JBEI Highlights May 2016

1. Phosphorylation of a NAC transcription factor
by ZmCCaMK regulates abscisic acid-induced
antioxidant defense in maize
Outcomes
• Functional analysis reveals that ZmNAC84 is essential for
ABA-induced antioxidant defense in a ZmCCaMK-
dependent manner.
• Moreover, overexpression of ZmNAC84 in tobacco can
improve drought tolerance, and alleviate drought-induced
oxidative damage of transgenic plants.
Zhu et al. (2016) ”Phosphorylation of a NAC transcription factor by ZmCCaMK regulates abscisic acid-
induced antioxidant defense in maize.” Plant Physiol. DOI: http://dx.doi.org/10.1104/pp.16.00168
Background
• Calcium/calmodulin-dependent protein kinase (CCaMK) has
been shown to play an important role in abscisic acid (ABA)-
induced antioxidant defense and enhance the tolerance of
plants to drought stress. CCaMK is also central to the
interactions with symbiotic microbes.
• However, its downstream molecular events are poorly
understood.
Significance
• These results define a mechanism for ZmCCaMK function in ABA-
induced antioxidant defense, where ABA-produced H2O2 first induces
expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK,
and subsequently the activated ZmCCaMK phosphorylates
ZmNAC84 at S113, thereby inducing antioxidant defense by
activating downstream genes.
Approach
• Here, we identify a NAC transcription factor, ZmNAC84,
in maize, which physically interacts with ZmCCaMK in
vitro and in vivo.
CCaMK is a key signalling component in plants.
CCaMK is involved in symbiosis with mycorrhizal fungi and
nitrogen fixing bacteria. It is also involved in the response to abiotic
stress. How the kinase can have these multiple function sis not
understood at the molecular level. Figure is from Oldroyd (2013,
Nat Rev Microbiol).
CCaMK phosphorylates the transcription factor
ZmNAC84. The transcription factor is known to directly
activate a number of stress response genes. Serine-113 is
phospphorylated by CCaMK and this activates the transcription
factor. When Ser-113 is mutated to Ala, the signal transduction
is inhibited. When Ser-113 is mutated to the phosphorylation
mimim Asd, the activation is constitutive.

2. Defective Pollen Wall 2 (DPW2) Encodes an
Acyl Transferase Required for Rice Male
Reproduction
Outcomes
• Compared with the wild type (WT), dpw2 anthers have
increased amounts of cutin and waxes and decreased levels
of lipidic and phenolic compounds. DPW2 encodes a
cytoplasmically localized BAHD acyltransferase.
• In vitro assays demonstrated that recombinant DPW2
specifically transfers hydroxycinnamic acid moieties, using ω-
hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-
CoAs as acyl donors.
Pollen development is impacted in
dpw2 mutants. The Pollen in dpw2-1
(E) and dpw2-2 (F) are collapsed
compared to the wild-type pollen (D).
Scale bars are 10 µm.
Xu et al. (2016) “Defective Pollen Wall 2 (DPW2) Encodes an Acyl Transferase
Required for Rice Male Reproduction.” Plant Physiol. DOI: 10.1104/pp.16.00095
Background
• Aliphatic and aromatic lipids are both essential structural components
of the plant cuticle, an important interface between the plant and
environment and critical for drought tolerance.
• Although crosslinks between aromatic and aliphatic or other moieties
are known to be associated with the formation of leaf cutin, root and
seed suberin, the contribution of aromatic lipids to the biosynthesis of
anther cuticles and pollen walls remains elusive.
Significance
• DPW2 plays a fundamental role in pollen development via the
biosynthesis of key components of the anther cuticle and pollen
wall.
DPW2 is a feroloyl: ω-
hydroxy fatty acid
transferase. Heterologously
expressed protein was
incubated with feruloyl-CoA
and ω-hydroxy palmitic acid.
The product ester was
identified by mass
spectrometry. The enzume can
also use sinapoyl-CoA but not
coumarouyl-CoA or caffeoyl-
CoA. C15 and C17 ω-hydroxy
fatty acids are also good
substrates, but longer or
shorter compounds are not.
Approach
• In this study, we characterized the rice male sterile
mutant, defective pollen wall 2 (dpw2), which showed an
abnormal anther cuticle, a defective pollen wall, and
complete male sterility.
DPW2 is specifically expressed in
anthers. Rice plants were
transformed with PPW2promoter-GUS
constructs and stained for expression
at different time points of
development.

3. Characterizing strain variation in engineered
E. coli using a multi-omics based workflow.
Brunk et al. (2016) “Characterizing strain variation in engineered E. coli using a
multi-omics based workflow”, Cell Systems, DOI: 10.1016/j.cels.2016.04.004
Background
• Understanding complex metabolic interactions in engineered
microbes is one of the major challenges in biofuel R&D.
• The development of omics technologies, such as metabolomics and
proteomics, and systems biology has dramatically enhanced our
ability to understand biological phenomena, but the interpretation of
large omics data into meaningful ‘knowledge’ is still very challenging.
Significance
• By integrating the strengths of synthetic biology (metabolic
engineering) and systems biology, we can drive the transition from
vision to conception of a designed working phenotype.
• We shared this workflow as an open-source tool in the form of
iPython notebooks, and it will allow anyone in microbial engineering
field to apply this workflow to their system
Approach
• We present a workflow that integrates various omics data and
genome-scale models (Fig 1)
• We obtained and analyzed large omics data set from eight
engineered strains producing three different biofuels (Fig 2) for the
workflow demonstration
Fig 1. Overall workflow presented in this work
Fig 2. Biofuels pathways used for this work
Outcomes
• From the workflow, we identified the roles of candidate genes,
pathways, and biochemical reactions in observed experimental
phenomena, which helps studying the effects of biofuel production in a
microbial host.
• We finally used this approach to facilitate the construction of a mutant
strain with improved productivity

4. A genetic screen identifies a requirement
for cysteine-rich–receptor-like kinases in
rice NH1 (OsNPR1)-mediated immunity
Outcomes
• We identify the snim1 mutant and demonstrate that cysteine-rich-receptor-
like kinase CRK6 and CRK10 complement the snim1 mutant.
• Silencing of CRK6 and CRK10 individually recreates the snim1 phenotype;
a crk10 frameshift mutant displays compromised immunity.
• Elevated NH1 levels enhance CRK10 expression while reduced NH1
levels decrease CRK10 expression.
• Rice TGA2.1 protein binds to the CRK10 promoter.
Chern et al. (2016) “A genetic screen identifies a requirement for cysteine-rich–receptor-like kinases in
rice NH1 (OsNPR1)-mediated immunity.” PLoS Genetics, DOI: 10.1371/journal.pgen.1006049
Background
• Systemic acquired resistance, mediated by Arabidopsis NPR1
and rice NH1, confers broad-spectrum immunity to diverse
pathogens.
• NPR1 and NH1 interact with TGA transcription factors to activate
downstream signaling; the signaling components downstream of
NPR1/NH1 and TGA proteins are poorly defined.
Significance
• These experiments demonstrate a requirement for CRKs in NH1-mediated
immunity and establish a molecular link between NH1 and induction of
CRK10 expression.
• CRKs are newly identified genes that can be used to engineer biofuel
crops for enhanced disease resistance and sustainability.
Approach
• Forward genetics approach to identify rice mutants (snim) that
block the NH1-mediated immunity.
• Comparative genome hybridization to identify the gene(s).
NH1ox snim1
Xoo induced
disease lesions
A
NH1ox snim1
BTH induced
lesion mimics
B
1) The snim1 mutant is
compromised in immunity
to Xoo and BTH-induced
necrotic lesion formation.
Two representative leaves each are
displayed for the NH1ox parent and
the snim1 mutant in (A) and (B).
Inoculation with Xoo was carried out
with the scissor-dip method. (A)
Xoo-induced, long water-soaked
disease lesions in the snim1 mutant.
(B) Lesion mimic necrotic spots. The
necrotic spots (marked with
arrowheads) developed one week
after application of 1 mM BTH.
BTH
CRK10 gene
Immunity
NH1
TGA
CRK10
Plasma
membrane
2) Working model:

5. Tissue-specific distribution of hemicelluloses
in six different sugarcane hybrids as related to
cell wall recalcitrance
Outcomes
• Evaluation of the digestibility of sugarcane polysaccharides by
commercial enzymes indicated that the cell wall recalcitrance varied
considerably along the internode regions and in the sugarcane
hybrids.
• Pith regions of the hybrids with high MLG and low-lignin contents
reached up to 85 % cellulose conversion after 72 h of hydrolysis,
without any pretreatment.
The six sugarcane hybrids differ substantially in their
cell wall composition. Here is shown mixed-linkage glucan
content. Lignin, cellulose crystallinity, hemicellulose conposiition,
etc., were also analyzed biochemically and by immunofluorescene
imaging (not shown). Example of immunofluorescence labeling of
xylan in interface (upper) and pith (lower) is shown to the right.
Costa et al. (2016) ”Tissue-specific distribution of hemicelluloses in six different sugarcane hybrids as related to cell
wall recalcitrance.” Biotechnology for Biofuels, DOI:10.1186/s13068-016-0513-2
Background
• Grasses are lignocellulosic materials useful to supply the billion-
tons annual requirement for renewable resources that aim to
produce transportation fuels and a variety of chemicals.
• However, the polysaccharides contained in grass cell walls are
present in a recalcitrant composite. Deconstruction of these cell
walls is still a challenge.
Significance
• The collective characteristics of the internode regions were related to
the varied recalcitrance found in the samples.
• Components such as lignin and GAX were critical for the increased
recalcitrance, but low cellulose crystallinity index, high MLG contents,
and highly substituted GAX contributed to the generation of a less
recalcitrant material.
Saccharification differs between the sugarcane hybrids.
Biomass samples were extracted to remove sucrose, milled and
digested with Ctec2 for 72 hrs at 45 C.
Approach
• Six different sugarcane hybrids were used as model
grasses to evaluate the tissue-specific distribution of
hemicelluloses and the role of these components in cell
wall recalcitrance.

6. From Sugars to Wheels: The Conversion
of Ethanol to 1,3-Butadiene over Metal-
Promoted Magnesia-Silicate Catalysts
Outcomes
• A process for the conversion of ethanol to 1,3-BD, which uses our catalyst
and accounts for separations and product logistics
• High 1,3-BD selectivity (60-75%) in a one-step process, with minimal
conversion of ethanol to butenes and ethylene, and no production of
diethyl ether and acetylene.
• Net carbon-negative process relative to petroleum (155% GHG reduction)
Shylesh et al. (2016) “From Sugars to Wheels: The Conversion of Ethanol to 1,3-Butadiene over Metal-
Promoted Magnesia-Silicate Catalysts”, ChemSusChem, doi: 10.1002/cssc.201600195
Background
• 1,3-Butadiene (1,3-BD) is a high-value chemical intermediate produced from
naphtha crackers and used mainly as a monomer for the production of
synthetic rubbers
• 1,3-BD prices have fluctuated in recent years, reaching $4740/ton
• 1,3-BD can be produced renewably by chemically converting bio-based
ethanol, but reported yields have been low (10-30% for MgO-SiO2 catalysts)
Significance
• One-step, low-carbon bio-based 1,3-BD production process capable of
competing with fossil-based pathways, particularly when market conditions
necessitate energy-intensive on-purpose 1,3-BD production
Simplified process flow diagram that depicts the
various steps in the conversion of ethanol to 1,3-BD.
Approach
• Development and investigation of a new catalyst and process for the
one-step conversion of ethanol to 1,3-BD
• Chemical process and life-cycle GHG modeling of pathways using
Au/MgO-SiO2 catalysts starting from corn grain-, sugarcane-, and
corn stover-derived ethanol
Life-cycle GHG Emissions per kg of 1,3-BD.

7. Fractional pretreatment of raw and calcium
oxalate-extracted agave bagasse using ionic
liquid and alkaline hydrogen peroxide
Outcomes
• IL pretreatment had more pronounced impact on cellulose crystallinty for
all samples and conditions studied
• Calcium oxalate extraction significantly improved saccharification yields
after IL pretreatment
• Calcium oxalate extraction significantly reduced saccharification yields
after AHP pretreatment
Perez-Pimienta et al. (2016) “Fractional pretreatment of raw and calcium oxalate-extracted agave bagasse using
ionic liquid and alkaline hydrogen peroxide.” Biomass and Bioenergy, DOI:10.1016/j.biombioe.2016.05.001.
Background
• Agave bagasse (AGB) is a promising bioenergy feedstock in
Mexico and Central America
• Previous studies have shown that the presence of calcium oxalate
can adversely impact pretreatment efficiency
Significance
• Highlights the importance of understanding of all components found in
plant cell walls and how they impact deconstruction
• Ionic liquids generated highest sugar yields observed
Impact of ionic liquid (IL) and alkaline hot peroxide (AHP)
pretreatments on (top) cellulose crystalllinity index and
(bottom) sugar yields from intact (AGB) and extracted
(EAB) agave bagasse.
Approach
• Compared alkaline hot peroxide (AHP) and ionic liquid (IL)
pretreatment before and after calcium oxalate extraction
• Monitored crystallinity index and sugar yields as a function of process
severity