Overexpression of four previously uncharacterized poplar transcription factors produced enhanced saccharification of plant biomass in poplar. Two transcription factors, PtxtERF123 and PtxtZHD14, significantly improved both glucan and xylan release from plant cell walls. These transcription factors regulate groups of genes involved in xylem cell wall biosynthesis and are potential targets for improving biomass digestibility in poplar and other bioenergy feedstocks.

1. Plant Biosystems Design Research Roadmap 1.0
Background
• Various plants have been genetically improved mostly through
breeding, along with limited modification via genetic engineering,
yet they are still not able to meet the ever-increasing needs, in
terms of both quantity and quality, resulting from the rapid increase
in world population and expected standards of living.
Approach
• This review highlights how plant biosystems design seeks to
accelerate plant genetic improvement using genome editing and
genetic circuit engineering or create novel plant systems through
de novo synthesis of plant genomes.
Outcomes and Impacts
• We highlight current challenges, future opportunities, and research
priorities, along with a framework for international collaboration,
towards rapid advancement of this emerging interdisciplinary area
of research.
• We discuss the importance of social responsibility in utilizing plant
biosystems design and suggest strategies for improving public
perception, trust, and acceptance.
Yang et al. (2020) BioDesign Research, doi: 10.34133/2020/8051764

2. Genome sequencing adds to the synthetic biology
“parts list”
Background
• Ochratoxin (OTA) is a secondary metabolite produced by
Aspergillus species
• This study focuses on the characterization of a novel cyclase
involved in tailoring the polyketide synthase backbone of OTA
Approach
• JBEI’s Whole Aspergillus Genus Genome Sequencing project aims
to sequence one strain from every Aspergillus species (~300) in
order to uncover new biomass deconstruction enzymes and
biochemical pathways
Outcomes and Impacts
• Comparative genomic analysis identified an additional gene
encoding a cyclase putatively needed for the biosynthesis of
ochratoxin, a polyketide
• Polyketides and other secondary metabolites have potential as
enzymes to produce bioproducts or biofuel molecules
• Understanding the biosynthetic pathway, including tailoring
enzymes such as the cyclase uncovered here, adds to the
synthetic biology “parts list” needed for design and synthetis of
biofuel and bioproduct molecules
https://doi.org/10.3389/fmicb.2020.581309
The ochratoxin biosynthetic gene cluster is found in several
species of Aspergillus. Comparative genomic analysis of these
clusters revealed the presence of an novel polyketide cyclase
encoding gene.

3. The TaCslA12 gene expressed in the wheat grain
endosperm synthesizes wheat-like mannan when
expressed in yeast and Arabidopsis
Background
• Mannan is a class of cell wall polysaccharides ubiquitous in the
plant kingdom. Mannan structure and properties vary according to
species and organ.
• The cell walls of cereal grains have been extensively studied due
to their role in cereal processing and to their beneficial effect as
dietary fiber.
• Recently, we showed that mannan in wheat (Triticum
aestivum) grain endosperm has a linear structure of β-1,4-linked
mannose residues.
Approach
• The aim of this work was to study the biosynthesis and function of
wheat grain mannan.
• We showed that mannan is deposited in the endosperm early
during grain development, and we identified candidate mannan
biosynthetic genes expressed in the endosperm.
• The mannan synthase genes were expressed in heterologous
systems to study their activity.
Outcomes and Impacts
• The endosperm-specific TaCslA12 gene expressed in Pichia
pastoris and in an Arabidopsis thaliana mutant depleted in
glucomannan led to the production of wheat-like linear mannan
lacking glucose residues and with moderate acetylation.
• Different mannan synthases produce pure mannans or
glucomannans. The product is not determined by substrate
availability as previously thought.
• Mannan synthases may be useful to increase the C6/C5 sugar
ratio in bioenergy crops.
Verhertbruggen et al. (2021) Plant Science, doi: 10.1016/j.plantsci.2020.110693
Subcellular localization in tobacco leaves show that wheat
CslA12 colocalizes with the Golgi marker, as expected for an
enzyme involved in hemicellulose biosynthesis
(A) Wheat TaCSLA12 is active when expressed in Pichia.
AkCSLA3 is a positive control from konjac. (B) Linkage analysis
shows that the mannan synthesized by the wheat enzyme is
pure mannan, unlike the glucomannan synthesized by the
konjac homolog. Expression in Arabidopsis confirmed these
results (not shown)

4. Cell Wall β-1,4-galactan Regulated by BPC1/BPC2-
GALS1 Module Aggravates Salt Sensitivity
in Arabidopsis thaliana
Background
• Salinity severely reduces plant growth and limits agricultural
productivity.
• Dynamic changes and rearrangement of the plant cell wall is an
important response to salt stress, but relatively little is known
about the biological importance of specific cell wall components in
the response.
Approach
• Salt stress induced the accumulation of β-1,4-galactan in root cell
walls by up-regulating the expression of GALACTAN SYNTHASE
1 (GALS1), which encodes β-1,4-galactan synthase.
• The role of GALS1 in salt response was investigated in mutants
and overexpressors.
• A yeast-1-hybrid screen was used to identify transcription factors
controlling GALS1 expression in response to salt stress.
Outcomes and Impacts
• Exogenous application of D-galactose causes an increase in β-
1,4-galactan levels in plants, especially in GALS1 overexpressors,
which correlated with the aggravated salt hypersensitivity.
• Further analysis revealed that the BPC1 and BPC2 transcription
factors affected the salt tolerance by repressing GALS1
expression and β-1,4-galactan accumulation.
• Our results reveal a new regulatory mechanism where β-1,4-
galactan regulated by BPC1/BPC2-GALS1 module aggravates salt
sensitivity in Arabidopsis thaliana.
• The study may help to identify tools to increase salt tolerance of
bioenergy crops, which is especially important on some marginal
lands.
Yan et al. (2020) Molecular Plant, doi: 10.1016/j.molp.2020.11.023
GALS1 expression is increased in response to NaCl treatment.
NaCl suppresses root growth in wild type. Mutants gals1
respond much less, whereas GALS1 overexpressors respond
more strongly
Galactan in the root affects
cellulose content as shown with a
fluorescence assay. Salt treatment
reduces cellulose synthesis, but
less so in gals1 mutants and more
so in GALS1 overexpressors
Schematic model of the interaction between BPC1/2, GALS1
and cellulose synthase. Decreased cellulose synthesis during
salt stress is likely the direct cause of decreased root growth.

5. Identifying transcription factors that reduce wood
recalcitrance and improve enzymatic degradation of
xylem cell wall in Populus
Background
• Fast growing Populus spp. (poplar) trees are promising sources
of woody biomass for the production of biofuels and bioproducts
• The biomass is highly recalcitrant, and efforts to improve
saccharification efficiency by altering the activity of single
metabolic enzymes has had limited success.
Approach
• Here, in work led by Hokkaido University researchers, we
overexpressed 33 poplar transcription factors (TFs, proteins
which control the expression of groups of genes) in hybrid aspen
(Populus tremula × Populus tremuloides) that were predicted to
have a role in regulating xylem cell wall biosynthesis.
Outcomes and Impacts
• Of the 33, four previously uncharacterized transcription factors
produced enhanced saccharification in the initial screen of
material grown in tissue culture.
• Lines of the two best performing TFs (PtxtERF123 and
PtxtZHD14) were grown to maturity in the greenhouse.
• Lines showed both increased glucan release as well as xylose
from xylan release.
• These TFs are widely distributed in angiosperms, and so are a
breeding target in other woody biomass species, such as willow
and eucalyptus
Hori et al. (2020) Sci Reports, doi: 10.1038/s41598-020-78781-6
Fig. 1: Gene expression and saccharification data from
the transformed poplar generated in this study