This study evaluated the use of Raman spectroscopy and partial least squares modeling to predict lignin syringyl-to-guaiacyl (S/G) ratios in Acacia and Eucalyptus plants. Pairwise comparisons found predicted S/G ratios from Raman spectra matched those measured using pyrolysis/molecular beam mass spectrometry. The Raman spectroscopy method provides a high-throughput way to evaluate lignin composition without destructive analytical techniques, showing potential for phenotypic screening of plant cell wall traits.

1. Expression of a Bacterial 3-dehydroshikimate dehydratase
(QsuB) Reduces Lignin Content and Improves Biomass
Saccharification Efficiency
Outcomes
• Arabidopsis plants expressing QsuB show drastic lignin reductions (~50%), enrichment of H-units, and reduced lignin DP.
• The biomass from the engineered Arabidopsis lines display a two-fold increase of saccharification efficiency.
Eudes, A., Noppadon, S., Baidoo, E., George, A., Liang, Y., Yang, F., Singh, S., Keasling, J., Simmons, B., Loque, D. (2014).
Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification
efficiency. Plant Biotechnology Journal.
Background
• Lignin is a polymer that
confers recalcitrance to plant
biomass.
• Novel approaches to reduce
lignin in bioenergy crops
without affecting biomass
yield are desired for
economical production of
second-generation biofuels
Approach
• Identify enzymes whose
expression in Arabidopsis
leads to lignin reduction.
Such enzymes will be
expressed in a tissue-specific
manner in bioenergy crops to
avoid the adverse effects of
lignin reduction.
Significance
• Expression of 3-dehydroshikimate dehydratase is a novel strategy to reduce lignin.
• Crops expressing QsuB under the control of tissue-specific promoters are being developed.
3-dehydroshikimate
shikimate
phenylalanine
p-coumaroyl-CoA
p-coumaroyl-shikimate
feruloyl-CoA
p-coumaryl alcohol
coniferyl alcohol
sinapyl alcohol
H-unit
G-unit
HCT shikimate
PCA
QsuB
phenylalanine
coniferaldehyde
sinapaldehydeS-unit
phosphoenol pyruvate
+
p-coumaraldehyde
erythrose-4-phosphate
1) The lignin pathway and expression of QsuB
PCA, protocatechuate
0
2
4
6
8
10
12
14
16
18
WT
qsuB‐1
qsuB‐3
qsuB‐6
qsuB‐7
3) Lignin content, composition (2D‐NMR), and structure (SEC) in qsuB transgenic lines
qsuB-1 WT qsuB-3 qsuB-6 qsuB-7
2) QsuB plants do not show drastic biomass reduction
Lignin (% Cell wall)
H: 3.8%
S: 20.0%
G: 76.2%
H: 27.2%
S: 30.2%
G: 42.6%
0
50
100
150
200
250
300
350
400
450 WT qsuB‐1
qsuB‐3 qsuB‐6
qsuB‐7
4) Biomass saccharification
Sugars (µg mg‐1biomass)
0.00
0.05
0.10
0.15
0.20
0.25
5 10 15 20 25
Elution time (min)
Normalized intensity
m > 22 kDa 22 kDa > m > 0.74 kDa
WT
qsuB-1
m < 0.74 kDa

2. OsSERK1 Regulates Rice Development but not
Immunity to Rice Bacterial Blight or Fungal Blast
Outcomes
• OsSERK1 silencing results in a reduced angle of the lamina
joint, but does not affect immunity.
• OsSERK1 overexpression complements brassinosteroid (BR)
signaling defects of OsSERK2-silencing plants.
Zuo, et al.,OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or
Magnaporthe oryzae. JIPB 56:1179-1192 (2014). DOI: 10.1111/jipb.12290
Background
• Somatic embryogenesis receptor
kinase (SERK) proteins play pivotal
roles in regulation of plant
development and immunity.
• The rice genome contains two SERK
genes, OsSERK1 and OsSERK2.
We previously demonstrated that
OsSERK2 is required for rice
immunity and for normal
development. Here we report the
function of OsSERK1.
Approach
• We use a transgenic approach to
silence or over-express OsSERK1
in rice and investigate its influence
on plant immunity and
development.
Significance
• Unlike OsSERK2, OsSERK1 contributes to plant development, but not immunity.
Kitaake
Xa21-Kit
Xa21kit-
OsSerk2Ri
#A814
Xa21kit-
OsSerk1ox
Xa21kit-
OsSerk2Ri
OsSerk1ox
Xa21-kit
Xa21kit
OsSerk2Ri
#A814
Xa21kit
OsSerk1Ri
Xa21kit
OsSerk2Ri
oSSerk1ox
• Transgenic rice over-expressing OsSERK1 are
semi-dwarf, but complement OsSERK2-silencing
effects on BR-signaling.
• OsSERK1-silencing results in reduced
lamina joint angles.
Xa21-Kit
Kittake
Xa21kit-
OsSerk2Ri
#A814
Xa21kit-
OsSerk1Ri
#1602
Xa21kit-
OsSerk1Ri
#1603
• OsSERK1 silencing does
not affect rice immunity to
Xanthomonas oryza pv
oryzae.

3. Background
• Root hairs are model single cells that develop by tip growth, a
process shared with pollen tubes, axons, and fungal hyphae.
• Prolyl-4-Hydroxylases (P4H) are enzymes that modify
proteins prior to glycosylation. Many plant proteins have
glycans attached to HyP and P4Hs are important for root hair
growth.
Approach
• To understand how different P4H proteins work together in
cell wall protein hydroxylation and root hair growth, we
analyzed P4H localization, protein-protein interactions, and
mutant phenotypes.
Outcomes
• Among of Prolyl-4-Hydroxylases, P4H5 was pivotal for root
hair tip growth.
• P4H5 had in vitro preferred specificity for hydroxylation of
extensin, a cell wall protein essential for root hair expansion.
• P4H2 and P4H13 are present in root hair cells and have
interchangeable functions but cannot replace P4H5.
• The P4Hs are shown to be targeted to the secretory pathway,
where P4H5 forms dimers with P4H2 and P4H13.
Complex Regulation of Prolyl-4-Hydroxylases
Impacts Root Hair Expansion
Velasquez, Silvia M., Ricardi, Martiniano M., Poulsen, Christian P., Oikawa, A., Dilokpimol, A., Halim, A., Mangano, S., Denita
Juarez, Silvina P., Marzol, E., Salgado Salter, Juan D., Dorosz, Javier G., Borassi, C., Möller, Svenning R., Buono, R., Ohsawa, Y.,
Matsuoka, K., Otegui, Marisa S., Scheller, Henrik V., Geshi, N., Petersen, Bent L., Iusem, Norberto D., & Estevez, José M. (2014).
"Complex Regulation of Prolyl-4-Hydroxylases Impacts Root Hair Expansion". Molecular Plant(0). doi, 10.1016/j.molp.2014.11.017
Significance
• The effect of protein-protein interactions on localization of
proteins in Golgi is an important phenomenon that must to be
understood for engineering of biomass traits in crops for
biofuel production
Deficient extensin proline
hydroxylation by P4H5 impacts
on root hair cell wall architecture.
P4H2 localization can shift by
associating with P4H5.
P4H-P4H protein interaction assessed by Bimolecular
Fluorescence Complementation

4. Background
• To optimize greenhouse and field breeding trials, we used a variety
of compositional assays to identify biomass compositional trends
between rice varieties and tissue types that translate successfully
from greenhouse to field grown plants.
Approach
• Researchers from GLBRC and JBEI participated in a study that
examined 16 compositional traits of stem and leaves from 20
diverse rice varieties
• An array of compositional assays were used to identify
compositional trends between varieties and tissue types across
growth conditions. Compositional assays include analysis of cell
wall polysaccharides (cellulose and hemicellulose), lignin, ash,
enzyme saccharification, hydroxyproline, and bulk density.
Outcomes/Impacts
• Rice variety had the least effect on traits measured in this study,
while growth environment (greenhouse versus field) had the
greatest effect.
• Yield of greenhouse-grown biomass was significantly higher than
field grown biomass, which indicates that studies utilizing
greenhouse grown biomass overestimate yield.
• The authors conclude that glucose yield of greenhouse-grown
plants is a good predictor of glucose yield in field grown plants and
could be used to optimize greenhouse and field breeding trials.
Cell Wall Composition and Bioenergy Potential of
Rice Straw Tissues Are Influenced by Environment,
Tissue Type, and Genotype
Tanger, P., Vega-Sánchez, M. E., Fleming, M., Tran, K., Singh, S., Abrahamson, J. B., Jahn, C. E., Santoro, N., Naredo, E. B.,
Baraoidan, M., Danku, J. M. C., Salt, D. E., McNally, K. L., Simmons, B. A., Ronald, P. C., Leung, H., Bush, D. R., McKay, J. K., &
Leach, J. E. (2015). "Cell Wall Composition and Bioenergy Potential of Rice Straw Tissues Are Influenced by Environment, Tissue
Type, and Genotype". BioEnergy Research, 1-18. doi, 10.1007/s12155-014-9573-y

5. Background
• Reduced cell wall recalcitrance and increased C6
monosaccharide content are desirable traits for future
biofuel crops
• New approaches are needed to achieve this goal with
minimal impact to phenotype
Approach
• Mixed-linkage glucans (MLGs), are comprised of glucose
monomers linked by both β-1,3 and β-1,4 bonds
• Previous studies that expressed in planta have shown
decreased recalcitrance, but can negatively impact plant
growth
• Use of senescence-associated promoters may yield higher
MLG content while maintaining desirable plant growth
phenotype
Outcomes
• 4x more glucose in the matrix cell wall fraction
• 42% increase in saccharification yields
Engineering Temporal Accumulation of a Low
Recalcitrance Polysaccharide leads to Increased
C6 Sugar Content in Plant Cell Walls
Vega-Sanchez, M. E., Loque, D., Lao, J., Catena, M., Verhertbruggen, Y., Herter, T., Yang, F., Harholt, J., Ebert, B., Baidoo, E. E.,
Keasling, J. D., Scheller, H. V., Heazlewood, J. L., & Ronald, P. C. (2015). "Engineering temporal accumulation of a low
recalcitrance polysaccharide leads to increased C6 sugar content in plant cell walls". Plant Biotechnol J. doi, 10.1111/pbi.12326
Significance
• Induction of cell wall polysaccharide biosynthesis in
senescing tissues offers a novel engineering alternative
to enhance cell wall properties of lignocellulosic biofuel
crops
Accumulation of MLG in Arabidopsis lines expressing OsCslF6 via the
senescence‐associated promoter SAG12 without causing growth
defects. (a) Morphology of mature rosettes (top) and whole plants
(bottom) of Col‐0 and three independent pSAG12::CslF6 lines. Scale
bars: 20 mm (rosettes), 15 mm (whole plants). (b) Cell wall
immunolabelling of senescing leaf cross sections using an anti‐MLG
monoclonal antibody; scale bar: 10 lm; m: mesophyll; sm: spongy
mesophyll; vb: vascular bundle. (c) MLG content of senesced rosettes

6. High-throughput Prediction of Acacia and
Eucalypt Lignin Syringyl/Guaiacyl Content using
FT-Raman Spectroscopy and Partial Least Squares Modeling
Summary/Impacts
• This study examined the usefulness of Raman model
to gauge the lignin S/G ratio in a large unknown data set. Specifically, we
demonstrated the application of a partial least squares model comprised of
Raman spectral data and lignin S/G ratios measured using pyrolysis/molecular
beam mass spectrometry (pyMBMS) for the prediction of S/G ratios in an
unknown data set.
• Pairwise comparisons within each data set were employed to assess statistical
differences between each biomass species.
• Predicted S/G ratios of Acacias and eucalypts were statistically similar with
those measured using pyMBMS.
• This study shows the power of using Raman spectroscopy to replace tedious,
destructive methods for the evaluation of the lignin S/G ratio of diverse plants.
Background
• HTP method to evaluate
phenotypic traits that correlate to
plant cell wall structure and
recalcitrance are needed.
• The ratio of lignin syringyl (S)-to-
guaiacyl (G) moieties has been
shown as an indirect means to
evaluate biomass recalcitrance.
• Mid-IR and Raman spectroscopy
models are shown to be capable
of predicting lignin S/G ratios.
However, more robust models to
accurately predict S/G are
needed.
Comparison of the first derivative + EMSC Raman spectra of low S/G Acacia
microbotrya (black spectrum, S/G=1.2) and higher S/G Eucalyptus globulus subspecies
maidenii (red spectrum, S/G=2.8), as measured by pyrolysis/molecular beam mass
spectrometry (A), Scores plot showing classification by plant genus (B). The x and y-
axes represent the second and third factors respectively. The blue squares represent
the genus Acacia, the red circles depict the genus Corymbia, and the green triangles
show the genus Eucalyptus
Lupoi, J. S., Healey, A., Singh, S., Sykes, R., Davis, M., Lee, D. J., Shepherd, M., Simmons, B. A., & Henry, R. J. (2015). "High‐Throughput Prediction of Acacia and
Eucalypt Lignin Syringyl/Guaiacyl Content Using FT‐Raman Spectroscopy and Partial Least Squares Modeling". BioEnergy Research. doi, 10.1007/s12155‐015‐9578‐1
Reference versus predicted lignin S/G ratios using FT-
Raman spectra and pyMBMS reference data
A B

7. Background
• Isoprenoids have been identified and used as
natural pharmaceuticals, fragrances, solvents,
and, more recently, advanced biofuels.
• Although isoprenoids are most commonly
found in plants, researchers have successfully
engineered both the eukaryotic and prokaryotic
isoprenoid biosynthetic pathways to produce
them in microorganisms at high yields.
Approach
• In this review, we highlighted the metabolic
engineering strategies that were used to
successfully produce valuable isoprenoid
compounds including antimalarial drug
artemisinin and various biofuel compounds in
microbial hosts.
• In addition, we presented a current outlook on
microbial isoprenoid production, with an eye
towards the many challenges that must be
addressed to achieve higher yields and
industrial-scale production.
Significance
• This review provides a good summary of the
synthetic biology efforts for microbial
isoprenoids production for drugs, biofuels, and
chemicals
Isoprenoid Drugs, Biofuels, and Chemicals-
Artemisinin, Farnesene, and Beyond
George, K. W., Alonso-Gutierrez, J., Keasling, J. D., & Lee, T. S. (2015). Isoprenoid Drugs, Biofuels, and
Chemicals-Artemisinin, Farnesene, and Beyond Adv Biochem Eng Biotechnol (2015 Jan 11 ed.): Springer
Berlin Heidelberg