A-SPS
1.
2.
3.
4.
5.
1. DE52
Molecular
size were
2.
DEAE Toyo-pearl
3. SourceQthan 120 kDa
shorter
4. cOmplete Histag
5. Superdex 200
DE52
DEAE Toyo-pearl
Butyl Toyo-pearl
Superdex200
Blue-sepharose
Gene construct into
expression vector
Construct Preparation :
N-term.
F6P
UDP-G
C-term.
pTrc99A-SPS A
pTrcHis-SPS
6xHis-tag
Construct: 6xHis-tag at N-terminus
Host: E. coli BL21 (DE3)
B
Expression :
SPS recombinant E. coli was cultured at
20oC + 0.5mM IPTG
Expression of recombinant SPS protein in E. Coli under limited-proteolysis condition
N-term.
F6P
UDP-G
C-term.
pTrc99A-SPS A
Preparation under carefully regulated condition :
Anti SPS
The A form was close
in size with SPS from
sugarcane leaves
Two major bands were detected in SPS
gene transformed cells around
120kDa (A Form ) and 100 kDa (B Form )
Characterization of two forms of SPS through enzymatic activity
135 kDa
100 kDa
Form A
Form B
Recombinant SoSPS1 (B Form)
[S6P formed] (mM/10min)
The total extracts prepared from the
comparable amounts of bacterial cells :
0.8
0.6
0.4
20mM F6P
0.2
0
10mM F6P
5mM F6P
0
2
4
[G6P] (mM)
6
8
• The SPS activity profile from time course of E. coli cultivation was increased
according to the accumulation profile of B Form
• B Form was successfully purified by several steps of chromatography systems
• Addition of allosteric effector of G6P did not increase SPS B activity and the
allosteric property was not observed in B Form of SPS.
• So, the truncated SPS or B form SPS is not regulated by allosteric effector of G6P
Production of the N-terminal truncated forms of SPS
6xHis
N-term. F6P
UDP-G
C-term.
HisFull
His∆N1
His∆N2
His∆N3
His∆N4
His∆N5
His∆N6
Trimmed site
Expression of the N-terminal truncated forms of SPS in E. coli
150
100
75
Expression and purification of full-length SPS from insect Sf9 cells
GFP Nanotrap purification of Full length SPS
[imidazole]
0 mM
20 mM
250 mM
SoSPS1-EGFP
Full (Sf9)
The full-length SPS was produced in insect cells and purified using GFP
nanotrap system. The full length SPS was resulted after treatment with TEV
protease.
Effect of the N-terminal truncation on SPS spesific activity
Preparation of SPS forms by partially purification
Specific activity of full-length and truncated form of SPS
• Full-length SPS has the lowest
specific activity
• ∆N6 (without N-terminal
region) increased in the
activity of SPS by 10-fold than
full-length
• Longer truncated of Nterminal region enhances the
specific activity
Kinetic properties of SPS forms, full length and series N-terminal truncation
• The N-terminal region give significant influence on the SPS activity. Addition of G6P
increased activity full length SPS, but the activity of N-terminal truncated SPS was not
significantly influenced by addition G6P
• The full-length SPS activity is regulated by allosteric effector G6P, but the N-terminal
truncated SPS is not effected by the effector G6P.
• So, the N-terminal domain play a significant role on the regulation of SPS
Proposed model the regulation of plant SPS
Full-length
UDP-G
N
UDP-G
N
N
UDPG
UDP-G
N
+ G6P
N
N
UDP-G
UDP-G
UDP-G
UDP-G
Less Activate
N-terminal
truncated
form
N
N-terminal region
as a suppressor for
enzyme activity
N
UDP-G
UDP-G
G6P
Activated
N
G6P
N
G6P
N
G6P
UDP-G
UDP-G
UDP-G
UDP-G
+ G6P
N
UDP-G
N
UDP-G
Activated
G6P
N
N
Activated
G6P
N
IN PLANTA STUDY
Overexpresssion of engineered SPS1-cDNA in tomato plants
(A)
(B)
(C)
Fig. 3. Growth of two weeks tomato seedling (A), meristematic tomato shoot tips during 2
days co-cultivation (B), and tomato shoots in selection MS media containing antibiotic
kanamycin (C). Putative transformant tomato shoot survived in the selection media, but
the non-transformant shoot turned to chlorosis, browning and died.
On going experiment
Purification and trial crystallization to determine biological structure SPS protein
Sample : His∆N6
The result of size exclusion chromatography :
Vector: pTrcHisA
Host: E.coli BL21 (DE3)
Medium: LB medium
Expression: 16L (+0.06mM IPTG); 20oC
- Resuspend in extraction buffer
- Sonication
- Centrifuge 15,000rpm at 4°C, 15
min
Purification steps:
1. DE52
2. DEAE
3. SourceQ
4. cOmplete His-tag
5. Superdex200 (+5mM F6P)
Crystallization trial
Difficult to obtain the crystal
Problems :
• Easy to precipitate
• Not enough sample for crystallization
• High percent polydispersity (over than 40%)
Further purification is necessary
Conclusion
1. Sucrose-phosphate synthase (SPS) is a key enzyme for sucrose
synthesis and accumulation in sugarcane leaves. Enhancement of
SPS activity increases sucrose accumulation.
2. The mechanism of allosteric regulation in plant SPS may involve
the upstream N terminal region. Truncation of N terminal
resulted in an increased of SPS spesific activity.
3. We propose to redesign SPS protein through genome editing for
improving in sucrose accumulation and plant growth
THANK VERY MUCH
TERIMA KASIH
Acknowledgments :
• Professor Tatsuo SUGIYAMA and Professor H. SAKAKIBARA from Nagoya University,
Professor T. HASE, Professor A. NAKAGAWA, Osaka University, Japan
• My Collegues at CDAST Jember University, PT. Perkebunan Nusantara XI, Indonesia
• These research activities were supported in part by grants from Indonesian Ministry of
Research, Technology and Higher Education , PT. Perkebunan Nusantara XI