Note: Descriptions are shown in the official language in which they were submitted.
~1 93~61
W096/007X9 I~l/r
TRANSGENIC PLANTS PRODUCING T~R~AT.~C~
FIE~D OF T~ INVENTION
The present invention relates to the genetic engineering of
plants to introduce a capacity to synthesise trehalose. In
particular, the invention concerns plants having an
increased trehalose content and methods for producing them.
The present invention also relates to methods for increasing
the tolerance oi plants towards stresses such as cold and
drought, as well as to methods for pr~oducing trehalose.
BAC~GROUND OF THE INVENTION ~
Trehalose (~-glucopyranosyl-~-D-glucopyranose) is a dimer of
glucose molecules linked through their reducing groups.
Because of its unusual combination of chemical properties
compared to other sugars, including its lack of reducing
groups, slow hydrolysis and ability to form a non-
20 AP1 ;~l~cent glass, it is one of the most effective knownpreservatives of proteins, cellular membranes and other
biological ~ ds in vitro. Also, living organisms that
contain large amounts of trehalose are characteristically
those often exposed to osmotic, dehydration and heat
stresses, such as insects, certain litoral animals and many
microorganisms, including yeasts and bacteria. There is
circumstantial evidence (summarised by Wiemken [1990]
Antonie van ~eeuwenhoek 58, 209-217) that the primary role
of trehalose in baker's yeast is to confer resistance to
these stresses. However, it has also been suggested (Nwaka
et al [1994] FEBS ~etters 344, 225-228; Van Dijk et al
[1995] Applied Environ Microbiol. 61, 109-115) that the
accumulation of trehalose in baker~s yeast is not, by
itself, suificient to confer stress-tolerance.
~ = ~ . . . ~ =
High levels of trehalose occur in the so-called resurrection
plants, such as the pteridophyte, Selaqinella le~ido~hvlla,
which can survive prolonged desiccation and heat exposure
W096io0789 2 1 9 3 ~ 6 ~ p~ l lrL ~
(reviewed by Avigad [1982] in Encyclopedia of Plant Research
(New Series) 13 A, pp 217-347~. The great majority of
vascular plants, however, are unable to synthesise
trehalose. Such plants often accumulate other "compatible'~
solutes, including glycine betaine, proline ana various
polyols, in response to stresses such as drought that
decrease the availability of intracellular water ~reviewed
by McCue ~ Hanson [1990] Trends in Biotechnology 8, 358-
362).
There are very few reports of trehaIose in angiosperms, and
these usually describe small amounts that could reflect
microbial cnnt~m' n~tion (e.g., Kandler ~ Senser [1965] Z.
Pflanzenphysiol. 53, 157-161; Oesch ~ Meier [1967]
Phytochemistry 6, 1147-1148). Indeed, it has been suggested
that trehalose is toxic to many plant tissues ~Veluthambi et
al. [1981] Plant Physiol. 68, 1369-1374), especially those
with little or no trehalase activity (trehalase is the
enzyme that converts trehalose to glucose). ~owever, at
least one angiosperm, Mvrothamnus flabellifolia (another
~resurrection" plant), accumulates significant amounts of
trehalose (Bianchi et al. [1993] Physiologia Plantarum 87,
223-226), showing that there is not an absolute
compatability barrier between trehalose and angiosperms.
The absence of trehalose from most angiosperms and reported
toxicity in some suggests that introduction of a trehalose
synthetic pathway into these plants might sometimes have
deleterious effects. On the other hand, successful
production of trehalose in plants would have substantial
advantages. Trehalose accumulated in, e.g., the storage
organs of sugar beet, potato, onion etc, could be
commercially extracted to provide trehalose at costs that
make it competitive with sucrose in certain applications.
These applications include the manufacture of dried foods
(milk and egg powders, soups, fruit purées! etc), because
trehalose preserves the flavour and texture of many food
stuffs through economically attractive drying procedures,
2 l 9386 1
~ W096/00789 r~l/r~
and is much superior in thiq-regard to sucrose (cee, e.g.,
Roser [1991] Trends in Food Science h Ter~no,logy, July
issue, pp. 166-169; Roser & Colaço [1993] New Scientist, May
issue, pp. 25-28). Compared to sucrose, the non-sweetness of
trehalose is a further advantage in many cases (soups, egg
powders), as is the fact that it does not yield fructose,
which is perceived as a health risk. However, the high price
of trehalose makes its use in the dried food industry
prohibitively expensive. Secondly, production of trehalose
in the edible portion of certain plants could extend the
shelf life of products such as tomatoes. Thirdly,
accumulation of trehalose in sensitive tissues could
increase the tolerance of plants towards frost, drought,
high balinity and similar stresses. :
: _
S~MMARY OF INVENTION _
Based on what has been stated above, it is an object of the
invention to provide plants having a novel capacity to
synthesise trehalose. In particular, it is an object of the
invention to provide controlled A . 1 Ation of trehalose in
plant tissues as a commercial source of trehalose or to
provide stress-tr,lPrAnre or both, whilst m;nim;q;nrJ
pctrnt;Ally deleterious effects of the trehalose on plant
growth.
The present invention i8 based on the concept of trans-
forming the plants of interest with structural genes for
trehalose synthesis under the control of appropriate plant
promoters in order to produce transgenic plants having
increased trehalose r~nt~ntq~ In particular, the coding
sequences of one or more genes encoding polypeptides of
enzymes producing trehalose 6-phosphate or trehalose itself
are, according to the inver,tion, expressed in plants under
the control of specific kinds of plant promoters.
Thus, the plant of interest is transformed at least with the
coding ser~uence of a gene for trehalose-6-phosphate synthase
W096l00789 2 1 9 3 8 6 1 J ~ l lrL ~
(Tre6P synthase) fused appropriately to a plant promoter 80
that full expression of the gene is realised only as the
plant matures or when it encounters specific environmental
conditions. The promoters are therefore preferably non-
constitutive and chosen to allow temporal (e.g., diurnal),
topological (e.g., tissue-specific) or stress-activated (or
~stress-induced") control over the expression of the genes.
The plant may be also transformed with one or more genes
encoding a trehalose-6-~phosphata~e (Tre6Pase) or a
regulatory polypeptide that interacts with the Tre6P
synthase or the Tre6Pase or both.
Transformation of the plant may be done by any of the
methods available in the art, including infection with
tranformed Acrobacter tumefaciPnc and the direct
introduction of foreign DNA by microinjection,
electroporation, particle bombardment and direct DNA uptake.
The structural genes are preferably selected from the group
comprising the yeast genes TPS1, TPS2 and TSL1 encoding
respectively the 56 kDa Tre6P synthase, 102 kDa Tre6Pase and
123 kDa regulatory subunits of yeast trehalose synthase.
It is another object of the invention to provide a method
for producing transgenic plants with increased trehalose
rrntPnt~, which method comprises the steps of transforming a
plant of interest with the structural genes for trehalose
synthesis, in particular a gene for Tre6P synthase, as
mentioned above, and expressing those genes under the
control of a suitable promoter to allow temporal,
topological, or stress-induced~control over the expression
of the genes.
It is a third object of the invention to produce trehalose,
which comprises the steps of
- transforming a plant with at least one structural gene
for Tre6P synthase in order to produce a transgenic
plant,
- cultivating the transgenic plant under conditions which
~ ~W096i00789 ~ q38 6~ P~l/r. ~ Il
will induce the expression of trehalose synthesis in
the plant, and
- extracting the trehalose from the tissues of the plant.
A fourth object of the invention is to provide a method of
protecting plants against adverse conditions such as
drought, high salinity, temperature extremes and other
stresses, which method comprises transforming the plant with
at least the coding sequence of a gene for a trehalose-6-
phosphate synthase fused appropriately to a plant promoterso that full expression of the gene is not realised until
the plant encounters adverse conditions. The plant may
optionally be also cotransformed with one or more genes
encoding trehalose-6-phosphatase and regulatory proteins
that interact with the trehalose-6-phosphate synthase or
trehalose-6-phosphatase or both. For example, the invention
provides a method of protecting plants bearing berries or
other fruits against frost damage to blossom, which
comprises transforminq the plant with a gene for trehalose-
6-phosphatase fused appropriately to a plant promoter so
that full expression of the gene i8 not realised until the
plant ~nrmlnt~rs low temperatures.
A fifth object of the invention i5 to provide a method of
producing transformed ornamental plantb that do not reriuire
auch intensive and expert care as the untransformed plant,
which method comprises transforming the plant with a gene
for trehalose-6-phosphate synthase appropriately fused to a
plant promoter so that the plant cnnt~;n~ trehalose in some
of its tissues.
BRIEF DESCRIPTION OF FI~URES _ _
Figure l depicts the schematic structure of the chimeric
gene construct containing the A. th~ n~ Rubisco small
subunit promoter (patslA) fused to the TPS1 yeast gene,
~nrn~1ng the Tre6P synthase subunit, and the transcription
stop signal from the nopaline synthase gene of AcrQbacterium
W096/00789 2193861 r~llr~-~ "
tumefaciens (nos). Only the part of the plasmid pKOH51 with
the chimeric gene is shown. Unique restriction enzyme
cleavage sites for the chimeric gene construction are shown.
Figure 2 gives the results of Western blot analysis of
transgenic tobacco plants showing the 56 kDa TPS1 product
indicated by an arrow. Proteins were extracted from the
transformed tobacco plants cnnt~;nin~ (lines 1, 1, 4, 5, 6,
8, lO, 12, 15, 16 and 19) the construct described in Fig 1,
or (GUS) another chimeric gene with the Cauliflower mosaic
virus 35S promoter fused to the ~-glucuronidase gene (UIDA)
in the same vector or (SRl) from untransformed tobacco
plants. Equal amounts of protein were loaded in each lane.
The antiserum used (anti-57) was raised against the 5Ç kDa
Tre6P synthase subunit from yeast.
Figure 3 shows the chromatographic identification of
trehalose. Samples (20 ~l) of water extracts of tobacco
leaves were analyzed by HPLC as described in General
Materials and Methods. Extracts A and B rnnt~;nPd 192 mg
fresh weight of greenhouse-grown Transformant 19 ml-1 (A)
before and (B) after treatment with trehalase. Extracts C
and D rnnt~i nPd 149 mg leaf ml-1 from (C) Transformant 4 or
(D) a control plant transformed with plasmid pDE1001 lacking
the TPS1 gene, both grown under sterile conditions.
Trehalose peaks are indicated with T. The two large peaks at
about 25 min (peaks 7 and 8 in A) are glucose and sucrose.
Figure 4 Protein i Innhl ot showing tissue localization of
TPS1-subunit analyzed in the atsl-TPSl transformed line 4.
~eaves of wild-type tobacco (SR1) were used as a negative
control and 0.1 ~g of Tre6P synthase subunit purified from
yeast (TPS1) as a positive control. The trehalose content of
the same tissues is indicated in mg/g dry weight above the
immunoblot. nt, not tested. The following abbreviations are
used: FB for flower buds, U~ for upper leaves, ML for middle
leaves, ~ for lower leaves, US for upper stem, R for roots
and EC for enzyme control.
21 938~1
~ W09~00789 P~/r~
Fig. S. Enhanced drought tolerance in trehalose-producing
plants. Detached leaves of same developmental stage from six
to eight week-old in vitro propagated plants were exposed to
air-drying ~25 ~RH). (A) At times indicated the leaves were
weighed and the results presented as relative fresh weight
of the leaves at each time point. The dry weight of each
leaf was obtained following drying of the leaves at + 60~C
for 48 hr~. The wild-type control tobacco is indicated by
SR1 and the trehalose-producing transgenic lines l, 4 and 8
by their respective line numbers. (B) Appearance of the
detached leaves from transgenic line 4 and control plants at
selected time points during the stress treatment.
Fig. 6. Drought survival of control and trehalose-producing
tobacco plants. ~A) Six to eight weeks' old in vitro
propagated whole plants were exposed to air-drying (D) for
the times indicated. After 17 h of stress-treatment, the
nontransformed control plants (SR1) and trehalose-producing
transgenic plants of lines 4 and 8 were rehydrated (R) by
placing them in water. (B) Three week old seedlings of
transgenic line 8 and both nontransformed (SR1) and vector-
transformed (C~ control plants were exposed to air-drying
(D) for the time~ indicated. The s~l;ngc were subsequently
rehydrated (R) after 7 h of stress by placing them in water.
Figure 7. SDS-PAGE analysis of fractions ~nt~;nlng M.
smeqmatis Tre6P synthase eluted from the first Heparin-
Sepharose column. ~anes l to 4 contain, respectively,
samples of H33 (7.0 mU), H35 (20 mU), H36 (21 mU) and H37
(12 mU) (Table 3). After SDS-PAGE on 8 ~ acrylamide, the
gels were stained with Coomassie Brilliant Blue. The sizes
of the molecular mass standards in lane 5 are shown in kDa.
The position of the 55 kDa Tre6P synthase polypeptide is
shown with an arrow.
Figure 8. SDS-PAGE analysis of the peak fraction of M.
smeqmatis Tre6P synthase eluted from the second Heparin-
Sepharose column. Fraction T21 (Table 3) was concentrated in
W096l00789 2 1 93~6 1 ~I/rl C.~ "
a Centricon 10 tube and then mixed with half its volume of
3-fold concentrated SDS-PAGE sample buffer. The overall
concentration was 6-fold. Samples containing (l~nç_1) 2.9 mU
and (l~açl) 8.7 mU of Tre6P synthase were subiected to SDS-
PAGE on 8 ~ acrylamide and stained with Coomassie Brilliant
Blue. The molecular mass standards in lane 2 are, from top
to bottom, 109, 84, 47, 33, 24 and 16 kDa.
Figure 9. Western analysis of Tre6P synthase purified from
M. smeqma~is. An 8 ~ acrylamide gel was loaded with
prestained molecular mass markers (l~aç_1 200, 117, 80 and
47 kDa; lane 10: the only yisible marker is 84 kDa), pure
Tre6P synthase from fraction T21 (Table 3) (lanes 7 and 8:
2.9 mU; lanes 3,4 and 9: 8.7 mY) and the pooled active
fractions from G100 Sephadex (Table 3) (la~es 6: 7.8 ~g
total proteini lanes 2 ~n~ 5- 23 ~g to~al protein). After
electrophoresis and blotting to nitrocellulose, the
nitrocellulose membrane was cut between lanes 3 and 4 and
between lanes 7 and 8. ~anes 1 to 3 were stained with
Coomassie Brilliant Blue, lanes 4 to 7 were probed with
anti-57 serum raised against the purified 56 kDa Tre6P
synthase subunit from yeast and lanes 8 to 10 were probed
wi~h preimmune serum. Immunoreactive bands were visualised
using goat anti-rabbit IgG-~1k~1; n~ phosphatase conjugate
from Promega according to the r-nnfarturer~s instructions,
with colour development times of 2.8 minutes in both cases.
The diagonal lines across lanes 4 to 7 are due to acri~nt~
creasing of the membrane during transfer to nitrocellulose.
Fig 10. Western analysis of 8 transgenic lines of
Arabido~sis thaliana. Plants grown from seed of primary
transformants were extracted and analyzed with antiserum
raised against the 56 kDa Tre6P synthase gubunit from yeast
essentially as described in Fig. 2. Tre6P synthase subunit
purified from yeast (0.1 ~g) was used a positive control.
Untransformed A. th~ n~ gives no signal.
2l q3861
~ W096/00789 p~llrL ~
DETAILED DESCRIPTION OE T~E INVENTICN
In the follow'ng description, the phrase 'lrnnrtitutive plant
promoter" refers to plant promoters that cause the
rnnt;n~lnus and general expression of their associated coding
eriuences~ 80 that the products of~these ser~uences are found
in all cells of the plant and at all phases of growth. Non-
cnnqtitutive promoters, in contrast, are activated by
specific internal or external events, such as the
differe~tlation of cells to form distinct tissues as a plant
developes and matures or changes in the plant~s environment.
Many kinds of envirnn~nt~l change are known to activate
particul ~r promoters. Examples include the light induced
activation of promoters for the small subunit of ribulose-
1,5-bisphosphate carboxylase (Krebbers et al. [1988] Plant.
Mol. Biol 11, 745-759), such as the atslA promoter used in
Examples 1-3, and the activation by various stresses of
promoters such as LTI78 (Nordin et al. [1993] Plant Mol.
Biol. 21, 641-653) and R~B18 (Lang & Palva [1992] Plant Mol.
Biol 20, 951-962). ~owever, our invention is not limited to
these examples of non-constitutive promoters. Rather, an
important and novel part of the invention is the concept
that the con8titutive synthesis of trehalose by a plant will
always be wasteful and F ti ~ deleterious, so that the
benefits of trehalose production disclosed in this
application can best be realised by the use of non-
constitutive promoters. By means of these non-constitutive
promoters, the biosynthesis of trehalose in transgenic
plants can be sub]ected to temporal control (i.e., it only
occurs at certain times), topological control (i.e., it is
limited to certain parts oi the plant) or both.
The names of plant organs used in this description are
generally those applied by the ordinary greengrocer and her
customer. Thus, for example, "fruit" includes the parts of
an apple or strawberry plant that are sold to be eaten,
although these storage tissues rrnt~in;nr, the seeds are not
themselves fruits in a strict botanical sense.
W096l007X9 21 9386~ r~l/r~ ~- "
It has already been mentioned that nearly all species of
vascular plants appear to lack the ability to synthesise
trehalose, 80 that the term ~~trehalose-producing~ plants
does not re~uire r~uantitative definition. ~owever, the
pr~;h;lity that some ordinary plants (other than the 80-
called resurrection plants) might produce trehalose during
stress appears not to have been investigated. The present
invention is concerned with the genetic engineering of
plants to introduce a heterologous capacity for trehalose
synthesis (in the present context also called a '~novel~
capacity for trehalose synthesis~. The increase in trehalose
rer~uired, compared to that in the untransformed plant grown
under identical conditions, is that which either causes a
useful ; __uv~ ~nt in stress-tolerance or can be profitably
extracted from the plant for commercial use.
Many microbial organisms contain enzyme systems that produce
trehalose-6-phosphate ~Tre6P) and hydrolyse it to trehalose.
For example, SacrhAromyces cerevisiae crntA;n~ a trehalose
synthase complex comprising 56, 102 and 123 kDa subunits
(Londesborough & Vuorio [1993] Eur. J. Biochem. 216, 841-
84B) which condenses uri~;np~;phrl~phoglucose (UDPG) and
glucose-6-phosphate (Glc6P) first to Tre6P (the Tre6P
synthase reaction) and then to free trehalose (the Tre6Pase
reaction). Tre6P syntha8e and Tre6Pase activities reside in
the 56 and 102 kDa subunits, respectively, and the 123 kDa
subunit confers regulatory properties on the complex and
appears to stabilise it. ûther microbial systems include
those of ~An~;da utilis (Soler-et al [1989~ FEMS Microbiol
Letters 61, 273-278), E. coli ~Glaever et al [1988] J.
Bacteriol. 170, 2841-2849), Dictvostelium ~;rcoideum
(Killick [1979] Arch. Biochem. Biophys. 196, 121-133) and
Mvcobacterium ~ -7m-t; q (Lapp et al [1971] J. Biol. Chem.
246, 4567-4579), the latter two systems being able to use
~Pn;nP~;phrrphoglucoge (ADPG) as an alternative to UDPG.
Little information is available about the subunit structure
of these other microbial enzyme systems. Multicellular
organisms that make trehalose, including nematodes, insects
~ ~096/00789 11 P~llr~SI~
and resurrection plants, may al~o be a~umed to contain
enzymes with Tre6P synthase and Tre6Pase activity.
In principle, transfer of any of these Tre6P synthases to
plants would give the plants the novel capacity to
synthesise Tre6P. It is disclosed that some plants, such as
tobacco, have an inherent capacity to convert Tre6P to
trehalose, so that, surprisingly, transformation of, e.g.,
tobacco with a gene for Tre6P synthase alone leads to
e~ficient production of trehalose itself. However, if the
endogenous conversion o~ Tre6P to trehalose is slow or
absent, a Tre6Pase from any suitable source may be also
transferred. With systems such as the yeast enzyme, where
the Tre6P synthase and Tre6Pase are subunits of a trehalose
synthase, it may advantageous to cotransform plants with
genes for Tre6P synthase and Tre6Pase from the same source,
so that the natural enzyme complex can be formed. Whatever
the source of the enzymes, the trehalose formed in the
transgenic plants can then be extracted or could confer
increased tolerance to certain stresses. The production of
trehalose in plants transformed in this way with certain
yeast genes has already been described (PCT/~I93/00049).
However, although the accumulation of trehalose at certain
times (e.g., during exposure to stress or in a mature plant)
and in certain tissues (e.g. storage organs or, at
~ Liate times, frost-sensitive tissues) is expected to
be bPnP~;ci~l/ or at least harmless to a plant, there is a
distinct possibility that at other times and in other
tissues the accumulation of trehalose may be harmful to a
plant (VPlnth~hi Ç~_al- [1971] loc ci~). It would therefore
be advantageous to use a plant promoter (a promoter is a
part of a gene that promotes transcription of the coding
se~uence) that does not permit full expression of the
gene(s) causing trehalose synthesis until the plant is
mature or encounters envi~ 1 conditions, including
drought and low temperature, in which the benefits of
trehalose outweigh its possible disadvantages to the plant.
W096l00789 2 1 9 3 ~ 6 1 P~l/rL C ~ "
12
Several examples of such non-c~n~titutive ~lant=promoters
are known to those familiar with the art, including the
small subunit ribulose-1,5-bisphosphate carboxylase
~Rubisco) promoter, which drives the light-induced
expression of the small subunit of RUBISCO (Krebbers
[1988] Plant Mol. Biol. 11, 745-759).
Tobacco and Arabido~sis plants transformed with the coding
sequence (open reading frame, ORF) of the yeast TPS1 gene
correctly fused to the atslA promoter of a Rubisco small
subunit gene are disclosed. TPS1 encodes the 56 kDa subunit
of yeast trehalose synthase. The transformed plants are
healthy and fertile and contain trehalose in their leaves.
Untransformed tobacco or tobacco transformed with a similar
vector lacking the TPS1 gene do not contain trehalose. The
disclosed transgenic plants were obtained using A. ;=
tun~faciens to mediate the transformation, but any method
available in the art may be ex~loited, including the direct
introduction of DNA by microinjection, electroporation or
particle ~: ' ,' t (Gasser & Fraley [1989] Science 244,
1293).
One of these transformed tobacco plants (Transformant 4) is
shown to contain Tre6P synthase activity. The free 56 kDa
subunit is known to be unstable when isolated from the
intact trehalose synthase complex of yeast (~nn~hnrough &
Vuorio [1993] loc cit). Methods are described by which a
person skilled in the art can co-transform plants with the
TPS1 gene and one or both of the other yeast trehalose
synthase genes (TPS2 and TSL1) under the control of plant
promoters, e.g. the atslA promoter or some convenient
promoter that may be constitutive. Such co-transformation
may increase the trehalose content of the plants compared to
that of plants cnnt~in;ng only TPS1, because the subunits
encoded by TPS2 and TS~1 will stabilise the 56 kDa subunit.
In plants cotransformed with a Tre6P synthase gene
controlled by an inducible promoter together with one or
~ W096/00789 l3 I~/rL~
more genes for Tre6Pase~or regulatory proteins (such as the
TSL1 product) controlled by constitutive promoters, the
production of trehalose will be regulated by the ;n~n~;hle
promoter, since Tre6P synthase catalyses the first unique
step of trehalose biosynthesis. ~ =
It is disclosed that the transformed tobacco plants
producing trehalose demonstrate PnhAnr~d drought tolerance.
This is shown by experiments both with ~tArh~ leaves and
with whole plants. The improved drought tolerance is
exhibited by mature plants and by young seedlings of self-
pollinated progeny of the transformants. In these progeny,
drought tolerance co-segregated with the TPS1~ character as
revealed by the presence of the 52 kDa Tre6P synthase
subunit in green tissue of the progeny.
The water-stress tolerance of the disclosed transgenic
plants is surprising, because the amounts of trehalose found
in their tissues seem too small to provide osmotic buffering
of the intracellular contents. Possibly the trehalose acts
by protecting specific sites, such as membranes, or possibly
trehalose or its precursor, Tre6P, perturb the plant's
metabolism to cause sec~n~Ary changes that protect the cells
against stress.
~
It is disclosed that the mild morphological changes seen in
the primary transformants containing the chimeric TPS1 gene,
such as lancet-shaped leaves, reduced apicaI ~I ;nAn~ and
reduced height mainly disappeared in progeny still producing
trehalose. These alteratio~s appear to be mainly artefacts
due to tissue culture. Lowever, it is disclosed that
trehalose-producing tobacco plants, including self-
pollinated progeny, grow a little slcwer, lagging the
control plants by 1 to 2 weeks after 8 weeks' growth under
optimal conditions. This indicates that the synthesis of
trehalose in the plants, even under the control of the atslA
promoter, may slow growth. Thus, stress-induced promoters
such as LT178 (Nordin et al [1993] Plant Mol. Biol. 21, 641-
W096/00789 2 1 9 3 8 6 1 r~ llrL ~
653) may be used to further advantage to maintain normalgrowth rates and yields under non-stressing conditions
whilst providing trehalose-induced protection under stress,
as explained in Example 5.
These transgenic plants synthesised trehalose although they
were transformed with only the yeast TPSl gene encodi~g
Tre6P synthase, and not with the TPS2 gene, encoding
Tre6Pase. Thus, some plants have an endogenous capacity to
convert Tre6P into trehalose. It may be advantageous in some
cases to cotransform the plant also with a gene, such as
TPS2, encoding a Tre6Pase, to increase the rate of
conversion of Tre6P to trehalose. Furthermore, it is obvious
that similar results may be obtained with genes for Tre6P
synthase (and Tre6Pase) obtained from organisms other than
yeast. Many of these genes will be homologous to TPSl (and
TPS2) and may be readily cloned using immunological or
oligonucleotide probes derived from the enzymes and genes of
the yeast system. For example, the Tre6P synthase from
Mvcobacterium smeqmatis is disclosed to be an about 55 kDa
polypeptide that cross-reacts with anti-serum raised against
the purified 56 kDa Tre6P synthase subunit of yeast
trehalose synthase. Amino acid sequences of tryptic peptides
from the M. smeqmatis enzyme are disclosed, revealing close
sequence homology to the yeast enzyme. A person skilled in
the art can readily clone genes for Tre6~ synthase and
Tre6Pase from many organisms using these approaches. Our
invention embraces the transformation of plants with genes
for Tre6P synthase and Tre6Pase obtained from any suitable
organism, and fused to appropriate plant promoters.
Plants containing trehalose as a result of transformation
with one or more genes for trehalose synthase can be used in
several ways. For example, trehalo~e can be extracted from
the plants on a commercial scale. Such trehalose would be
cheap enough to be used in bulk applications, including the=
preservation of the flavour ana structure of food stuffs
during drying. For this application the trehalose would
21 93861
W096/00789 l~1/~L~
preferably be accumulated in a storage organ, such as the
tuber of a potato, the root of a sugar beet or turnip or the
bulb of an onio~. Methods have been described for the
transformation of these example~ and many other crop plants
(Lindsey [1992] J. Biotechnol. 26, 1-28), but our invention
is not limited to those plants that are frequently used as
models by molecular biologist6. The methods developed for
model plants can be adapted by one skilled in the art to
other plants. Thus, the present application could also be
realised for example, in the stem and leaves of a
transformed sugar cane or the fruit of a banana. Plant
promoters are known in the art (e.g., the patatin promoter)
that cause expression specificalIy in a storage organ. In
one aspect of the present invention, the coding sequence of
a gene for Tre6P synthase, such as TPSl, is fused to such a
promoter in the same way as the TPSl coding sequence was
fused to the atslA promoter, and suitable plants are
transformed with these D~A constructions. The trehalose
accumulated in the storage organs may then be extracted.
In another aspect of the invention, the trehalose
accumulated in the tissues of a plant may improve the
storage properties after harvesting. Thus, the detached
leaves of transformed tobacco lost water more slowly than
those of untransformed tobacco, and even after loosing water
did not become discoloured ~Fig. 5). This aspect is
applicable both to edible plants and to ~rni Lals.
Regarding edible plants, the shrivelling and discolouration
of the various kinds of commercial lettuces that occur after
harvesting is a serious economic burden especially to retail
outlets. The ultimate cost is passed on to the consumer.
Trehalose-producing lettuces will provide the consumer with
both cheaper and more attractive salads. Similar
r~nr;~rr~ ns apply to other food plants, especially leafy
products such as cabbages, broccoli, spinach, dill or
parsley and other green vegetables such as peas and runner
beans. Many orn ~l~, such as roses, tulips, daffodils,
are transported after cutting. Fven with expensive
W096/00789 2 ~ 9 3 8 6 ~ r~l/rL _.- / I ~
16
~ refrigerated, air freight) transport, much wastage occurs.
Again, the cost is carried by the consumer, who will ~e
provided with both cheaper and more delightful products by
trehalose-producing orn t~l.s that better retain their
water content and are less susceptible to discolouration.
In this aspect of.our invention, the Tre6P synthase gene is
fused to a plant promoter chosen 80 that trehalose
accumulates in the plant parts to be harvested. For example,
the atslA promoter causes expression of Tre6P synthase in
the leaves, upper stem and flower buds of tobacco, and
trehalose accumulates in these:parts. However, the inventors
disclose that pIants such as tobacco transport trehalose
from its site of synthesis to tissues that do not synthesise
trehalose. Thus, smaller amounts of trehalose were also
found in the roots of these transgenic tobacco plants,
although the non-constitutive atslA promoter did not cause
expression of Tre6P synthase in the roots.
In a related aspect of the invention, edible parts of
transformed plants c~nt~;n;ng trehalose, such as tomatoes,
berries and other fruits, are processed to make purees,
pastes, jellies and jams that have a fresher and richer
flavour because of their trehalose content. It has been
shown (WO 89/00012) that adding trehalose to such food
stuffs improves the preservation of their flavour,
particularly when the processing involves a dr,ving ste~. The
present invention circumvents the need to add trehalose, by
providing a plant that already cont~;nc trehalose. It may be
an advantage in this aspect of our invention to use
promoters, such as the patatin promoter, that direct the
synthesis of trehalose primarily to the storage organ of the
target plant.
It is disclosed that transformed tobacco that produ~es
trehalose has increased drought resistance. In general~
transformed plants containing trehalose may be more
resistant to drought, frost, high salinity and other
stresses than the untransformed plants. Thus, drought, frost
~ W096/00789 2 ~ ~ 3 8 b ~ ~l/rL~
and osmotic stress all-distress plants primarily by
withdrawing water from within cells, so causing damage to
membranes and proteins that trehalose is known to alleviate
in vitro (Çrowe_et al tl992] Annu. ReY. Physiol 54, 579).
In this aspect of the invention, the plant promoter used may
be one that is induced by stress. Such promoters are known
in the art, e.g. LTI78 (Nordin et al. [1993] Plant Mol.
Blol. 21, 641-653) and RAB18 (~ang & Palva [1992] Plant Mol.
Biol. 20, 951-962). This will prevent the synthesis of
trehalose until it is needed. For plants that grow well
whilst ~nta;n;ng trehalose, this aspect of the invention
can be achieved without resource to a stress-induced
promoter. However, the use of stress-induced promoters to
prevent trehalose production until it ig needed has the
additional advantages of avoiding the yield penalty that
would otherwise result from the diversion of photosynthetic
capacity to trehalose synthesis, and avoiding a possible
retardatio~ o~_growth, such as is disclosed for tobacco
harbouring the patslA-TPSl chimera. This aspect has
applications not only for food plants, but also for
, t~l plants intended for gardens or indoor display.
The transformed stress-tolerant orn t~l plants would
require less intensive and less expert care than the
corr~cp~n~;ng untransformed plants. The slower growth of
some trehalose-producing plants and minor morphological
changes observed at least in the primary transformants may
not be a disadvantage with ~rn -nt~l plants: slower growth
and novel appearance can be attractive characteristics
especially for indoor orn: ~l a,~
In yet another aspect of the invention, a gene for Tre6P
synthase is appropriately fused to a plant promoter (e.g.,
78 or RAB13) that is activated by a specific event or set
of conditions (e.g. cold or drought stress) so that
accumulation of commercially extractable amounts of
trehalose in the plant can be triggered to occur in the
mature plant shortly before harvesting, avoiding any
deleterious effects of trehalose on the early development of
W096l00789 2 ~ 9 3 8 6 1 r~llrL - ~ "
18
certain plants.
Based on the above disclosure, the transgenic plants
according to the invention can be monocotyledonous plants,
such as corn, oats, millet, wheat, rice, barley, sorghum,
amaranth, onion, asparagus or sugar cane, or dicotyledonous
plants such as alfalfa, soybean, petunia, cotton,
sugarbeet, sunflower, carrot, celery, cabbage, cucumber,
pepper, tomato, potato, lentil, flax, broccoli, tobacco,
bean, lettuce, oilseed rape, cauliflower, spinach, brussel
sprout, artichoke, pea, okra, squash, kale, collard greens,
tea or coffee_ _ _ _ _ _
It is disclosed that the yeast gene TPS1 and its product are
compatible with the biochemical machinery of tobacco: the
gene was highly expressed and ~he 56 kDa subunit caused the
appearance of trehalose. ~owever, it is known in the art
that plant genes often have lower A+T ratios than, e.g.,
microbial genes, and that the expression level of
heterologous genes in plants can be increased by altering
the codon usage, particularly near the start of the coding
sequence, towards that found in plants ~Perlak Q~_3l.=[1991]
88, 3324-3328). We envisage that these and similar
modifications of genes may be useful in the present
invention.
It is also well known that natural mutatio~s occur in genes.
These and artifical changes in the DNA seguence may alter
the amino sequences of the encoded polypeptides. As used
herein, the term TPS1 (or TPS2 or TSL1) includes all DNA
sequences homologous with TPS1 (or TPS2 or TSBl) that encode
polypeptides with the desired functional or structural
properties of the 56 kDa (or 102 kDa or 123 kDa) subunit of
yeast trehalose synthase. Similarly, the term "genes for
Tre6P synthase (or Tre6Pase or~regulatory polypeptides) n
includes not only such genes as may be readily isolated from
natural organisms (for example, by using nucleotide probes
designed from the known sequences of TPS1 or of TPS2 or
~096/00789 19 ~ L ~C/~ I /
TSL1) but also natural and arti~icial variants that encode
polypeptides with the desired functional and structural
properties of the polypeptides encoded by the originally
isolated genes.
EX~MP~ES _ _ _ _
General MAteri~l~ and Methods.
Materials. The plants used were Nicotiana tabacum cv. SR1
and Arabidspsis thaliana L. Eeynh. ecotype C-24.
The yeast genes TPSl (formerly called TSS1), encoding the 56
kDa Tre6P synthase subunit of trehalose synthase, and TS~l,
encoding the 123 kDa regulatory subunit of trehalose
synthase, were obtained from the plasmids pALK752 and
pALK754 described by Vuorio et al. ([1993] Eur. J. Biochem.
216, 849-861). The yeast gene TPS2 was obtained from a
plasmid supplied by Drs Claudio De Virgilio and Andres
Wiemken, Botanisches Institut der Universitat Basel,
Switzerland and rrnt~;ning the TPS2 gene cloned into the
SacI site of plasmid Y~pl~c1--. This gene (described in De
Virgilio et al. [1993] Eur. J. Biochem. 212, 315-323)
encodes amino acid sequences derived from the 102 kDa
subunit as shown in Table 1 of Vuorio et al, (1993, loc
cit). Antisera prepared against yeast trehalose synthase
(anti-TPS/P) and the 56 kDa subunit (anti-57K) and general
biochemicals were from the sources cited in Vuorio çt al.
(1993, loc cit). Vacuolar trehalase wae partially purified
as described by Londesborough & Varimo ([1984] Biochem. J.
219, 511-518) from a suc~gal~mel~mal~ yeast strain (A~K02967)
and did not hydrolyse sucrose, maltose or melibiose.
DNA Mani~ulations. All DNA manipulations were carried out
accordi~g to established laboratory procedures (Sambrook et
al [1989] in Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, New York~. The E. coli strains DH5~ and
W096/00789 2 19 3 8 6 1 r~llL~
MC1061 were used for plasmid preparations The promoter used
to control TPS1 expression came from the atslA gene of _
~hAli~n~, which encodes the small subunit of Rubi~co
(Krebbers et al [1988] loc cit).
To construct a chimeric atslA-TPS1 gene the atslA promoter
fragment, lacking the sequence for the transit peptide, was
amplified by PCR from the plasmid pGSFR401 Synthetic
oligonucleotide primer~ were used to create an EcoRI site at
the 5'end and an XbaI site at the 3'end of the amplified
fragment. The PCR amplification product was digested with
the appropriate restriction enzymes and, following
purification on an agarose gel, ligated into an EcoRI and
MluI digested pUC19 plasmid. The yeast TPS1 gene was
amplified from the plasmid pALR752 described above. The
resulting fragment contained 5' MluI and 3' XbaI sites.
After digestion and purification the fragment was ligated
behind the patslA in pUC19. A fragment with the promoter-
TPS1 construct was cut out with EcoRI and XbaI and then
inserted into a pBluescriptII SK' (Stratagene) derived
plasmid carrying the 3'end of the T-DNA gene G7 includlng
its polyadenylation signal and the T-DNA right border.
Finally, the entire chimeric gene was inserted as an EcoRI-
SacI fragment into the plant transformation vector pDE1001
(Denecke ç~_~Ll [1992] EMBO ~. 11, 2345-2355) r~nt~ining the
chimeric kanamycin resistance gene pNOS-NEO-3'0CS as a
selective marker. This resulted in the plasmid pROH51
carrying the chimeric patslA-TPS1-3'G7 gene. Constructions
were cloned in E. coli strain DH5~ by transformation, and
then transferred by electroporation (Dower, Miller &
Ragsdale [1988] Nucl. Acids Res. 16, 6127-6145) to
Aqrobacterium tumefaciens (C58C1 rif~) containing the non-
oncogenic Ti plasmid pGV2260 (Deblare et al Ll995] Nicl.
Acids Res. 13, 2777-2788).
Other constructs were made in similar ways.
Growth of ~lant material. For axenic growth, sterilised
~ W096t00789 2 ~ 9 3 8 ~ ~ I ~llrL ~ "
explants from Nicotiana t~ha~nm [SR1) were planted in glass
jars containing solidified MS (Murashige & Skoog [1962]
Physiol. Plant 15, 473-497) medium supplemented with 2 ~
sucrose (MS-2). These jars were then placed in a controlled
growth environment in a culture chamber where plants were
allowed to grow at 22 ~C with a 16 h photoperiod. Explants
were regularly transferred to new jars and MS-2 medium for a
~n~;nn~ growth of axenic material. Greenhouse plants were
grown in soil in pots and watered daily. Transformed A.
th~ n~ plants were first grown axenically in baby-food
jars in a controlled environment as described for tobacco
above, but were later transferred to soil in pots in the
greenhouse for Geed production. Seeds from the primary
transformants were either directly planted in soil for new
seed pro~n~t;~n or surface sterilised and grown axenically
in 24-well tissue culture plates for molecular analysis.
P~nt transformation. Tobacco and A. thaliana were both
transformed according to the following protocol, with
starting material being excised leaves of tobacco and roots
of A. thaliana. The transformation and tissue culture were
essentially as described by Valvekens et al ([1988] Proc.
Natl. Acad. Sci. U.S.A. 85, 5536-5540) with the following
modifications. Isolated roots or leaves were pr~;ncllhat~d on
801;~;f;~ callus-in~ ;ng medium (CIM) for 4 days, roots
were cut into small segments (1 - 2 mm) and leaves were cut
into larger pieces (10 - 20 mm) and transferred into 20 ml
liquid CIM. 3',5'-Dimethoxy-4'hydroxyacetophenone was added
(0.2 mg/l) prior to Aqrobacterium (C58C1 rif~) infection. The
bacteria used for infection were propagated overnight in YEB
medium (Vervliet et al. L1975] J. Gen. Virol. 26, 33-48)
~nt~;n;ng a~ riate antibiotics at 28 ~C, and collected
by centrifugation. The bacterial pellet was then resuspended
in 10 mM MgS04, added to the plant tissue and mixed gently
for about 15 min. Excess liquid was poured off and the roots
blotted on sterile filter paper. After co-cultivation for 2
days on solid CIM, the plant tissue was rinsed 3 - 4 times
with liquid CIM to wash off bacteria, and transferred to
W096l00789 2 1 ~ 3 ~ 6 1 P~llrL ~ "
selective shoot induction medium (SIM). After 7 days of
growth, explants with differentiated morphogenic sectors
were transferred to fresh SIM.
Protein extra~tion f~r Western ~n~lysis~ Plant sample6 of
100 - 200 mg fresh weight were homogenised-with a glass rod
in 1.5 ml micro centrifuge tubes with 100 ~l of protein
extraction buffer (50 mM Tris/HCl pH 7.2, 250 mM sucrose, 5
mM EDTA, 10 mM MgClz, 1 mM CaCl2, 10 mM ~-mercaptoethanol, 1
mM phenylmethylsulfonylfluoride (PMSF), 30 ~M pepstatin, 50
~M leupeptin and 15 ~M aprotinin. Insoluble material was
removed by two centrifugations (13,000 g for 10 min). The
protein concentration in the supernatants was measured
according to Bradford ([1976] Anal. Biochem. 72, 248-254)
using bovine albumin as standard. E~ual amounts of soluble
proteins were loaded onto SDS-PAGE for immunological
studies.
SDs-pAGE ~n~; nlo~i~l technioues. Protei~s were
separated by SDS-PAGE (Laemmli [1970] Nature 227, 680-685).
For detection of proteins ; n~l otting was made with the
antiserum anti-57 (diluted 1/1000) and an anti-rabbit
alkaline phosphatase conjugated second antibody. For Western
blotting the proteins were electrophoretically transferred
to nitro-cellulose filters and stained with 0.5 ~ Ponceau
Red in 5 ~ acetic acid to confirm e~ual loading of the
samples and success of transfer. The filters were
subsequently blocked overnight with 5 ~ fat-free dry milk
powder and probed and stained by standard methods.
Tre6P svnthase Assaye. About 500 mg of frozen plant material
was weighed and then ground to a fine powder with a mortar
and pestel on solid CO2. The powder=was transferred to 0.7 ml
of 50 mM HEPES/KOH pH 7.0 containing 1 mM benzamidine, 2 mM
MgCll, 1 mM EDTA and 1 mM dithiothreitol (HBMED) ~nnt~;n;ng 1
mM PMSF, and 10 ~g/ml each of p~epstatin A and leupeptin and
allowed to melt. The resulting homogenate was centrifuged 10
min at 17 000 g and the supernatant assayed for Tre6P
2 1 938~ 1~ ~096/00789 Pcl/r
23
syntha~e essentially according to Lapp et al. ([1971] ~.
Biol Chem. 246, 4567-4579). Sample ~10 ~l) was added to 90
~l of reaction mixture cnnt~;ning 40 mM HEPES/KOH pH 7.0, 10
mM MgCl2, 10 mM glucose 6-phosphate, 5 mM fructose 6-
phosphate, 5 mM uri~;nP~iphnsphoglucose (~DPG) and 1 mg/mlbovine albumin and ;nr~h~te~ at 3Q ~C for the required time.
The react;on ~as stopped by 2 min at 100 ~C. Sugar
derivatives (;n~ ;ng any sucrose formed) except for.
trehalose and trehalose 6-phosphate were destroyed by adding
50 ~l 0.6 M HCl and heating for 5 min at 100 ~C and then 50
~l of 8 % NaOH and heating for 15 min at 100 ~C. ~, ~;n;ng
carbohydrate (i.e., trehalose and trehalose 6-phosphate)
were then determined with the anthrone assay (Trevelyan &
Harrison [195Ç] Biochem. J. 63, 23-33).
_ ~
T~eh~1nse As8aYs. About 500 mg of frozen plant material was
quickly weighed into a glass tu~e. Hot, distilled water (1
ml) was added and the mixture was boiled for 20 min, the
leaf material being broken up at intervals with a blunt
glass rod. The liquid phase was ~oll ~t~ with a pasteur
pipette and the solid re-extracted with 0.5 ml of water. The
combined liquid phases were clarified by centrifugation.
Combined solid residues were dried to constant weight at 107
~C (the dry weights of leaves averaging 5.1 % of the fresh
weights). The supernatant was analysed using a Dionex DX-300
liquid chromatograph equipped with a Dionex pulsed
electrochemical detector (PBD-2). Samples (20 ~l; in
triplicate) were in~ected via a Carbopac PA-1 (4 x 50 mm)
pre-column onto a Carbopac PA-1 (4 x 250 mm) column and
eluted with water at 1 ml/min. The eluate was mixed with
post-column reagent (0.6 ml/min of 0.3 M NaOH). Trehalose
emerged at about 3 min, well before the glucose and sucrose
peaks, which emerged at ca 20 min.
W096l00789 21 93~6 I r~ Lc~
24
E le 1. ~rehalo$e ~roduction bv tobacco=~lants
transformed with the Yeast TPS1 qene under-the ats1
~romoter.
Tobacco transformants and control plants were grown up both
in sterile, "in vitro'~, conditions and in a greenhouse
Mature transformants had no obvious phenotype compared to
the untransformed controls or controls transformed with the
vector pDE1001 (lacking TPS1). Leaves were=collected at 0900
h, frozen and stored at or below -70 ~~ prior to analysis.
Out of 26 kanamycin-resistant transformants tested, 20 were
found to produce detectable levels of immunoreactive
polypeptides of the expected size when probed with an
antiserum raised against the purified 56 kDa Tre6P synthase
subunit of yeast trehalose synthase. Examples are shown in
Fig. 2. Table 1 summarises the trehalose contents of the
leaves.
21 938~
~ 'W09~00789 r~ J 5'~ J
~hl~ 1~ Trehalose contents of TPS1-tra~ormants of tobacco
Tobacco pl antS~ecial Treatmen~ Trehaloce
(mq/q fresh leaf)
I~ vitro ~lants
Untransformed SR1 - s 0.002
pDE1001 Control - s 0.002
~ ~ U~Il~llL 1 0.02
I._ll~ ULll~llL 3 _ 0 0O9
T,_l.~ .,.. ,_,.1 4 - 0.067
Tr~nc~nrr~nt 8 - 0 075
Trans~ormant 8 EthaLol extraction iLstead of water 0.055
~ro~nhnl~c~ ~l~ntq
pDE1001 Control - s 0.002
I.~u~ ULIl~llL 1 - - 0.16
Trana nrr-nt 4 - 0.16
T.~ .l 4 Alkaline rh~cphAtAce- 0.13
Trnnc nrr-nt 5 _ 0.052
Trans ormant 6 - 0 044
T,_.. ~ r... 1.l 8 ~ ~'
T,_.. ~ .... .. 1 8 Specific trehalase 0.021
Trans:ormant 19 _ 0 053
Trancinrr~nt 19Alkaline rhncphAtAc~' 0.060
I.~,~.ULIl~llL 19Specific trehalase 0.016
T,~"_ .. ",_.,1 25 - 0.036
~ 26 - 0.11
' T_e extract was treated with alkaline ll,.,~under conditions such
that carrier rl'C]trehalose 6-phosphate was ~ h~ h~ylated~
These results disclose that the yeast TPS1 gene is
efficiently expressed in tobacco when its promoter is
replaced by the atslA promoter. The specific signal observed
on Western blots has the correct molecular weight. The
strongest signals (e.g., that from Transformant 4 grown in
vitro) were only slightly weaker per unit of protein applied
to the gel than the signals obtained from stationary phase
yeast. Expression of TPS1 was accompanied by the appearance
of trehalose in the leaf tissue, identified both by its HPLC
behaviour and by the fact that it was degraded by a highly
specific trehalase (see also Fig 3). Different transformants
expressed the TPS1 product to different levels for reasons
that have not yet been established, and (with the apparent
exception of Transformant 8) the amount of trehalose found
in the leaves roughly correlated with the strength of the 56
W096/00789 2 1 9 3 8 6 1 l~l/r~8 ~ "
kDa signal in the Westerns ~compare Fig. 2 with Table 1).
Although these transformants did not carry a gene encoding a
recombinant Tre6Pase, no evidence was found that the plants
accumulated more.Tre6P than trehalose Apparently, tobacco
possesses phosphatases capable~of converting Tre6P into
trehalose, disclosing that for~at least this plant, the key
enzyme required to introduce a trehalose synthetic pathway
is Tre6P synthase, and introduction of this enzyme alone is
adequate, though not n~c~ rily optimal.
Determination of the tissue distribution of the 56 kDa Tre6P
synthase subunit in transgenic line 4 showed that
substantial amounts of this polypeptide could be found in
all green parts of the plant except the lower stem, but not
in the roots (Fig. 4). This distribution is in accord with
the tissue specificity of atslA gene expression (De Almeida
~_a~ [1989] Mol. Gen. Genet. 218, 78). Fig 4 also shows
that tissues in which Tre6P synthase was expressed also
contained trehalose. In addition, smaller amounts of
trehalose were found in the roots, indicating that
transgenic tobacco transports trehalose from its site of
synthesis to other tissues.
The results also disclose that tobacco plants expressing
TPS1 under the atslA promoter and accumulating trehalose in
their green tissues during daylight are healthy and normal
in appearance. Although primary transformants c~nt~;ning the
chimeric TPS1 gene had some minor morphological alterations,
such as lancet-shaped leaves, reduced apical ~ ;n~n~e and
reduced height (see Figs 5 & 6), most of the changes were
not exhibited in self-pollinated TPS1-positive progeny that
still produced trehalose (see Fig 6). Thus, the
morphological changes in the primary transformants appear to
be artefacts of the tissue culture rather than results of
trehalose production. ~igh level production of the Tre6P
synthase subunit and the presence of trehalose did, however,
lead to a 20 to 50 ~ decrease in the growth rate under
normal conditions of the transgenic plants compared to
~ 'W096/00789 27 F~llr~
untransformed control~. The apparently normal phenotype of
these transgenic plants is in contrast to the reported
toxicity of exogenously added trehalose to certain plants
(Veluthambi ç~_al [19811 loc.cit.). These results disclose
that in ~lanta production of trehalose under the control of
the atslA promoter is not toxic to tobacco, although it may
contribute to some reduction in growth rate. Tn~nr;hle
promoters triggered by specific eveLts, including drought or
cold, may be used to minimise the reduction in growth rate,
by causing the production of trehalose only when it is
needed. ~
On a protein basis, the trehalose contents of the best
transformants in Table 1 (e.g. 2 16 mg/g protein for
Transformant 4~ are already at least 20 ~ of the level at
which a clear i~ v~ t in thermotolerance is observed in
yeast (De Virgilio ~et al [1990~ FEBS Letters 273, 107-110).
Some TPS1-transformants and controls were assayed for Tre6P
synthase activity. The results shown in Table 2 are means
the extreme ranges from duplicate zero and 15 or 30 min
assays. For the controls, Tre6P synthase activity did not
differ from zero. For Transformant 4, an acid- and alkali-
stable carbohydrate AC' 1 Ated in the presence of UDPG and
Glc6P. This ~ Ation required Glc6P, but not fructose 6-
phosphate (Fru6P; this hexose phosphate activates the native
trehalose synthase complex of yeast) and was prevented when
~DPG was replacea by ADPG (the enzyme purified from yeast
also cannot use ADPG). The ac~ t~ carbohydrate is
presumably trehalose or Tre6P, because other possible
products are destroyed by the hydroIyses. HPLC analysis
showed it was not trehalose. Thus, under these in vitro
assay conditions, Tre6P is synthesised by extracts of
Transformant 4 faster than it is converted to trehalose.
This shows that the overall rate of trehalose synthesis in
the leaves may be increased by cotransformation with TPS2,
which encodes the Tre6Pase subunit.
W096l00789 2 1 ~ 3 8 6 1 l~llr~s ~ "
28
The Tre6P synthase activity of yeast extracts found by the
method used in Table 2 agreed with that found by measuring
the appearance of UDP as described by Tnn~lmchnrnugh & Vuorio
([1991] J. Gen. Microbiol. 137, 323-330). Furthermore, yeast
5 extracts measured in the presence of extracts of tobacco
plants were not inhibited. Thus, the absence of activity in
the control plants in Table 2 is not due to interference by
some factor present in tobacco extracts.
'rslhle 2. Tre6P svnthase activit~f of Tpsl-tL~ r~Ll ~ Ond control tobacco
leave6 (All results are with plants ~rown under sterile conditions in
vitro).
~ransformant ~ A88av Mixture Tre6P svnthase Activitv
15 min 3 0 min
UILLoll~L~Ll.~d Control Complete 3 :F 47 3 :F71
PDEloo1 Control Complete 22 ~ 35 7 :F 8
ILOII5LULIIICIIIt 4, ~Cxpt.1 Complete 2ss :F147 60 ~ 6
Tr~ncfnrr~n~ 4, sxpt.2 Complete 128 :F 39155 :F22
~ess Glc6P 12 ~ 19 -1 ~ 12
Less Fru6P 153 1: 10144 :F 3
ADPG instead Cf UDPG O 1: s7 4 :F 21
The Tre6P synthase activity found in Transformant 4 was
35 labile. With some extracts, the activity disappeared during
a few hours storage on ice. However, the specific band seen
in Western analyses was still present at nearly its original
strength in extracts stored for 24 h at room temperature.
Thus, it is probable that the conformation of the Tre6P
40 synthase subunit changes during storage of tobacco extracts.
These results indicate that increased Tre63~ synthase
activity will be achieved by transforming the tobacco
simultaneously with TPSl and one or more of the other
subunits of yeast trehalose synthase, thereby increasing the
45 conformational stability of the Tre6P synthase subunit.
~ 'W0 96100789 ~ ~ 9 3 ~ 6 1 F~,l/rL _. l l
F le 2. T~ansqenic Arabido~sis thallAn~.
A. thaliana plants cnnt~in;ng TPSl~under the at~IA promoter
were constructed in the same way as the tobacco
transformants described above. These transformed Ar~hido~sis
plants are also healthy and normal in appearance and
produced fertile seed. Western analysis of plants grown up
from seeds of the primary transformants showed that they
cnnt~;n~ the 56 kDa subunit of yeast trehalose synthase
(Fig 10). It is an obvious expectation that these plants
accumulate trehalose in their green tissues.
Exam~le 3. Drv resistance o~ t~ehalose-Producinq tobacco
~lants.
To determine whether the amounts of trehalose produced in
the transgenic tobacco were sufficient to enhance their
drought tolerance, detached leaves from in vitro propagated
control and transgenic-lines were subjected to air drying
(25~ relative humidity, RH) (Fig. 5). Whereas detached
leaves of control plants rapidly lost water and showed clear
signs of browning after 3 hours of stress, the leaves of
trehalose-producing plants remained green up to 24 hours
with initially clearly decreased water 1O8s. Thus, the
protective effect of trehalose appears two-fold: First
detached leaves from transgenic plants synth~ci 7i ng
trehalose appeared initially to have improved water
retention. Only after prolonged drought treatment did this
difference between trehalose-producing and control plants
disappear. Secondly, the control leaves exhibited clear
signs of senescence and browning. In contrast, the leaves
from trehalose-cnnt~;n1ng plants appeared green at least for
24 hours and only ~t~n~d drought exposure (for several
days) resulted in some browning. There was a clear
difference between trehalose-producing plants and control
plants, even at the same water content. Thus, trehalose
appears to protect the plant tissue from the effects of
W096l00789 21 9 3 8 6 1 I cl/r~l~ "
dehydration, as well a6 decreasing the rate of water Ioss
The browning could at least partly be due to the Maillard
reaction where reducing sugars react with free-amino groups
of polypeptides and amino acids producing a brown colour. As
a nonreducing sugar trehalose does not participate in thi~
reaction and it even inhibits the reaction between other
sugars and proteins (Roser ~ Colaco [1993]) New ~cientist
1873, 24).
The protection by trehalose of excised leaves from drought
injury indicated~that trehalose could have a similar effect
even at the whole plant level. To assess whether trehalose
could enhance the drought tolerance of intact plants we
exposed in vitro propagated whole plants to air drying (30
R~, Fig. 6A). Within 3 to 4 h the control plants had lost
turgor and wilted. In contrast, the trehalose-producing
transgenic plants only showed signs of turgor loss after
prolonged air drying. After 17 hours of desiccation
treatment the plants were rehydrated and the survival of the
plants du~ ted (Fig. 6A). Nontransformed control plants
did not survive this treatment, as no recovery was detected
even after prolonged rehydration. In co~trast, trehalose-
producing transgenic lines, although clearly wilted and
having lost most of their tissue water (down to 30 ~
residual fresh weight), survived 17 hours of desiccation.
Enhanced drought survival was~also manifested :in young
seedlings (Fig. 6B). Exposure of 3-week old sPP~l ;ng~ from
transgenic line 8 together with nontransformed and vector
transformed control seedlings to air drying (50 ~ RH)
demonstrated clear differences in drought tolerance between
the trehalose-producing and control plants. The transgenic
trehalose-pnc; t; ~r~ line 8 showed both delayed loss o~ turgor
and ~n~r~n~P~ survival of dehydration stress as compared with
the control plants (Fig. 6B).
~ W096/00789 2 ~ 9 3 8 ~ r~ . "
31
R~ le 4. Trehalgse production bY ~lants co-transformed
wi~h a qene enco~;nq a Tre6P svnthase and one or more qenes
ençodinq a Tre6Pase or requlatorY ~oly~e~tide.
A person skilled in the art can pre~are vectors ~nt~;n;ng
the coding sequences of the yeast genes TPS2 and TS~1 under
the control of the atslA promoter and use them to transform
tobacco, Arabidg~sis and other plants ~y the methods
described in General Materials and Methods and Example 1.
Plants simultaneously transformed with TPS1 and one or both
of the other genes, TPS2 and TS~1, can be obtained by cross-
breeding of individual transformants, by further
transformation of one transformed plant with a second gene,
or by transformation with vectors c~nt~;n;ng two or three of
the genes linked to appropriate promoters: for example, TPSl
can be linked to the non-constitutive atslA promoter, to
provide control over trehalose synthesis, and the other
gene(s) driven by constitutive promoters.
It is expected that the controlled expression of genes for
two or more subunits of the yeast trehalose synthase
complex, at least one being the 56 kDa subunit, will result
in increased accumulation of trehalose in green tissues of
the plant, because the 56 kDa subunit will be stabilised by
the presence of theiother subunit~s). Furth~ 'e,
introduction of the 102 kDa, Tre6Pase subunit will be
beneficial because it will decrease the potential
tion of Tre6P expected when the stability of the
Tre6P synthase subunit is increased.
It is obvious that for this type of construction the TPSl
coding sequence can be replaced by the coding sequence of
some other Tre6P synthase structural gene, the TPS2 sequence
by that of some other Tre6Pase gene and the TS~1 sequence by
that of some other gene Gnr~;ng a polypeptide that confers
regulatory properties or stability upon the Tre6P synthase
and Tre6Pase in the same way as the TSL1 product confers
such properties upon the other subunits of native yeast
W096/00789 21 938~ ~ r~ s~
trehalose synthase.
Exam~le 5. Transformation of ~lants with qenes for Tre6P
svntha8e under the control of stress-induced ~romoters.
Plant promoters, such as LTI78 (Nordin et al [1993] Plant
Mol. Biol. 21, 641-653) and R~B18 (~ang & Palva [1992] Plant
Mol. Biol. 20, 951-962), are known that are induced in
response to drought and cold stress. By transforming
tobacco, Arabido~sis and other plants with the_coding_
sequence of a gene ior Tre6P synthase, such as TPSl under
the control of such a stress-induced promoter, alone or
together with the coding sequences of ~enes for Tre6Pase or
a regulatory polypeptide or both, such as TPS2 and TS~l
under the control of ary conveniçnt plant promoter, the
accumulation of trehalose in plant tissues can be made to
occur only in response to these stresses. The advantage i8
that levels of trehalose that might be ~ terious to
certain tissues of certain plants and which can also
represent a yield-decreasing diversion of photo-synthetic
capacity and possibly retard the growth of the plant, would
arl lAte only (1) when the plant is exposed fortuitously
to stress (the benefits of the protection afforded by the
trehalose then overcoming any deleterious effects) or ~2)
when the plant is deliberately exposed to stress in order to
cause the ~r( lAtion of trehaloge which will thçn be
r~trArt~ from the harvested plant.
Exam~le 6. Purification, analvsis and cloninq of othçr Tre6P
svnthases
Whereas there may be other metabolic routeg to trehalose, it
seem8 clear that the main synthetic pathway is via Tre6P, as
described by Cabib & ~eloir ~958, loc. cit.). The key
enzyme in this pathway is Tre6P synthase, since, as
disclosed above, once Tre6P has been made, many cells will
~ W096/00789 2 ~ 9 3 8 6 ~ P_llr~_ ~ "
33
be~capable of dephosphorylating it to free trehalose. Thus,
the key concept in the present invention is to introduce
Tre6P synthase activity into the target plant. It is not of
primary importance where this activity comes from. We have
used the yeast enzyme, which happens to be a subunit of the
yeast trehalose synthase complex, which ~nnt~;n~ at least
two other subunits. In cases like this, it is possible that
optimum activity of the Tre6P synthase subunit may require
the presence of one or more of the other subunits, though
evidently the 56 kDa Tre6P synthase subunit from yeast
functions effectively in tobacco, without the 102 and 123
kDa subunits.
It is well known that enzymes catalysing the same reaction
in widely differe~t organisms are often homologous, so that
once one member of the family has been cloned, the task of
cloning other members is facilated. Since the yeast Tre6P
synthase was cloned (Londesborough & Vuorio, [1992] USPA
07/836,021) it has become clear that there is a family of
homologous proteins, including Tre6P synthase from ~, coli
and a protein from Meth~nnha~Grium th~ Lotrophicum
(McDougall et al, [1993] FEMS Microbiology Letters 107, 25-
30). We decided to test whether other organisms also
~nnt~;n~ homologous Tre6P synthases.
Mvcgbacterium smeqma~is ront~;nq a heparin-activated Tre6P
synthase which has been partially purified and studied by
Elbein's group (Liu et al, [1969] J. Biol. Chem. 244, 3728-
3791; Lapp et al [1971] J. Biol Chem. 246, 4567-4579;
Elbein & Mitchell [1975] Arch. Biochem. Biophys. 168, 369-
377; Pan et al, [1978] Arch. Biochem. Biophys. 186, 392-
400). The enzyme purified by these workers had a specific
activity of 0.8 U/mg protein at 37 ~C, with Glc6P and UDPG as
substrates (the enzyme can use a spectrum of nucleoside-
diphospho-glucose derivatives) and in the presence of
optimal heparin. The preparation cnnt~;n~d two polypeptides
with SDS-PAGE molecular weights of about 45 and 90 kDa. We
modified the authors~ purification procedure by including
W096l00789 2 1 ~ 3 ~ 6 1 P~l/rL ~ ~ "
34
protease inhibitors and other protein p~otecting agents in
the buffers used, and adding a~final chromatographic~step in
the presence of the non-ionic detergent, Triton X-100. Our
final procedure was as follows:
1) M. smeqmatis cells~(28 g fresh weight grown for 3 ~ays in
~uria broth and stored frozen) were allowed to melt in 40 ml
of 50 mM HEPES/KOH pH 7.5 containing 1 mM benzamidine, 2 mM
MgCll, 1 mM EDTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonylfluoride IPMSF) and 10 ~g pepstatin
A/ml, and then broken in a French press. The homogenate was
centrifuged for 20 min at 28 OOog. (NH4)2SO~ (30 g for every
100 ml) was added to the supernatant The precipitated
protein was collected, dissolved in 20_mM HEPES/KOH pH 7.5
containing 0.1 mM EDTA and 0.2 mM dithiothreitol (HED
buffer) c~nt~;n;~g 1 mM PMSF and 10 ~g pepstatin A/ml and
dialysed overnight against HED buffer.
2) The dialysate was applied to a 3.4 x 25 cm column of
DEAE-cellulose and the enzyme eluted at 20 ml/h with a
linear gradient to 0.9 M NaCl in 1.2 1 of HED buffer.The
peak enzyme fractions (at 0.2 M NaCl) were pooled (33 ml),
and adjustea to 0.5 mM PMSF and 5 ~g pepstatin A/ml. Protein
was precipitated by addition of 9.9 g of (NH4)2SO~ and
dissolved in 50 mM Tris/HCl pH 7.5 r~t~;n;ng 50 mM NaCl,
0.1 mM EDTA and 0.2 mM dithiothreitol (TNED buffer).
3) The dissolved proteins (3.7 ml) were run through a 2.8 x
34 cm column of Sephadex G100 equilibrated with TNED buffer
at 36 ml/h. Peak enzyme fractions were immediately applied
to a 1.5 x 8.5 cm column of Heparin Sepharose in TNED
buffer. The column was developed with a g~ nt to 1.0 M
NaCl in 100 ml at 5 ml/h. Enzyme eluted at about 0.5 M NaCl.
4) A sample of the Heparin Sepharose eluate (fraction H37 in
Table 3) was transferred to TNED/O.1 ~ (v/v) Triton X100
over a PM10 membrane in an Amincon cell and then applied to
a 0.7 x 8 cm column of Heparin Sepharose equilibrated with
~ ~096/00789 21 9386~ r~llrl ~ "
TNED containing 0.1 ~ (v/v) Triton X100 and eluted ~ith a
gradient to 1.0 M NaCl in 50 ml of the same buffer.
TAhle 3. Purification Df Tre6P Svnthase from M. sme~r~tis.
Tre6P syntha5e was a9sayed as described by Londesborough and
Vuorio (1993, loc.cit.) but at 35 CC and in the presence of
0 25 ~g heparin~ml.
Fraction Vol~me Sp. Activit,y Total Activitv
(ml) (mU/mg) (U)
23 000g Sup~nAtAnt 154 9 25 0
~
Dialysed (NH~)zSO,
precipitate 52 .21 35.2
DE52 eluate 33 139 23.4
G100 eluate 21 385 14.6
First Heparin Sepharose eluate
Fraction H33 2.7 1880 0.9
Fraction H34 2.7 4380 2.1
Fraction E35 2.7 6190 2.7
Fraction H36 2.7 7890 2.8
Fraction H37 2.7 6340 1.6
Second Heparin Sepharose eluate
(Only H37 applied)
Fraction T21 1.4 ND 0.08
Fraction T22 1.4 1YD 0.14
Fraction T23 1.4 1~D 0~06
W096l00789 21 93861 l~l/r~ ~ "
Although the speci~ic activities a~ter the ~irst Heparin
Sepharose step were much higher than reported by Pan ~
(1978), SDS-PAGE (Fig 7) showed that these fractions were
not pure. Surprisingly, a band at about 55 kDa was the only
major band for which the intensity correlated well with
enzyme activity. The 55 kDa band was excised and digested in
the gel with trypsin and the tryptic peptides were separated
by HPLC and sequenced as described (Londesborough and
Vuorio, 1993 loc. cit,.). Seq~uences are shown in Table~4 and
in the Sequence Listings. For peptide peaks 29 and 31, which
gave double sequences, the amino acids have been arbitrarily
assigned to SEQ ID NOs 3 & 4 (from peak 29) and to SEQ ID
NOs 6 ~ 7 (from peak 31) in the same way as shown in Table
4. The first 9 residues of Peptide 13 are 39 ~ ent;r~l to
residues 250-258 (VGAFPIGID; see Vuorio et. sl,, 1993~loc.
cit.; EMBL accession no. X67499) of the 56~kDa Tre6P
synthase from yeast.
Table 4. Amino acid sequences of internal trvPtic PePtides
obtained from Tre6P svnthase Purified from M. smeamatis.
PePtide PeakSeauence Seauence Listinq
13 ~~ VGAFPISIDSAEL SEQ ID NO:1
21AT/GFLDALAATGETGDsGvT SEQ ID NO:2
29 (double) ~vv Vi~ SEQ ID NO:3
YLEGAR SEQ ID NO:4
QVLAHDVDR SEQ ID NO:5
31 (double) IGGAQPAD SEQ ID NO:6
VGALQVLL SEQ ID NO:7
43 ~v~v~ SEQ ID NO:8
To confirm that this about 55 kDa band represents the Tre6P
synthase, further purification was attempted. The enzyme
bound strongly to a ~DP-glucuronate agarose column, but
could not be recovered. There~ore, fraction H37 was
~ W096/00789 2, 9 3 8 ~ /rL l "
37
transferred to a buffer rnnt~in;ng 0.1 ~ Triton Xloo (two
thirds of the activity was lost during this transfer) and
rechromatographed on Heparin Sepharose in the presence of
Triton X100. Fig 8 shows that the about 55 kDa band was the
only Coomassie blue reactive material present in the active
fractions, apart from two faint bands smaller than
cytochrome c. Evidently, the 9Q kDa polypeptide reported by
Pan et al (1978; loc. cit.) is not an essential component of
this Tre6P synthase from M. smeqmati6, and the size of the
essential polypeptide, about 55 kDa, is notably bigger than
that of the smaller, 45 kDa, component reported by these
authors. Fig 9 shows that this polypeptide is recognised by
antiserum raised against the 56 kDa Tre6P synthase subunit
of yeast trehalose synthase but not by pre-immune serum,
showing that the Tre6P synthase of M. smeqmatis shares
antigenic ~t~rm;r~nt~ with the yeast enzyme. The nature of
the other immunoreactive bands present in relatively crude
preparations of the enzyme (see lanes 5 and 6 of Fig 9) has
not been investigated: not all of them arc artefacts due to
the large protein load, but represent related proteins.
Nevertheless, the antiserum raised against the yeast enzyme
can be used to detect and isolate positive clones from host
cells transformed with a M. smeqmatis gene bank. Similarly,
the amino acid sequence data in Table 4 can be used to check
the sequence of the isolated gene. The immunological and
amino acid sequence similarities between the enzymes from
yeast and M, smeqmatis indicate that nucleotide probes
designed from the TPS1 gene may also be successfully used to
screen for the M. smea~matis gene.
In conclusion, immunological and sequencing studies of the
M. smeqmatis enzyme confirm that the Tre6P synthases from
different organisms are members of a family. The genes of
these enzymes may be used in the same way as TPS1 to make
transgeni~ plants that synthesise trehalose and have
improved stress-tolerance.
W 096~00789 ~ ~ 9~8~ I r~ c /I
38
SEQUENCE LISTING
(1~ GENER~L INFORMATION
(i) APPLICANTS: Alko Group Ltd
and
L~.~de~L~L~yll, John
Tunnela, Outi
Holmstrom, ~jell-Ove
Mantyl~, Einar
Welin, Bjorn
Mandal, Aoul
Palva, E. Tapio
15 lii) TITLE OF INVENTION TrAn~n;~ plants producing trehalose.
(iii) NUMBER OF SEQUENCES: 8
(1V) UU~S~U. JL~1~'~ ADDRESS-
20 A) ADDRESSEE: Alko Group Ltd
3) STREET: Salmisaarenranta 7
C) CITY: Helsinki
D) STATE:
E) COUNTRY: Einland
25 F) POSTAL CODE: FIN-00180
(v) COMPUTER REA~ABLE FORM:
(A) MEDIUM TYPE: Diskette, 1.5 inch, 720 Kb
(B) COMPUTER: IBM PC/XT/AT
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: WP'i.1 file exported as DOS text file
(vi) CJRRENT APPLICATION DATA:
(A) APPLICATION NUMBER: ?
(B) FILING DATA: 29 June 1995
(C) CLASSIFICATION: ?
(vii) PRIOR APPLICATIQN DATA:
(A) APPLICATION NUMBER: FI 943133
(B) FILING DATE: 29-JUNE-199
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE r~T~rTT~TcTIcs
(A) LENGTF: 13 amino acids
(B) TYPE- ~ nn~ri ~
(D) TOPOLOGY: Linear
(ii) MOLECULAR TYPE: Peptide
(iii) HYPOTHETICAL: no
(iv) FRAGMENT TYPE: N-terminal
55 (v) SEQUENCE DESCRIPTION: SEQ ID NO:1
Val Gly Ala Phe Pro Ile Ser Ile Asp Ser Ala Glu Leu
(2) INFORMATION FOR SEQ ID NO:2
(i) SEQUENCE r~rT~RT.CTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: aminoacid
(D) TOPOLOGY: Linear
(ii) MOLECULAR TYPE: Peptide
21 q386~
~ W096/00789 , r~l/r 't. Il
39
(lil) HYPOTHETICAL: no
(iv~ FRAGMENT TYPE: N-terminal
(v) SEQ~ENCE DESCRIPTION POR SEQ ID NO:2
- Ala Xaa Phe Leu Asp Ala Leu Ala Ala Thr Gly Glu Thr Gly Asp
Ser Gly Val Thr
(2) INFORMATION FOR SEQ ID NO:3
(i) SEQUENCE ~DD~ TcTIcs
(A) LENGTH: 9 amino acids
(B) TYPE: aminoacid
(D) TOPOLOGY: Linear
~ii) MOLECULAR TYPE: Peptide
(iii) ~Y~u~ CAL: yes
(iv) FRAGMENT TYPE: N-terminal
(v) SEQ~ENCE ~9UKl~JlUN: SEQ ID NO:3
Arg Val Val Val Asn Asn Thr Ser Alg
(2) INPORMATION FOR SEQ ID NO:4
(i) SEQUENCE ~
(A) LENGTH: 6 amino acids
~ (B) TYPE: ~ ' n~
(D) TOPOLOGY: Linear
(ii) MOLECULAR TYPE: Peptide
(iii) nY~ulr~llu~L: yes
(iv) FRAGMENT TYPE: N-terminal
(v) SEQUENCE ~S~ .lUl~ FOR SEQ ID NO:4
Tyr Leu Glu Gly Ala Arg
(2) INFORMATION FOR SEQ ID NO:5
(i) SEQUENCE ~M~ DrT~T.cTTrc
(A) LENGTH: 9 amino acids
~(B) TYPE: aminoacid
(D) TOPOLOGY: Linear
~ (ii) MOLEC~LAR TYPE: Peptide
(iii) ~Y~l~llCAL: no
(iv) FRAGMENT TYPE: N-terminal
(v) SEQUENCE DESCRIPTION: SEQ ID NO:5
Gln Val Leu Ala ~is Asp Val Asp Arg
W 096l00789 2 ~ 93~ ~ r~llr~-~ "
4Q
(2) INFORMATION FOR SEQ ID NO:6
(i) SEQUENCE r~rr~RTcTIcs
(A) LENGTH: 8 amino aclds
(B) TYPE: aminoacid
(D) TOPOLOGY: Linear
(ii) MOLEC~LAR TYPE: Peptide
(iii) HYPOTHETICAL: yes
(iv) FRAGMENT TYPE: N-terminal
(v) SEQBENCE DESCRIPTION FOR SEQ ID NO 6
Ile Gly Gly Ala Gln Pro Ala Asp
(2)INFORMATION FOR SEQ ID NO:7
(i) SEQUENCE r~rT~rcTI
(A) LENGTH: S amino acids
(B) TYPE: aminoacid
(D) TOPOLOGY: Linear
(ii) MOLECULAR TYPE: Peptide
(iii) HYPOTHETICAL: yes
(iv) FRAGMENT TYPE: N-terminal
(v) SEQ~ENCE DESCRIPTION: SEQ ID NO:7
Val Gly Ala Leu Gln Val Leu Leu ~~
(2) INFORMATION FOR SEQ ID NO:8
(i) SEQUENCE r~Ti~rT~TcTIcs:
(A) LENGTH: 8 amino acids
(B) TYPE: aminoacid
(D) TOPOLOGY: Linear
(ii) MOLECULAR TYPE: Peptide
(iii) ~Y~Ul~hll~ML: no
(iv) FRAGMENT TYPE: N-terminal
(v) SEQUENCE DESCRIPTION FOR SEQ ID NO:8
Gly Glu Val Gln Val Gly Phe Arg
s
