Towards Tailor-Made Crops And Compounds
Many attempts have been made to convert plants into efficient factories.
The ability to modify genome sequences in plant cells is fundamental to
modern agriculture, but current methods - classical and moleculular - are extremely inefficient. To overcome the biotechnological barriers to
precise genome editing, we developed MemoGene technology for the creation of
commercially successful cultivars in leading high-value plants.
MemoGene (commercially developed by Danziger Innovations Ltd.) is a
viral-based tissue-culture-independent technology that bypasses traditional
genetic engineering for precise plant-genome modification in all plants.
It is based on highly efficient viral vectors for DNA delivery and
targeted endonucleases for nuclear and plastid genome manipulations:
site-specific genetic modifications can be applied to plants and cell
cultures, to meet the rapidly shifting trends in the areas of field and
vegetable crops, horticulture, woody crops, bio-fuels, etc.
Secondary metabolites determine color, flavor, fragrance and the
health-beneficial nutritional/pharmacological value of foods,
beverages, detergents, cosmetics and pharmaceutical products.
Tools allowing efficient metabolic engineering of these products have a
major impact on the almost limitless world bio-agriculture market.
Our characterization of novel plant-regulatory factors allowed us to
genetically engineer petunia flowers with ca. 10-fold higher scent
production/emission, and to transform naturally white flowering gypsophila,
sold worldwide, into a novel cut-flower crop with purple flowers (currently
being commercially developed by Danziger "Dan" Flower Farm). Our studs in
yeast of factors regulating production of the secondary metabolite
artemisinin, the most important antimalarial drug today, aim to boost
artemisinin production in host Artemisia annua plants, and to produce it in
tobacco and aspen. The drug will be produced at high levels in planta by
inducing the expression of five genes necessary for its synthesis in
specific tissues, cell types and intracellular compartments at specific
developmental stages. The development of yeast/plant-based approaches
(supported by Isaac Kaye award) for this drug's cost-effective production
will bring this much awaited remedy to developing nations.
Yeast and plants as factories for anti-malarial drugs
Malaria causes more than 500 million infections and approximately 3
million deaths annually, the vast majority of which are of children
under the age of 5 in developing countries. Artemisia annua is an annual
herb native to Asia (India, China, Vietnam), but has become naturalized
in America and northern Europe. In the early '70s, artemisinin was
identified as the principal compound in A. annua extract with
anti-malarial activity. In view of the low concentrations of artemisinin
produced naturally by plants, many attempts have been made to increase
its yield.
Our studies in yeast of factors regulating production of artemisinin,
the most important antimalarial drug today, aim to boost artemisinin
production in host A. annua plants, and to produce it in tobacco and
aspen. The drug will be produced at high levels in planta by inducing
the expression of five genes necessary for its synthesis in specific
tissues, cell types and intracellular compartments at specific
developmental stages. The development of yeast/plant-based approaches
(supported by Isaac Kaye award) for this drug's cost-effective
production will bring this much awaited remedy to developing nations.
Nontransgenic Genome Modification in Plant Cells
Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in
eukaryotic cells. ZFNs have been used for targeted mutagenesis in model
and crop species. In animal and human cells, transient ZFN expression is
often achieved by direct gene transfer into the target cells. Stable
transformation, however, is the preferred method for gene expression in
plant species, and ZFN-expressing transgenic plants have been used for
recovery of mutants that are likely to be classified as transgenic due
to the use of direct gene-transfer methods into the target cells. Here
we present an alternative, nontransgenic approach for ZFN delivery and
production of mutant plants using a novel Tobacco rattle virus
(TRV)-based expression system for indirect transient delivery of ZFNs
into a variety of tissues and cells of intact plants. TRV systemically
infected its hosts and virus ZFN-mediated targeted mutagenesis could be
clearly observed in newly developed infected tissues as measured by
activation of a mutated reporter transgene in tobacco (Nicotiana
tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to
move to developing buds and regenerating tissues enabled recovery of
mutated tobacco and petunia plants. Sequence analysis and transmission
of the mutations to the next generation confirmed the stability of the
ZFN-induced genetic changes. Because TRV is an RNA virus that can infect
a wide range of plant species, it provides a viable alternative to the
production of ZFN-mediated mutants while avoiding the use of direct
plant-transformation methods.
EOBII, a Gene Encoding a Flower-Specific Regulator of Phenylpropanoid
Volatiles' Biosynthesis in Petunia
Floral scent, which is determined by a complex mixture of low molecular
weight volatile molecules, plays a major role in the plant’s life cycle.
Phenylpropanoid volatiles are the main determinants of floral scent in
petunia (Petunia hybrida). A screen using virus-induced gene silencing
for regulators of scent production in petunia flowers yielded a novel
R2R3-MYB-like regulatory factor of phenylpropanoid volatile
biosynthesis, EMISSION OF BENZENOIDS II (EOBII). This factor was
localized to the nucleus and its expression was found to be flower
specific and temporally and spatially associated with scent
production/emission. Suppression of EOBII expression led to significant
reduction in the levels of volatiles accumulating in and emitted by
flowers, such as benzaldehyde, phenylethyl alcohol, benzylbenzoate, and
isoeugenol. Up/downregulation of EOBII affected transcript levels of
several biosynthetic floral scent-related genes encoding enzymes from
the phenylpropanoid pathway that are directly involved in the production
of these volatiles and enzymes from the shikimate pathway that determine
substrate availability. Due to its coordinated wide-ranging effect on
the production of floral volatiles, and its lack of effect on
anthocyanin production, a central regulatory role is proposed for EOBII
in the biosynthesis of phenylpropanoid volatiles.
Navigating the network of floral scent
production
Flower fragrance is a composite character determined by secondary
metabolites of diverse biosynthetic origin. Together with other
traits,
such as flower color, it is used by plants to lure pollinators
and seed
dispersers, thus ensuring plant survival. Research into the
regulatory
mechanisms leading to floral scent production/emission is still
in its
infancy and even less is known regarding flow within and cross-talk
between secondary metabolic pathways leading to floral scent
production. Using transgenic plants modified in anthocyanin
production,
we revealed an intriguing interrelationship between the branches
of the
phenylpropanoid pathway leading to the production of anthocyanins and
volatiles. Specifically, we recorded five- to sevenfold higher levels
of the volatile phenylpropanoids methyl benzoate and 2-hydroxymethyl
benzoate in flavanone 3-hydroxylase (F3h)-suppressed
carnation
flowers with dramatically reduced anthocyanin levels, as compared to
control non-transgenic flowers. Furthermore, overexpression in
petunia
flowers of the transcriptional regulator Pap1
(production of
anthocyanin pigment 1), which activates the phenylpropanoid pathway,
led to increases in both anthocyanin accumulation and volatile
phenylpropanoid emission. Using virus-induced gene silencing
(VIGS) for
large-scale identification of floral scent genes, we further
characterized metabolic flow within the pathway. The advantages
of VIGS
and of petunia as a model plant create a solid infrastructure for the
future isolation of regulatory factors involved in floral scent
production/emission. Knowledge gained from an understanding of
mechanisms leading to floral scent production/emission should provide
us with better insight into nature's way of ensuring evolutionary
success, as well as with advanced tools for the metabolic engineering
of fragrance.
Generation of phenylpropanoid pathway-derived volatiles in
transgenic plants: rose alcohol acetyltransferase produces
phenylethyl
acetate and benzyl acetate in petunia flowers
Esters are important contributors to the aroma of numerous
flowers and
fruits. Acetate esters such as geranyl acetate, phenylethyl
acetate and
benzyl acetate are generated as a result of the action of alcohol
acetyltransferases (AATs). Numerous homologous AATs from various
plants
have been characterized using in-vitro assays. To study the
function of
rose alcohol acetyltransferase (RhAAT) in planta, we generated
transgenic petunia plants expressing the rose gene under the
control of
a CaMV-35S promoter. Although the preferred substrate of RhAAT in
vitro
is geraniol, in transgenic petunia flowers, it used phenylethyl
alcohol
and benzyl alcohol to produce the corresponding acetate esters, not
generated by control flowers. The level of benzyl alcohol emitted by
the flowers of different transgenic lines was ca. three times higher
than that of phenylethyl alcohol, which corresponded to the ratio
between the respective products, i.e. ca. three times more benzyl
acetate than phenylethyl acetate. Feeding of transgenic petunia
tissues
with geraniol or octanol led to the production of their respective
acetates, suggesting the dependence of volatile production on
substrate
availability.
Flower proteome: changes in protein spectrum during the
advanced stages of rose petal development
Flowering is a unique and highly programmed process, but hardly
anything is known about the developmentally regulated proteome
changes
in petals. Here, we employed proteomic technologies to study petal
development in rose (Rosa hybrida). Using
two-dimensional polyacrylamide gel electrophoresis, we generated
stage-specific (closed bud, mature flower and flower at anthesis)
petal-protein maps with ca. 1,000 unique protein spots. Expression
analyses of all resolved protein spots revealed that almost 30%
of them
were stage-specific, with ca. 90 protein spots for each stage.
Most of
the proteins exhibited differential expression during petal
development, whereas only ca. 6% were constitutively expressed.
Eighty-two of the resolved proteins were identified by mass
spectrometry and annotated. Classification of the annotated proteins
into functional groups revealed energy, cell rescue, unknown function
(including novel sequences) and metabolism to be the largest classes,
together comprising ca. 90% of all identified proteins.
Interestingly,
a large number of stress-related proteins were identified in
developing
petals. Analyses of the expression patterns of annotated proteins and
their corresponding RNAs confirmed the importance of proteome
characterization.
Synthesis of the food flavoring methyl benzoate by genetically
engineered Saccharomyces cerevisiae
Current means of production for plant-derived aroma compounds include
chemical synthesis and extraction from plant material. Both
methods are
environmentally detrimental and relatively expensive: plant
material is
only seasonally available and only a small subset of the plant
biomass
produces the desired aroma compounds, while organic synthesis
inevitably involves waste byproducts with a negative ecological
impact.
Benzenoids are a class of plant metabolites that includes a
number of
aroma compounds. This research explores, for the first time, the
feasibility of producing benzenoids in yeast. We elucidated a method
for the production of the phenylpropanoid methyl benzoate in
Saccharomyces cerevisiae using benzoic acid as the
substrate,
via heterologous expression of Antirrhinum majus benzoic
acid
methyl transferase. Production was pH-dependent with a maximal
yield of
approximately 50 micrograms of methyl benzoate per liter of
culture per
hour, and with linear kinetics for at least 24 h. In addition, we
analyzed two alternative expression vectors for the production of
benzoic acid methyl transferase in S. cerevisiae: a
constitutive triosephosphate isomerase promoter-based system was
compared with a copper-inducible CUP1 promoter system. We found major
differences in the amounts of methyl benzoate produced by these
respective systems.
Expression and functional analyses of the plastid
lipid-associated protein CHRC suggest its role in chromoplastogenesis
and stress
Chromoplastogenesis during flower development and fruit ripening
involves the dramatic overaccumulation of carotenoids sequestered
into
structures containing lipids and proteins, termed PAPs (plastid
lipid-associated proteins). CHRC, a cucumber (Cucumis
sativus)
PAP, has been suggested to be transcriptionally activated in
carotenoid-accumulating flowers by gibberellic acid (GA). Mybys, a
MYB-like trans-activator identified in this study, may represent a
chromoplastogenesis-related factor: its expression is flower-specific
and parallels that of ChrC during flower development; moreover, as
revealed by stable ectopic and transient-expression assays, it
specifically trans-activates ChrC promoter
in flowers accumulating carotenoids and flavonoids. A detailed
dissection of ChrC promoter revealed a GA-responsive element,
gacCTCcaa, the mutation of which abolished ChrC activation by GA.
This
cis-element is different from the GARE motif and is involved in ChrC
activation, probably via negative regulation, similar to other
GA-responsive systems. The GA responsiveness and MYBYS floral
activation of the ChrC promoter do not overlap with respect to
cis-elements. To study the functionality of CHRC, which is
activated in
vegetative tissuessimilar to other PAPsby various biotic and abiotic
stresses, we employed a tomato plant system and generated
RNAi-transgenic lines with suppressed LeCHRC. Transgenic flowers
accumulated ca. 30% less carotenoids per unit protein than controls,
indicating an interrelationship between PAPs and flower-specific
carotenoid accumulation in chromoplasts. Moreover, the transgenic
LeCHRC-suppressed plants were significantly more susceptible to
Botrytis cinerea infection, suggesting CHRC's
involvement in
plant protection under stress conditions and supporting the general,
evolutionarily preserved role of PAPs.
CHRD, a plant member of the evolutionarily conserved YjgF
family, influences photosynthesis and
chromoplastogenesis
As noted above, studies on the carotenoid-overaccumulating structures
in chromoplasts led to the characterization of PAPs, involved in the
sequestration of hydrophobic compounds. Here we characterized the PAP
CHRD, which, based on sequence homology, belongs to a highly
conserved
group of proteins, YER057c/YjgF/UK114, involved in the regulation of
basic and vital cellular processes in bacteria, yeast and
animals. Two
nuclear genes were characterized in tomato plants: one (LeChrDc) is
constitutively expressed in various tissues and the other
(LeChrDi) is
induced by stress in leaves and is upregulated by developmental
cues in
floral tissues. Using RNAi and antisense approaches, we showed their
involvement in biologically significant processes such as
photosynthesis. The quantum yield of photosynthetic electron flow in
transgenic tomato leaves with suppressed LeChrDi/c expression was
30 to
50% of that in control, non-transgenic counterparts and was
ascribed to
lower PSI activity. Transgenic flowers with suppressed LeChrDi/c also
accumulated up to 30% less carotenoids per unit protein as
compared to
control plants, indicating an interrelationship between PAPs and
floral-specific carotenoid accumulation in chromoplasts. We suggest
that CHRD's role in the angiosperm reproductive unit may be a rather
recent evolutionary development; its original function may have
been to
protect the plant under stress conditions by preserving plastid
functionality.
