Plant Molecular Biology 17: 1013-1021, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.

1013

Expression of antifreeze proteins in transgenic plants Robin Hightower, Cathy Baden, Eva Penzes, Peter Lund and Pamela Dunsmuir*

DNA Plant Technology Corporation, 6701 San Pablo Avenue, Oakland, CA 94608, USA (*author for correspondence) Received 14 May 1991; accepted in revised form 8 July 1991

Key words: transformed plants, antifreeze protein, ice recrystaUization

Abstract

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystaUization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. The afa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.

Introduction

Several proteins that adsorb to ice are known to cause a freezing point depression when present at high concentrations ( > 10 mg/ml), and an inhibition of ice recrystallization at much lower concentrations ( < 100#g/ml). The antifreeze proteins (AFPs) and glycoproteins (AFGPs) from polar fish [9], and the thermal hysteresis proteins (THPs) from insects [ 11 ] are the best characterized proteins of this kind. The AFPs in the blood of the northern hemisphere winter flounder (Pseudopleuronectes arnericanus) are a heterogeneous mixture of related proteins containing repeats of an alanine-rich 11 amino acid unit [6, 8, 10]. The two predominant AFP species in the winter flounder each contain three imperfect repeats of this unit [ 16]. A detailed study on the

recrystallization inhibition (RI) activity of several antifreeze protein analogues has recently shown that RI activity increases progressively for chimeric proteins whose AFP portions contain three, four and five 11 amino acid repeats [27]. We are interested in the possibility that the presence of AFPs in plant tissue could improve the freezing properties of harvested produce. At present, there are many examples of foodstuffs that cannot be frozen without unacceptable changes in quality, particularly fruits and berries, where texture is an important part of their perceived quality [2, 19]. It is known that extracellular and intracellular ice formation results in undesirable changes in texture due to the dehydration of tissue constituents, concentration of solutes, and mechanical factors that can collectively lead to destruction of the plasma mem-

1014 brane [4, 36]. Ice formation in frozen foods is influenced by both rate of freezing and storage temperature; when plant tissues are frozen slowly, ice crystals fill the extracellular spaces and are generally large and randomly distributed. In contrast, small intracellular and extracellular ice crystals form when tissues are frozen rapidly. During storage, fluctuations in temperature can result in an increase in ice crystal size as well as a redistribution of ice in food [ 19]. If the food thaws, a deterioration of quality from subsequent refreezing at a slow rate in storage can also occur [3]. The aim of this research is to determine whether the properties of frozen and thawed plant tissue can be modified by the expression of genes which affect ice crystal size and structure. We have focused upon the winter flounder AFP analogues, and have used two forms that have been characterized with respect to RI actitivity. The afa3 gene, a chemically synthesized sequence, contains three repeats of an alanine-rich 11 amino 'acid repeat. The spa-afa5 gene encodes a fusion between a truncated staphylococcal protein A [ 28 ] and afa5, a derivative of afa3 which contains five repeats. These genes have been engineered for expression in plant cells by fusion to a plant promoter and polyadenylation region. Additionally, we have prepared fusions of the afa3 and spa-afa5 gene to a gene fragment which encodes a plant signal sequence in order to determine whether intracellular or extracellular expression of AFP alters freezing properties differentially. Here we report expression of the afa3 and spaafa5 genes at the transcriptional level in transgenic tobacco or tomato plants. Furthermore, we have detected the chimeric antifreeze protein Spa-Afa5 as well as ice recrystallization inhibition, corresponding to antifreeze protein activity, in transgenic tomato extracts. Materials and methods

Plasmid constructions The synthetic antifreeze gene afa3 (identical to afa3 SB/R, [27]) contains Sma I and Nco I sites at the 5' end, and a Bam HI site downstream of

the translation termination site [27]. Ligation of the Nco I-Barn HI afa3 fragment into the pChiA plasmid (cut with Nco I and Bam HI) [23] produces pAF1, a plasmid with the antifreeze gene plus an initiator methionine residue fused to the CaMV 35S promoter upstream of the Nco I site, and the nos polyadenylation site [7] downstream of the Bam HI site. Ligation of the Sma I-Barn HI afa3 fragment into pPRSSChiA [23] results in pAF3 with the antifreeze gene fused (in frame) to codons for the tobacco P R l b gene signal sequence together with plant expression signals. Spa-afa5, a protein A-antifreeze gene fusion, has been described previously [24]. In order to clone this fusion into the above vectors, the Hind III site downstream of afa5 was converted to a Barn HI site using linkers, and the Bcl I-Barn HI fragment containing spa-afa5 [24] was subcloned into the commercially available vector pGEM7Zf( + ). The Nco I-Bam HI or Sma I-Bam HI fragments from this plasmid were then cloned into pChiA or pPRSSChiA plasmids. The resultant pAAF1 encodes a non-secreted form of spaafa5 and pAAF3 contains spa-afa5 in the correct reading frame relative to the P R l b signal sequence. D N A fragments encompassing plant expression signals fused to afa3 were transferred to binary vectors in a single step by virtue of unique restriction sites upstream in the CaMV 35S promoter (Bgl II) and downstream of the nos 3' end (Hind III). This BgllI-Hind III fragment was cloned into pJJ2964 (KanR), a binary vector with unique Bam HI and Hind III sites between the T-DNA borders. To subclone spa-afa5 from the pAAF plasmids int0 pJJ2964, it was necessary to isolate Hind III partial-Bgl II fragments because there are two additional Hind III sites within spaafa5. Gene fusions were cloned into the p J J2964 binary vector for subsequent transfer into plant cells.

Production of transgenic tobacco and tomato plants The binary constructions described above, and p J J3499 as a negative control, were individually transformed into Escherichia coli strain JM83 and

1015 then introduced into Agrobacterium tumefaciens strain LBA4404 [ 17] by conjugal transfer. Triparental matings were conducted with HB101/ pRK2013 [ 12] providing the mobilization functions. Tobacco [18] and tomato [30] were transformed by cocultivation of sterile leaf discs derived from SR1 and Bonny Best varieties, respectively. Individual transformed shoots arising on leaf discs were selected by their capacity to form roots on media containing 150/~g/ml kanamycin. The afa3 constructions (AF1, AF3) were introduced into both tobacco and tomato, while the spa-afa5 fusions (AAF1, AAF3) were introduced only into tomato.

mRNA expression analysis Total leaf RNA was isolated from transformed plants as described [15], and the steady-state m R N A levels were evaluated by RNase protection [25]. For analysis of plants which carry the afa3 gene, a 208 nucleotide radio-labeled antisense afa3 transcript was prepared in vitro by T7 polymerase from plasmid pB S(-)AF (Bluescript). 10 #g of leaf RNA was annealed with the labeled RNA probe and RNase-digested products were analyzed on a denaturing acrylamide gel. The predicted protected fragment sizes in pAF1 and pAF3 transformants are 134 and 141 nucleotides, respectively. For the spa-afa5 RNase protection experiments, a 255 nucleotide radio-labeled antisense afa5 transcript was prepared in vitro by T7 polymerase from plasmid pBS(-) afa5. The predicted protected fragment size for pAAF1 and pAAF3 transformants is 204 nucleotides.

Protein analysis Total soluble protein extracts were prepared from leaves by grinding the tissue to a fine powder in liquid nitrogen and resuspending in 0.2 M Tris pH 7.5, 15~o fl-mercaptoethanol. Samples were vortexed well, boiled for 10 min, and centrifuged at 12000 x g for 15 min at 4 °C. Protein concentrations in the supernatants were estimated by the

Bradford method [ 1]. Total soluble extracts were eletrophoresed on 12.5 ~o SDS-polyacrylamide gels and electroblotted onto nitrocellulose. Immunodetection was performed with a monoclonal antibody to protein A (Sigma), using the Protoblot Immunoblotting System (Promega Biotec).

Antifreeze activity analysis Antifreeze protein activity in transgenic plants was assayed with the 'splat' assay [21] which is based on the property of antifreeze proteins, at low concentrations, to inhibit recrystallization of ice (RI activity). In this assay, a 5/A drop of total soluble protein extract is rapidly frozen by impacting upon a polished metal plate maintained at -70 ° C, then the frozen drop is transferred to a cryostage at - 7 ° C. Ice crystal growth is monitored and photographed after 1-2 h at low power through cross-polarizing lenses.

Overexpression of Spa-Afa5 in E. coli The spa-afa5 gene was subcloned into pKK2332, a bacterial overexpression vector, for high-level expression of the protein in E. coli [37].

Results

Construction of chimeric antifreeze genes Evaluation of RI activity has shown that the purified Spa-Afa5 fusion protein is 10-fold more active than purified Afa3 protein [27]. The afa3 and spa-afa5 genes were each used in plant transformation experiments; afa3 was introduced into tobacco and tomato, and spa-afa5 was introduced into tomato. The predicted sequences of the Afa3 and Afa5 proteins are shown in Fig. la. Figure lb diagrams the afa3 (AF1, AF3) and spa-afa5 (AAF1, AAF3) chimeric genes that were transferred to plants. The AF1, AAF1 constructs contain the CaMV 35S promoter fused to the afa3 or hybrid spa-afa5

1016 a,

Afa3 MD-TASDAAAAAAL-TAANAAAAAKLTADNAAAAAAA-TAR-COOH Afa5 HAAD-TASDAAAAAAL-TAANAAAAAAL-TAANAAAAAALTAANAAAAAAL-TAANAAAAAAA-TAA-COOH

b. N B

Bg I CaMV 35Sp

Bg I

H 1 ~fa3 nos 3'

~

AF1

N

B

] , spa

NS

H I

===d

AAF1

afa5

B

I

I

AF3

afa3

Bg

1

NS

B

~I spa

~ afa5

H

I

AAF3

~2~ PRI-b signal sequence

Fig. 1. a. One-letter amino acid code for the protein sequences encoded by the afa3 and the afa5 genes. The carboxyl terminus of Afa3 and Spa-Afa5 is COOH. b. Scheme of the afa3 and spa-afa5 genes introduced into plants. B, Barn HI; N, Nco I; Bg, Bgl II; S, Sma I; H, Hind III.

gene followed by the nopaline synthase polydenylation region. The AF3, AAF3 genes differ from AF1, AAF1 by the insertion of a signal sequence encoding fragment which should direct the secretion of the contiguous protein to the extracellular space in plant tissue. Based on the predicted cleavage site of this signal peptide, the processed form of Afa3 will contain 5 additional residues at the amino terminus relative to the nonsecreted form of Afa3. As a consequence of cloning, the spa-afa5 gene in the AAF1 and AAF3 constructs encodes Spa-Afa5 protein containing 10 additional amino-terminal residues. In view of the amino terminal differences between the Spa-Afa5 protein which we were at-

tempting to express in plants and the one characterized to be highly active after expression in E. coli [27], we prepared the altered form (Spa-Afa5) in E. coli to determine its activity. The spa-afa5 genes encoding secreted and nonsecreted forms of Spa-Afa5 were subcloned into a bacterial overexpression vector, and the RI activity in E. coli extracts was compared to equivalent amounts of purified Mpa-Afa5 protein. The Mpa-Afa5 protein is a derivative of Spa-Afa5 with equivalent antifreeze activity but a smaller protein A portion. Comparable RI activity occurred in total soluble protein extracts from E. coil cells expressing the spa-afa5 'plant' fusion and extracts containing an equivalent amount of Mpa-Afa5, confirming that

1017 the altered protein expressed in plants was not compromised in activity (data not shown). The afa3 gene was not expressed in E. coli since it encodes a very small protein which is thought to be subject to selective degradation [22, 34].

Expression of the chimeric antifreeze genes in plants RNase protection analysis was used to measure

afa3 and spa-afa5 steady-state m R N A levels in transgenic tobacco and tomato [25]. The RNase protection analysis for R N A from leaves of 6 independent tobacco plants transformed with AF 1 (afa3 with no signal sequence) is shown in Fig. 2. The undigested R N A probe (lane 1) and two negative controls (the probe hybridized to t R N A or to R N A from AF1- plants; lanes 2, 3) are also shown. In each of the AF1 transformants, there is evidence of afa3 transcription as indicated by the presence of a 134 base fragment (lanes 4-9). In addition to these tobacco transformants, fourteen independent tomato transformants containing the AF 1 gene were analyzed and similar levels of afa3 expression were observed in these plants (data not shown). Fifteen transgenic tobacco plants containing the AF3 gene (afa3 with the P R l b signal sequence) were also analyzed by

RNase protection, and expression in thirteen transformants was observed as a 141 base protected fragment (data not shown). The predicted RNase protected fragment size in AAF1 and AAF3 (apa-afa5) transformants is 204 nucleotides. Figure 3 shows RNase protection on R N A isolated from the leaves of 10 independent AAF1 transformants (spa-afa5, no signal sequence), and 10 AAF3 transformants (spa-afa5, P R l b signal sequence). The protected fragments which occur approximate the expected size (frequently R N A migrates as if it is 5-10~o larger than D N A of the same size on denaturing gels). These RNase protection data indicate that many of the transgenic plants contain high levels of afa3 or spa-afa5 mRNA; no significant difference in m R N A levels occurred between afa3 or spa-afa5 transformants. The plants with the highest levels of afa3 and spa-afa5 m R N A were submitted to protein and/or activity assays.

Expression of the chimeric antifreeze protein in plants Where possible, we have used immunoblotting of proteins extracted from transgenic plants to determine whether antifreeze protein is being ex-

Fig. 2. RNase protection analysis on transgenic tobacco plants (afa3). Steady-state afa3 mRNA levels in AF1 trans-

Fig. 3. NRase protection analysis on tomato plants containing spa-afa5. Steady-state spa-afa5 mRNA levels in AAF1

formants were measured using RNase protection analysis; the predicted protected fragment size is 134 bases. Lane 1, undigested RNA probe; lane 2, 20 #g tRNA; lane 3, 20 #g RNA from AF-plant; lanes 4-9, 20/lg RNA from independent transformants, b = bases.

and AAF3 transformants were measured using RNase protection analysis; the predicted protection fragment size is 204 bases. *indicates the plants which were analyzed by western analysis and the splat assay, b--bases. 20 #g RNA in each lane.

1018 pressed. We do not currently have antibodies which cross-react with Afa3 protein hence we were unable to assay protein levels directly in the AF1 or AF3 transformants. However, we have used a monoclonal antibody against protein A to measure levels of Spa-Afa5 fusion protein in the AAF1 and AAF3 transformants. Total soluble protein was isolated from selected AAF1 and AAF3 tomato transformants and western analysis was performed (Fig. 4). No cross-reacting proteins were detected in the AF- plants (lane A); a doublet of 35 and 37 kDa was observed in extracts of some of the AAF3 plants (Fig. 4, AAF3 plants 9, 8, 6) while only the faster migrating band (35 kDa) was detected in selected AAF1 plants (Fig. 4, AAF1 plants 3, 8, 6). Since the predicted molecular weight of the Spa-Afa5 protein is 35 kDa, these data suggest that a full-length Spa-Afa5 protein is being synthesized in planta. Compared to the 100 ng of Mpa-Afa5 protein (lane B), we estimate that the Spa-Afa5 level in the transgenic plant extract AAF3 No. 9 approaches 100 ng or 0.1 ~o of total soluble leaf protein.

total soluble protein extracts from selected transformants were used in a 'splat' assay and recrystallization inhibition (RI) was measured [21]. This technique allows direct visual examination of ice crystals while recrystallization occurs. Splats on leaf extracts from AF1 and AF3 transformants and AF-plants showed a parallel increase in ice crystal size with time, suggesting that RI activity was not present in the plants which carry the afa3 gene. In contrast, splats from the AAF1 and AAF3 (spa-afa5) transformants displayed a crystal size that increased less than the AF- control in the same time period (Fig. 5). These results indicate that the Spa-Afa5 protein expressed in the transgenic plants was active in recrystallization inhibition. Purified Mpa-Afa5 protein, a derivative of Spa-Afa5 with equivalent antifreeze activity, was added to an AF- extract to demonstrate that it was functional in the presence of the plant extract. Furthermore, comparable levels of antifreeze activity were observed for Mpa-Afa5 (10 ng/#l) and AAF3 No. 9, the transgenic plant containing the highest Spa-Afa5 levels by western analysis. Using Mpa-Afa5 as a

Recrystallization inhibition activity of the chimeric antifreeze proteins in plants To determine whether the antifreeze protein which is expressed in transgenic plants is active,

Fig. 4. Western analysis of tomato plants containing SpaAfa5. 100 #g protein from indicated transgenic plants. Lane A, AF-plant. Lane B, 100 ng Mpa-Afa5. *indicates the extracts shown in Fig. 5 splat analysis.

Fig. 5. Ice recrystallization inhibition by Spa-Afa5 protein in transgenic tomato leaf extracts. 'Splats' were prepared as described in Materials and methods. Photographs of splats derived from extracts of AAF1 # 8, AAF3 # 9, AF-, and AFcontaining 10 ng/#l Mpa-Afa5. All samples contained 7.5 mg/ ml protein.

1019 standard of comparison, we estimate that the antifreeze activity observed in extracts from the other transgenic plants corresponds to less than 10 ng/#l, consistent with the immunoblotting data which showed that these plants contain significantly less Spa-Afa5 protein than AAF3 No. 9.

Discussion

This research provides the first demonstration that antifreeze genes are expressed and that a functional antifreeze protein can be produced in transgenic plants. Both the gene which encodes antifreeze protein alone (afa3), as well as the gene which encodes the antifreeze fusion (spa-afa5), were transcribed to high steady-state m R N A levels in tobacco and tomato. Antifreeze protein activity, measured by ice recrystallization inhibition, was detectable only in transgenic plants containing spa-afa5. Since the Afa3 protein is 10-fold less active than the Spa-Afa5 protein, it may be at levels in the plants that are below the limit of detection in the splat assay. In the absence of antibodies specific for antifreeze protein, it is not possible to determine the level of Afa3 protein in these plants. The larger and more active SpaAfa5 fusion protein confers RI activity to plant extracts; it is possible that the size and nature of the protein A portion protects the small antifreeze protein from proteolytic degradation in plants. Based upon our measurements of steady-state transcript levels for the different AAF1 and AAF3 transformants, we selected 'high expressors' for protein and activity analyses. Although the selected transformants had m R N A levels which differed by approximately 50~o, the relative AF protein levels differed much more based on western analysis (Fig. 4). We do not yet understand this lack of correlation between m R N A and protein levels in the selected AAF3 transformants which we have analyzed. However, for all transgenic plants, the levels of protein measured by immunoblotting and by activity assay are consistent, suggesting that a full-length, functional protein A-antifreeze protein fusion is present in the transgenic plants.

Genes that encode secreted and nonsecreted forms of Spa-Afa5 have been expressed in transgenie plants. In AAF3 transformants with the spa-afa5 gene plus a secretion signal, the SpaAfa5 protein is presumably targeted to the extracellular space where it can adsorb to ice associated with extracellular ice formation. In AAF1 plants expressing a non-secreted form ofspa-afa5, the Spa-Afa5 protein remains within the cell where it can inhibit intracellular ice recrystallization. Two proteins, 35 and 37 kDa, were detected in AAF3 transformants whereas only the 35 kDa protein was observed in AAF1 plants. It is possible that the multiple size classes result from post-translational modification of Spa-Afa5 protein in the AAF3 transformants since there are four putative N-glycosylation sites (Asn-X-Thr/ Ser) in the protein A portion of the protein [ 13]. Alternately, the 37 k D a band may represent the unprocessed protein although this is unlikely since the P R l b signal sequence is known to direct efficient secretion of several foreign proteins in plant cells [23, 35]. Additional experiments must be done to confirm that Spa-Afa5 is in the extracellular space in the AAF3 transgenic plants. Recently, it has been shown that antifreeze protein has the potential for improving plant cold hardiness by lowering the freezing temperature of leaves that have been infiltrated with antifreeze protein [5, 14]. In contrast, our goal is to develop fruit and vegetable products with improved postharvest freeze/thaw quality based on the property of antifreeze protein to inhibit ice recrystallization. Changes in the quality of produce as a result of freezing and thawing can occur in several ways [20, 32]: there will be membrane damage, with consequent loss of the liquid-retaining properties of cells, and therefore succulent plant tissues lack turgor upon thawing [26]. In addition, the dehydration of cellulose during freezing may account for changes in textural qualities of thawed fruits and vegetables [31]. The freezing rate influences freezing damage, with the extent of damage being influenced by the structural complexity and the water content of the plant tissue [29]. In foods that are frozen slowly, ice crystals are large and randomly distributed and ice fills the extracellu-

1020 lar space causing dehydration of the cell and forcing cells apart. When plant tissues are frozen rapidly, water does not translocate across the plasma membrane and small uniformly distributed ice crystals are formed within the cell [ 19]. The expression of A F proteins in plant tissue could impact only part of the multidimensional freezing damage process, but it may still be able to improve the overall quality of frozen plant products. A recent observation that A F G P s protect membranes at hypothermic temperatures [ 33 ] suggests that there may be additional properties of these proteins significant in reducing freezing damage. To extend our research, we will determine whether whole tissue from these transformants has improved freezing properties. With the availability of purified Spa-Afa5 protein, we will also establish the extracellular levels of antifreeze protein that are necessary to alter the freezing properties of the tissue by vacuum infiltration of the fruit with this protein.

6. 7.

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9. 10.

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14.

Acknowledgements 15.

We thank the greenhouse staff for maintenance of plants in the greenhouse; Joyce Hayashi, Rob Narberes and Lily Kruger for help with manuscript preparation, and Bob McKown and Gary Warren for helpful discussions during the course of this research.

16.

17.

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Expression of antifreeze proteins in transgenic plants.

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze prot...
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