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£ngineering High Starch Corn Leaves

Sean L. welse, Zachary 1. 1arou, Thomas D. Sharkey
Mlchlgan State Unlverslty
Department of 8lochemlstry and Molecular 8lology
RationaIe
Thls subproìect focuses on lncreaslng the yleld of easlly degraded polymers,
such as starch, ln corn leaves. The goal ls to make corn leaves a better
feedstock for a celluloslc ethanol fermentor. The maìor advantages of the
approach ln thls subproìect ls (l) potentlal for hlgher yleld because energy ls
not used exportlng sugars from leaves to make cellulose and (2) ease and
completeness of fermentatlon. The maìor bottleneck ls reduced yleld ln most
starch accumulatlng mutants.
|n most plants transltory starch ls produced ln the chloroplasts of leaves
durlng the day and broken down durlng the nlght. |n the past l0 years our
lab and labs around the world have made slgnlñcant progress ln
understandlng the pathway of transltory starch degradatlon (Plg 2, welse et
al. 2004, Lu et al. 2006, Smlth et al. 2007). we can now make educated
declslons about where to modlfy thls pathway ln order to dlsrupt the starch
degradatlon process thereby lncreaslng the amount of starch ln the leaves.
Complete converslon to fuels
l00% cellulose 50% converslon
¬ 7.5 tons equlvalent
75% cellulose, 25% starch,
l0% reductlon ln total yleld
¬ (75%`0.5 + 25%`l)`0.9
¬ 8.4 tons equlvalent
l5 tons dry matter / ha
Figure 1. HypotheticaI Increase in ßiofueI YieId with a PartiaI conversion
of CeIIuIose to Starch
Chloroplast
Starch Sucrose
Cytosol
Maltodextrin
B-Amylase
Maltose Heterogl ycan Maltose
D
-E
n
z
y
m
e
Glucose Glucose
Glucanotransferase
GWD
Sex4
Starch Starch Sucrose
Cytosol
Maltodextrin
B-Amylase
Maltose Heterogl ycan Maltose
D
-E
n
z
y
m
e
Glucose
D
-E
n
z
y
m
e
D
-E
n
z
y
m
e
Glucose Glucose
Glucanotransferase Glucanotransferase
GWD
Sex4
Figure 2. Pathway of 7ransitory Starch Degradation
There are many potentlal targets to cause starch accumulatlon. we thlnk the
glucan water dlklnase (GwD) enzyme and the starch excess 4 (Sex4) proteln
are the best targets to provlde the greatest lncrease ln the yleld of starch
whlle mlnlmlzlng downstream chemlcal and slgnallng enects of maltose and
glucose accumulatlon.
W7 Starch £xcess
Figure 3. WiId type and a Starch AccumuIating Mutant of Arabidopsis
A maìor obstacle to overcome ls reduclng or ellmlnatlng the yleld penalty
observed ln most starch accumulatlng mutants. (ñgure from Nlttyla et al. 2004)
Dvercoming the YieId PenaIty
To attempt to overcome the yleld penalty we wlll block starch degradatlon at
a speclñc developmental tlme polnt late ln the llfe cycle of the plant. Larly ln
plant growth carbon, from starch, relnvested ln leaves causes exponentlal
growth. Once sumclent plant mass ls present to contaln the carbon,
metabollsm can be swltched to cause accumulatlon of starch.
To do thls we wlll use PNA lnterference (PNAl) agalnst the glucan water
dlklnase enzyme (GwD) and the Sex4 proteln. These PNAl constructs wlll be
coupled to a senescence lnduced promoter (SAGl2) or an alcohol lnduclble
promoter (Plg 4). Constructs for the varlous promoters and lnverted repeats
were made by 8lo-8aslc ln Markham Ontarlo Canada (www.blobaslc.com).
Ethanol
added
E
RISC
ALCR
E
ALCR
E
Sex 400bp
Sex 400bp
Sex 400 bp
Sex 400 bp
Target Sex mRNA
for degradation
35S alcR alcA Sex 400bp Intron
Dicer
Sag12 Sex 400bp Intron
Ethanol
added
E
RISC
ALCR
E
ALCR
E
ALCR
E
Sex 400bp
Sex 400bp
Sex 400bp
Sex 400bp
Sex 400 bp
Sex 400 bp
Target Sex mRNA
for degradation
35S alcR alcA Sex 400bp Intron
Dicer
Sag12 Sex 400bp Intron Sag12 Sex 400bp Intron
Figure 4. Senescence InducibIe and AIcohoI InducibIe RNAi Constructs
The promoter of the SAGl2 gene, a cystelne protease, dlscovered by ¥oo-Sun
Noh and Plck Amaslno (l999) ls actlve only at the tlme of senescence ln
Arabldopsls and wlll be used to drlve PNAl targeted to the GwD enzyme or
the Sex4 proteln. An Alcohol lnduclble promoter wlll also be used to drlve
PNAl of GwD and Sex4. The advantage of the alcohol lnduclble promoter ls
that the developmental tlmlng of starch accumulatlon can be preclsely
controlled and thls promoter wlll work ln corn.
RNAi ls a mechanlsm that lnhlblts gene expresslon at translatlon or by
hlnderlng transcrlptlon. Double stranded PNA ls generated by constructlng
an lnverted repeat wlth 400 bp of DNA ldentlcal to target gene ln the forward
orlentatlon and 400 bp of DNA ln the reverse orlentatlon separated by an
lntron. The double stranded PNA ls cleaved lnto short double stranded PNA
molecules by the enzyme dlcer. Slnge stands of PNA are then lncorporated ln
the PNA-lnduced sllenclng complex (P|SC). The P|SC targets the
compllmentary mPNA or DNA to prevent transcrlptlon and/or translatlon.
First Arabidopsis then Corn
Uslng Arabldopsls ñrst presents several advantages
l. The starch metabollc pathway ls well characterlzed
2. Knock outs of GwD and Sex4 have been characterlzed (¥u et al 200l, Zeeman et al l998)
3. The senescence lnduclble promoter, SAGl2p, ls characterlzed (Noh and Amaslno l999)
4. A fast llfe cycle wlll glve us and early "proof of conceptª
5. Arabldopsls ls very amenable to genetlc manlpulatlon allowlng us to ñne tune
what we learn from lnltlal experlments.
Concurrent wlth the Arabldopsls work, PNAl constructs targeted to the corn
homologue of GwD and Sex4 have been made. These wlll be placed behlnd the
alcohol lnduclble promoter, and behlnd the constltutlve promoter of Ublqultln.
The transformatlons are belng carrled out by Dr. Heldl Kaeppler at the Unlverslty
of wlsconsln-Madlson.