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Photosynthesis: Assimilate partitioning
Getting the building blocks for growth and cell wall biosynthesis from the leaf tissue to actively growing organs.
Bob Turgeon
Water
proton-coupled symporters
amino acids amino acids
PM-ATPase
chloroplast
SucroseSucrose
Vacuole
Plasmodesmata
mass flow
ATP
pH = 7.3∆E = -180 mV
CO2
Phloem
Mesophyll pH = 5.5
H+
SucroseH+
Apoplastic phloem loading
pH jump forms trans-membrane pH gradient
potassium & valinomycinform defined membrane potential
Κ
14C-substrate
+pH = 8.0
pH = 6.014C-substrate
pHi = pHo
Negative membrane potential
6.04.02.00.00.00
400
800
1200
Time (min)
Sucr
ose
Tra
nspo
rtpm
ol/ m
g p
CCCP
Sucrose
Cytoplasm Extracellular
Plasma Membrane
+H
C
CHSucCHSuc
CSucrose
H+
(∆ sensitive?)Ψ
(∆ sensitive?)Ψ
DEPC
SH
PCMBS
Binds to a substrate protectable site
Properties of the Proton-Sucrose Symporter
m
5050
Apparent KmSucrose 1.0 mMProtons 0.7 µM
InhibitorsDEPC I = 750 µMPCMBS I = 30 µM
pH Optimum < 6.0
Stoichiometry 1:1
Location Plasma Membrane
Specificity Low Affinity For:glucose, fructoseraffinose, maltose mannose, lactoseand melibose
5050
Proton-sucrosesymporter
Amino Acid Import
Amino Acid Import
Amino Acid ImportAmino Acid ExportPrimary AssimilationAmino Acid Cycling (phloem to xylem)
Amino Acid ExportPrimary AssimilationAmino Acid Cycling (xylem to phloem)
Amino Acid Import
Mature leaves
Developing leaves, fruit, and seed
Root
Developing rootsand meristem Phloem
Xylem
Amino acids are the currencyof nitrogen allocation in
multicellular plants.
Strategies for identifying symporter proteins and genes ….
• BiochemicalDifferential labelingPhotoaffinity labelingSolubilization & reconstitution
• MolecularPCRMutantsFunctional complementation
Biochemically limited andtransport incompetent
Yeast Mutant
MolecularCloning via Functional Complementation
Transform mutants with a plant cDNAlibraryconstructed in a yeast expression vector
Screen for restored growthon a limiting medium
H
ExpressionVector
+
Limiting Substrate
Positive Transformant
With successful transcription, translation, and insertion, we select for plant transporter-dependent growth.
ATP
6.04.02.00.00.0
1.0
2.0
Time (min)
Ala
nine
Tra
nspo
rt (n
mol
/ mg
cells
)
B
16.012.08.04.00.00.0
0.2
0.4
0.6
0.8
Time (min)
His
tidin
e Tr
ansp
ort
(nm
ol/ m
g ce
lls)
A
100
100
200
200
300
300
400
400
3 3 2 2 1 1 0 0 -1 -1-2 -2-3 -3* * *
Amino Acid Position, N to C Terminal
Hydrophobic
Hydrophilic
Structure no. 1
N
1 2 3 4 5 6 7 8 9 10
C
CYTOPLASMStructure no. 2
N
1 2 3 4 5 6 7 8 9 10 11
C
CYTOPLASM
Proton-Sucrose Symporter
CYTOPLASMN C
1 2 3 4 5 6 7 8 9 10 11 12
Cytoplasmic side
Random Mutagenesis
silent mutations, nonsense mutations, substitutions, premature stops
Transform into yeast
Screen for altered transport phenotypes
Construct
0.1
0.2
0.3
0.4
0.5
0.6
0Wt Mal9 (G334S)
Mal
tose
tran
spor
t (n
mol
/ m
g FW
. min
)
0.2 mM
0.5 mM
1 mM
2 mM
H65
CytoplasmicN C
1 2 3 4 5 6 7 8 9 10
L461
11 12
N155
G334
Q249W250
Plasmodesmata
Sucrose
Sink
Tissu
e
Phloem
Sucrose
Mesophyll
amino acids
chloroplast
Sucrose
amino acids
amino acids
H+
hexose
sucrose
H+
Vacuole
Vacuole
Vacuole
Amino acids
Amino acids
Sucrose
amino acids
ATP
symplastic
NO3
mass flow
water
water
CO2
1. Sucrose symporters serve both source and sink tissues
2. BUT, there is at least one other way to generate the hydrostatic pressure difference
Phloem Loading in a “Symplastic System”
Also depends on accumulating high concentrations of solutes, but in this case, the “sugar” is synthesized in place, not transported into the CC/SE complex.
X
Synthesize high concentrations of raffinose &/or stachyose in the companion cell (intermediary cell). Raffinose is too large to diffuse back into bundle sheath cell via plasmodesmata. This generates a high osmotic concentration that results in water influx and high hyrodstatic pressure that drives phloem transport to sinks.
H2O
Sucrose diffuses into the companion cell
Direct evidence of pressure flow: the aphid.
Sapdroplet
Aphid feeding Stylet in sieve-tubemember (LM)
Stylet
Severed stylet exuding sap
Sap droplet
Sieve-tubemember
25 µm
What happens when sucrose gets to the import-dependent organs?
It is unloaded from the companion cell/ sieve element complex via apoplastic and symplasticpathways.
The movement of sucrose from sources to sinks is called assimilate partitioning. Controlling this process could have a profound impact on crop yield.
Plasmodesmata
Sucrose
Sink
Tissu
e
Mesophyll
amino acids
chloroplast
sucrose
Phloem
SucroseSucrose
amino acids
amino acids
H+
H+
hexose
Vacuole
Vacuole
Vacuole
Amino acids
Sucrose
ATP
symplastic
NO3
mass flow
water
Apoplastic model of phloem loading
?
?
TP
H+
water
Amino acids
CO2
H+
amino acids
SUCROSESYMPORTER
PHOTOSYNTHETICACTIVITY SINK ACTIVITY
H2O
CO2
time
transpirational feeding
Tran
spor
t Act
ivity
(pm
ol /
min
/ mg
prot
ein)
0
500
1000
1500
2000
2500
0 100 200
mM Sucrose Fed 24 Hours
SucGlucAla
• Are changes in sucrose transport activity due to osmotic effects?
No. KCl, sorbitol, mannitol have no effect.
• Is the observed regulation sucrose-specific?
Yes. Hexoses and hexose analogs are not effective.
• What happens to sucrose transport activity?
Km remains unchanged. Vmax decreases.
• How?
BvSUT1 mRNA and protein decrease with 2 hr half-livesTranscription is controlled by a sucrose sensing protein-phospho relayand overall transport capacity is directly proportional to transcription
Jen Chiou, Matt Vaughn, Wendy Ransom-Hodgkins, Greg Harrington
Sucrose SensorSucrose
Protein Turnover
Sucrose Symporter
Transcription
mRNA Turnover
A
Kinase
Phosphatase
B
PhloemLoading
All within the companion cell
Sucrose
H+
High rate of symporter transcription
High rate of sucrose export
Abundant symporter protein
Sucrose
Low Sink Demand
H+
Sucrose accumulation in the phloem
Low rate of sucrose export
Down-regulation of symporter transcription
Less symporter protein
PH
LOE
M
PH
LOE
M
Sucrose-signalingresponse pathway
High Sink Demand
How can we identify players in the sucrose signaling pathway?
1. Mutagenize SUT1promoter::reporter plants and screen for mutants that lack the sucrose signaling pathway
2. Use expression profiling of companion cells to identify specific kinases and phosphatases, then look at phenotypes of knockouts
3. Look for a sucrose response that is more tractable for genetic analysis
Four day old seedlings induced with 90 mM sugar treatments and photographed three days later.
Water
Anthocyanin Content
012345678
wat
er
sucr
ose
gluc
ose
fruct
ose
gluc
+fru
c
man
nose
mal
tose
sorb
itol
A53
0-A
657/
gFW
H20
Suc
rose
PAP1
ACTING
luco
se
Fruc
tose
Glu
c +
Fruc
Man
nose
Mal
tose
Sor
bito
l
PAP1 (Production of Anthocyanin Pigment 1) Transcription factor ‐ known “master”regulator of anthocyanin biosynthesis and part of a multi‐peptide complex
Pro 8 Pro 62 Pro 63
H2O SUC H2O SUC H2O SUC
Col-0 35S-GUS
1 mm
H2O SUC H2O SUC
H2O SUC H2O SUC H2O SUC
Pro 8 Pro 62 Pro 63
Wg 34 Wg 35 Wg 43
H2O SUC H2O SUC H2O SUC
Col-0 35S-GUS
H2O SUC H2O SUC1 mm
H2O SUC H2O SUC
ΔIntron1 2A ΔIntron1 3B
ΔIntron2 3A ΔIntron2 5B
H2O SUC H2O SUC
ΔIntron1&2 4A ΔIntron1&2 6A
H2O SUC H2O SUC
SURE-1
5’-upstream sequence
200 bps = exon = intron
ATG
I II III
ISURE-2
PAP1 gene
PAP1wg_mut1-GUSGUSI II III
SURE-1 (TTTTCTATT) mutated to TTTGAGATT
PAP1wg_mut2-GUSGUSI II III
SURE-1 (TTTTCTATT) mutated to SURE-2 (AATACTAAT)
PAP1wg_mut3-GUSGUSI II III
205 bp deletion (includes SURE-1)
PAP1wg_mut4-GUSGUSI II III
205 bp deletion (excludes SURE-1)
H20
SU
C
∆ SURE-1
PAP1
GUS
ACTIN
GLU
H20
SU
C
∆ SURE-1
GLU
H20
SU
C
Minus SURE-1
GLU
H20
SU
C
+ SURE-1, - 5’
GLU
4 day old plants, 4 hr induction
Biotech Approach to Increase Yield: Use sugar beet as a model biofuel crop by by manipulating sugar allocation in the plant’s vascular system.
Why sugar beet as a model for carbon partitioning
• Root yields of over 40 tons per hectare at 15.5-18% sucrose content (6-7 tons of sugar per hectare)
• Record yields approach 25% sucrose, so there is somewhere to put “extra” sucrose
Approach: Constitutively express hyper-active symporter
Hypothesis: This will draw down sugars in photosynthetic cells which will,1) Increase photosynthesis 2) Delay senescence
Leaf mesophyllPlasmodesmata
Sucrose
Phloem
Sucrose
chloroplast
SucroseH+
H+
vacuole
Sucrose
CO2
ATP
mass flow
?
?
AtSUC1H65K TTCmGAS1p
The modified SUT is expressed in the transgenic lines
Independent transgenic lines
Con
trol 1
Con
trol 2
13 121
8 43 46 139
202
226
230
239
248
257
Average tuber FW of control and transgenic lines
0100200300400500600700800900
aver
age
tube
r FW
(g)
Con
trol
SU
T-8
SU
T-13
SU
T-43
SU
T-46
SU
T-12
1
SU
T-13
9
SU
T-20
2
SU
T-22
6
SU
T-23
0
SU
T-23
9
SU
T-24
8
SU
T-25
7
**
*
Independent transgenic lines
p-value ≤ 0.01
Average above ground biomass of control and transgenic lines
05
101520253035
** * *
aver
age
abov
e gr
ound
bi
omas
s (g
)
Con
trol
SU
T-8
SU
T-13
SU
T-43
SU
T-46
SU
T-12
1
SU
T-13
9
SU
T-20
2
SU
T-22
6
SU
T-23
0
SU
T-23
9
SU
T-24
8
SU
T-25
7
Independent transgenic lines
p-value ≤ 0.05p-value ≤ 0.01
aver
age
tube
r FW
/leaf
DW
Average tuber FW/leaf DW of control and transgenic lines
Con
trol
SU
T-8
SU
T-13
SU
T-43
SU
T-46
SU
T-12
1
SU
T-13
9
SU
T-20
2
SU
T-22
6
SU
T-23
0
SU
T-23
9
SU
T-24
8
SU
T-25
7
Independent transgenic lines
05
101520253035404550
Rice as a model crop for identifying biomass genes
Rationale:The Green Revolution was about seed production, not biomassRice is a great model for new energy crops because ……….
Simple grassGene syntenyAg-infrastructure well establishedPowerful genetic, molecular, and genomic tools
OryzaSNP set20 diverse rice lines• resequenced for SNP discovery
PNAS 106:12273-12278 2009
Approach:• Define morphological and physiological differences
• Forward genetic screens
• QTL mapping
• Association mapping
• Transgenic manipulation of candidate genes
knockout, over‐expression, truncated proteins
OryzaSNP set• Sampling
– Morphological• leaf length & width, plant height, tiller number, and total above ground biomass (3‐fold) and seed yield (inverse of biomass)
– Physiological• Leaf area index, photosynthetic rates, cellulose and lignin content of leaves and stems
OryzaSNP set
• Significant genetic variation for both morphological and physiological data
• Heritability• Photosynthesis
– M2O2 and Cypress• Highest photosynthetic rate
– Pokkali• Lowest photosynthetic rate, largest biomass
– Why PS not correlated to total biomass
Mutant populations: chemical and fast neutron
Amino Acid Transporters & Nitrogen regulation of gene expressionLishan ChenHui-Chu ChangAdriana Ortiz-LopezAaron SchmitzEkrem DundarMengjuan GuoXianan LiuVince StoergerChristian HermansSilvana Porco
Sucrose Transporters and gene regulationTzyy-Jen ChiouJade LuMatt VaughnWendy Ransom-HodgkinsGreg HarringtonAnshuman Kumar
RICEJan LeachJohn McKayHei Leung - IRRIBettina BroecklingCourtney Jahn
USDA-ARS, DOE, & NSF