Ethanol

added
E
RISC
ALCR
E
ALCR
E
GWD 400bp
GWD 400bp
GWD 400 bp
GWD 400 bp
Target GWD DNA or mRNA
35S alcR alcA GWD 400bp Intron
Dicer
Sag12p Intron a.
b.
GWD 400bp
ngineering the 7iming of Leaf Starch AccumuIation
Sean L. welse, Zachary 1. 1arou, Thomas D. Sharkey
Mlchlgan State Unlverslty
Department of 8lochemlstry and Molecular 8lology
RationaIe
Thls subpro[ect focuses on lncreaslng the yleld of easlly degraded polymers,
such as starch, ln leaves. The goal ls to make leaves a better feedstock for a
celluloslc ethanol fermentor. The ma[or advantages of the approach ln thls
subpro[ect ls (l) potentlal for hlgher yleld and (2) ease and completeness of
fermentatlon. The ma[or bottleneck ls reduced yleld ln most starch
accumulatlng mutants.
Dvercoming the YieId PenaIty
To attempt to overcome the yleld penalty we blocked starch degradatlon at a
speclc 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 used PNA lnterference (PNAl) agalnst the glucan water dlklnase
enzyme (GwD) and the phosphoglucan phosphatase. 8oth enzymes are
necessary for starch degradatlon ln leaves. These PNAl constructs wlll be
coupled to a senescence lnduced promoter (SAGl2) or an alcohol-lnduclble
promoter (Plg l). Constructs for the varlous promoters and lnverted repeats
were made by 8lo-8aslc ln Markham Ontarlo Canada (www.blobaslc.com).
Figure 1. Senescence InducibIe and AIcohoI InducibIe RNAi Constructs
a. 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 "SBCJEPQTJT and wlll be used to drlve PNAl targeted to the GwD enzyme or
the phosphoglucan phosphatase.
b. An alcohol-lnduclble promoter wlll also be used to drlve PNAl. 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 other specles besldes "SBCJEPQTJT.
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.
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Figure 2. Senescence Induced RNAi Starch AccumuIating pIants
Photographs were taken and leaves were stalned wlth |K| for starch ln the
mornlng when starch levels are the lowest. The GwD knock out plant has hlgh
starch levels but a slgnlcant growth penalty. The plant wlth the senescence
lnduced PNAl gene agalnst GwD does not exhlblt a decrease ln growth and
after 2 months contalns hlgh leaf starch levels comparable to the GwD KO llne.
First Arabidopsis then Corn
Figure 3. AIcohoI Induced RNAi Starch AccumuIation
wlld type, Prlmary transformant plants contalnlng the alcohol lnduclble PNAl
agalnst the glucan water dlklnase (GwD) or the phosphoglucan phosphatase,
both necessary for leaf starch degradatlon, and the correspondlng knock out
llnes were grown for 27 days. Alcohol lnduclble plants were then sprayed wlth
2% ethanol. Leaves were taken for starch determlnatlons ln the mornlng. The
alcohol lnduclble plants qulckly accumulate as much starch as the knock out
llne. |n the case of the Alc lnduclble PNAl agalnst the phosphoglucan
phosphatase the starch levels excede those of the knock out llne.
Starch AccumuIation in 7ransgenic PIants
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