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 particular, 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, texture 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 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 food product comprising the dried rehydratable food and methods for manufacturing the dried rehydratable food are also provided.

Description:
[0001]    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. 
         [0002]    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. 
         [0003]    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. 
         [0004]    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 
       [0005]    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/T/V,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. 
         [0006]    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. 
         [0007]    Derivatives thereof include glycosylated dehydrins and truncated dehydrins, wherein truncated dehydrins still contain the critical K segment (see below). 
         [0008]    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. 
         [0009]    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. 
         [0010]    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. 
         [0011]    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. 
         [0012]    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  FIG. 3   b ), preferably at least 90%, 95% or 99% identical thereto. It was observed that the dehydrin of SEQ. ID NO. 1 was 47× 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. 
         [0013]    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  FIG. 4   b ), preferably at least 90%, 95% or 99% identical thereto. 
         [0014]    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  FIG. 5   b ), preferably at least 90%, 95% or 99% identical thereto. 
         [0015]    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. 
         [0016]    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. 
         [0017]    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. 
         [0018]    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. 
         [0019]    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/T/V,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.   
 
         [0022]    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. 
         [0023]    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. 
         [0024]    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/T/V,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.   
 
         [0029]    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. 
         [0030]    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. 
         [0031]    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 
         [0032]    The invention will now be exemplified with reference to the following figures in which: 
           [0033]      FIG. 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 φ 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 α-helix forming domain (the sequences of letters refer to the corresponding amino acid sequence); 
           [0034]      FIG. 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 KCl gradient (the arrow indicates the position of the dehydrin band); 
           [0035]      FIG. 3  shows a tea dehydrin gene in schematic form in  FIG. 3   a  and in the form of a nucleotide and deduced amino acid sequence in  FIG. 3   b  (SEQ. ID NO. 1); 
           [0036]      FIG. 4  shows the tea dehydrin of  FIG. 3  in truncated form (truncate 122-201 of SEQ. ID NO. 1) in schematic form in  FIG. 4   a  and in the form of a nucleotide and deduced amino acid sequence in  FIG. 4   b  (SEQ. ID NO. 2); 
           [0037]      FIG. 5  shows the tea dehydrin of  FIG. 3  in truncated form (truncate 163-201 of SEQ. ID NO. 1) in schematic form in  FIG. 5   a  and in the form of a nucleotide and deduced amino acid sequence in  FIG. 5   b  (SEQ. ID NO. 3); 
           [0038]      FIG. 6  shows the nucleotide and deduced amino acid sequence (SEQ. ID NO. 4) of the  Forsythia suspensa  dehydrin gene; 
           [0039]      FIG. 7  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; 
           [0040]      FIG. 8  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; 
           [0041]      FIG. 9  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; 
           [0042]      FIG. 10  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; 
           [0043]      FIG. 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;    
           [0044]      FIG. 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 Plt” 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; 
           [0045]      FIG. 13  shows load (N) versus distance (mm) graphs for pieces of green pepper ( Capsicum ) which have not been infused, dried and rehydrated ( FIG. 13   a ), which have not been infused but nevertheless dried and rehydrated in accordance with this example 6 ( FIG. 13   b ), which have been infused with water, dried and rehydrated in accordance with example 6 ( FIG. 13   c ); and which have been infused with  Selaginella lepidophylla  dehydrin, dried and rehydrated in accordance with example 6 ( FIG. 13   d ); 
           [0046]      FIG. 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); 
           [0047]      FIG. 15  shows a visible light micrograph (×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 
           [0048]      FIG. 16  shows visible light micrographs (×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 
       [0049]    Dehydrin genes are composed of distinct domains that exhibit high levels of conservation across plant species.  FIG. 1  shows a schematic diagram of the generalised architecture of dehydrins. Each protein is comprised of multiple copies of K, φ, 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, φ, 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, I/V/M/L/F, M/L/V, D/E, K, I, K, E/D, K, L/I, P, G. 
       Example 1 
     Extraction and Purification of Resurrection Plant  Selaginella lepidophylla  Dehydrin 
       [0050]    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. 
         [0000]    a) Preparation of Whole Protein Extract from  Selaginella lepidophylla  Tissue 
         [0051]    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-(N-morpholino)ethanesulfonic acid (MES), 20 mM NaCl 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. 
         [0000]    b) Detection of Dehydrin Proteins in  Selaginella lepidophylla  Whole Protein Extracts by Western Blotting 
         [0052]    A ˜20 μL aliquot of  Selaginella lepidophylla  total protein extract was loaded onto an electrophoresis gel (12% Novex bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bis-tris)) 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). 
         [0000]    c) Purification of Dehydrin Protein from  Selaginella lepidophylla  Tissue Whole Protein Extracts. 
         [0053]    20 mL  Selaginella lepidophylla  total protein extract was dialysed over night in 10 mM tris.HCl, 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 100 mL 0.02M:1 M KCl gradient. Pure dehydrin containing fractions were identified by sodium dodecyl sulphate/polyacrylamide (SDS-PAGE) gel electrophoresis, illustrated in  FIG. 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    
       [0054]    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
   
 
         [0061]    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&#39;s instructions. 
         [0000]    Synthesis of cDNA from mRNA to form cDNA Library 
         [0062]    mRNA was purified from 500 μg total RNA using a polyadenylic acid isolation kit (Qiagen) in accordance with the manufacturer&#39;s instructions. 5 μg of the polyadenylic acid-mRNA molecule was heated to 72 degrees centigrade for 5 minutes together with 2.8 μg 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 μL 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×RT buffer (Stratagene), 2.5 mM deoxyribonucleotide triphosphates (dNTP&#39;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). 
         [0063]    Second strand cDNA was synthesised using 45 μL of the first strand synthesis reaction in a 200 μL reaction for 150 minutes at 16 degrees centigrade by adding 11 μL DNA polymerase 1 (9 units/μl) (Stratagene), 20 μL 10× second strand synthesis buffer (Stratagene), 6 μL dNTP&#39;s (40 mM) (Amersham-Pharmacia) and 2 μL RNAse H (1.5 units/μL) (Stratagene). 180 μL of the second strand synthesis reaction was blunted for 30 minutes at 72 degrees centigrade by adding 20.7 μL dNTP&#39;s (10 mM) and 1.8 μl cloned Pfu DNA polymerase (Stratagene) (an enzyme found in the hyperthermophilic archaeon  Pyrococcus furiosus ) (2.5 units/μL). 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 μL 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× 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 μl reaction at 37 degrees centigrade for 30 minutes by adding 1 μl T4 polynucleotide kinase (10 units/μl) (Stratagene), 1 μl ligase buffer (10×) and 1 μl rATP (10 mM). 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 12×100 uL fractions by passing it through a 1 mL (flat bed volume) column (Chroma Spin-400 from Clontech) using 1×STE (100 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane-HCL (pH 7.5), 10 mM EDTA) (Stratagene) as the column buffer. 5 μL 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 μL 10 mM tris(hydroxymethyl)aminomethane (pH 7.6). 
         [0064]    10 ng of the pooled cDNA were ligated into 20 ng pBluescript SK+Xho1/EcoR1 digested vector (Stratagene) in a 5 μL reaction at 12 degrees centigrade overnight using 2 units T4 DNA ligase in 1× ligation buffer+1 mM rATP. The cDNA library was then transformed into XL10-Gold ultracompetent cells (Stratagene) according to the manufacturer&#39;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. 
         [0000]    Multiplication of cDNA for Sequencing 
         [0065]    Colonies were grown up overnight in 2.5 mL of 2TY broth (16 g tryptone, 10 g NaCl, 10 g yeast extract (pH 7.3) per litre) (made in house using Sigma reagents) containing 100 μg/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&#39;s instructions. The DNA was then quantified and diluted to 50 ng/μL using PicoGreen DNA quantitation kit (Molecular Probes BV) in accordance with the manufacturer&#39;s instructions. 
       Sequencing of Expressed Sequence Tags, Compilation of Contigs and Identification of Homologues 
       [0066]    DNA sequencing was carried out on an Applied Biosystems Genetic Analyser 3100 using 5 μL of template DNA at 0.1 μg/μL and 1 μL of primer at 1 pmol/μL 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). 
         [0067]    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-11). 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, φ segments and two K segments near the C-terminal end as shown schematically and in actuality (SEQ. ID. 1) in  FIGS. 3   a  and  3   b  respectively. No other single contig represented as many independent cDNA clones. 
         [0000]    Cloning of Full Length and Truncated Tea Dehydrins into pDEST17 Expression Vector 
         [0068]    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 pDONR221 vector (Invitrogen). The pDONR221 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. 
         [0069]    Tea dehydrin truncate (deoxynucleotide numbers 364-606) was designed to encompass the C-terminal φ 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 φ and one K segment only.  FIGS. 4   a  and  5   a  show respectively the schematic nucleotide sequence of each tea dehydrin truncate and  FIGS. 4   b  and  5   b  the actual nucleotide and deduced amino acid sequences of each tea dehydrin truncate (SEQ. ID. 2 and 3 respectively). 
         [0000]    Isolation and Cloning of  Forsythia Suspensa  Dehydrin cDNA into pDEST17 
         [0070]    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 ‘Y-segment’ dehydrin domain T/V,D,E,Y,G,N,P (see  FIG. 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  FIG. 1 ) in accordance with Close (Physiologica Plantarum, 100, 291-296 (1997)). In the aforementioned primer sequences, the letter “I” 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). 
         [0071]      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).  FIG. 6  shows the  Forsythia  nucleotide and deduced amino acid sequences. Table 1 shows the primers used to clone the  Forsythia  dehydrin sequence into pDEST17. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Polymerase chain reaction (PCR) primers used to generate attB 
               
               
                 (engineered foreign region from  E .  coli ) recombination target 
               
               
                 site flanked dehydrin sequences 
               
             
          
           
               
                   
                   
                 Sequence 
               
               
                 Primer 
                 Sequence 5′ to 3′ 
                 Generated 
               
               
                   
               
               
                 12attB1 TD 
                 AAAAAGCAGGCTTCATGGCACATAACAGCAAC 
                 Full length tea 
               
               
                 (SEQ. ID. 8) 
                   
                 dehydrin 
               
               
                   
               
               
                 12attB2 TD* 
                 AGAAAGCTGGGTTTTATTTATTAGTGGTGGTGTG 
                   
               
               
                 (SEQ. ID. 9) 
                   
                   
               
               
                   
               
               
                 12attB1 TD 
                 AAAAAGCAGGCTTCATGGAGGATGATGGTCAAG 
                 Truncated tea 
               
               
                 (364-606) 
                   
                 dehydrin 
               
               
                 (SEQ. ID. 10) 
                   
                   
               
               
                   
               
               
                 12attB1 TD 
                 AAAAAGCAGGCTTCATGGCAGCCACCACCGGT 
                 Truncated tea 
               
               
                 (487-606) 
                   
                 dehydrin 
               
               
                 (SEQ. ID. 11) 
                   
                   
               
               
                   
               
               
                 12attB1 FOR 
                 AAAAAGCAGGCTTCCTGCACTACTGAACAAACTTAG 
                 
                   Forsythia 
                 
               
               
                 (SEQ. ID. 12) 
                   
                 dehydrin 
               
               
                   
               
               
                 12attB2 FOR 
                 AGAAAGCTGGGTTCATAAACTCGACTCAGACGCATG 
                 
                   Forsythia 
                 
               
               
                 (SEQ. ID. 13) 
                   
                 dehydrin 
               
               
                   
               
               
                 *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 
       [0072]    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 
       [0073]    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 μg/mL ampicillin and incubated at 37 degrees centigrade overnight. 2.5 mL of LB medium containing 100 μg/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 μg/mL ampicillin and shaken at 37 degrees centigrade until the A 600  (absorbance at 600 nm) of the culture was 0.6. Dehydrin protein expression was then induced by the addition of isopropyl 6-D-1-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 
       [0074]    Cell pellets were vigorously resuspended in 8 mL Binding Buffer (50 mM NaPO4, 0.5 M NaCl, 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 
       [0075]    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 NaPO4, 0.5 M NaCl, 100 mM imidazole). The histidine tagged protein was eluted from the resin by the addition of 8 mL of Elution Buffer (50 mM NaPO4, 0.5 M NaCl, 250 mM imidazole). Fractions were collected in 1 mL aliquots and analysed by SDS-PAGE gel electrophoresis ( FIGS. 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 
       [0076]    Expression of the full length tea dehydrin in  Pichia Pastoris  and subsequent purification was conducted by Invitrogen Corporation (1600 Faraday Avenue, Carlsbad, Calif. 92008, USA) as a fully-funded toll manufacture of the protein. 
       Example 4 
     Effects of Dehydrin Infusion 
     Observations of Rehydrated Tissue 
       [0077]    Red pepper ( Capsicum ) pieces (1 cm 3 ) 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. 
         [0078]      FIG. 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 
       [0079]    To assess the effect of different dehydrins on red pepper ( Capsicum ) tissue dimensions, post-rehydration, 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  FIG. 12  wherein “Res Plt” 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. 
         [0080]    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 
       [0081]    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 ( FIG. 13   c  for water infused pieces and  FIG. 13   d  for  Selaginella lepidophylla  dehydrin infused pieces) variants are shown in  FIG. 13  together with controls for raw green pepper pieces which have not been infused but nevertheless dried and rehydrated in accordance with this example ( FIG. 13   b ), and raw green pepper tissue pieces which have not been infused, dried and rehydrated ( FIG. 13   a ). 
         [0082]    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 
       [0083]    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. 
         [0084]    Images of the dried (left hand images) and rehydrated leaves (middle images) are shown in  FIG. 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 
       [0085]    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 50 mM tris(hydroxymethyl)aminomethane, pH7.4, 250 mM NaCl, 5 mM EDTA, 2 mM Na 3 VO 4 , 1 mM NaF, 20 mM Na 4 P 2 O 7 , 0.02% NaN 3 , detergent and 0.5 mM 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). 
         [0086]    10 μL of the protein extract was loaded onto a 4-12% bis-(2-hydroxy-ethyl)-amino-tris(hydroxymethyl)-methane SDS-PAGE gel. 10 μL 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. 
         [0087]    The proteins were transferred from the SDS-PAGE gel onto a 0.2 μm 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 (SMP) 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+SMP. After incubation the membrane was washed 6×4 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). 
         [0088]    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. 
         [0000]    b) Infusion of Red Pepper with Exogenous Dehydrin and Estimation of Levels Thereof. 
         [0089]    Red pepper pericarp was cored using a circular 1 cm diameter cork borer, the pieces rinsed in water and blotted dry on tissue paper. A 1 mg/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. 
         [0090]    2 μL of the total protein extract was loaded onto two a 4-12% bis-(2-hydroxy-ethyl)-amino-tris(hydroxymethyl)-methane SDS-PAGE gel. An extract from a non-infused red pepper was loaded as a control. 10 μL 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. 
         [0091]    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 μg of pure dehydrin standard loaded onto the gel and thus more than 1 μg, but less than 5 μg per 2 μL 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 
       [0092]    Onion ( Allium cepa ) epidermal peels were used as a model plant cell monolayer system. Epidermal cells (1 cm×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. 
         [0093]    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× magnification. A digital colour camera (JVC KY-F75U) was used to capture images (JVC KY-LINK Software). 
         [0094]    Trypan blue is a vital dye used for visualising cell viability.  FIG. 15   a  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  FIG. 15   b.    
         [0095]      FIG. 16   a  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 ( FIGS. 16   b - 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 ( FIG. 16   b ) and buffer ( FIG. 16   c ) controls was lower than the dehydrin infused sample indicating a limited uptake of water on rehydration. 
         [0096]    The swelling of the BSA infused control ( FIG. 16   d ) was intermediate between dehydrin infused tissue ( FIG. 16   a ) and water/buffer controls ( FIGS. 16   b  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.