Effect of Phenolic Glycosides on Agrobacterium tumefaciens virH Gene Induction and Plant Transformation

Article abstract of DOI:10.1021/np030281z

O-Aryl-D-glucoside (4-7) and D-xyloside (8-10) derivatives were synthesized and tested on Agrobacterium virH gene induction and plant transformation. α- or β-Glycosides enhanced vir activity at concentrations above 250 μM. The highest vir activity was obs

Full text of DOI:10.1021/np030281z

348
J . Nat. Prod. 2004, 67, 348-351
Effect of P h en olic Glycosid es on Agr oba cter iu m tu m efa cien s vir H Gen e
In d u ction a n d P la n t Tr a n sfor m a tion
Philippe J oubert,† Daniel Beaupe`re,‡ Anne Wadouachi,*,‡ Sophie Chateau,† Rajbir S. Sangwan,† and
Brigitte S. Sangwan-Norreel†
Laboratoire Androgene`se et Biotechnologie, Universite´ de Picardie J ules Verne, 33 Rue Saint-Leu,
80039 Amiens Cedex, France, and Laboratoire de Chimie Organique, Universite´ de Picardie J ules Verne, 33 Rue Saint-Leu,
80039 Amiens Cedex, France
Received J une 19, 2003
O-Aryl-D-glucoside (4-7) and D-xyloside (8-10) derivatives were synthesized and tested on Agrobacterium
virH gene induction and plant transformation. R- or â-Glycosides enhanced vir activity at concentrations
above 250 µΜ. The highest vir activity was observed with â-glucoside derivative 4 at 10 mM. A marked
difference between phenol glucoside derivative 4 and the corresponding free phenol on the growth of
transformants was observed. The regenerated transgenic tissues, after transformation on medium
containing acetosyringyl â-glucoside 4, grew at twice the rate of those on medium containing only free
acetosyringone (AS). Compound 4 was less toxic for tobacco explants compared to the corresponding free
phenol. However, the xyloside derivatives tested (8-10) were less effective for gene induction compared
with corresponding free phenols.
Several compounds are known to influence gene expres-
sion. A plant cell becomes susceptible to Agrobacterium
when it is wounded or is precultured on a phytohormone-
containing medium.1-6 Wounded plant cells produce an
abundance of low molecular weight phenolic compounds7
that are implicated in lignin biosynthesis or its degradation
and act as specific inducers of vir gene expression.2 Once
the vir genes are expressed, the encoded protein products
facilitate T-DNA transfer to the cytoplasm and nucleus,8,9
which is followed by integration of T-DNA into the plant
genome. The role of some phenolic compounds and of VirA-
VirG activation to obtain high levels of vir gene induction
and gene transfer have been reported.7,10-12 Recently we
have studied the effects of nine phenolic compounds on
Agrobacterium virulence gene induction, on Agrobacterium-
mediated gene transfer, and on both transient and stable
transformation rates on Petunia and tobacco. We confirmed
that virulence induction and transformation rates are
increased by the use of phenolic vir inducers bearing an
unsaturated lateral chain.13 It has also been demonstrated
that some monosaccharides such as D-glucose or D-galactose
can interact with constitutive gene products, and activation
of surface proteins leads to a synergestic vir gene induction
in the presence of phenolic compounds.14 Delmotte and co-
workers synthesized phenolic glycosides and tested their
effect on virulence of Agrobacterium.15,16 They showed that
O-aryl â-glycosides derived from L-fucoside, D-galactoside,
D-glucoside, and D-maltoside were much less effective than
the corresponding free phenols. Nevertheless, the â-mal-
toside derivative of syringic acid showed induction capacity
equivalent to that of the free acid at 500 µM and had
induction capacity higher than that of syringaldehyde
â-glucoside or â-galactoside derivatives.15 These facts
strongly suggest that the sugar residue may be a key
determinant of the virA-virG virulence gene induction. It
has been further shown that the toxicity of free phenolics
on Agrobacteria was considerably reduced by glycosyla-
tion.17
In this paper, we report the effect of new O-aryl-D-gluco-
and D-xylosides on Agrobacterium tumefaciens virH gene
induction and plant transformation.
Resu lts a n d Discu ssion
Previously, we have observed a higher activity of 4-(4-
hydroxy-3,5-dimethoxyphenyl)but-3-en-2-one (2) and 1-(4-
hydroxy-3,5-dimethoxyphenyl)-3-(4-hydroxyphenyl)prop-2-
enone (3) than acetosyringone (1), which was attributed
to a better stabilization due to the mesomeric forms
involved in the molecular mechanism of VirA activation
by phenols.13 Here, we have synthesized their glycosidic
derivatives following three methods of O-aryl â-glycosyla-
tion (Figure 1). The â-D-glucosides derivatives 4,18 5, and
6 were synthesized according to Michael reaction,19 the R-D-
glucoside 7 was prepared from a D-glucopyranosyl phenyl-
sulfoxide,20 and the â-D-xylosides 8-10 were prepared via
a stereoselective one-pot synthesis involving 1,2-cyclic
sulfite derivatives from unprotected xylose.21
Action of Glycosyla ted Com p ou n d s on vir H Gen e
In d u ction . At a concentration of 100 µM, acetosyringone
(1) and benzalacetone (2) have a high vir gene induction
(Figures 2 and 3). At the same concentration O-aryl
â-xyloside derivatives (8, 9, and 10), acetosyringyl R-glu-
coside (7), and â-glycosides (4, 5) (Figure 1) showed a strong
inhibition of vir gene induction (Figures 2 and 3). The three
acetosyringyl glycosides (4, 7, and 8), the â-xyloside deriva-
tives (9 and 10), and the â-glucoside (5) gave vir induction
equal to the control. This was probably due to the masked
phenol function in the glycosides.
We have previously shown17 that a â-glucosyl ester of
syringic acid has the same vir induction effect at a
concentration of 100 µM as syringic acid. The loss of an
active phenol function appeared to be the most important
parameter in the inhibition of vir induction observed by
the three forms of glycosidic phenolic compounds. We have
also shown that â-glycosides of phenolic compounds have
* To whom correspondence should be addressed. Tel: +33 03 22 82 75
27. Fax: +33 03 22 82 7560. E-mail: anne.wadouachi@u-picardie.fr.
† Laboratoire Androgene`se et Biotechnologie.
‡ Laboratoire de Chimie Organique.
10.1021/np030281z CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/28/2004

Agrobacterium virH Gene Induction
J ournal of Natural Products, 2004, Vol. 67, No. 3 349
F igu r e 3. Effect of various concentrations of phenolic glycosides 4, 7,
and 8 on gene virH induction compared to AS. Values represent the
average of 5 repetitions (mean ( SE). The â-galactosidase activity units
were calculated using the formula A420 × 1000/A600 and are described
as units per bacteria cells.22
F igu r e 1. Structure of tested phenolic compounds: 4-6, â-glucosides;
7, R-glucoside; 8-10, â-xyloside derivatives.
F igu r e 4. Effect of phenolic compounds (1-3) and aryl D-glycosides
(4-8) on tobacco transformation, based on number of calli and buds
regenerated per leaf explant on selection medium 4 weeks after co-
cultivation. Compounds were tested at 100 µM in the co-culture
medium. 30 to 40 leaf explants were used for each measurement
(n ) 3). Values are average ( SE. Probability showing differences
between media was >0.9. Newman-Keuls test classification of phenolic
compounds’ statistical effect is indicated by letters A, B, and C.
Delay et al.16 investigated the activities of endogenous
glycosylhydrolases of Agrobacterium tumefaciens A348
pSM243 cd and A348 pSM358 harboring virB::lacZ and
virE::lacZ fusion plasmids and showed that the naturally
occurring R- and â-glucosidases have high activities and
â-xylosidase a low activity. If similar glycosidase activities
in our A348 pSM219 strain are present, it may explain the
high vir activities observed at 2.5-15 mM glycosides
(Figure 3): these compounds could have been hydrolyzed
and thus liberate free acetosyringone 1 and sugar moieties.
It is known that a synergistic effect exists between some
monosaccharides such as glucose and simple phenolic
compounds.14 These free molecules can cooperate and
generate the high vir induction rates as observed in the
present study.
F igu r e 2. Effect of aryl D-glycoside (â-glucosides
4 and 5) and
â-xyloside (8, 9, and 10) derivatives on virH gene induction compared
with free phenols AS (1) and 2. All compounds were tested at 100 µM.
Values are averages (n ) 9) ( SE. Probability showing differences
between media was 1.00. The â-galactosidase activity units were
calculated using the formula A420 × 1000/A600 and are described as
units per bacteria cells.
no effect on vir gene induction at an effective concentration
of the free forms (100 µM) and a relatively strong action
at a high concentration (∼10 mM).17
Action of Differ en t Con cen tr a tion s of Glycosyla ted
P h en olic Com p ou n d s on vir H In d u ction . As shown in
Figure 3, when the concentration of acetosyringone (1) was
increased, the vir gene induction decreased after 250 µM
and a toxicity and null effect were observed at 5 mM.
Comparatively, the vir activity of the three tested glyco-
sides, R- and â-glucosides and â-xyloside, on vir genes was
very different at concentrations between 0.25 and 20 mM.
At 0.5 mM, the effect of glycosides derivatives 7 and 8 on
vir induction reached about 25% of maximal action of 1.
At 1 mM, the vir activity of 8 reached 50% of the max 1.
The â-glucoside 4 had a marked effect on vir induction at
10 mM. However, the maximal activity of these compounds
varied with their concentrations: about 2.5-5.0 mM for
â-xyloside (8) and R-glucoside (7) and about 10.0-15.0 mM
for â-glucoside (4).
Action of Glycosyla ted P h en olic Com p ou n d s on
P la n t Tr a n sfor m a tion . Glycosylation led to a complete
loss of vir induction property of the compounds at the
concentration of 100 µM in Figure 1. A similar result on
plant transformation with glycosides could be expected. We
tested some of these glycosides on Agrobacterium-mediated
genetic transformation of tobacco. Indeed, we observed
(Figure 4) that these glycosides significantly increased
transformation rates as compared with the control. Com-
paratively, these transformation rates were nearly the
same as those obtained with the corresponding free com-
pounds. Many secondary metabolites of plants are trans-
ported and stored in tissues as glycosides, and plants
contain the necessary enzymes to hydrolyze these glyco-
sides and then release the active metabolite forms.22 We
measured (data not shown) the natural activity of glyco-

350 J ournal of Natural Products, 2004, Vol. 67, No. 3
J oubert et al.
F igu r e 5. Effect of acetosyringyl â-D-glucoside (4) (9) and aceto-
syringone (1) (0) on the mortality of leaf explants. Values represent
average (2 repetitions of 40-45 explants) mortality (%) of leaf explants,
measured 7 days after bacterial co-culture.
F igu r e 7. Influence of acetosyringyl glycosides (4, 7, and 8) and free
AS (1) on transformation frequency of tobacco leaf explants. Trans-
formation frequency is expressed as number (0s0) and the size of calli/
explant. Sizes are represented by measures of callus growth (shaded)
(dry weight in mg) after 21 days in culture. Compounds were tested
at 100 µM. Weight values are averages of 310-574 putative trans-
formed calli.
concentrations. Whereas 2 mM 1 killed all leaf explants
(within 3 to 4 days), the explants survived for several weeks
at the same concentration of â-glucosylated (4). While at
500 µM, compound 4 had no significant effect on transfor-
mation rate (Figure 6), the same concentration of free 1
had a detrimental effect. Nevertheless, compound 4 at
500 µM had a qualitative positive effect on transformed
calli compared to the control, as they were greener and
more vigorous.
Effect of Glycosyla tion on Ca llu s F or m a tion a n d
P la n t Regen er a tion . Explants of tobacco leaves co-
cultivated on media with acetosyringyl â-glucoside (4)
formed a transgenic callus more quickly, and buds/plantlets
were developed more rapidly. In addition, the transformed
calli were bigger than those on medium with free 1. The
difference between the two media was further confirmed
from dry weight analysis of transformed calli. For example,
transformed calli obtained on medium with acetosyringyl
â-glucoside (4) weighed the same as those on the control
medium (without any compound) (Figure 7), but had twice
the dry weight of those obtained on other media. Thus, leaf
explant survival and transgenic callus development (and
consequently transformation efficiency of tobacco leaf ex-
plants) were strongly influenced by the phenolic com-
pounds.
Furthermore, to compare the effect of 1 and its glycoside
derivatives on Agrobacterium tumefaciens-mediated trans-
formation, these compounds were tested for vir gene
induction. Although the tested phenolic compounds had
poor effect on vir gene induction, these compounds had
significantly promoted transformation rates in tobacco leaf
explants (Figure 6). Two hypotheses could best explain the
results obtained by glycosylated phenolic compound on
transformation rates. First, plant glycosylhydrolases re-
lease in the culture medium free phenolic compounds from
the glycosides, may induce significant bacteria virulence,
and avoid a toxic effect on plant cells of a high initial
concentration of free phenolics. Second, the plant glyco-
sylhydrolases also release free monosaccharides such as
â-glucose, which can be directly assimilated by plant cells.
In conclusion, our investigations showed that some of
these new phenolic compounds were very effective in
Agrobacterium-mediated transformation. We observed that
acetosyringyl â-D-glucoside (4) promotes a better growth
of transformed calli and shoots as compared to acetosyrin-
gone and has a highly reduced toxicity at high concentra-
tions. Therefore, its quantitative and qualitative effect on
F igu r e 6. Effect of acetosyringyl â-D-glucoside (4) (9) and aceto-
syringone (1) (0) on plant transformation using increased phenolic
compound concentrations. Transformation frequency is expressed as
the number of calli/explant. Values are average (3 repetitions of 40-
45 explants) of callus on selection medium measured 4 weeks after
bacterial co-culture. Control explants were cultivated on a medium
without phenolic compound.
sylhydrolases in tobacco and observed a high activity of
â-glucosidase and â-xylosidase and low activities of R-glu-
cosidase. Therefore, during co-culture, â-glycosides could
be hydrolyzed and released as free active phenols. However,
we observed a notable increase in transformation rate in
the presence of acetosyringyl R-glucoside (7), with an
intermediate result between control and acetosyringyl
â-glucoside (4).
Delay et al. studied glycosylhydrolase activity in Agro-
bacterium strains and made a correlation between these
activities and the strain virulence.16 No glycosylhydrolase
activity was detected in culture supernatants of the various
strains they investigated. They suggested that there was
a specific transport through the outer membrane and the
periplasmic space.15,16 Our study may suggest that the host
plants also have the capacity to release free active vir
inducers from their glycosylated forms. This enzymatic
activity in plants could have an impact on the susceptibility
of tobacco to Agrobacterium-mediated transformation.
P r otective Effect of Glycosyla tion a ga in st Toxicity
of P h en olic Com p ou n d s. One of the general effects of
glycosylation is to protect biological systems against toxicity
of metabolites such as free phenolic compounds.15,16 We
observed a possible protection effect on Agrobacterium and
also in tobacco plants.
The study of different concentrations of 1 and the
corresponding acetosyringyl â-glucoside (4) on the number
of surviving leaf explants (after 7 days of culture) and on
transformation rates (after 4 weeks of culture on selection
medium) showed that efficiency of leaf protection by
glucosylation of 1 was high (Figures 5 and 6). At 500 µM,
1 showed a high toxicity, whereas compound 4 was less
toxic. Compound 4 induced high toxicity only at higher

Agrobacterium virH Gene Induction
J ournal of Natural Products, 2004, Vol. 67, No. 3 351
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NP030281Z