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Plant Physiol. (1998) 116: 1133-1144
Genetic Manipulation of Condensed Tannins in Higher
Plants1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We have
produced and analyzed transgenic birdsfoot trefoil (Lotus
corniculatus L.) plants harboring antisense dihydroflavonol reductase (AS-DFR) sequences. In initial experiments the effect of
introducing three different antisense Antirrhinum majus
L. DFR constructs into a single recipient genotype (S50) was assessed. There were no obvious effects on plant biomass, but levels of condensed
tannins showed a statistical reduction in leaf, stem, and root tissues
of some of the antisense lines. Transformation events were also found,
which resulted in increased levels of condensed tannins. In subsequent
experiments a detailed study of AS-DFR phenotypes was carried out in
genotype S33 using pMAJ2 (an antisense construct comprising the 5
half
of the A. majus cDNA). In this case, reduced tannin
levels were found in leaf and stem tissues and in juvenile shoot
tissues. Analysis of soluble flavonoids and isoflavonoids in tannin
down-regulated shoot tissues indicated few obvious default products.
When two S33 AS-DFR lines were outcrossed, there was an
underrepresentation of transgene sequences in progeny plants and no
examples of inheritance of an antisense phenotype were observed. To our
knowledge, this is the first report of the genetic manipulation of
condensed tannin biosynthesis in higher plants.
Condensed tannins are polymeric flavonoid molecules that are found
in a range of higher plant species. These secondary compounds are of
particular value in forage crops because their presence in the foliage
of herbage legumes at levels of up to 3 to 4% dry weight is regarded
as being a beneficial agronomic trait. The accumulation of tannins in
shoot tissues is beneficial for grazing livestock from the perspective
of the prevention of pasture bloat (Waghorn et al., 1990 The focus of this paper, however, is to explore the possibility of
decreasing the levels of condensed tannins in crop species using
antisense technology. Lowering the amounts of condensed tannins in
animal feedstuff is important both for ruminant and nonruminant
(monogastric) livestock. At high levels (>3-4% dry weight) tannins
in forage and fodder are deleterious for use with ruminants, and such
levels reduce both palatability and nutritive value (for review, see
Mueller-Harvey and McAllan, 1992 The genetic control of tannin biosynthesis has been elucidated in
recent years in the studies of tannin mutants in barley (Jende-Strid,
1991 DFR is a particularly attractive target with reference to tannin
biosynthesis. The gene corresponding to this enzyme activity has been
cloned from a number of plant species, including maize, barley,
Gerbera sp., and Antirrhinum majus L. DFR is the
most distal gene in the tannin biosynthetic pathway that has been
cloned and identified to date, and it shows high levels of sequence
homology when comparisons are made between clones isolated from a range of higher plant species (Charrier et al., 1995
In previous work (Carron et al., 1994 Source Material
Growth of Plants
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
); as these
highly hydroxylated polymers collapse, protein foams within the rumen
in a dose-dependent manner (Tanner et al., 1995). Another advantage of
condensed tannins is that they increase the levels of bypass protein,
passing through the rumen of grazing animals (Barry and Duncan, 1984),
and therefore the accumulation of these end products can increase
livestock productivity in an environmentally sensitive manner. Because
the major European forage legumes white clover and lucerne do not contain tannins in leaf tissues, strategic work aims at transferring the foliar tannin trait from birdsfoot trefoil (Lotus
corniculatus L.) to these major pasture crop species (Morris and
Robbins, 1997).
). This phenomenon mitigates against
the effective use of a number of potential forage crop species that
have good levels of primary productivity, often in nonoptimal
environments, but are of limited nutritive value because of
accumulation of high levels of antinutritive secondary compounds. For
monogastric species such as pigs and chickens, tannins in feedseeds are
generally detrimental at both high and low levels (Griffiths, 1989).
Methods of reducing levels of condensed tannins in tanniferous forages,
fodders, and feedseeds are therefore of considerable interest (Bavage
et al., 1997a; Morris and Robbins, 1997).
) and Lotus japonicus (Regel) Larsen (M.P. Robbins and
T.E. Evans, unpublished data). One particular feature of interest is
that the initial enzymatic steps of the anthocyanin pathway are also
common with condensed tannin biosynthesis. Therefore, the cloning of
genes involved in anthocyanin and flavonoid biosynthesis (for review,
see Forkmann, 1993) has direct significance for workers studying the
agronomically important pathways that lead to tannins and related
polyphenolics.
). Therefore, if one
wishes to make specific modifications to the tannin biosynthetic pathway, manipulative strategies aimed at DFR appear to be of potential
value. The position of DFR with respect to other enzymes and related
secondary pathways is shown in Figure 1.
View larger version (43K):
[in a new window]
Figure 1.
Interrelationship of different classes of
flavonoids in higher plants. Letters beside arrows indicate enzymes
catalyzing conversions between classes. CHI, Chalcone isomerase; F3OH,
flavanone 3-hydroxylase; FDR, flavan 3,4-diol reductase; FS, flavone
synthase; FlS, flavonol synthase; AS, anthocyanin synthase; 3GT,
3-glycosyl transferase; CE, condensing enzyme; and PE, polymerizing
enzyme. Additional enzymatic steps exist in this pathway that alter
B-ring hydroxylation and the stereospecificity of monomer units, but
these have been omitted for the sake of clarity.
) we reported the introduction of
three different antisense A. majus DFR constructs into three
recipient Lotus corniculatus genotypes. In genotypes that accumulated low (S33) or medium (S50) levels of tannin, antisense root
culture lines were produced with decreased levels of condensed tannin
accumulation. In genotype S33 three lines were found, RFD8, RFD19, and
RFD38, in which 80% reductions of tannin accumulation were noted
relative to controls. In genotype S50 low tannin lines were also
produced with reductions of up to 70%, RFD40 and RFD28. Although root
cultures have been subjected to study, no results have been previously
presented regarding a detailed analysis of AS-DFR plants. In this paper
we report the analysis of cotransformed S50 and S33 plants harboring
these AS-DFR constructs. Condensed tannin levels in leaf, stem, and
root tissues under a variety of environmental growth conditions have
been determined, and in addition to PCR and RT-PCR analysis we report,
to our knowledge, the first preliminary analysis of default
products in tissues of AS-DFR plants. Progeny were also produced by
outcrossing control and antisense lines, and we report data regarding
inheritance of transgene sequences.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
). After 4 to 6 weeks of growth in the dark, plates were transferred to light (80 µmol m
2 s1) and
shoots started to appear after 1 to 2 weeks. After 1 month in the
light, shoots, together with basal nodes, were excised and transferred
to 50-mL Sterilin tubes (Bibby Sterilin Ltd., Stones, Staffordshire,
UK) containing solid Murashige and Skoog medium (Imperial Laboratories,
Ltd.) supplemented with 1% agar and 3% Suc and then allowed to root.
In genotype S33 control transformed lines (C26, C28, and C32) and
vector controls (C33/3, C33/5, and C33/10) regenerated shoots poorly
when grown on solid medium. These lines were therefore cultured in
liquid B5 basal medium plus 3% Suc in the light, and the
resultant shoots were then detached and transferred to solid
Murashige and Skoog medium for rooting.
View this table:
Table I.
Control and RFD lines in the S50 genotype analyzed
in this study and a summary of PCR analysis
Details of control and antisense constructs (pROK2, pMAJ1, pMAJ2, and
pMAJ3) and derived amplification products are shown in Figure 2.
Amplification products with nptII primers confirm the
transfer of the selectable marker from binary vector T-DNA to L. corniculatus genomic DNA. CaMV-nos amplification products confirm
the presence of promoter and terminator sequences and produce
characteristically sized products from each of the four constructs in
this study. +, Product noted; 
, no product after amplification.
View this table:
Table II.
Control and RFD lines in the S33 genotype analyzed
in this study and a summary of PCR analysis
Details of control and antisense constructs (pROK2, pMAJ1, pMAJ2, and
pMAJ3) and derived amplification products are shown in Figure 2.
Amplification products with nptII primers confirm the
transfer of the selectable marker from binary vector T-DNA to L. corniculatus genomic DNA. CaMV-nos amplification products confirm
the presence of promoter and terminator sequences and produce
characteristically sized products from each of the four constructs in
this study. +, Product noted; , no product after amplification.
2 s1 light from
"white" fluorescent tubes (General Electric). Plants were gradually
hardened off in plastic bags and then nodulated with
Rhizobium loti Lc 3011 (Institute of Grassland and
Environmental Research nomenclature) over a 3-week period. Replicate,
whole-shoot cuttings were taken from 10-week-old plants and rooted in
sand as described above. Established plants were grown under
containment-B greenhouse conditions (June-July, 1995) at an
average day temperature of 25 ± 4°C and a minimum night
temperature of 15°C, and leaf and stem tissues were harvested and
analyzed for condensed tannins after 10 weeks of growth. Rooted and
nodulated cuttings were also transferred and grown in vermiculite
medium in 7.5-cm pots in a growth room (20°C, 18-h day, 530 µmol
m2 s1 fluorescent
light). Replicated plants were harvested after 6 weeks (harvest 1) or
cut back to 5 cm, grown for another 6 weeks, and harvested (harvest 2).
Chemical Analysis of Plant Tissues
Condensed Tannins
Plant tissues were harvested, freeze dried, and powdered before the measurement of condensed tannins. Juvenile shoot tissues were harvested directly, whereas stem and leaf tissues from older plants were subjected to botanical separation after freeze drying. Roots from vermiculite-grown plants were washed vigorously and then used for tannin analysis; however, it was not possible to make accurate determinations of tannin levels in roots from soil-grown plants. All tannin determinations were carried out in duplicate on 30 to 50 mg dry weight of freeze-dried powdered tissues using the butanol/HCl-hydrolysis method of Terrill et al. (1992), and levels were
determined by summing values derived from residue and aqueous
hydrolysates. Statistical analysis of tannin levels in transgenic
plants was made by comparing levels of condensed tannins in individual
AS-DFR lines with those in corresponding controls derived from
the parental genotype (S33 or S50), wild-type Agrobacterium
rhizogenes transformants, and vector transformants of these
genotypes (Tables I and II).
Leaf Flavonoids
Freeze-dried powder samples of leaves (100 mg) were extracted three times with 25 mL of 80% MeOH for 2 h with shaking at 20°C. After filtration and MeOH removal under a vacuum at 50°C, the remaining aqueous extracts were concentrated on an activated C18 Sep-Pak cartridge (500 mg) (Waters) and bound phenolic compounds were eluted with 4 mL of 100% MeOH. Flavonoid profiles were obtained by gradient HPLC onto a µNovapak C18 RCM cartridge (Waters) with a linear MeOH:acetic acid (5%) gradient from 0 to 100% MeOH in 50 min at a flow rate of 2 mL min1. Eluting peaks were
monitored with a diode array detector (model 990, Waters) at 350 nm,
and spectra were recorded between 240 and 400 nm to obtain their
UV/visible absorption spectra. Acid, alkali, and enzymic hydrolysis was
carried out on 1-mL extracts with 1 m HCl at 100°C for
1 h, with 0.5 m NaOH at 50°C for 30 min, or with 100 units of 
-glucosidase (sweet almond), pH 5.0, at 37°C for 2 h. Hydrolyzed samples were diluted to 5 mL, adjusted to pH 6.0 with
NaOH or HCl, and concentrated on an activated C18 Sep-Pak cartridge, and the flavonoids were eluted with 4 mL of MeOH.
Flavonoid glycosides and aglycones were identified by their retention
times and UV/visible spectra compared with authentic standards obtained
from Sigma-Aldrich and Apin Chemicals, Ltd. (Oxford, UK).
PCR Analysis of Transgenic Lines
In this study plants were produced that harbored a range of transgene constructs (Table I). PCR from genomic DNA derived from leaf tissues was employed using well-characterized PCR primers (see Fig. 2). Genomic DNA was extracted from leaf tissues of control and antisense plants using the method of Dellaporta et al. (1983), as modified by
Robbins et al. (1991). Amplification of nptII sequences used
a primer pair (nptIIa: 5 GAG GCT ATT CGG CTA TGA CGT; and nptIIb: 3 ATC GGG AGC GGC GAT ACC GTA), which amplified a
700-bp sequence from the nptII gene in the T-DNA of
constructs derived from pROK2. Antisense sequences were amplified using
CaMV-specific (5 CTG ATA TCT CCA CTG AC) and nos-specific (3 TCA TCG
CAA GAC CGG C) primers, which amplified characteristic fragments from different antisense constructs. Amplified fragment sizes were 200 bp
for pROK2, 500 bp for pMAJ1, 1 kb for pMAJ2, and 900 bp for pMAJ3.
Similar methods were used when analyzing for the presence of the
transgene in T1 progeny derived from original
transgenic lines.
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Transcript Analysis
RNA was extracted from Lotus corniculatus shoot and root tissues using the method of Ougham and Davies (1990). Plant
material was harvested, frozen in liquid nitrogen, and stored at
70°C before extraction. Nucleic acid was quantified on a UV
spectrophotometer (model PU 8720, Pye Unicam Ltd., Cambridge, UK) and
10-µg samples were run on 1% formaldehyde gels. RNA was blotted onto
Hybond N membranes (Amersham) and probed using a labeled L. corniculatus fragment, pLCDFRA (accession no. X97576), which
contains 683 bp of an endogenous DFR gene amplified from genomic DNA
(Bavage and Robbins, 1994; Bavage et al., 1997b).
.
Total RNA was extracted from leaf tissues using the
cetyl-trimethyl-ammonium bromide method of Chang et al. (1993), and
mRNA was isolated using Dynal oligo(dT) Dynabeads (Dynal AS, Oslo,
Norway). Then, first-strand cDNA was synthesized from 0.5 µg of mRNA
using Moloney murine leukemia virus RT and oligo(dT) primer
(Stratagene) in a total reaction volume of 50 µL. Five-microliter aliquots of resultant cDNA were diluted one-tenth with sterile, distilled water and used as a target for PCR analysis.
CAA GCA AAA ACC GTC AAG and 3
GCG ATA TCA TCA TAA ATT GTT GC; predicted product size was 450 bp. For
L. corniculatus DFR, primers were 5 ATA AAC GGG GTG CTA GAC
and 3 ACA ACA AGG GAT GGA ATG; predicted product size was 300 bp.
Cycling conditions for RT-PCR were 94°C for 3 min; 10 cycles of
94°C for 1 min, 53°C for 1 min, and 72°C for 1.5 min; 20 cycles
of 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min; followed
by a final extension of 72°C for 3 min. Products of PCR amplification
were analyzed by electrophoresis of 20-µL aliquots of reactions on
0.7% agarose gels in Tris-borate-EDTA buffer and visualized with
ethidium bromide. This protocol has been used successfully to detect
and discriminate between A. majus and L. corniculatus DFR transcripts in cDNA from "hairy root" cultures (Bavage et al., 1997b).
Southern-Blot Analysis of Parental Lines
For lines selected for progeny analysis transgene copy numbers were estimated using the following method. Genomic DNA was isolated as for PCR analysis and after fluorimetric quantification using bisbenzimide (Brunk et al., 1979), 11 µg of L. corniculatus genomic DNA was cut with appropriate restriction
enzymes: HindIII and XbaI. After ethanol
precipitation, digests of genomic DNA were run on a 0.8% gel and
transferred to Zetaprobe membrane by alkaline blotting. The membrane
was probed with the 1.6-kb A. majus DFR fragment from
pJAM212 (Beld et al., 1989) and insert copy numbers were estimated by
counting numbers of hybridizing border fragments noted in
XbaI digests.
Production of Progeny and Analysis of T1 Generation
For production of progeny, S33, C26, RFD8, and RFD19 plants were grown to flowering in a containment-B greenhouse. Lines were then manually outcrossed to genotype S50. This protocol was necessary because L. corniculatus is a typical outbreeding forage species and because Agrobacterium rhizogenes-transformed plants exhibit reduced female fertility (Webb et al., 1990).
Statistical Analysis of Condensed Tannin Levels in Transgenic Plants
The data were assessed for normality using the Shapiro-Wilk test (Shapiro and Wilk, 1965). In most of the experiments described in this
paper, the Shapiro-Wilk test indicated that data were from a nonnormal
population, which could not be corrected using data transformations.
Therefore, we used the sign test, which is thought to be robust to
nonnormality and asymmetric data (Sprent, 1993). The sign test was used
to construct 85% (maximum possible from the available tables)
confidence limits for the control lines. Any antisense line that had
all of its values outside of the confidence interval of the control
lines was defined as having a statistically different condensed tannin
content in comparison with the control lines. Such lines are marked
with asterisks in Figures 3-6.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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PCR Analysis and Initial Characterization of Control and AS-DFR Lines
The lines in this study are outlined in Tables I and II. There were six classes of lines in the S50-line background and these corresponded to the original recipient genotype, an A. rhizogenes control line, vector control lines, and transgenic lines harboring three types of the AS-DFR gene construct. In the S33-line background, the recipient genotype was analyzed together with A. rhizogenes control lines, vector control lines, and lines harboring pMAJ2, which was the construct that produced optimal antisense phenotypes in cotransformed root cultures (Carron et al., 1994).
). The binary
vector used in these experiments was pROK2, which includes the CaMV and
nos sequences within its T-DNA. A. majus DFR sequences
resided between these sequences in an antisense orientation relative to
the CaMV. To verify the cotransformed nature of the lines in this
study, PCR analysis was carried out on genomic DNA extracted from
control and antisense plants. We used both nptII and CaMV
plus nos primer pairs to detect transgene sequences inserted into
genomic DNA in these lines. The generation of predictable amplification
fragments is shown schematically in Figure 2B.
Analysis of S50 AS-DFR Lines Grown in Controlled Environments
Our initial observations concentrated on the effects of AS-DFR constructs in an S50-line background. Replicate plants were set up and grown in vermiculite under growth-room conditions. Leaf, stem, and root tissues were harvested and subjected to analysis. No obvious effects were noted on biomass (data not shown) or plant morphologic characteristics, although some of the antisense lines appeared to be paler green and had a more erect phenotype.
S50 AS-DFR Phenotypes in Greenhouse-Grown Plants
Analysis of S33 AS-DFR Lines
Molecular Analysis of S33 AS-DFR Lines
Chemical Analysis of S33 AS-DFR Lines
Genetic Analysis of Parental Lines and Inheritance of Transgene in
Progeny
Antisense is a powerful technology that may be useful for the
analysis of plant metabolic pathways. Secondary pathways are an area of
metabolism that is particularly attractive for plant genetic
manipulation (Bavage et al., 1997a Received September 18, 1997;
accepted November 24, 1997.
Abbreviations:
AS-DFR, antisense dihydroflavonol reductase.
CaMV, cauliflower mosaic virus promoter.
CHS, chalcone synthase.
DFR, dihydroflavonol reductase.
MAJ, antisense DFR construct.
MeOH, methanol.
nos, nopaline synthase terminator.
RFD, antisense DFR line.
RT, reverse transcriptase.
Work on genetically manipulated plants was carried out under
Ministry of Agriculture, Fisheries, and Food license 162A/61/80. We
thank a number of people for assistance with this study: Wendy Thornley, Ian Davies, Eunice Carter, and Jenny Phillips (Terrill tannin
determinations) and Teri Evans (molecular analysis of parental and
progeny lines). We also thank Dan Dhanoa for critical comments on the
manuscript.
Barry TN,
Duncan SJ
(1984)
The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. 1. Voluntary intake.
Br J Nutr
51:
485-491
[CrossRef][Medline]
Bavage AD, Davies IG, Robbins MP, Morris P (1997a) Progress and
potential for the genetic manipulation of plant quality. In
G Cadisch, KE Giller, eds, Driven by Nature: Plant Litter Quality and
Decomposition. CAB International, Wallingford, CT, pp 201-211
Bavage AD,
Davies IG,
Robbins MP,
Morris P
(1997b)
Expression of an Antirrhinum dihydroflavonol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus (bird's foot trefoil).
Plant Mol Biol
35:
443-458
[CrossRef][Web of Science][Medline]
Bavage AD,
Robbins MP
(1994)
Dihydroflavonol reductase a Lotus corniculatus L. tannin biosynthesis gene: isolation of a partial gene clone by PCR.
Lotus Newsletter
25:
37-40
Beffa RS,
Neuhaus J-M,
Meins F
(1993)
Physiological compensation in antisense transformants: specific induction of an "ersatz" glucan endo-1,3-glucosidase in plants infected with necrotizing viruses.
Proc Natl Acad Sci USA
90:
8792-8796
Beld M,
Martin C,
Huits H,
Stuitje AR,
Gerats AGM
(1989)
Flavonoid synthesis in Petunia hybrida: partial characterisation of dihydroflavonol 4-reductase genes.
Plant Mol Biol
13:
491-502
[CrossRef][Web of Science][Medline]
Brunk CF,
Jones KC,
James TW
(1979)
Assay for nanogram quantities of DNA in cellular homogenates.
Anal Biochem
92:
497-500
[CrossRef][Web of Science][Medline]
Carron TR,
Robbins MP,
Morris P
(1994)
Genetic modification of condensed tannin biosynthesis in Lotus corniculatus. 1. Heterologous antisense dihydroflavonol reductase down-regulates tannin accumulation in "hairy root" cultures.
Theor Appl Genet
87:
1006-1015
Carter EB, Morris P, Theodorou MK (1995) Environmental effects on
the chemistry and nutritive value of Lotus corniculatus
(birdsfoot-trefoil). In GE Pollott, ed, Grassland into the
21st Century: Challenges and Opportunities. British Grassland Society
Symposium No. 29. British Grassland Society, Harrogate, UK, pp 166-168
Carter EB,
Theodorou MK,
Morris P
(1997)
Responses of Lotus corniculatus to environmental change. 1. Effects of elevated CO2, temperature and drought on growth and plant development.
New Phytol
136:
245-253
[CrossRef]
Chang S,
Puryear J,
Cairney J
(1993)
A simple and efficient method for isolating RNA from pine trees.
Plant Mol Biol Rep
11:
113-116
Charrier B,
Coronado C,
Kondorosi A,
Ratet P
(1995)
Molecular characterisation and expression of alfalfa (Medicago sativa L.) flavanone 3-hydroxylase and dihydroflavonol 4-reductase encoding genes.
Plant Mol Biol
29:
773-786
[CrossRef][Web of Science][Medline]
Colliver SP,
Morris P,
Robbins MP
(1997)
Differential modification of flavonoid and isoflavonoid biosynthesis with an antisense chalcone synthase construct in transgenic Lotus corniculatus.
Plant Mol Biol
35:
509-522
[CrossRef][Web of Science][Medline]
Dellaporta SL,
Wood J,
Hicks JB
(1983)
A plant DNA minipreparation: version II.
Plant Mol Biol Rep
1:
19-21
Dwivedi UN,
Campbell WH,
Yu J,
Datla RSS,
Bugos RC,
Chiang VL,
Podila GP
(1994)
Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus.
Plant Mol Biol
26:
61-71
[CrossRef][Web of Science][Medline]
Forde BG,
Day HM,
Turton JF,
Wen-Jun S,
Cullimore JV,
Oliver JE
(1991)
Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants.
Plant Cell
1:
391-401
Forkmann G
(1993)
Genetics of flavonoids.
In
JB Harborne,
eds, The Flavonoids: Advances in Research since 1986.
Chapman & Hall, London, pp 538-564
Griffiths DW
(1989)
Polyphenols and their possible effect on nutritive value.
Aspects of Applied Biology
19:
93-103
Jende-Strid B
(1991)
Gene-enzyme relations in the pathway of flavonoid biosynthesis in barley.
Theor Appl Genet
81:
668-674
Lees GL,
Hinks CF,
Suttill NH
(1994)
Effect of high temperature on condensed tannin accumulation in leaf tissue of bigfoot trefoil (Lotus uliginosus).
J Sci Food Agric
65:
415-421
[CrossRef]
Luo G,
Hepburn AG,
Widholm JD
(1992)
Preparation of plant DNA for PCR analysis: a fast, good and reliable procedure.
Plant Mol Biol Rep
10:
319-323
Morris P, Robbins MP (1997) Manipulating condensed tannins in
forage legumes. In BD McKersie, DCW Brown, eds, Biotechnology and the Improvement of Forage Legumes. CAB International, Wallingford, CT, pp 147-173
Mueller-Harvey I,
McAllan AB
(1992)
Tannins: their biochemistry and anti-nutritional properties.
Adv Plant Cell Biochem Biotechnol
1:
149-213
Ougham HJ,
Davies TGE
(1990)
Leaf development in Lolium temulentum: gradients of RNA complement plastid and non-plastid transcripts.
Physiol Plant
79:
331-338
[CrossRef]
Raynaud J,
Jay M,
Raynaud J
(1982)
Flavonoid glycosides of Lotus corniculatus (Leguminosae).
Phytochemistry
21:
2604-2605
[CrossRef]
Robbins MP,
Bavage AD,
Morris P
(1997)
Options for the genetic manipulation of astringent and antinutritional metabolites in fruit and vegetables.
In
FA Tomas-Barberan,
RJ Robins,
eds, Phytochemistry of Fruit and Vegetables.
Clarendon Press, Oxford, UK, pp 251-261
Robbins MP,
Evans TE,
Morris P,
Carron TR
(1991)
Lotus Newsletter
22:
18-21
Shapiro SS,
Wilk MB
(1965)
An analysis of variance test for normality (complete samples).
Biometrika
52:
591-611
Sprent P
(1993)
Applied Nonparametric Statistical Methods, Ed 2.
Chapman & Hall, London
Tanner GJ,
Moate PJ,
Davis LH,
Laby RH,
Yuguang L,
Larkin PA
(1995)
Proanthocyanidins (condensed tannin) destabilise plant protein foams in a dose dependent manner.
Aust J Agric Res
46:
1101-1109
[CrossRef]
Terrill TH,
Douglas GB,
Foote AG,
Purchas RW,
Wilson GF,
Barry TN
(1992)
Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains.
J Sci Food Agric
58:
321-329
van der Krol AR,
Lenting PE,
Veenstra J,
van der Meer IM,
Koes RE,
Gerats AGM,
Mol JNM,
Stuitje AR
(1988)
An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation.
Nature
333:
866-869
[CrossRef]
Waghorn GC,
Jones WT,
Shelton ID,
McNabb WC
(1990)
Condensed tannins and the nutritive value of herbage.
Proceedings of the New Zealand Grassland Association
51:
171-176
Webb KJ,
Jones S,
Robbins MP,
Minchin FR
(1990)
Characterisation of transgenic root cultures of Trifolium repens, Trifolium pratense and Lotus corniculatus and transgenic plants of Lotus corniculatus.
Plant Sci
70:
243-254
[CrossRef]
Webb KJ,
Robbins MP,
Mizen S
(1994)
Expression of GUS in primary transformants and segregation of GUS, TL and TR-DNA in the T1 generation of hairy root transformants of Lotus corniculatus.
Transgenic Research
3:
232-240
. However,
statistically significant differences were apparent between control and
AS-DFR lines. Levels of tannins in leaf tissues were reduced in RFD63
and RFD64 (in both harvests), whereas levels in RFD40 were lower than
levels in controls at harvest 1 only. Phenotypes were also noted in
stem tissues at the second harvest, with reductions in condensed
tannins being detected in RFD60, RFD61, RFD62, RFD63, RFD64, and RFD66.
Because these plants had been grown in vermiculite, it was also
possible to estimate the tannin levels in root tissues of S50 control
and AS-DFR lines (Figs. 3C and 4C). Root results were difficult to
fully interpret, but statistically significant reductions in RFD64,
RFD65, and RFD40, and an increase in condensed tannin levels in the
roots of RFD28 were apparent.
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Figure 4.
Levels of condensed tannin (CT) in S50 plants
harboring control and AS-DFR construct at harvest 2. Bars represent
mean values, and control bars correspond to the mean of lines S50, C22,
C50/1, and C50/2. For each control and antisense line three replicate plants were analyzed. Arrows indicate 85% confidence limits for the
median of the control condensed tannin values using the sign test.
Vertical lines indicate the range of values noted for each antisense
line. Asterisks indicate antisense ranges outside of the control
confidence intervals. A, Leaves, growth room (harvest 2); B, stems,
growth room (harvest 2); and C, roots, growth room (harvest 2). DW, Dry
weight.
; Carter et al., 1995). S50 AS-DFR lines were therefore grown in soil as described in ``Materials and Methods'' under summer
greenhouse conditions. In this case low-tannin phenotypes were also
noted in leaf tissues, with levels below those found in control tissues
being observed in RFD60, RFD62, and RFD64 (Fig.
5A). No lines were detected that showed
statistically reduced tannin levels in stem tissues (Fig. 5B), but
RFD31 had significantly elevated tannin levels in both leaves and
stems.
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Figure 5.
Levels of condensed tannin (CT) in S50 control and
AS-DFR plants grown in a greenhouse. Bars represent mean values, and
control bars correspond to the mean of lines S50, C22, C50/1, and
C50/2. For each control and antisense line three replicate plants were analyzed. Arrows indicate 85% confidence limits for the median of the
control condensed tannin values using the sign test. Vertical lines
indicate the range of values noted for each antisense line. Asterisks
indicate antisense ranges outside of the control confidence intervals.
A, Leaves; B, stems. DW, Dry weight.
View this table:
Table III.
Summary of RFD phenotypes in the S50 background
Results summarize changes in condensed tannin levels in leaf, stem, and
root tissues and under different growth conditions. Transgenic lines
analyzed harbored the pMAJ1 construct (RFD60, 61, 62, 63, 64, 65),
pMAJ2 (RFD28, 31, and 40), or pMAJ3 (RFD9 and 66). 
, Statistically
significant reduction in condensed tannin levels relative to four
independent control lines using the sign test under a given
environment; 
, a statistically significant increase relative to
control lines; -, not statistically different from control lines.
), and there were a number of examples (RFD8,
RFD19, and RFD38) that showed large reductions in condensed tannin
levels relative to S33 control lines when analyzed as root cultures.
Therefore, we analyzed tannin levels in juvenile shoots (Fig.
6A), leaves (Fig. 6B), and stems (Fig.
6C) in control and AS-DFR plants. In juvenile tissues, RFD7, RFD8, and
RFD38 had tannin levels lower than the control lines. In leaves and
stems of 6-week-old plants of RFD7, RFD8, and RFD50, tannin levels were
reduced relative to controls. These results are summarized in Table
IV. Although none of the reductions in the quantity of condensed tannins was on the order of 80%, as found in
root cultures, reductions in levels of condensed tannins were similar.
In juvenile shoot tissues reductions were in the order of 4 mg/g dry
weight, and in leaves and stems they were in the order of 2 mg/g dry
weight. Corresponding tannin reductions in root cultures for effective
antisense lines were on the order of 0.25 mg/g fresh weight, which
translates to approximately 2.5 mg/g dry weight.
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Figure 6.
Levels of condensed tannin (CT) in S33 control
plants and lines harboring the pMAJ2 construct. Bars represent mean
values, and the control bars correspond to the mean of lines S33, C26, C28, and C32. For each control and antisense line three replicate plants were analyzed. Arrows indicate 85% confidence limits for the
median of the control condensed tannin values using the sign test.
Vertical lines indicate the range of values noted for each antisense
line. Asterisks indicate antisense ranges outside of the control
confidence intervals. A, Juvenile growth; B, leaves; and C, stems. DW,
Dry weight.
View this table:
Table IV.
Summary of RFD phenotypes in the S33 background
Results summarize changes in condensed tannin levels in leaf, stem, and
juvenile shoot tissues. All transgenic lines harbor the pMAJ2 construct
(RFD4, 7, 8, 19, 38, 49, and 50). , Statistically significant
reduction in condensed tannin levels relative to four independent
control lines using the sign test. -, Not statistically different from
control lines.
), we found it straightforward to detect antisense transcript in S33 and S50 root cultures. However, when using either the
A. majus DFR sequence or a L. corniculatus DFR
probe (Bavage et al., 1997b) on RNA blots from these plant tissues, no
transcript was detectable in either case. One possible explanation
might be that flavonoid gene and antisense transcripts are at very low levels in regenerated plant tissues compared with corresponding root
cultures. Alternatively, the high levels of soluble and insoluble condensed tannins in regenerated plants may compromise conventional northern blotting. Therefore, we tried to detect transcripts using RT-PCR from cDNA derived from control and antisense lines. We have
found this method to be convenient for the detection of endogenous and
introduced A. majus DFR genes in L. corniculatus
root cultures (Bavage et al., 1997b). Using this method we obtained the
results shown in Figure 7. No antisense
amplification product was detectable in S33 (recipient genotype) or C26
(control line); however, a 450-bp fragment was noted in RFD7, RFD8, and
RFD19, but not in RFD38. The faintness of these amplification products
was consistent with low levels of expression of the antisense
transgene, and it is interesting to note the detectable product in both
effective RFD7, RFD8, and ineffective RFD19 lines.
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Figure 7.
RT-PCR analysis of cDNA prepared from selected S33
control and pMAJ2 lines. Amplifications from selected lines were
performed as outlined in ``Materials and Methods''. The Plasmid lane
is the amplification product from 20 pg of pJAM212 (A. majus DFR cDNA). The arrow indicates the predicted size of the
amplification product derived from pMAJ2. DNA-marker lane is 2.5 µg
of a 100-bp ladder (Pharmacia-Biotech).
) indicated the presence of 13 kaempferol and quercetin
glycosides, with kaempferol- and quercetin-3,7-dirhamnoside, and the
corresponding 3-rhamnoside-7-glucosides, as the major components.
However, we were able to detect only kaempferol glycosides (Fig. 8, 
max band I, 260 nm) in extracts of genotype S33. This was confirmed by
acid hydrolysis of extracts that yielded only kaempferol (peak 13). A
combination of alkali and enzymatic hydrolysis indicated other peaks as
kaempferol-3,7-dirhamnoside (peak 10),
kaempferol-3-rhamnoside-7-glucoside (peak 7), kaempferol-3-glucoside (peak 8), kaempferol-7-rhamnoside (peak 11), and
kaempferol-3-rhamnoside (peak 9). Both kaempferol and quercetin
glycosides, however, were found in leaves and stems of genotype S50.
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Figure 8.
HPLC profile and UV/visible spectra of MeOH
extracts of 6-week-old shoot tissues from control (S33) and AS-DFR
lines (RFD7 and RFD 8) of L. corniculatus. Condensed
tannin contents of these lines were: S33, 10.7 mg/g dry weight; RFD7,
7.1 mg/g dry weight (66% of control level); and RFD8, 5.9 mg/g dry
weight (55% of control level). Extracts from equivalent amounts of
tissue were subjected to HPLC with diode-array detection. HPLC profiles
were monitored at 350 nm and spectra were recorded between 240 and 400 nm. Chemical analysis indicated that the majority of peaks were either
kaempferol or kaempferol glycosides (peaks 1-3 and 5-13). Identified
peaks are: kaempferol (peak 13), kaempferol-3-rhamnoside-7-glucoside (peak 7), kaempferol-3-glucoside (peak 8), kaempferol-3-rhamnoside (peak 9), kaempferol-3,7-dirhamnoside (peak 10), and
kaempferol-7-rhamnoside (peak 11).
) and are
typical for binary T-DNA sequences introduced into L. corniculatus using A. rhizogenes.
View this table:
Table V.
PCR analysis of progeny from an effective RFD (RFD8)
Parental lines were analyzed together with 24 progeny lines (1-24).
Amplification products with nptII primers confirm the presence of the selectable marker from binary vector T-DNA in genomic
DNA of parental and progeny lines. CaMV-nos amplification products
confirm the presence of promoter and terminator sequences and the
antisense DFR fragment from pMAJ2. +, Product noted; , no product
noted after amplification.
View this table:
Table VI.
PCR analysis of progeny from an ineffective
RFD (RFD19)
Parental lines were analyzed together with 21 progeny lines (1-22).
Amplification products with nptII primers confirm the presence of the selectable marker from binary vector T-DNA in genomic
DNA of parental and progeny lines. CaMV-nos amplification products
confirm the presence of promoter and terminator sequences and the
antisense DFR fragment from pMAJ2. +, Product noted; , no product
noted; +(), product not consistently observed after amplification.
). Another
possibility is that in at least some of these lines there has been
truncation of transgene sequences at the left-hand border of binary
vector T-DNA.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Robbins et al., 1997). The results
presented in this paper can be compared with other examples of
heterologous antisense in higher plants. Notable examples include
the expression of antisense sequences of petunia (Petunia hybrida) CHS genes in tobacco (van der Krol et al., 1988) and the
antisense expression of a lignin-specific
O-methyltransferase from Populus tremuloides in
tobacco (Dwivedi et al., 1994). In the experiments reported here, an
A. majus DFR gene has been expressed in antisense
orientation in L. corniculatus and phenotypes have been
noted in leaf, root, and stem tissues.
; Webb et al., 1994). The data presented here are at variance with
these observations. One possible reason is that the CaMV promoter is
positioned next to different tissue-specific or development-specific enhancers in different transformation events, and that each transgenic plant therefore may show differences in tissue-specific expression even
though all plants in the study share a common promoter sequence. Another possibility is that a number of different DFR genes are expressed in L. corniculatus, and that different antisense
transformation events suppress DFR genes that are expressed in
individual tissues.
-1,3 glucanase in tobacco (Beffa et
al., 1993) and in antisense expression of CHS in L. corniculatus root cultures (Colliver et al., 1997). However, to
fully test this hypothesis one would need to clone all of the expressed
members of the DFR gene family in S50 and analyze their expression in
control and antisense up-regulated lines.
reported
underrepresentation of Agrobacterium-derived TR-DNA
sequences and of GUS expression in the progeny of L. corniculatus, and further studies of the inheritance of transgenes
in forage species may be of considerable interest.
1
The Institute of Grassland and Environmental
Research is grant funded by the Biotechnology and Biological Sciences
Research Council (BBSRC). This work was supported by the BBSRC Plant
Molecular Biology Initiative (parts 1 and 2).
FOOTNOTES
2
Present address: John Innes Research Centre,
Colney Lane, Norwich NR4 7UH, UK.
*
Corresponding author; e-mail mark.robbins{at}bbsrc.ac.uk; fax
44-1970-828357.
ABBREVIATIONS
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
Copyright Clearance Center: 0032-0889/98/116/1133/12
© 1998 American Society of Plant Physiologists
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3
.
B, Schematic representation of T-DNA of control and antisense gene
constructs. RB, Right border of T-DNA; LB, left border of T-DNA.
Positions of nptII gene and AS-DFR constructions within
T-DNA are indicated. nptII, CaMV, and nos primer
positions are marked with > and <. Sizes of predicted PCR
products from the control construct and from the three AS-DFR constructs are indicated.
