(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(10) International Publication Number
(43) International Publication Date i i
15 December 2011 (15.12.2011) WO 2U11/154253 A l
(51) International Patent Classification: Colworth, Sharnbrook, Bedford Bedfordshire MK44 1LQ
A23L 1/212 (2006.01) C12N 15/82 (2006.01) (GB). REDFERN, Sally, Pamela [GB/GB]; Unilever
C07K 14/415 (2006.01) A23L 1/305 (2006.01) R&D Colworth, Sharnbrook, Bedford Bedfordshire
MK44 1LQ (GB). SILVA DE, Jacqueline [GB/GB];
(21) International Application Number: Unilever R&D Colworth, Sharnbrook, Bedford Bedford
PCT/EP201 1/058539 shire MK44 1LQ (GB). WILKINSON, Joy, Elizabeth
(22) International Filing Date: [GB/GB]; Unilever R&D Colworth, Sharnbrook, Bedford
25 May 201 1 (25.05.201 1) Bedfordshire MK44 1LQ (GB).
(25) Filing Language: English (74) Agent: ACHAM, Nicholas, Clive; Unilever PLC,
Unilever Patent Group, Colworth House, Sharnbrook,
(26) Publication Langi English Bedford Bedfordshire MK44 1LQ (GB).
(30) Priority Data: (81) Designated States (unless otherwise indicated, for every
10165272.5 8 June 2010 (08.06.2010) EP kind of national protection available): AE, AG, AL, AM,
(71) Applicant (for AE, AG, AU, BB, BH, BW, BZ, CA, CY, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
EG, GB, GD, GH, GM, IE, IL, KE, KN, LC, LK, LS, MT, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
MW, MY, NA, NG, NZ, OM, PG, SC, SD, SG, SL, SZ, TT, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
TZ, UG, VC, ZA, ZM, ZW only): UNILEVER PLC [GB/ HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
GB]; a company registered in England and Wales under KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
company no. 41424, Unilever House, 100 Victoria Em ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
bankment, London Greater London EC4Y 0DY (GB). NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
(71) Applicant (for all designated States except AE, AG, AU, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
BB, BH, BW, BZ, CA, CY, EG, GB, GD, GH, GM, IE, IL,
IN, KE, KN, LC, LK, LS, MT, MW, MY, NA, NG, NZ, (84) Designated States (unless otherwise indicated, for every
OM, PG, SC, SD, SG, SL, SZ, TT, TZ, UG, US, VC, ZA, kind of regional protection available): ARIPO (BW, GH,
ZM, ZW): UNILEVER N.V. [NL/NL]; Weena 455, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
NL-3013 AL Rotterdam (NL). ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
(71) Applicant (for IN only): HINDUSTAN UNILEVER EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
LIMITED [IN/IN]; Hindustan Lever House, 165/166 LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
Backbay Reclamation, Maharashtra, Mumbai 400 020 SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
(IN). GW, ML, MR, NE, SN, TD, TG).
(72) Inventors; and Published:
(75) Inventors/ Applicants (for US only): BERRY, Mark,
John [GB/GB]; Unilever R&D Colworth, Sharnbrook, — with international search report (Art. 21(3))
Bedford Bedfordshire MK44 1LQ (GB). CASEY, John — before the expiration of the time limit for amending the
[GB/GB]; Unilever R&D Colworth, Sharnbrook, Bedford claims and to be republished in the event of receipt of
Bedfordshire MK44 1LQ (GB). GUNGABISSOON, amendments (Rule 48.2(h))
Ravine, Anthony [GB/GB]; Unilever R&D Colworth, — with sequence listing part of description (Rule 5.2(a))
Sharnbrook, Bedford Bedfordshire MK44 1LQ (GB).
ORMEROD, Andrew, Paul [GB/GB]; Unilever R&D
(54) Title: REHYDRATABLE FOOD
(57) Abstract: Use of dried rehydratable food, such as in a dried soup, a dried beverage, a breakfast cereal, a yoghurt and a dried
sauce, is widespread. However it has been observed that when the dried components are fruit and/or vegetable, the components,
on rehydration, do not resemble the fruit and/or vegetable before desiccation. That is to say they no longer have a fresh appearance
but are discoloured and lack firmness. This transformation is due to cellular damage which occurs during desiccation. In particu
lar, it is thought that phospholipid membranes are destabilised by insertion of cellular amphiphiles, phase transition into the gel
phase and membrane fusion. This invention seeks to solve the above-mentioned technical problem by providing, amongst other
things, a dried rehydratable food which is a fruit, vegetable or part thereof which, on rehydration, has improved appearance, tex
ture and rehydration properties. In particular, a dried rehydratable food is provided, the food comprising less than 10% w/w water
and at least 0.02% w/w of a dehydrin protein and derivatives thereof, the dehydrin protein and derivatives thereof comprising an
o amino acid sequence selected from the group consisting of K,I,K,E,K,L,P,G; K,I,K,E/D,K,L/I,P,G; and K,I,K,E/D,K,L/I/TA/,P/H/
S,G, and wherein the dried rehydratable food is unbroken tissue of a vegetable or part thereof and/or a fruit or part thereof, and not
o a seed, wherein the unbroken tissue has a shortest linear dimension of at least 0.5 millimetres, preferably a shortest linear dimen
sion of 0.5 to 25, more preferably 0.5 to 10 millimetres. A food product comprising the dried rehydratable food and methods for
manufacturing the dried rehydratable food are also provided.
REHYDRATABLE FOOD
This invention relates to dried rehydratable food, in particular to a food which is a fruit,
vegetable or part thereof. This invention also relates to methods of manufacture thereof
and a food product, such as a dried soup, a dried beverage, a breakfast cereal, a yoghurt
and a dried sauce, comprising the aforementioned dried rehydratable food.
Use of dried rehydratable food, such as in a dried soup, a dried beverage, a breakfast
cereal, a yoghurt and a dried sauce, is widespread. However it has been observed that
when the dried components are fruit and/or vegetable, the components, on rehydration, do
not resemble the fruit and/or vegetable before desiccation. That is to say they no longer
have a fresh appearance but are discoloured and lack firmness. This transformation is due
to cellular damage which occurs during desiccation. In particular, it is thought that
phospholipid membranes are destabilised by insertion of cellular amphiphiles, phase
transition into the gel phase and membrane fusion.
Serrano et al (Food Sci. Tech. Int., 9(3), 157-161 (2003)) discusses potential applications
to food dehydration and discloses that it has been observed that late embryogenesis
abundant (LEA) proteins are abundant at high levels during the maturation process of seed
embryogenesis and also in all plant tissues experiencing water stress. LEA proteins
comprise, amongst others, the dehydrins. The authors state that LEA proteins may
stabilise proteins and membranes during desiccation by a similar mechanism to osmolytes.
That is by conferring preferential hydration of the cellular structures, then actually replacing
water during desiccation. Osmolytes also contribute to osmotic adjustment and act as
efficient hydroxyl radical scavengers during desiccation. The authors also disclose that
three biological systems seem to act in concert to achieve desiccation tolerance: enzymes
involved in osmolyte synthesis; proteins specialised in desiccation protection of
membranes and proteins (LEA proteins); and antioxidant enzymes and molecules.
This invention seeks to solve the above-mentioned technical problem by providing,
amongst other things, a dried rehydratable food which is a fruit, vegetable or part thereof
which, on rehydration, has improved appearance, texture and rehydration properties.
Summary of the invention
In a first aspect of the invention, a dried rehydratable food is provided, the food comprising
less than 10% w/w water and at least 0.02% w/w of a dehydrin protein and derivatives
thereof, the dehydrin protein and derivatives thereof comprising an amino acid sequence
selected from the group consisting of K,I,K,E,K,L,P,G; K,I,K,E/D,K,L/I,P,G; and
K,I,K,E/D,K,L/I/TA/,P/H/S,G, and wherein the dried rehydratable food is unbroken tissue of
a vegetable or part thereof and/or a fruit or part thereof, and not a seed, wherein the
unbroken tissue has a shortest linear dimension of at least 0.5 millimetres, preferably a
shortest linear dimension of 0.5 to 25, more preferably 0.5 to 10 millimetres.
By the term "rehydratable" is meant that the food may be rehydrated to at least 50 %,
preferably at least 60 %, most preferably at least 70 % w/w of the water content of a fully
hydrated vegetable or part thereof and/or fruit or part thereof. Preferably the term
"rehydratable" means that the food may be rehydrated to no more than 99 %, preferably no
more than 97 %, most preferably no more than 95 % w/w of the water content of a fully
hydrated vegetable or part thereof and/or fruit or part thereof.
Derivatives thereof include glycosylated dehydrins and truncated dehydrins, wherein
truncated dehydrins still contain the critical K segment (see below).
Adjacent positions in the amino acid sequence are separated by a comma and the most
frequently observed amino acid listed first with each amino acid at a single position
separated by a forward slash.
The inventors have surprisingly observed that by raising the concentration of dehydrin
proteins in fruit and/or vegetable tissue above that found naturally, dried rehydratable food
is obtained which on rehydration has improved appearance, texture and rehydration
properties. Whilst the prior art does suggest that LEA proteins may play a role in protecting
plants from water stress, it is surprising that the use of dehydrin alone has resulted in this
improvement in the aforementioned performance properties of dried rehydratable fruit
and/or vegetable because three biological systems seem to act in concert to achieve
desiccation tolerance.
From data provided by Roberts et al. (The Plant Cell, 5, 769-780 (1993)), it has been
calculated that seeds can comprise about 0.5% of dry weight levels of dehydrin. There is,
however, no consensus in the literature as to why the levels are this high. Seeds, as such,
are not the subject of the invention and are therefore excluded.
Preferably the dried rehydratable food comprises 0.02 to 20, preferably 0.1 to 5, most
preferably 0.2 to 2.5 % w/w dehydrin protein and derivatives thereof. Natural levels of
dehydrin protein are below 0.02 % w/w. The dehydrin protein and derivatives thereof
preferably have a molecular weight of 1 to 150, preferably 5 to 100, most preferably 5 to 50
kD. Dehydrin proteins and derivatives thereof of lower molecular weight are preferable
because they more easily infuse into the fruit or vegetable tissue.
The dehydrin and derivatives thereof are preferably derived from the group consisting of
Camellia sinensis, Forsythia and Selaginella, more preferably from Camellia sinensis, in
particular with an amino acid sequence at least 80% identical to SEQ. ID NO. 1 (see figure
3b), preferably at least 90%, 95% or 99% identical thereto. It was observed that the
dehydrin of SEQ. ID NO. 1 was 47x up-regulated when tea plant tissue was withered,
therefore it was postulated that this particular dehydrin had an important role to play in
protecting the plant tissue during desiccation. This has subsequently been proven to be
the case from the results set forth hereinafter in the detailed description of the invention.
Another preferred Camellia sinensis derived dehydrin protein (a truncate form) has an
amino acid sequence at least 80% identical to SEQ. ID NO. 2 (see figure 4b), preferably at
least 90%, 95% or 99% identical thereto.
Yet another preferred Camellia sinensis derived dehydrin protein (another truncate form)
has an amino acid sequence at least 80% identical to SEQ. ID NO. 3 (see figure 5b),
preferably at least 90%, 95% or 99% identical thereto.
It was thought that truncated versions of tea dehydrin SEQ. ID NO. 1 still containing the
critical K segment (see below) and labelled SEQ. ID NO. 2 and 3 would infuse more easily
and deeply into fruit and/or vegetable tissue and thus provide enhanced protective activity
when compared to the full length form (SEQ. ID NO. 1) . The truncated versions may also
have a higher functional activity as some of the regulatory areas of the protein would be
removed.
The rehydratable food may additionally comprise a compound selected from the group
consisting of trehalose, sucrose, glucose, fructose, raffinose, an enzymatic antioxidant or a
non-enzymatic reactive oxygen species scavenger. The foregoing species are thought to
be able to protect, by a variety of different mechanisms, the integrity of the fruit or
vegetable tissue when it undergoes dehydration. For example the sugars are thought to
protect cell membranes during desiccation firstly by inducing preferential hydration of the
cellular structures, then by actually replacing water protecting the cellular structure. Also
the sugars may act as efficient hydroxyl radical scavengers controlling the increased
production of reactive oxygen species seen during desiccation. Cellular electron transport
chains are impaired upon dehydration and hence generate increasing amounts of reactive
oxygen intermediates. Thus enzymatic antioxidant or a non-enzymatic reactive oxygen
species scavenger would be expected to improve the ability of fruit or vegetable tissue to
resist desiccation with reduced cellular damage. Suitable enzymatic antioxidants include
catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase. Suitable
non-enzymatic reactive oxygen species scavengers include ascorbate, glutathione and
carotenoids.
The vegetable may be selected from the group consisting of spinach, broccoli, onion,
aubergine, courgette, potato, pumpkin, mushroom, carrot, tea, asparagus, turnip, leek,
beetroot, cauliflower, celeriac, artichoke, mint, thyme, oregano, rosemary, parsley, sage,
chives, marjoram, basil, bay leaf, tarragon, celery and garlic and the fruit may be selected
from the group consisting of lemon, raspberry, red currant, blackberry, berry, blueberry,
strawberry, pineapple, banana, peach, apricot, lychee, apple, pear, tomato, capsicum,
cucumber and mango.
In a second aspect of the invention, a food product is provided, the food product
comprising a dried rehydratable food according to the first aspect of the invention. The
food product may be selected from the group consisting of a dried soup, a dried beverage,
a breakfast cereal, a yoghurt and a dried sauce. All of the foregoing food products are
characterised in including a dried fruit or vegetable component which is rehydrated on use.
In a third aspect of the invention, a method for manufacturing a dried rehydratable food
according to the first aspect of the invention, the method comprising the steps of:
(a) Infusing a vegetable or part thereof, or a fruit or part thereof excluding a seed, with
a dehydrin protein and derivatives thereof, the dehydrin protein and derivatives
thereof comprising an amino acid sequence selected from the group consisting of
K,I,K,E,K,L,P,G; K,I,K,E/D,K,L/I,P,G; and K,I,K,E/D,K,L/I/TA/,P/H/S,G to produce an
infused food; and
(b) Drying the infused food thereby to produce a dried rehydratable food according to
the first aspect of the invention.
The surprising observation of the third aspect of the invention is the fact that a dried
rehydratable food is obtained which on rehydration has improved appearance, texture and
rehydration properties by simple diffusion of dehydrin into the fruit and/or plant tissue.
Preferably step (a) of the third aspect of the invention is carried out under a vacuum. It is
anticipated that under vacuum, infusion is faster. Step (a) of the third aspect of the
invention may be carried out at a temperature of 3 to 70, preferably 10 to 50, most
preferably 15 to 30 degrees centigrade. At too low a temperature, the infusion process is
too slow, and at too high a temperature, the tissue structure is damaged.
In a fourth aspect of the invention, a method for manufacturing a dried rehydratable food
according to the first aspect of the invention is provided, the method comprising the steps
of:
(a) Cloning a gene into a plant expression vector thereby to produce a modified plant
expression vector, wherein the gene encodes a dehydrin protein and derivatives
thereof, wherein the dehydrin protein and derivatives thereof comprises an amino
acid sequence selected from the group consisting of K,I,K,E,K,L,P,G;
K,I,K,E/D,K,L/I,P,G; and K,I,K,E/D,K,L/I/TA/,P/H/S,G;
(b) Introducing the modified plant expression vector into a target crop by plant
transformation thereby to produce a transgenic target crop;
(c) Growing the transgenic target crop thereby to express the dehydrin protein and
derivatives thereof; and then
(d) Drying the transgenic target crop thereby to produce a dried rehydratable food
according to the first aspect of the invention.
In a fifth aspect of the invention is provided a dehydrin protein with an amino acid
sequence identical to SEQ. ID NO. 1, and derivatives thereof.
In a sixth aspect of the invention is provided a dehydrin protein with an amino acid
sequence identical to SEQ. ID NO. 2, and derivatives thereof.
In a seventh aspect of the invention is provided a dehydrin protein with an amino acid
sequence at least 80% identical to SEQ. ID NO. 4, preferably at least 90%, 95% or 99%
identical thereto, and derivatives thereof.
Brief description of the figures
The invention will now be exemplified with reference to the following figures in which:
Figure 1 shows the characteristic structure of a generalised dehydrin protein
molecule showing conserved sequence motifs wherein the Y segment is
typically located towards the nitrogen terminus, the F segments are
repeated regions mostly made up of glycine and polar amino acids, the S
segment contains a tract of phosphorylatable serine residues and the K
segment is rich in lysine and constitutes the putative amphipathic a-helix
forming domain (the sequences of letters refer to the corresponding amino
acid sequence);
Figure 2 shows a sodium dodecyl sulphate / polyacrylamide gel electrophoresis
(SDS-PAGE) plate of Selaginella lepidophylla dehydrin fractions following
purification by a DEAE-Sepharose CL-6B ion exchange column where
column ( 1 ) are the molecular weight marker (in kiloDaltons), column (2) is
the total Selaginella lepidophylla extract and columns (3) to (15) are the
fractions of the extract eluted with a 0.02 M:1 M KCI gradient (the arrow
indicates the position of the dehydrin band);
Figure 3 shows a tea dehydrin gene in schematic form in figure 3a and in the form of
a nucleotide and deduced amino acid sequence in figure 3b (SEQ. ID NO.
1) ;
Figure 4 shows the tea dehydrin of figure 3 in truncated form (truncate 122-201 of
SEQ. ID NO. 1) in schematic form in figure 4a and in the form of a
nucleotide and deduced amino acid sequence in figure 4b (SEQ. ID NO. 2);
Figure 5 shows the tea dehydrin of figure 3 in truncated form (truncate 163-201 of
SEQ. ID NO. 1) in schematic form in figure 5a and in the form of a
nucleotide and deduced amino acid sequence in figure 5b (SEQ. ID NO. 3);
shows the nucleotide and deduced amino acid sequence (SEQ. ID NO. 4) of
the Forsythia suspensa dehydrin gene;
shows SDS-PAGE gel plate of recombinant tea dehydrin wherein the arrow
indicates the position of the dehydrin fusion protein band, column ( 1 ) the
molecular weight markers (kiloDaltons), column (2) the cell lysate
supernatant of IPTG induced E. coli harbouring the pDEST 17-dehydrin
construct, column (3) unbound proteins eluted from the Ni-NTA column after
sample addition, columns (4) to (8) the contaminant proteins eluted after
successive column washes, and columns (9) to (15) the fractions collected
following Elution Buffer application;
shows SDS-PAGE gel plate of recombinant tea dehydrin (364-606) wherein
the arrow indicates the position of the dehydrin fusion protein band, column
( 1 ) the molecular weight markers (kiloDaltons), column (2) the cell lysate
supernatant of IPTG induced E. coli harbouring the pDEST 17-dehydrin
construct, column (3) unbound proteins eluted from the Ni-NTA column after
sample addition, columns (4) to (8) the contaminant proteins eluted after
successive column washes, and columns (9) to (15) the fractions collected
following Elution Buffer application;
shows SDS-PAGE gel plate of recombinant tea dehydrin (487-606) wherein
the arrow indicates the position of the dehydrin fusion protein band, column
( 1 ) the molecular weight markers (kiloDaltons), column (2) the cell lysate
supernatant of IPTG induced E. coli harbouring the pDEST 17-dehydrin
construct, column (3) unbound proteins eluted from the Ni-NTA column after
sample addition, columns (4) to (8) the contaminant proteins eluted after
successive column washes, and columns (9) to (15) the fractions collected
following Elution Buffer application;
shows SDS-PAGE gel plate of recombinant Forsythia dehydrin wherein the
arrow indicates the position of the dehydrin fusion protein band, column (1)
the molecular weight markers (kiloDaltons), column (2) the cell lysate
supernatant of IPTG induced E. coli harbouring the pDEST 17-dehydrin
construct, column (3) unbound proteins eluted from the Ni-NTA column after
sample addition, columns (4) to (8) the contaminant proteins eluted after
successive column washes, and columns (9) to (15) the fractions collected
following Elution Buffer application;
Figure 11 shows fully dried and rehydrated red peppers following infusion (A) with
water, (B) Selaginella lepidophylla dehydrin, and (C) tea dehydrin expressed
from Pichia pastoris;
Figure 12 shows a graph of the length versus breadth of rehydrated red pepper tissue
pieces infused with various dehydrins or water and controls, wherein "Res
Pit" refers to the Selaginella lepidophylla dehydrin of example 1, "TD" refers
to the full tea dehydrin of example 2, "TD (364-606)" refers to a truncated
tea dehydrin of example 2, "Forsythia" refers to the Forsythia dehydrin of
example 2, "Raw" refers to raw red pepper tissue pieces which have been
dried and rehydrated in accordance with example 5, "Water-VI" refers to raw
red pepper tissue pieces which have been vacuum infused with water, dried
and then rehydrated in accordance with example 5, and "Totally raw" refers
to raw red pepper tissue pieces;
Figure 13 shows load (N) versus distance (mm) graphs for pieces of green pepper
(Capsicum) which have not been infused, dried and rehydrated (figure 13a),
which have not been infused but nevertheless dried and rehydrated in
accordance with this example 6 (figure 13b), which have been infused with
water, dried and rehydrated in accordance with example 6 (figure 13c); and
which have been infused with Selaginella lepidophylla dehydrin, dried and
rehydrated in accordance with example 6 (figure 13d);
Figure 14 shows spinach leaves infused with Pichia pastoris expressed tea dehydrin
(upper images) or water (lower images) after being dried (left hand images)
and then rehydrated (middle images), and optical microscopy of a section of
one of the leaves shown in the middle images (right hand images);
Figure 15 shows a visible light micrograph (x 10 magnification) of in (a) of live onion
epidermal peel dyed with trypan blue and in (b) onion epidermal peel which
has been blanched and then dyed with trypan blue; and
Figure 16 shows visible light micrographs (x 10 magnification) of dried and rehydrated
onion epidermal peels stained with the uptake of trypan blue following
infusion with (a) the full-length tea dehydrin of example 2, (b) deionised
water, (c) bis-tris trehalose buffer, and (d) BSA in bis-tris trehalose buffer.
Detailed description of the invention
Dehydrin genes are composed of distinct domains that exhibit high levels of conservation
across plant species. Figure 1 shows a schematic diagram of the generalised architecture
of dehydrins. Each protein is comprised of multiple copies of K, F, S and Y segments. For
example, the K segment can occur up to 11 times per polypeptide whereas the Y segment
is normally found in 1 to 3 tandem repeats near the N-terminus. The four main segments
are interspersed by other lesser conserved and usually repeated regions. The strict
conservation of the K, F, S and Y segments during evolution indicates that they define
functional units within these proteins. Dehydrins can be characterized by the
K,I,K,E,K,L,P,G amino acid sequence found near the carboxy terminus which is usually
repeated within the protein. This amino acid sequence forms part of the K segment. More
generally the carboxy terminal peptide of dehydrins that emerges from an alignment of
available published data is: E, K, K, G/S, IA//M/L/F, M/L/V, D/E, K, I, K, E/D, K, L/l, P, G.
Example 1: Extraction and purification of resurrection plant Selaginella lepidophylla
dehydrin
Protein extracts from Selaginella lepidophylla were probed with a dehydrin anti-body to
detect dehydrin-like proteins in Selaginella lepidophylla (a type of Resurrection plant which
is a plant known for showing remarkable tolerance to drought) tissues. Once identified the
proteins were purified by ion exchange chromatography.
a) Preparation of whole protein extract from Selaginella lepidophylla tissue
A fully hydrated entire Selaginella lepidophylla plant which had been dehydrated at room
temperature for five hours (so it is partially dehydrated) was ground to a flour-like
consistency in a coffee grinder. The powder was suspended at a concentration of 200 g/L
in a pre-chilled (4 degrees centigrade) pH 6.0 extraction buffer containing 25 mM 2-(Nmorpholino)
ethanesulfonic acid (MES), 20 mM NaCI and 1 mM phenylmethylsulfonyl
fluoride (PMSF). The suspension was stirred for 3 hours at 4 degrees centigrade before
being mixed in a blender for 1 minute after which the homogenate was stirred for a further
12 hours at 4 degrees centigrade. Insoluble material was pelleted by centrifugation at
10,000 rpm for 30 minutes at 4 degrees centigrade. The supernatant was subsequently
filtered through two separate layers of cheesecloth. Non-heat stable proteins were
denatured by incubation at 70 degrees centigrade for 10 minutes with occasional shaking.
The solution was rapidly cooled on ice and filtered through Whatman Paper No. 1. Any
remaining insoluble material was pelleted by centrifugation at 30,000 rpm for 1 hour.
b) Detection of dehydrin proteins in Selaginella lepidophylla whole protein extracts by
Western Blotting
A -20 m I_ aliquot of Selaginella lepidophylla total protein extract was loaded onto an
electrophoresis gel (12% Novex bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bistris))
and run for approximately 40 minutes at 200V in a MES and sodium dodecyl sulphate
(SDS) running buffer. The proteins on the gel were blotted onto a nitrocellulose sheet for 1
hour at 30 V. Unbound sites on the nitrocellulose were blocked by immersion in
tris(hydroxymethyl)aminomethane (tris) buffered saline (TBS) containing 5 % dried milk
powder (blocking buffer) for 1 hour. The blot was then incubated in rabbit polyclonal
dehydrin anti-serum (Stressgen Bioreagents) diluted 1:1000 in the blocking buffer for 1
hour. Following four consecutive 5 minutes washes with TBS containing 0.05 %
polysorbate 20 (Tween 20), the nitrocellulose was soaked in alkaline phosphatase
conjugated goat anti-rabbit antibody (Invitrogen). After four consecutive 5 minute washes
with TBS containing 0.05 % polysorbate 20 (Tween 20), conjugate bound dehydrin was
detected by the addition of 5 mL 5-bromo-4-chloro-3-indolyl phosphate / nitro blue
tetrazolium (BCIP/NBT) solution (Sigma).
c) Purification of dehydrin protein from Selaginella lepidophylla tissue whole protein
extracts.
20 mL Selaginella lepidophylla total protein extract was dialysed over night in 10 mM
tris.HCI, 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT) pH 8.0
buffer at 4 degrees centigrade. A 20 mL diethylaminoethyl cross-linked agarose based ion
exchange column (DEAE-Sepharose CL-6B) was equilibrated with 500 mL of the same
buffer. The dialysed extract was passed through the column at a rate of approximately 1
drop per second. Following sample addition the column was washed with 20 mL of column
buffer. Proteins were then eluted in 4 mL fractions by the application of a 10OmL 0.02M:1 M
KCI gradient. Pure dehydrin containing fractions were identified by sodium dodecyl
sulphate / polyacrylamide (SDS-PAGE) gel electrophoresis, illustrated in figure 2, and
fractions 9 to 15 (containing pure dehydrin) pooled.
Example 2 : Isolation of Camellia sinensis dehydrin cDNA, cloning of full length and
truncated dehydrin sequences and expression and purification of recombinant
dehydrin proteins in E.coli
The procedure involved the following steps:
(a) A complementary deoxyribonucleic acid (cDNA) library was constructed from
withered and non-withered tea shoots (Camellia sinensis);
(b) cDNA for a tea dehydrin contig present only in the withered cDNA library and
represented by 47 independent cDNA clones (more than any other dehydrin
detected) was selected;
(c) The selected cDNA dehydrin sequence was cloned into the Escherichia coli (E.coli)
expression vector pDEST17 and transformed into E.coli;
(d) The transformed E.coli was multiplied in culture and induced to express the
corresponding tea dehydrin protein;
(e) The E.coli cells were lysed and the expressed dehydrin protein purified using nickel
ion-nitrolotriacetic acid resin (Ni-NTA); and
(f) The purified dehydrin was dialysed with water prior to use.
Total RNA isolated from withered tea shoots
Tea shoots (two leaves and a bud) from Camellia sinensis variety assamica were
harvested and withered for 19 hours (in a partially sealed plastic bag). Total ribonucleic
acid (RNA) was isolated using a plant RNA isolation kit (Qiagen) in accordance with the
manufacturer's instructions.
Synthesis of cDNA from mRNA to form cDNA library
mRNA was purified from 500 mg total RNA using a polyadenylic acid isolation kit (Qiagen)
in accordance with the manufacturer's instructions. 5 mg of the polyadenylic acid - mRNA
molecule was heated to 72 degrees centigrade for 5 minutes together with 2.8 mg
polyadenylic acid linker primer containing an Xho1 restriction site (Stratagene), then snap
cooled on ice for two minutes. The mRNA was reverse transcribed in a 50 m I_ reaction for
60 minutes at 42 degrees centigrade after an initial incubation at room temperature for 10
minutes, using 75 units of reverse transcriptase (Stratascript RT from Stratagene) in 1 x RT
buffer (Stratagene), 2.5 mM deoxyribonucleotide triphosphates (dNTP's) (comprising
deoxyadenosine triphosphate (dATP), thymidine triphosphate (dTTP), 5-methyl
deoxycytidine triphosphate and deoxyguanosine triphosphate (dGTP)) (Amersham-
Pharmacia) and 40 units ribonuclease (RNAse) block (Stratagene).
Second strand cDNA was synthesised using 45 m I_ of the first strand synthesis reaction in a
200 m I_ reaction for 150 minutes at 16 degrees centigrade by adding 11 m I_ DNA
polymerase 1 (9 units/ m I) (Stratagene), 20 m I_ 10x second strand synthesis buffer
(Stratagene), 6 m I dNTP's (40mM) (Amersham-Pharmacia) and 2 m I_ RNAse H ( 1 .5
units/ m I ) (Stratagene). 180 m I_ of the second strand synthesis reaction was blunted for 30
minutes at 72 degrees centigrade by adding 20.7 m I_ dNTP's ( 1OmM) and 1.8 m I cloned Pfu
DNA polymerase (Stratagene) (an enzyme found in the hyperthermophilic archaeon
Pyrococcus furiosus) (2.5 units/m I-). The reaction was terminated by extracting once with
an equal volume of 1: 1 v/v phenol/chloroform and once with an equal volume of chloroform
before precipitating the cDNA overnight at -20 degrees centigrade with 0.1 volume of 3M
sodium acetate solution (pH 5.2) and 2 volumes of ethanol. The cDNA was resuspended in
9 m I_ EcoR1 (an endonuclease enzyme isolated from strains of E. coli) adapters
(Stratagene) and incubated at 8 degrees centigrade overnight with 4 units of T4 DNA
ligase (Stratagene) (T4 is a bacteriophage of E. coli) in 1 x ligase buffer (Stratagene) + 1
mM ribonucleotide ATP (rATP) (Stratagene). The ligation reaction was terminated by
heating to 70 degrees centigrade for 30 minutes and then snap cooled on ice for two
minutes. The cDNA ends were then phosphorylated in a 22 m I reaction at 37 degrees
centigrade for 30 minutes by adding 1 m I T4 polynucleotide kinase (10 units/ m I)
(Stratagene), 1 m I ligase buffer (10x) and 1 m I rATP (10mM). The phosphorylation reaction
was terminated by heating the reaction to 70 degrees centigrade for 30 minutes. The cDNA
was then digested with Xho 1 restriction enzyme (Stratagene) using standard molecular
biology procedures. The cDNA was size fractionated into 12 x 100 uL fractions by passing
it through a 1 mL (flat bed volume) column (Chroma Spin-400 from Clontech) using 1x STE
(100mM NaCI, 20mM tris(hydroxymethyl)aminomethane - HCL (pH 7.5), 10mM EDTA)
(Stratagene) as the column buffer. 5 m I_ of each fraction was then visualised on an ethidium
bromide-1 .2% agarose tris(hydroxymethyl)aminomethane-borate-EDTA (TBE)
electrophoresis gel (made in house using Sigma reagents). The first four fractions
containing cDNA were then pooled, ethanol precipitated and resuspended in 10 m I_ 10mM
tris(hydroxymethyl)aminomethane (pH 7.6).
10 ng of the pooled cDNA were ligated into 20 ng pBluescript SK+ Xho1/EcoR1 digested
vector (Stratagene) in a 5 m I_ reaction at 12 degrees centigrade overnight using 2 units T4
DNA ligase in 1x ligation buffer +1mM rATP. The cDNA library was then transformed into
XL10-Gold ultracompetent cells (Stratagene) according to the manufacturer's instructions.
The size of the primary cDNA library was estimated at 250,000 clones and the average
insert size, 1072bP (base pairs). 2592 clones were hand picked and DNA prepared for
sequencing.
Multiplication of cDNA for sequencing
Colonies were grown up overnight in 2.5 mL of 2TY broth ( 6g tryptone, 10g NaCI, 10g
yeast extract (pH 7.3) per litre) (made in house using Sigma reagents) containing 100
mg mL carbenicillin (Sigma) at 225 r.p.m. and 37°C. Plasmid DNA was isolated using
montage plasmid miniprep (96) kit (Millipore) in accordance with the manufacturer's
instructions. The DNA was then quantified and diluted to 50 ng/ m I using PicoGreen DNA
quantitation kit (Molecular Probes BV) in accordance with the manufacturer's instructions.
Sequencing of expressed sequence tags, compilation of contigs and identification of
homologues
DNA sequencing was carried out on an Applied Biosystems Genetic Analyser 3100 using 5
m I_ of template DNA at 0.1 mg mL and 1 m I_ of primer at 1 prmol/m I- according to standard
fluorescence dideoxy sequencing procedures. In total 1971 expressed sequence tag (EST)
clones were sequenced and 1772 of these yielded good quality sequence data. These EST
sequences were compiled into contigs. The consensus sequence of each of these contigs
was used for identification of gene function by blasting against the following EMBL public
databases 'plantdna', 'em_pl', 'emnew_pl', 'em_est_pl', 'em_gss_pl', 'em_nonpl',
'emnew_nonpl', 'em_est_nonpl' and 'em_gss_nonpl'. TblastX and blastN search programs
were used and the -500,000 results parsed into a database. A single (best annotated)
homologue was identified for each of the tea genes (automated for single EST genes).
A single contig represented by 47 independent cDNA clones exhibited significant homology
to AF220407, a Vitis riparia dehydrin-like protein (Dhn) mRNA (expectation score 3e-1 1).
The tea dehydrin protein exhibited all the typical traits associated with a member of the
dehydrin family. The sequence contained an N-terminal Y segment, an S segment, F
segments and two K segments near the C-terminal end as shown schematically and in
actuality (SEQ. ID. 1) in figures 3a and 3b respectively. No other single contig represented
as many independent cDNA clones.
Cloning of full length and truncated tea dehydrins into pDEST17 expression vector
Full length wild type tea dehydrin protein and two truncated tea dehydrins were produced in
accordance with the Gateway Expression System (Invitrogen) instructions. attB sequences
(engineered foreign region from Escherichia coli) (Invitrogen) were added to either end of
the dehydrin sequence from the cDNA vector (pBluescript from Invitrogen) in a two step
polymerase chain reaction (PCR) process by using dehydrin template specific primers
containing 12 attB nucleotides (Invitrogen) (see table 1) . The specific primers were
designed around full length and truncated tea dehydrin sequences to generate either full
length or truncated DNA. The second step added the Universal attB sequence (Invitrogen)
to the full length or truncated tea dehydrin sequence allowing it to be inserted into the
pDONR 221 vector (Invitrogen). The pDONR 221 vector was then recombined with
pDEST17 bacterial expression vector (with T7 promoter and ribosome binding site)
(Invitrogen) which codes for the histidine residues to be added to the dehydrin allowing for
purification of the protein.
Tea dehydrin truncate (deoxynucleotide numbers 364-606) was designed to encompass
the C-terminal F and the two K segments of the protein. The highly conserved K segment
domain is believed to be vital for dehydrin activity. The smaller tea dehydrin truncate
(deoxynucleotide numbers 487-606) was comprised of the C-terminal F and one K
segment only. Figures 4a and 5a show respectively the schematic nucleotide sequence of
each tea dehydrin truncate and figures 4b and 5b the actual nucleotide and deduced amino
acid sequences of each tea dehydrin truncate (SEQ. ID. 2 and 3 respectively).
Isolation and Cloning of Forsythia suspensa dehydrin cDNA into pDEST17
In addition to the tea dehydrin, another dehydrin gene was isolated by real time
polymerase chain reaction (RT-PCR) on polyadenylated RNA isolated from Forsythia
suspensa bark. The lack of N-terminal Forsythia sequence meant that an alternative
strategy was required to clone the Forsythia dehydrin. From amino acid sequence data, a
short peptide T,D/E,E,Y,G,N,P,V,Q,H with homology to dehydrins was identified. The
presence of this short sequence together with data in the literature, for example conserved
amino acid sequences of angiosperm dehydrins disclosed in table 1 of Close (Physiologica
Plantarum, 100, 291-296 (1997)), offered an alternative approach to clone the Forsythia
dehydrin. To achieve this goal two degenerate forward primers (FOR-D4 and FOR-D5)
were designed to the Ύ -segment' dehydrin domain TA/,D,E,Y,G,N,P (see figure 1) in
accordance with Close (Physiologica Plantarum, 100, 291-296 (1997)). These would
account for variation in the residues of the N-terminal consensus region between a
threonine and valine. Therefore the primer FOR-D4 (ACIGAYGARTAYGGIAAYCC) (SEQ.
ID. 5) utilised threonine as the first amino acid, whilst FOR-D5
(GTIGAYGARTAYGGIAAYCC) (SEQ. ID. 6) utilised valine as the first amino acid. The
antisense primer FOR-R2 (ARYTTYTCYTTDATYTTRTCCAT) (SEQ. ID. 7) was designed
to the 'K-segment' domain (E,K,K,G,I,M,D,K,I,K,E,K,L,P,G) (see figure 1) in accordance
with Close (Physiologica Plantarum, 100, 291-296 (1997)). In the aforementioned primer
sequences, the letter " represents inosine (which bonds with any base) and has been
replaced, in the attached formatted sequence listings, with "N" which leads to the same
technical effect as inosine. These primers were then used to synthesise the Forsythia
dehydrin cDNA sequence using total polyadenylated RNA extracted from Forsythia bark as
the template by RT-PCR. Primers were then designed to capture the 3' and 5' ends of the
Forsythia cDNA using the GIBCO 5' RACE (Rapid Amplification of cDNA Ends) system kit
version 2.0 (Life Technologies).
Forsythia cDNA was inserted into a vector pGEM®-T Easy vector from Promega) and
cloned into pDEST17 as described above, using the Gateway Expression System
(Invitrogen). Figure 6 shows the Forsythia nucleotide and deduced amino acid sequences.
Table 1 shows the primers used to clone the Forsythia dehydrin sequence into pDEST17.
Table 1: Polymerase chain reaction (PCR) primers used to generate attB
(engineered foreign region from E. coli) recombination target site flanked
dehydrin sequences
* 12attB2 TD also was used as the reverse primer for tea dehydrin (364-606) and tea
dehydrin (487-606)
Expression in E. coli and purification of recombinant dehydrin proteins
The pDEST 17 bacterial expression vector adds six consecutive histidine residues to the
C-terminal end of the expressed protein. The histidine tag allowed rapid isolation of the
protein from the soluble fraction of cell lysates by passage through a histidine tag binding
nickel -affinity matrix (Ni-NTA from Pro-Bond Purification System from Invitrogen). Histidine
fusion proteins expressed in pDEST 17 were purified from E. coli using the Pro-Bond
Purification System (Invitrogen). Further details are provided below.
a) Cell culture and protein expression
pDEST 17 carrying the various dehydrin sequences was transformed into E. coli strain
BL21 Star (DE3) One Shot (Invitrogen) (chemically competent BL21 hosts designed for
improving protein yield in a T7 promoter-based expression system). Cells harbouring the
pDEST 17 dehydrin constructs were spread onto lysogeny broth (LB) agar plates
containing 100 mg mL ampicillin and incubated at 37 degrees centigrade overnight. 2.5 mL
of LB medium containing 100 mg mL ampicillin was inoculated with a single colony of these
cells and shaken at 37 degrees centigrade overnight. The 2.5 mL culture was used to
inoculate 50 mL LB medium containing 100 mg mL ampicillin and shaken at 37 degrees
centigrade until the A6oo (absorbance at 600 nm) of the culture was 0.6. Dehydrin protein
expression was then induced by the addition of isopropyl b-D-l-thiogalactopyranoside
(IPTG) to a final concentration of 0.5 mM. Growth was continued for a further 5 hours
under the same conditions. The cells were harvested by centrifugation at 10,000 rpm for 10
minutes and stored at -20 degrees centigrade until required.
b) Cell lysis
Cell pellets were vigorously resuspended in 8 mL Binding Buffer (50 mM NaP04, 0.5 M
NaCI, 10 mM Imidazole pH 8.0). The protease inhibitor benzamidine was added to a final
concentration of 1 mg/mL. Lysis was carried out by five successive cycles of flash freezing
in liquid nitrogen followed by rapid thawing in a 30 degrees centigrade water bath.
Deoxyribonuclease I (DNase I (a non-specific endonuclease that degrades double- and
single-stranded DNA and chromatin)) was added to a final concentration of 1 ug/mL and
the lysate incubated on ice for 30 minutes. Insoluble material was pelleted by centrifugation
at 10,000 rpm for 1 hour at 4 degrees centigrade.
c) Protein purification and analysis
The 8 mL E. coli lysate supernatant was added to 2 mL of nickel ion-nitrolotriacetic acid
resin (Ni-NTA) which had been pre-equilibrated with Binding Buffer. The mixture was
incubated with mild agitation for one hour at room temperature to allow binding of the
histidine tagged protein. Unbound material in the soluble fraction was removed by
centrifuging the slurry at 800 g (800 times the force of gravity) for one minute and
decanting the supernatant. Further contaminants were removed with five consecutive
applications of Wash Buffer (50 mM NaP04, 0.5 M NaCI, 100 mM imidazole). The histidine
tagged protein was eluted from the resin by the addition of 8ml_ of Elution Buffer (50 mM
NaP04, 0.5 M NaCI, 250 mM imidazole). Fractions were collected in 1 mL aliquots and
analysed by SDS-PAGE gel electrophoresis (figures 7 to 10). SDS-PAGE gel
electrophoresis was carried out using the NuPAGE electrophoresis system (Invitrogen Ltd).
Dehydrin protein containing fractions were pooled accordingly. Relative protein
concentration was calculated using a Bradford Protein Assay (Bio-Rad).
Example 3 : Expression in Pichia Pastoris and purification of recombinant dehydrin
protein
Expression of the full length tea dehydrin in Pichia Pastoris and subsequent purification
was conducted by Invitrogen Corporation (1600 Faraday Avenue, Carlsbad, CA 92008,
USA) as a fully-funded toll manufacture of the protein.
Example 4 : Effects of dehydrin infusion - observations of rehydrated tissue
Red pepper (Capsicum) pieces (1cm3) were prepared and fully immersed in 0.1 mg/mL
dehydrin solution (Selaginella lepidophylla dehydrin obtained from example 1 or tea
dehydrin expressed from Pichia Pastoris as obtained from example 3) and vacuum infused
for two hours at room temperature. Water infused pieces were also prepared as controls.
The infused plant tissue was dried for six hours at 60 degrees centigrade and rehydration
carried out by immersion in water overnight at room temperature.
Figure 11 illustrates the greater rehydration of the red pepper pieces that were infused with
dehydrin, both that originating from Selaginella lepidophylla (B) and tea dehydrin
expressed from Pichia Pastoris (C) compared to the water-infused control (A).
Example 5 : Effects of dehydrin infusion - dimensions of rehydrated tissue
To assess the effect of different dehydrins on red pepper (Capsicum) tissue dimensions,
post-rehyd ration, pieces of red pepper were vacuum infiltrated with aqueous dehydrin
solution at 0.5 mg/mL or water alone for four hours at room temperature, dried for 16 hours
at 37 degrees centigrade, and rehydrated in water at room temperature for 4 to 5 hours.
Typically 10 to 15 pieces of plant tissue, prepared by using a circular 1 cm diameter cork
borer, were used for each dehydrin. As a control, raw red pepper tissue pieces were also
dried and rehydrated in accordance with this example. The length and breadth of
rehydrated samples are shown in figure 12 wherein "Res Pit" refers to the Selaginella
lepidophylla dehydrin of example 1, "TD" refers to the full tea dehydrin of example 2, "TD
(364-606)" refers to a truncated tea dehydrin of example 2, "Forsythia" refers to the
Forsythia dehydrin of example 2, "Raw" refers to raw red pepper tissue pieces which have
been dried and rehydrated in accordance with this example, "Water-VI" refers to raw red
pepper tissue pieces which have been vacuum infused with water, dried and then
rehydrated in accordance with this example, and "Totally raw" refers to raw red pepper
tissue pieces.
The measured dimensions of rehydrated tissue indicated greater rehydration of dehydrin
infused tissue compared to controls with no dehydrin infusion. In fact the dehydrin infused
tissue dimensions are closer to the dimensions of fresh pieces with no infusion, drying or
rehydration treatment.
Example 6 : Effects of dehydrin infusion - mechanical properties of rehydrated tissue
10 to 15 pieces of green pepper (Capsicum), prepared by using a circular 1 cm diameter
cork borer, were vacuum infused (overnight at room temperature) with either Selaginella
lepidophylla dehydrin of example 1 in the form of an aqueous solution of 0.05 mg/mL or
water. The infused pieces of green pepper were then dried at 45 degrees centigrade for 7.5
hours and rehydrated (3 hours at room temperature). The rehydrated pieces were then
subjected to compression tests using a Dartec Servohydraulic Mechanical Testing machine
to assess their mechanical properties. Specifically the pieces were compressed to a height
of 2 mm at a crosshead speed of 40 mm/sec and the load (N) in order to do this was
measured. The results for the two infused (figure 13c for water infused pieces and figure
13d for Selaginella lepidophylla dehydrin infused pieces) variants are shown in figure 13
together with controls for raw green pepper pieces which have not been infused but
nevertheless dried and rehydrated in accordance with this example (figure 13b), and raw
green pepper tissue pieces which have not been infused, dried and rehydrated (figure
13a).
Rehydrated Selaginella lepidophylla dehydrin-infused pieces required a greater force
during compression compared with non-infused and water-infused controls. This indicates
that the rehydrated dehydrin infused pieces were firmer than the rehydrated controls.
Infusion with the dehydrin gave results closest to those from raw green pepper pieces
which have not been infused, dried and rehydrated.
Example 7 : Effects of dehydrin infusion - observations of leaf tissue
Spinach leaves were immersed in a 0.2 mg/mL aqueous solution of Pichia Pastoris
expressed tea dehydrin in accordance with example 3, along with a water control, then
both vacuum infused for three hours at room temperature. The infused leaves were
dehydrated at 40 degrees centigrade overnight and then rehydrated for up to 3 hours at
room temperature.
Images of the dried (left hand images) and rehydrated leaves (middle images) are shown in
figure 14 along with optical microscope images of the internal structure of the rehydrated
tissues (right hand images). It can be observed that the dehydrin infused spinach (upper
images) had a larger leaf, after being both dried and rehydrated, compared to the water
infused control (lower images), and a more open, less collapsed tissue structure upon
rehydration. This indicated greater rehydration of the dehydrin infused leaves.
Example 8 : Detection and quantification of dehydrin in dry plant material
a) Extraction and estimation of dehydrin found naturally in red pepper
Red pepper pericarp were oven-dried at 60 degrees centigrade until constant weight,
weighed, ground in a mortar under liquid nitrogen, and the ground material placed in a 2
mL Eppendorf tube, extracted with Tissue Extraction Reagent 1 (Invitrogen) (containing
50mM tris(hydroxymethyl)aminomethane, pH7.4, 250mM NaCI, 5mM EDTA, 2mM Na3V0 4,
1mM NaF, 20mM Na4P20 7, 0.02% NaN3, detergent and 0.5mM phenylmethylsulfonyl
fluoride) in a 1 mL extraction per 100 mg of ground material by shaking for 1 hour at 4
degrees centigrade and centrifuging at 14,000 rpm, and the supernatant flash frozen and
stored at -80 degrees centigrade. Total proteins were quantified using the Bradford Protein
Assay (Bio-Rad).
10 m I_ of the protein extract was loaded onto a 4-12 % bis-(2-hydroxy-ethyl)-aminotris(
hydroxymethyl)-methane SDS-PAGE gel. 10 m I_ of the tea dehydrin of example 3 were
prepared at concentrations of 0.5, 0.1 and 0.05 mg/mL and loaded onto the gel. The gel
was run at 200 V for 30 minutes to separate the proteins. Protein standards (All Blue
Precision Plus Protein Standards (Bio-Rad)) were also run alongside the aforementioned
sample.
The proteins were transferred from the SDS-PAGE gel onto a 0.2 m h polyvinylidene
fluoride (PVDF) membrane ( Invitrogen) in a semi dry blotting module. The PVDF
membrane was then rinsed and dried between filter paper. The dry membrane with
transferred proteins was then incubated in blocking buffer (phosphate buffered saline
(PBS) solution with Polysorbate 20 (Tween 20) for use as a wash buffer and diluent
(PBST) + 4 % skimmed milk powder (SM P) solution, pH 7.2), for 30 minutes and then
removed. The membrane was then incubated for 2 hours with rabbit anti-dehydrin
polyclonal antibody (Agrisera, Vannas, Sweden) at a dilution of 1:1000 in PBST+SMP.
After 6 consecutive 4 minute washes in PBST, the membrane was incubated for 1 hour
with peroxidase-conjugated affinipure donkey anti-rabbit immunoglobulin G secondary
antibodies ( Jackson ImmunoResearch Laboratories, Baltimore, MD) at a dilution of 1:5000
in PBST+SM P. After incubation the membrane was washed 6 x 4 minutes, then drained
and developed using a chemiluminescent substrate (such as a Super Signal West Pico
chemiluminescent substrate) and imaged in a ChemiDoc-XRS (chemiluminescence)
Imaging System (Bio-Rad).
Intensity of the red pepper dehydrin bands on the membrane were compared to those of
the concentration gradient of the dehydrin standards and relative concentrations of red
pepper dehydrin in red pepper estimated by eye and back calculated to amounts present
per gram of dry red pepper core. The results indicated that natural levels of dehydrin in red
pepper vary from non-detectable to approximately 20 % of that of the lowest dehydrin
standard concentration , that is to say about 0.01 mg/mL equivalent to 0.1 mg dehydrin per
g dry weight, or 0.01 % w/w.
b) Infusion of red pepper with exogenous dehydrin and estimation of levels thereof
Red pepper pericarp was cored using a circular 1cm diameter cork borer, the pieces rinsed
in water and blotted dry on tissue paper. A 1mg/mL aqueous solution of the tea dehydrin of
example 3 was prepared and 2 mL of the solution used to cover 3 pepper cores in a 10 mL
beaker. These pieces were then vacuum infused for 4 hours at room temperature. After
infusion the pieces were rinsed three times in water to remove surface dehydrin and blotted
dry on tissue paper. The infused pieces were then placed in an air assisted fan oven to dry
until constant weight, starting at 40 degrees centigrade for 1 hour followed by 60 degrees
centigrade for 5 hours. Protein was extracted from the pieces as described above.
2 of the total protein extract was loaded onto two a 4-1 2 % bis-(2-hydroxy-ethyl)-aminotris(
hydroxymethyl)-methane SDS-PAGE gel. An extract from a non-infused red pepper
was loaded as a control. 10 m I_ of three standards of pure dehydrin (Invitrogen) were
loaded alongside at different concentrations (0.5, 0.1 and 0.05 mg/ml). Molecular markers
(All Blue Precision Plus Protein Standards from Bio-Rad) were also run. The gel was run at
200 V for 30 minutes to separate proteins by effective molecular weight. Proteins from one
gel were transferred to a PVDF membrane and probed with anti-dehydrin antibody using
Western Blotting as described above to confirm proteins observed in infused tissue
extracts at 37 kDa were dehydrin proteins. The other gel was stained for 1 hour (with
Simply Blue Safe Stain from Invitrogen) and then destained overnight before imaging.
Proteins on the dye stained SDS-PAGE gel were imaged using a gel imaging system (Gel
Logic 200 imaging system from Gel Logic) and the images adjusted using brightness,
contrast and inversion to remove background and highlight areas of intense protein
concentration to allow an estimation of the amount of dehydrin infused into red pepper
tissue. Estimation of concentration of dehydrin on the gel was back calculated to estimate
amounts present per gram of dry red pepper tissue.
The infused red pepper extract showed an intense protein band at -37 kDa, equivalent in
size to that of the pure dehydrin infused into the tissue and used for the concentration
gradient, this band was absent from the non-infused control. The intensity of the band was
between that of 1 and 5 mg of pure dehydrin standard loaded onto the gel and thus more
than 1 mg, but less than 5 mg per 2 m of extract. As the extract was 10 mL/gram dry tissue,
the amount of dehydrin infused was about 5 mg dehydrin per g of dry tissue, but not more
than 25 mg/g (0.5-2.5 % dehydrin per dry weight).
Example 9 : Effects of dehydrin infusion on onion monolayer
Onion (Allium cepa) epidermal peels were used as a model plant cell monolayer system.
Epidermal cells ( 1 cm x 2 cm) were prepared and fully immersed in 0.1 mg/mL full-length
Camellia sinensis dehydrin (obtained from example 2) solution in 50 mM Bis-Tris buffer
with 0.25 M trehalose (BTT buffer). The dehydrin was vacuum infused into onion tissue
for two hours at room temperature. Water, 0.1 mg/mL bovine serum albuinin (BSA) in BTT
buffer and BTT buffer only infused peels were also prepared as controls. 0.75 g of infused
onion tissue was dried overnight at 50 degrees centigrade and rehydration carried out by
immersion in 25 mL water for 2 hours at room temperature.
Rehydrated tissues were mounted on microscope slides and stained in-situ with 0.04%
(w/v) trypan blue. After rinsing with deionised water, cover slips were applied and slides
were observed with a light microscope (Leica DMRB) at 10 X magnification. A digital
colour camera (JVC KY-F75U) was used to capture images (JVC KY-LINK Software).
Trypan blue is a vital dye used for visualising cell viability. Figure 15a shows live cells or
tissues with intact cell membranes which exclude the dye as highlighted in the fresh tissue
control samples. The dye can enter cells with damaged cell membranes, for example in
blanched tissue (blanched 2 minutes, boiling water) making the nuclei clearly visible and
the membranes appear to detach from the cell walls as shown in figure 15b.
Figure 16a shows that dehydrin infused onion epidermal peel following drying and
rehydration showed little evidence of tissue damage and the cell nuclei are not visible. The
cells also appeared swollen compared to the controls with no dehydrin (figures 16b-d),
indicating greater rehydration. In particular, the controls with no dehydrin showed signs of
tissue damage and the nuclei are visible. The extent of cell swelling in the water (figure
16b) and buffer (figure 16c) controls was lower than the dehydrin infused sample indicating
a limited uptake of water on rehydration.
The swelling of the BSA infused control (figure 16d) was intermediate between dehydrin
infused tissue (figure 16a) and water / buffer controls (figures 16b and c) indicating
retention of water inside cells. However the membranes were not protected and allowed
the passage of vital dye inside the cells, evidenced by nuclear staining.
Claims
A dried rehydratable food comprising less than 10% w/w water and at least 0.02%
w/w of a dehydrin protein and derivatives thereof, the dehydrin protein and
derivatives thereof comprising an amino acid sequence selected from the group
consisting of K,I,K,E,K,L,P,G; K,I,K,E/D,K,L/I,P,G; and K,I,K,E/D,K,L/I/TA/,P/H/S,G,
and wherein the dried rehydratable food is unbroken tissue of a vegetable or part
thereof and/or a fruit or part thereof, and not a seed, wherein the unbroken tissue
has a shortest linear dimension of at least 0.5 millimetres, preferably a shortest
linear dimension of 0.5 to 25, more preferably 0.5 to 10 millimetres.
A dried rehydratable food according to claim 1 comprising 0.02 to 20, preferably 0.1
to 5, most preferably 0.2 to 2.5 % w/w dehydrin protein and derivatives thereof.
A dried rehydratable food according to claim 1 or claim 2, wherein the dehydrin
protein and derivatives thereof have a molecular weight of 1 to 150, preferably 5 to
100, most preferably 5 to 50 kD.
A dried rehydratable food according to any one of the preceding claims, wherein the
dehydrin protein and derivatives thereof is a dehydrin derived from the group
consisting of Camellia sinensis, Forsythia and Selaginella.
A dried rehydratable food according to any one of the preceding claims, wherein the
dehydrin protein and derivatives thereof has an amino acid sequence at least 80%
identical to SEQ. ID NO. 1, preferably at least 90%, 95% or 99% identical thereto.
A dried rehydratable food according to any one of the preceding claims, wherein the
dehydrin protein and derivatives thereof has an amino acid sequence at least 80%
identical to SEQ. ID NO. 2, preferably at least 90%, 95% or 99% identical thereto.
A dried rehydratable food according to any one of the preceding claims, wherein the
dehydrin protein and derivatives thereof has an amino acid sequence at least 80%
identical to SEQ. ID NO. 3, preferably at least 90%, 95% or 99% identical thereto.
8. A dried rehydratable food according to any one of the preceding claims, wherein the
rehydratable food additionally comprises a compound selected from the group
consisting of trehalose, sucrose, glucose, fructose, raffinose, an enzymatic
antioxidant or a non-enzymatic reactive oxygen species scavenger.
A dried rehydratable food according to claim 8, wherein the enzymatic antioxidant is
selected from the group consisting of catalase, superoxide dismutase, ascorbate
peroxidase and glutathione reductase.
A dried rehydratable food according to claim 8, wherein the non-enzymatic reactive
oxygen species scavenger is selected from the group consisting of ascorbate,
glutathione and a carotenoid.
A dried rehydratable food according to any one of the preceding claims, wherein the
vegetable is selected from the group consisting of spinach, broccoli, onion,
aubergine, courgette, potato, pumpkin, mushroom, carrot, tea, asparagus, turnip,
leek, beetroot, cauliflower, celeriac, artichoke, mint, thyme, oregano, rosemary,
parsley, sage, chives, marjoram, basil, bay leaf, tarragon, celery and garlic and the
fruit is selected from the group consisting of lemon, raspberry, red currant,
blackberry, berry, blueberry, strawberry, pineapple, banana, peach, apricot, lychee,
apple, pear, tomato, capsicum, cucumber and mango.
A food product comprising a dried rehydratable food according to any one of the
preceding claims.
A food product according to claim 12 which is selected from the group consisting of
a dried soup, a dried beverage, a breakfast cereal, a yoghurt and a dried sauce.
A method for manufacturing a dried rehydratable food according to any one of
claims 1 to 11, the method comprising the steps of:
(a) Infusing a vegetable or part thereof, or a fruit or part thereof excluding a
seed, with a dehydrin protein and derivatives thereof, the dehydrin protein
and derivatives thereof comprising an amino acid sequence selected from
the group consisting of K,I,K,E,K,L,P,G; K,I,K,E/D,K,L/I,P,G; and
K,I,K,E/D,K,L/I/TA/,P/H/S,G to produce an infused food; and
(b) Drying the infused food thereby to produce a dried rehydratable food
according to any one of claims 1 to 11.
A method for manufacturing a dried rehydratable food according to claim 14,
wherein step (a) of claim 14 is carried out under a vacuum.
A method for manufacturing a dried rehydratable food according to claim 14 or
claim 15, wherein step (a) of claim 14 is carried out at a temperature of 3 to 70,
preferably 10 to 50, most preferably 15 to 30 degrees centigrade.
A method for manufacturing a dried rehydratable food according to any one of
claims 1 to 11, the method comprising the steps of:
(a) Cloning a gene into a plant expression vector thereby to produce a modified
plant expression vector, wherein the gene encodes a dehydrin protein and
derivatives thereof, wherein the dehydrin protein and derivatives thereof
comprises an amino acid sequence selected from the group consisting of
K,I,K,E,K,L,P,G; K,I,K,E/D,K,L/I,P,G; and K,I,K,E/D,K,L/I/TA/,P/H/S,G;
(b) Introducing the modified plant expression vector into a target crop by plant
transformation thereby to produce a transgenic target crop;
(c) Growing the transgenic target crop thereby to express the dehydrin protein
and derivatives thereof; and then
(d) Drying the transgenic target crop thereby to produce a dried rehydratable
food according to any one of claims 1 to 11.
A dehydrin protein with an amino acid sequence identical to SEQ. ID NO. 1, and
derivatives thereof.
A dehydrin protein with an amino acid sequence identical to SEQ. ID NO. 2, and
derivatives thereof.
20. A dehydrin protein with an amino acid sequence at least 80% identical to SEQ. ID
NO. 4, preferably at least 90%, 95% or 99% identical thereto, and derivatives
thereof.