PHYSIOLOGY, BIOCHEMISTRY AND MOLECULAR BIOLOGY OF

A PREDOMINANT 18 kD PROTEIN IN THE ROOTS OF

DANDELION ( Taraxacum offinaale Weber)

A Tbesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

In partial fuifiIrnent of requirements

for the degree of

Doctor of Philosophy

August, 1998

6 Xiu-Ying Xu, 1998 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. nie ~drigtan OltawaON KlAONd OttawaON K1AW Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence dowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, disbr'buer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nùn, de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or othekse de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. PHYSIOLOGICAL, BIOCHIMICAL AND MOLECULAR BIOLOGY OF A PREDOMINANT 18kD PROTEIN IN THE ROOTS OF DANDELION (Tarmacum ofJieinaIe Weber)

Xiu-Ying Xu Advisors: University of Guelph, 1998 Dr. John S. Greenwood Dr. J. Derek Bewley

The root of dandelion (Taraxacum officinaleWeber) contains a prominent

18kD protein, which was previously thought to have a storage function. Since the characteristics of proteins can be determined from their sequence and expression of their genes, an isoform of the 18kD protein was purifed and its N-terminal amino acid sequence determined. Using this information oligonucleotide primers were constnicted and a full-length cDNA sequence obtained using RACE-PCR. The deduced full-length amino acid sequence of the protein showed that it has no signal peptide, it lacks serine, glutamine and tryptophan, has a pI of 5.6, two potential glycosylation and eight potential phosphorylation sites, and has a molecular mass of 16.91 1 W. The protein shows high homologies with a group of dergen and intracellular pathogenesis-related proteins with which it shares the common motif GXGGXG.It has two isoforms that are 85% homologous and three gene copies.

Although the 18kD protein is present throughout the year exclusively in the dandelion root and subterranean stem, with a small decline in amount in the summer, expression of its gene in these regions is greatly increased in the fall. This expression is affected by environmental perturbations involving temperature change, such as cold shock (20' to SOC) and heat shock (5' to 20°C). Leaf wounding and defoliation at SOC also stimulate both gene expression and protein synthesis, but not at 20°C. Thus the proteins may be more involved in a cold response than in having a storage role, and the signal to initiate gene expression may be through a systemic transduction pathway between the leaf and root. Although they share hornology to some pathogenesis-related proteins fimgal elicitor treatrnents of root discs do not cause an increase in gene expression. For my natural and adopted families ...... with love ACKNOWLEDGEMENTS

I am indebted to my Advisors, Drs. J. Derek Bewley and John S. Greenwood for their help and encouragement throughout this thesis work, and for their faith in my abilities. Without you, as friends and advisors, this would not have ken possible. My Advisory Cornmittee of Drs.

Annette Nassuth and Judith Strommer also helped me in my progress, and my daily contact with

Annette as a fnend and teacher was meaningfui for me.

My years in the Department were made happy by so many people (including Larry, Laurie and Sherry), in John and Annette's lab (Mike, Emily D., Shoba and Sandy K.), where 1 spent much of my working life, in Derek's lab (Emily W., Mitali, Rich, Ailan and Sandy R.), and in the little room where there were always fnendly faces and stimulating and fun conversations. You were al1 very special to me.

The time I spent working in Adelaide was generously made possible by Dr. Geoff

Fincher, and the help and fiendship of the lab members there was mernorable. Drs Heimo

Breiteneder and Karin Hoffman-Sommergmber from the University of Vienna kindly provided antibodies and clones that aided in the isolation of the L8kD protein message.

Thanks also to my many friends outside of the lab (in particular Wenjin and Ning, Li,

Yixin, Cuiwen, Jing, Xiuzhen, Huifang, Hailan, Weihai, Jian, Yuhai, Shujuan, Sen, and Mei) whose support over the years, and especially over the Iast few weeks helped me so much.

Finally a heartfelt thanks to my nahiral and to my adopted Chinese and Canadian families,

'Mum', 'Dad', Janette and Aiex, whose love and affection were so important to me

...... 1will miss you ail. CONTENTS

List of Figures ...... ix

List of Tables ...... xii

1. Literature Review ...... 1 1.1Introduction ...... 2 VSPidentification ...... 5 VSP identincation by organ removal ...... 6 VSP identification in bark and wood of deciduous trees according to their seasonal changes ...... 8 VSP identification in tuberous perennials according to their preferential synthesis during tuber development ...... 11 VSPs in roots of defoliated herbaceous perennids ...... 13 VSPgeneregulation ...... 14 The role of jasmonate (JA) and abscisic acid (ABA) in VSP gene expression ...... 16 The role of photopenod and temperature in VSP gene expression ...... 18 The role of water stress and wounding in VSP gene expression ...... 20 The role of nitrogen and sucrose in VSP gene expression ..... 24 The other possible roles of VSP ...... 26 Possible role of VSP in pathogen resistance ...... 26 Possible roles of VSP in frost hardiness ...... 27 Objectives ...... 29

2 . -cation of the 18 kD protein and the attempted isolation of a cDNA encoding the 18 kD protein ...... 30 2.1Introduction ...... 31 2.2 Materials and Methods ...... 34 2.2.1 Materials ...... 34 2.2.2 Methods ...... 34 2.2.2.1 Seasonal fluctuations of the 18 kD protein content in dandelion roots ...... 34 2.2.2.1.1Total soluble protein extraction from dandelion roots collected in different months ...... 34 2.2.2.1 2 Detennination of protein quantity ...... 3 5 2.2.2.1 -3 Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of the total soluble protein ...... 36 2.2.2.2 Detedation of the number of subunits or isofom of the 18 kD protein ...... 36 2.2.2.2.1 Total soluble protein extraction with buffer containing or not containing the reducing agent P-mercaptoethanol @-ME) . 37 2.2.2.2.2 Two-dimensional electrophoresis of the total soluble protein of dandelion root . 37 2.2.2.3 N-terminal-end sequencing of the 18 kD protein purified by two-&ensional electrophoresis . . . . . 39 2.2.2.3.1 Electrophoretic blotting for protein sequencing: ...... 3 9 2.2.2.4 cDNA library construction ...... 40 2.2.2.4.1 PolyA+ RNA isolation ...... 40 2.2.2.4.2 cDNA library construction using hgt 1 1 vector ...... 42 2.2.2.5 Screening of the cDNA library ...... 5 1 2.2.2.6 Protein purification by chromatography ...... 54 2.2.2.6.1 Determination of the optimal extraction buffer ...... 54 2.2.2.6.2 Total soluble protein extraction ...... 55 2.2.2.7 Cornparison of the N-terminal end sequence with other known proteins ...... 60 2.2.2.8 Northem blots using cDNAs encoding Mal d 1 and Bet v 1 allergen proteins as probes ...... 61 2.2.2.8.1 Electrophoresis of RNA through gels containing formaldehyde ...... 6 1 2.2.2.8.2 cDNA insert digestion and isolation fiom pMW 175 plasmid ...... 63 2.2.2.8.3 cDNA probe labelling and purification . . 65 2.2.2.8.4 Pre-hybridization and hybridization of the membrane with the cDNA probes . . . . . 66 2.2.2.9 Western blot using antibody raised against Bet v 1 allergen protein ...... 66 2.3 Results and Discussion ...... - ...... 69 2.3.1Resuh ...... 69 2.3.1.1 Seasonal changes in the 18 kD protein in dandelion roots during the year ...... 69 2.3.1.2 Subunit composition and number of isoforrns .... 69 2.3.1 .3 N-terminal-end sequence of the 18 kD protein purified by two-dimensional electrophoresis ..... 70 2.3.1.4 ScreeningthecDNA library ...... 71 2.3.1.5 Protein purification by chromatography ...... 72 2.3.1.6 Cornparison of the 40-amino-acid sequence of the N-terminal end of the 18 kD protein with known proteins ...... 74 2.3.1.7 Northern blots using the cDNAs encoding the Mal d 1 and Bet v l allergen proteins as probes ... 75 2.3.1.8 Western blots using polyclonal and monoclonal antibodies raised against Bet v 1 allergen protein ...... 75 2.3.2 Discussion ...... 77 2.3.2.1 Homologies of the 18 kD protein with other known proteins ...... 77

3. Construction and characterization of the full-len& cDNA sequence encoding the 18 kD protein and its genomic DNA-PCR products ...... 91 3.1Introduction ...... 92 3.2 Materials and Methods ...... 95 3.2.1 Materials ...... 95 3.2.2 Methods ...... 95 3.2.2.1 TotalRNAextraction ...... 95 3.2.2.2 Total poly A+ RNA isolation using an oligo (dT)-cellulose column ...... 96 3.2.2.3 Primer design for RACE-PCR ...... 97 3.2.2.4 3' end-RACE (rapid amplification of cDNA ends) PCR ...... 98 3.2.2.4.1 Reverse transcription of poly A+ RNA . . 98 3.2.2.4.2. 3' end-RACE-PCR ...... 99 3 .2.2.4.3 PCR product purification ...... 100 3.2.2.5 5' end-RACE PCR ...... 101 3.2.2.5.1 Reverse transcription of polyA+ RNA . 10 1 3.2.2.5.2 Purification of the reverse transcription reaction mumire ...... 101 3 .2.2.5.3 Polyadenylation at the 5' end ...... 102 3 .2.4.4 5'-end RACE-PCR ...... 102 3.2 2.6 Cloning of the RACE-PCR products into pGEM-T Easy vector ...... 103 3 -2.2.6.1Ligations of the RACE-PCR products to the pGEM-T Easy vector ...... 103 3.2.2.6.2 Transformations using the pGEM-T Easy vector iigation reactions ...... 104 3.2.2.7 Plasmid preparation for sequencing ...... 105 3 .2.2.7.1 Cnide plasmid extraction ...... 105 3 2.2.7.2 PIasrnid purification ...... 106 3.2.2.8 PCR amplification using genomic DNA as templates ...... 107 3.2.2.8.1 Genomic DNA isolation ...... 107 3.2.2.8.2 PCR amplification of the genomic DNA and its product sequencing ...... 108 3.2.2.9 Analysis of the cDNA encoding the 18 kD protein and its deduced amino acid sequence .... 110 3.2.2.9.1 Construction of the full-length cDNA sequence and its deduced amino acid sequence ...... 111 3.2.2.9.2 Some characteristics of the 18 kD protein ...... 111 3.2.2.10 Cornparison of the cDNA encoding the 18 kD protein and its deduced amino acid sequence with other known proteins ...... 111 3.2.2.11 Analysis of the PCR products arnplified using genomic DNA as templates ...... 112 3.3 Results and Discussion ...... 113 3.3.1 Results ...... ---...... 113 3.3.1.1 3'-endRACEPCR ...... 113 3.3.1.2 5'-end RACE PCR ...... 113 3 -3.1.3 Cloning of the PCR products into pGEM-T Easy vector and sequencing ...... 114 3.3.1.4 PCR amplifications using genomic DNA as templates ...... 114 3.3.1.5 The full-length cDNA sequence encoding the 18 kD protein and its deduced amino acid sequence ...... 114 3.3.1.6Analysisofthe 18 kDprotein ...... 115 3 .3 .L .7 Cornparison of the 18 kD protein with known proteins ...... 116 3 -3.1.8 The PCR products amplified using genomic DNA as templates and their deduced amino acid sequences ...... 117 3 -3.1.9 Cornparison of the nucleotide and amino acid sequences of the two PCR products using genomic DNA as templates with the Ml-length cDNA encoding the 18 kD protein and its deduced amino acid sequence ...... 118 3.4 Discussion ...... -O...... 120 4 . Seasonal, temporal and spatial expression of the gene encoding the 18 kD protein in dandelion and detection of homologous proteins in roots of other mernbers of Compositae ...... 138 4.1htroduction ...... 139 4.2 Materials and Methods ...... 141 4.2.1 Materials ...... 141 4.2.2Methods ...... 141 4.2.2.1 Seasonal, temporal, and spatial expression of the gene encoding the 18 kD protein in dandelion and detection of homologous proteins in the roots of other members of the Compositae ...... 141 4.2.2.1.1 SDS-PAGE ...... 141 4.2.2.1.2Northemblots ...... 142 4.2.2.2 Southern blots ...... 144 4.2.2.2.1 Genomic DNA isolation ...... 144 4.2.2.2.2Genomic DNA digestion with restriction enzymes ...... 145 4.2.2.2.3Electrophoresis of digested genomic DNA ...... 146 4.2.2.2.4Denaturation of the DNA gel and tramferring of the genomic DNA from gel to membrane ...... 146 4.2.2.2.5cDNA probe labelling and purification 147 4.2.2.2.6Pre-hybridization and hybridization of the membrane with the cDNA probe .... 147 4.3 Results and Discussion ...... 149 4.3.1 Results ...... 149 4.3.1.1 Seasonal changes in expression of the gene encoding the 1 8 kD protein ...... 149 4.3.1.2 Spatial expression of the gene encoding the 18 kD protein in other organs of dandelion ..... 149 4.3.1.3 Expression of the gene encoding the 18 kD protein during seedling establishment ...... 150 4.3.1 .4 Detection of homologous proteins in other Cornpositae ...... 151 4.3.1.5 The number of gene copies encoding the 18 kD protein ...... 152 4.3.2 Discussion ...... 154

5. Effects of environmental perturbations on the expression of the gene encoding the 18 kD protein and protein synthesis ...... 165 5.l.Introduction ...... 166 5.2 Materials and Methods ...... 168 5.2.1 Materials ...... 168 5.2.2. Methods ...... 168 5.2.2.1 Cold shock treatments ...... 169 5.2.2.2 Warm shock treatments ...... 169 5.2.2.3 Defoliation treatments ...... 169 5.2.2.4 Wounding treatments ...... 170 5.2.2.5 Water stress treatment ...... 170 5.2.2.6 Fungal eIicitor treatments ...... 171 5.2.2.7 SDS-PAGE ...... 171 5.2.2.8Northernblots ...... 171 5.3 Results and Discussion ...... 173 5.3.1 Results ...... 173 5.3.1.1 Effect of cold shock on the gene expression and on 18 kD protein synthesis ...... 173 5.3.1.2 Effects of warm shock on gene expression and 18 kD protein mobilization ...... 174 5.3.1.3 Effects of the defoliation on the 18 kD protein expression ...... 175 5.3.1.4 Effects of wounding on gene expression and 18 kD protein synthesis ...... 176 5.3.1.5 Effect of water stress on gene expression and 18 kD protein synthesis ...... 177 5.3.1.6 Effects of the fungal elicitor on gene expression and 18 kD protein synthesis ...... 177 5.3.2 Discussion ...... 179 Generaldiscussions ...... 195 Futurestudies ...... 203

References ...... 206 LIST OF FIGURES

Figure 2- 1. Seasonal changes in the 18 kD protein of dandefion roots ...... 79

Figure 2.2 . SDS-PAGE gel of dandelion root proteins stained with Coomassie BrilliantBlueR-250 ...... 80

Figure 2.3 . Two dimensional electrophoresis of dandelion root protein (silver stained) ...... 81

Figure 2.4 . Dandelion root proteins extracted with seven different buffers ...... 82

Figure 2.5 . 18 kD protein precipitated by five arnmoniuna sulfate concentrati~~.... 83

Figure 2.6 . SDS-PAGE gel of eluents derDE52 column chromatography @action 6 to 24) (stained with Coomassie Brilliant Blue R-250) ...... 81

Figure 2.7 . SDS-PAGE gel of the eluents afber gel filtration chromatography (fractions 1 to 48) (stained with Coomassie Brilliant Blue R-250) ...... 85

Figure 2.8 . Absorbance at 280 nm of the eluate after chromatofocusing chromatography (fiaction 110 to 156) ...... 86

Figure 2.9 . SDS-PAGE gel of the eluate after chromatofocusing chromatography (fiaction 1 1 0 to 156) (silver staining) ...... 87

Figure 2.10 . SDS-PAGE gel of the isoforms of pI 5.49 (a) and pI 5.56 (b) der the fmal gel filtration chromatography (silver staining) ...... 88

Figure 2-1 1. Cornparison of the 40 amino acid sequence with known proteins ...... 89

Figure 2- 12. Western blot of the 18 kD protein using antibody raised against .the Bet v 1 allergen protein fiom birch protein ...... 90

Figure 3.1 . Strategies for RACE-PCR ...... 124

Figure 3.2.5 pairs of primers used for genomic DNA-PCR ...... 125

Figure 3.3 . Products of 3'-end RACE-PCR agarose gel stained with ethidium bromide ...... 126 Figure 3.4 . Products of S'-end RACE-PCRagarose gel stained with ethidium bromide ...... 127

Figure 3.5 .RACE-PCR products digested fiom pGEMT Easy vector with EcoRI agarose gel stained with ethidium bromide ...... 128

Figure 3.6 . Products of PCR amplifkation using genomic DNA as template (agarose gel stained with ethidium bromide) ...... 129

Figure 3.7 . The cDNA sequence encoding the 18 kD protein and its deduced aminoacidsequence ...... 130

Figure 3.8 . pl value of the 18 kD protein ...... 131

Figure 3.9 . Charge density of the 18 kD protein ...... 132

Figure 3-1 0. Hydropathy profile of the 18 kD protein ...... 133

figure 3-1 1 . Cornparison of the 18 kD protein with other known proteins ...... 134

Figure 3.12 . Comparison of the nucleotide sequences of the genomic DNA-PCR products with the full-len-th cDNA sequence ...... 135

Figure 3- 13. Comparison of the arnino acid sequences deduced fhm genomic DNA-PCR products with the 18 kD protein deduced from the full-length cDNAsequence ...... 136

L Figure 4.1 . Seasonal regulation of expression of the gene encoding the 18kDprotein ...... 157

Figure 4.2 . Spatial expression of the gene encoding the 18 kD protein ...... 158

Figure 4.3 . Synthesis of the 18 kD protein in different parts of a dandelion ...... 159

Figure 4.4 . Expression of the gene encoding the 18 kD protein during seedling establishment ...... 160

Figure 4.5 . Synthesis of the 18 kD protein during seedling establishment ...... 161 Figure 4-6. SDS-PAGE of total soluble protein in roots of eight rnembers of the Compositae ...... 167

Figure 4-7. Absence of the 18 kD protein fiom roots of other members of the Compositae ...... 163

Figure 4-8. Southem blot of dandelion genomic DNA ...... 164

Figure 5-1. Effect of cold shock (20 OCto 5 OC) on expression of the gene and 18 kD protein synthesis ...... 184

Figure 5-2. Effect of warm shock (5 OCto 20 OC) on expression of the gene and 18 kD protein mobilization ...... 185

Figure 5-3. EEect of defoliation at 20 OC on expression of the gene and 18 kD protein synthesis ...... 186

Figure 5-4. Effect of defoliation at 5 OCon expression of the gene and 18 kD protein synthesis ...... 187

Figure 5-5. Effect of wounding at 20 OC on expression of the gene and 18 kD proteh synthesis ...... 188

Figure 5-6. Effect of wounding at 5 OCon expression of the gene and 18 kD protein synthesis ...... - - ...... 189

Figure 5-7. Effect of water stress at 20 OCon expression of the gene and 18 kD protein synthesis ...... - ...... 190

Figure 5-8. Effect of elicitor (homogenate of Phytophthora megasperma) on 18 kD protein gene expression at 20 OC ...... 19 1

Figure 5-9. EEect of elicitor (homogenate of Phytophthora megaspermn) on 18 kD protein synthesis at 20 OC ...... 192

Figure 5- 10. Effect of elicitor (homogenate of Phytophthora megasperma) on 18 kD protein gene expression at 5 OC ...... 193

Figure 5-1 1. Effect of elicitor (homogenate of Phytophthora megasperma) on 18 kD protein synthesis at 5 OC ...... 194 LIST OF TABLES

Table 3- 1. Amino acid composition of the 18 kD protein ...... - . . 137 If. Literature Review 1.1 Introduction

Storage proteins in plants are divided into two types according to their tissue localizations: seed storage proteins and vegetative storage proteins (VSPs). Seed storage proteins provide amino acids to the young growing seedhg (Bewley and

Black 1994, Pemollet and Mosse 1983), while vegetative storage proteins are the nitrogen source for the resumption of vegetative growth in spring in temperate plants

(Langheinrich and Tischner 1991, van Cleve et al. 1988, Wetzel and Greenwood

1991, Xu 1993), or when plants are recovering from other stresses, such as defoliation or environmental perturbations (Barber et al. 1996, Corre et al. 1996,

Hendershot and Volenec 1993% b), or they provide a source of nitrogen for new organ development, e. g. pods and seeds, in mature soybean and French bean plants

(Wittenbach 1982). While seed storage proteins have been extensively studied for about a century, VSPs have only been characterized du~glast decade.

As early as 1927, Graber et al. observed that both the stored carbohydrate and nitrogen content of &alfa taproots decreased during initial shoot growth in spring and during shoot regrowth after defoliation. This indicates that new tissue growth is at the expense of stored reserves. However, ment studies suggest that regrowth following defoliation of alfalfa, ryegrass and white clover is linked to the availability of nitrogen reserves in the roots rather than that of carbohydrate reserves (Barber et al. 1996, Corre et al. 1996, Ourry et al. 1989, 1994). Heilmeier et al. (1986) have found that the proportion of stored nitrogen remobilized for early sp~ggrowth is considerably higher than that of carbon in a biennial plant, Arctium tomentosum. Cyr and Bewley (1990~)also showed that hydrolysis of starch and partitiorhg of carbon into the soluble carbohydrate pool in leafy spurge roots were not affected by defoliation. However, unlike carbohydrate, nitrogen-containhg compounds responded significantly to the stress induced by defoliation, with reductions in al1 three pools (soluble protein, free amino acid, and nitrate) by mid-fall. In addition, removal of source leaves prior to senescence resulted in a severe limitation on the availability of remobilized nitrogen. As in herbaceous perennial plants, the availability of stored nitrogen is one of the most limiting factors to tree growth

(Harley et al. 1958, Keeney 1980, Oland 1959, Taylor and May 1967, Taylor et a1.1975, Tromp 1983). Tromp (1983) indicated that the initial amount of carbohydrate accumulated in woody tissues of a fnllt tree does not influence the amount of new growth in the spring, whereas the amount of nitrogen accumulated is closeiy related to the amount of growth and the vigour of newiy developed shoots.

These results indicate that attrogen compounds play a more dynamic role than carbohydrates in storage metabolism and may be a primary determinant of plant growth.

Increasing evidence shows that among the nitrogenous compounds studied

(free amino acids, nitrate, nitrite, ammonium, protein), proteins are the most predomlliaat nitrogen storage form (Barber et al. 1996, Corre et al. 1996, Culvenor and Simpson 1991, Hendershot and Volenec 1993 a, b, Kang and Titus 1980, Kim et al. 1991, Millard and Neiken 1989, 09Kennedyand Titus 1979, O'Kennedy et al.

1975, Ouny et al. 1989, Todd 1991, Tromp and Ovaa 1973, 1979, Wetzel et al.

1!Wb, Wetzel and Greenwood 1989). Todd (199 1) reported that for several deciduous trees, among the above five nitrogenous cornpounds, ody protein showed a seasonal pattern of a high amount in winter and low level in summer, and none of the other nitrogen compounds were abundant enough to be classified as primary cornpounds for ovemintering storage in several deciduous trees. Millard and Neilsen

(1 989) indicated that proteins account for approximately 90% of the total nitrogen in the shoots of apple trees over winter. During spring, approximately 80% of the amino acids in the branches are derived fiom the hydrolysis of uiese protek. Protein as a major storage fonof nitrogen was also dernonstrated in gras and legume plants

(Ourry et al. 1989, Culvenor and Simpson 1991, Kim et al. 1991). To date, VSPs have been detected in many annual and peremial plants, such as VSPa, VSPP, and lipoxygenase in leaves of soybean (Wittenback 1982), pod storage protein (PSI?) in

French bean, lectin-like VSPs in the bark and the wood of several hardwoods

(Greenwood et al. 1986, Hams and Sauter 1991, Langheinrich and Tischner 1991,

Peumans et al. 1986, Todd 1991, van Cleve et al. 1988, Wetzel and Greenwood

199 1, Xu 1993), patatin, proteinase inhibitor, and sporamin in tubers of potato (Paiva et al. 1983) and sweet potato (Hattori et al. 1985, Maeshima et al.1984), VSPs in tubers of Jerusalem artichoke (Mussigman and Ledoigt 1989) and yam (Harvery and

Boulter 1983), in roots of le@ spurge (Cyr and Bewley 1990~)~alfalfa (Hendershot and Volenec 1993a), and white clover (Corre et al. 1996).

VSP identification

The functions of storage proteins are evident. However, identiwg them as storage reserves is not easy since they are a heterogeneous group and no direct biochemical assay is available to detemetheir function. Generally speaking, seed storage proteins can be characterized by several main features: i) stored for nutritional needs during seedling establishment; ii) no other known function; iii) abundant among seed nitrogen compounds; iv) localized within storage organelles called protein bodies (Pemollet and Mosse 1983). However, there are exceptions, e.g. urease which is present in many legume seeds is considered as a storage protein

(Bailey and Boulter 1971, Pemollet and Mosse 1983), but it is localized in the cytosol instead of protein bodies (Faye et al. 1986). In addition, it is an enzyme, though it has no or very little enzymatic activity in soybean seed (Polacco et al.

1982). In vegetative organs, proteins which are abundant, show preferential synthesis after removal of developing organs [such as developing seeds (Wittenbach 1982,

Staswick 198911, or are preferentially synthesized during senescence and depleted during meristem reactivation (Langheihrich and Tischner 1991, Mussigmann and

Ledoight 1989, O'Kennedy and Titus 1979), are defrned as vegetative storage proteins (VSPs). As with the seed storage proteins, all VSPs so far detected have also been found to be localized in vacuoles or protein-rich organelles, which are structurally identical to the protein bodies of many dicotyledonous seeds (Franceschi et al. 1983, Greenwood et al. 1986, Hennan et al. 1988, Sauter and Wellenkamp

1988, Semerby-Forsse 1986, Wittenbach l983b). However, unlike seed storage proteins, many VSPs exhibit enzymatic activities, e.g. patatin is a lipid acyl hydrolase

(Andrews et al. 1988), the VSPa, VSPP, and 94 kD VSP in soybean leaves are acid phosphatases and lipoxygenase (DeWald et al. 1992, Tranbarger et al. 1991), and there is a VSP proteinase inhibitor in potato tubers (Pena-Cortes et al. 1988).

VSP identification by organ removal

Several VSPs were identZed by organ removal, such as the VSPs in soybean

(Staswick 1989, Wittenbach 1982,1983a, b) and in French bean (Zhong et al. 1997).

VSPs in soybean were fust demonstrated by Wittenbach (1982, l983a. b). His results indicated that three polypeptides in soybean Ieaves, 27 kD (VSPa), 29 kD (VSPB) and 94 kD (lipoxygenase), increase greatly after depodding. With continued pod removal these three polypeptides can accumulate to more than 45% of the total soluble leaf protein (Wittenbach l983b). They accumulate during normal vegetative growth, but accumulation is greatly increased by removing the pods. During the

pend of seed growth, the amounts of these proteins decrease rapidly. The 27 kD and

29 kD polypeptides are part of a single protein (Wittenbach 1983b), and both this

protein and the 94 kD protein are localized in the vacuoles of the paraveinal

mesophyU (PVM) and associated bundle sheath cells (Franceschi et al. 1983,

Tranbarger et al. 1991, Wittenbach 1983b). The PVM cells play an important role in

tramferring amino acids and carbohydrate between the vascular system and

mesophyll cells of the leaf (Franceschi and Giaquinta 1983%b, Franceschi et al.

1983, Franceschi 1986). The accumulation of the proteins in response to removal of

reproductive sinks and their localization in vacuoles of PVM ceUs suggest that they

are vegetative storage proteins and may be associated with seed development.

As with the VSPs in soybean, a sirnilar phenornenon has also been

demonstrated in French bean (Phaseolus vulgaris) plants (Zhong et al. 1997). When

French bean plants were depodded in the early stages of fnllt development, the

amount of a specific protein with a relative molecular mass of 28 kD was enhanced

greatly, by 32% to 59%, in young pods that forrned later. However, it was not detected in leaves of depodded plants. The protein was designated as pod storage

protein (PSP). The molecular mass of native PSP was estimated to be 67 kD by gel

ffitration and considered to be present as a dimer. Further evidence indicated that the deduced amino acid sequence from the PSP cDNA had 71 45 and 65% identiîy with VSPa and VSPP in soybean, respectively (Rhee and Staswick 1992a,b, Zhong et al.

1997). It seems that there is a close homology between the VSPs from the members of the sarne family. In addition, PSP, VSPa and VSPP share 40% and 44% sequence identity with tomato acid phosphatase-l (Exion et al. 1991, Williamson and Colwell

1991, Zhong et al. 1997). VSPa and VSPP have been demonstrated to exhibit acid phosphatase activity (Andrews et al. 1988). However, it is not known whether or not the PSP has a similar activity. hterestingly, although PSP shows very high homology with VSPa and VSPP, its synthesis in response to the plant hormone, jasmonic acid, is different. The syntheses of VSPa and VSPP are clearly induced by jasmonic acid and its methyl ester (Anderson et al. 1989, Staswick 1990); however, Zhong et al.

(1997) indicated that exogenously applied methyl jasmonate was not effective in enhancing the accumulation of PSP. This indicates that the PSP gene in French bean and VSP genes in soybean leaves may be regulated by different mechanisms.

VSP identificafion in bark and wood of deciduous trees uccording to their semonal clurnges

The vegetative storage proteins in deciduous trees play an important role in spring growth. Nitrogen is cycled fkom senescing foliage in the fall to ovemintering tissues, and later is remobilized for the next spring growth. In trees, proteins which are present in large quantities during winter and absent during the summer have been denned as storage proteins (O' Kennedy and Titus 1979). Several investigations have codirmed that there are some unique storage proteins in the wood and secondary phloem of trees, including apple (Malus) (Kang and Titus 1980, O' Kennedy and

Titus 1979), elder (Sambucus), black locust (Robinia) (Nsimba-Lubaki and Peumans

1986), maple (Acer)(Todd 1991, Wetzel et al. 1989b), willow (Salk)(Todd 1991,

Wetzel and Greenwood 1991, Xu 1993), poplar (Populus) (Sauter et al. 1988, van

Cleve et al. 1988), larch (Larix), pine (Pinus) (Wetzel and Greenwood 1989), cypress (Tarodium), redwood (Metasequoia) and yew (Taxus) (Harms and Sauter

1991). These specific proteins accumulate during Iate summer or early autumn, are highly abundant îhroughout the winter, and are mobilized after bud break. Using

SDS-PAGE, a prominent 32 kD polypeptide was detected both in the wood and bark of poplar and willow (van Cleve et al. 1988, Wetzel et a1.1989b, Wetzel and

Greenwood 199 1, Xu 1993). Two conspicuous polypeptides of 24 kD and 16 kD were present in maple bark (Todd 1991, Wetzel et al. 1989b). During the winter, the

32 kD polypeptides of wilIow and poplar, and the 24 kD and 16 kD polypeptides of maple together comprised approximately 30% of the total extractable bark proteins in each case. Peumans et al. (1986) also reported that specific storage proteins occurred in high concentrations in the bark of elder and black locust during winter.

In each, the protein was composed of two identical subunits of 34.5 kD (elder) and

37.5 kD (black locust). They represented up to 5% and 10% of the total protein content of the respective bark extracts. O'Kennedy and Titus (1979) identified three specific storage proteins in the bark of dormant apple shoots. Of four coniferous species studied by Hanns and Sauter (1991) only one did not contain a specifc storage protein in the fall. These results indicate that VSPs comrnonly exist in woody species, especially in hardwoods.

Further evidence to indicate the presence of VSPs in trees is the discovery of cellular inclusions similar to the protein bodies of seeds. Protein-rich organelles, which are stmcturally identical to protein bodies in many dicotyledonous seeds occur in cells of the cortical and secondary phloem parenchyma, vascular cambium, and xylem ray cells in overwintering shoots of a number of temperate deciduous perennials (Greenwood et al. 1986, Herman et al. 1988, Sauter and Wellenkarnp

1988, Sennerby-Forsse 1986). These proteinaceous organelles showed seasonal changes, and their formation and loss parallelled the changes in protein content as rneasured biochemically (Greenwood et al. 1986, Sauter and van Cleve 1990, Wetzel et al. 1989b). Using irnmunolocalization, several researchers determined that these vacuoles are the storage organelles for a specific protein (Herman et al. 1988, van

Cleve et al. 1988, Wetzel and Greenwood 199 1).

There also seems to be a close homology between the bark storage proteins of related species and genera. Antibodies raised against the 32 kD storage protein of poplar bind specifically to protein bodies in willow (Sauter and van Cleve 1989) and antibodies raised against the willow bark storage protein bind to those from poplar

(Wetzel and Greenwood 1991). Poplar and willow are both members of the

Salicaceae. In Harms and Sauter's investigation (199 1). they showed that an antibody raised to a 35 kD protein fkom cypress can react with proteins of 32 kD and 34 kD extracted kom the wood of redwood and with two polypeptides of 34 kD and 36 kD of yew. Thus there may be a widespread occurrence of closely related storage proteins within families.

VSP identification in fuberour perenniaLF according to their preferentùzl synthesis during tuber development

Tuberous perennials produce specialized organs, named tubers. They are vegetative tissues, present as either modified roots (sweet potato and yam) or moàified stems (Jerusalem artichoke, potato). They are reserve storage organs from which there is new growth of aerial tissues. Tuber-specific storage proteins have been characterized from Jenisalem artichoke (Mussigman and Ledoigt 1989), potato

(Paiva et al. 1983), sweet potato (Maeshima et al, 1984), and yam (Harvery and

Boulter 1983).

Potato tubers contain two major VSPs: patatin and proteinase inhibitor II (PI

II) (Paiva et al. 1983, Ryan et al. 19%). Patatins are a group of immunologically identical glycoproteins with a molecular mass of 40 kD. They comprise 40 to 45% of the soluble protein in tubers and are preferentially synthesized during tuber development Usually, patatins are present in only trace amounts, if at all, in leaves, stems or roots of potato plants (Paiva et al. 1983). PI II is also preferentidy synthesized during tuber development (Keil et al. 1989) and accounts for about 10% of total tuber protein (Ryan et al. 1976). However, its synthesis cm be induced in leaves (Pena-Cortes et al. 1988, Ryan and An 1988), but not in tuben (Sanchez-

Serrano et al. 1990) in response to damage caused by chewing insects or other severe mechanical damage. In contrast, patatins do not accumulate in response to these treatments in whole potato plants. Thus, despite the comrnon expressionof PI II and patatin gene families in tubers, their regulation is coupled to different signal pathways

(Pena-Cortes et al. 1992). The PI II gene family seems to be controlled by both developmental and environmental factors, while the patatin gene is controlled only by developrnental factors.

Yam tubers also contain a family of major proteins (about 85% of the soluble proteins) which consist principally of subunits of one size, 31 kD. They are recognized as the storage proteins because of their localization in organelles which are similar to seed protein bodies (Harvery and Boulter 1983). The tuberous roots, but not other organs, of sweet potato contained large quantities of two proteins which accounted for more than 808 of the total proteins. The two proteins, named sporamins A and B, are monomenc forrns with sirnilar molecular mass of 25 kD. These two proteins decrease in preference to other proteins du~gsprouting

(Maeshima et al. 1984). Mussigman and Ledoigt (1989) reported three tuber specific polypeptides (16 kD, 16.5 kD and 18 kD) in Jerusalem aaichoke. However, only the

18 kD protein is degraded during sprouting. Similar to patatin in potato, it is also not present in leaves, stems or roots.

VSPs in roots of defoliated herbaeous perenniaLr

Defoliation leads to a shortage of supply of current photosynthate and the plant must acquire nutrients from alternative sites to grow new leaves (Dankwerts and Gordon 1989). This results in the decline of nitrogen and carbohydrate reserves in the rernaining parts of the plant to support new shoot growth (Graber et al. 1927).

Defoliation, therefore, provides a framework to evduate the role of stored reserves, including proteins, in storage metabolism.

Hendershot and Volenec (1993a) have identified three proteins with molecular masses of 32 kD, 19 kD and 15 kD as VSPs in alfalfa taproots. They are the main components of a buffer-soluble fraction of proteins from alfalfa taproots and they accumulate during fall and early winter in taproot tissues and rapidly decline when shoot growth resumes in the spring. They are depleted fiom taproots during regrowth of defoliated alfalfa, suggesting that these proteins are indeed utilized as a source of nitrogen. These VSPs are taproot-specfic, and are not present in roots of young seedlings nor in seedlings or seeds, shoots, nodules, or stem tissues of aIfalfa

(Cunningham and Volenec 1996). In white clover, specific proteins of 17 kD in stolons, and 2 polypeptides of 15 kD in roots are also reported to behave as VSPs

(Corre et al. 1996). These three polypeptides, initially present at high concentrations, decrease sharply (by 41% in roots and 33% in stolons) during early regrowth of defoliated plants and accumulate again in roots and stolons once normal growth is re- established. In addition, their accumulation also follows a seasonal pattern with a peak of accumulation at the beginning of winter and spring degradation as for VSPs in woody species. These latter data provide further evidence that these three polypeptides act as VSPs. A root-specific 26 kD protein in leafy spurge was also ideneified as VSP by Cyr and Bewley (1990~).After defoliation in October, 26 kD protein synthesis was inhibited and a decline in that already stored was observed in

November. With recovery of above-ground activity from defoliation an increase in

the arnount of the 26 kD protein was detected in December. This pattern indicates

that nitrogen is remobW from the root by degradation of this protein, to provide

the nitrogen required by growing leaves. SDS-PAGE gel demonstrated that this

protein quantitatively changes in arnount throughout the year, with large quantities

present in the root from June to March, yet absent or present in reduced amounts

during April to June. Further investigation showed that formation of shoot buds is

temporally associated with a reduction in amount of the 26 kD protein. VSP gene regdation

To know more about the nature of VSPs and their gene expression rnechanisms, genes encoding VSPs in both annual and perennial plants were cloned and isolated, and regdation of their expression by either endogenous or environmental factors was studied (Anderson 199 1, Coleman et al. 1991, 1992,1993,

Creelman et al. 1990, 1992, Huang et al. 199 1, Langheinnch and Tischner 199 1,

Mason and Mullet 1990, Mason et al. 1992, Staswick 1990, Staswick et al. 199 1,

Todd 199 1, van Cleve and Apel1993,).

Synthesis and degradation of VSPs occur in annual plants in a single growing season. Their natural regulation is most likely controlled by endogenous developmental conditions. The plant hormones: jasmonic acid (JA) (or its ester form, methyl jasmonic acid NeJA]), and abscisic acid (BA) are considered to be plant growth regulators. Both can initiate leaf senescence (Parthier 1990, Weidhase et al.

1987) and inhibit plant growth (Anderson 1989, Dathe et al. 198 1). In addition, both have regulatory roles in the expression of many genes (see review by Chandler and

Robertson 1994, Parthier 1990). Hence, their effects on VSP gene expression in annual plants, especially in soybean, were extensively investigated (Anderson et al.

1989, Huang et al. 1991, Mason and MuUet 1990, Mason et al. 1992, Staswick 1990.

Staswick et al. 1991).

The synthesis and degradation of VSPs in bark, roots or tubers coincides with seasonal changes. It seems that, for perdal plants, seasonal changes, such as photoperiod and temperature, are possible regdators of VSP gene expression. Many studies have investigated the effects of photoperiod and temperature on VSP gene expression in trees and herbaceous perennials (Clausen and Apel 1991, van Cleve and Apel1993, Coleman et al. 1991, 1992, 1994, Langheinrich and Tischner 1991,

Luster and Farrell pers. commun., Todd 1991). Many researchers have also worked on the effects of environmental perturbations on VSP gene expression in these plants, such as water stress and wounding, as well as nitrogen and sugar supplements

(Datnrnann et al. 1997, Pena-Cortes et al. 1989,1991,1992, Farmer and Ryan l992b,

Parthier 199 1, Pena-Cortes et al. 1989,1992, Ryan 1992). AU this work has been to elucidate how environmental cues are perceived and interpreted by plants, and how

VSP gene expression is triggered. Among these, wounding effects have received the most extensive attention.

The role of ja~monate(JA) and ubscisic ucùi (ABA)in VSP gene expression

JA seems to play an important role in triggering VSP gene expression in soybean. Methyl jasmonate rapidly induces soybean VSP gene expression in the whole plant, leaf explants and in suspension cell cultures (Anderson et al. 1989,

Mason and Mullet 1990, Staswick 1990, Staswick et al. 1991). A few studies on the distribution of jasmonate within plant organs have demonshated a correlation between the location of JA/MeJA and VSP gene expression. Jasmonate is most abundant in vascular bundles of pericarp (seed pod) tissue in soybean (Parthier 1990), where VSP mRNA is also prevalent (Huang et al. 1991). In addition, the cell-specific pattern of VSP gene expression in leaves cmbe abolished by application of MeJA, which causes a high level of expression of VSP genes in all cell types, rather than in just the paraveinal mesophyll and bundle sheath cells (Huang et al. 1991). This indicates that although all cells are competent to express VSP genes, expression is nomdy limited to those tissues having higher concentrations of jasmonate, or greater sensitivity to endogenous jasmonate. Induction of VSP gene expression by jasmonate was also reported in potato (Pena-Cortes et al. 1992).

ABA is not as powerfùl an inducer of VSP as jasmonates. In contrast to jasmonates, ABA has ver- Little or no efiect on the amount of VSP in soybean suspension cultures, either in the presence or absence of JA (Anderson 1989, 199 1,

Mason and Mullet 1990). However, ABA has been reported to invoke VSP gene induction in potato (Dammam et al. 1997, Pena-Cortes et ai. 1989, 199 1). Potato mutants defcient in ABA synthesis fail to accumulate proteinase inhibitor II and its mRNA after wounding. This effect is correlated with a constant low level of endogenous ABA in the mutants, while in the wild-type, ABA increases upon wounding (Pena-Cortes et al. 1989). The role of photoperiod and temperature in VSP gene eqresswn

Results from studies on the roles of photoperiod and temperature on VSP gene expression are quite often in conflict. Coleman et al. (199 1, 1992) indicated that BSP

synthesis in Populus cm be induced by SD (short days; 8 hours photoperiod). The

accumulation of BSP was correlated with changes in the pool of translatable mRNA.

Similar results were also published by Clausen and Apel(1991) and Langheinrich

and Tischner (1991). However, Luster and Farrell (pers. commun.) reported that SD did not influence the synthesis of the 26 kD storage protein in 1- spurge roots. This may be due to inherent difference between the roots and stems. Roots, being underground tissues, receive very Little light, while the stem is an aerial tissue and perceives light fluctuations every day. Thus, changes in photoperiod may not have a sigiilficant direct effect on root biochemical processes. In contrast, temperature might have a si@icant effect on root protein accumuIation.

The role of temperature in poplar bark storage protein (BSP)gene expression has been demonstrated by van Cleve and Apel (1993). They reported that large amounts of BSP accumulate not only under short days but also under long-day conditions if poplar trees are exposed to a Iower temperature. This accumulation was dso correlated with a concomitant and massive increase in the amount of the corresponding &A.

However, studies by Todd (1991) presented conflicting results. She also investigated the effects of photoperiod and temperature on BSP accumulation in Salk and Acer. Her results showed that shorter photoperiods, especially when reduced at a rate similar to those occurring nanirally in the fall, seemed to have linle effect on the accumulation of BSP proteins. She suggested that the photoperiod used in the experiments of Coleman et al. (1991) may have elicited a stress response, since an eight-hour short photoperiod (SD) did not mimic conditions occurring in nature when

BSP accumulation is initiateci. Accumulation of the BSP in forest trees was observed to start by the middle of August, when daylength was about 14 hours (Coleman et al.

1991). This indicates that SD may not be the natural factor triggering BSP gene expression. Todd (1991) has also provided evidence that temperature is unlikely to be the natural inducer of BSP accumulation, either. She showed that gradually or drastically decreasing the temperature when the photoperiod was kept at 15 hours did not induce the accumulation of BSP in either Salk or Acer. In Acer, the BSP accumulation and degradation was seasonal, but the accumulation began pnor to any significant reduction in photoperiod and temperature and pnor to any visible signs of leaf senescence. Thus, she suggested that BSP gene expression in Acer is govemed by endogenous rhythm rather than external environmental cues.

Environmental conditions are not easily mimicked, especially the seasonal changes in photoperiod and temperature. Although Todd's experiments clearly dernonstratecl that changes in photoperiod and temperature do not tngger VSP gene expression, their roles in VSP gene regulation still cannot be excluded. Her experiments were designed to investigate the individual effixts of photoperiod and temperature on VSP gene expression. However, under natural conditions, changes in both photoperiod and temperature occur at the same time. Photoperiod and temperature may work together to trigger VSP gene expression.

The role of water stress and wounding in VSP gene expression

Water stress and womding are the two common environmental perturbations that plants often encounier. Elucidation of their effects on VSP gene expression may provide valuable information of the VSP regulation mechanisms. Thus, many researchers have paid attention to the responses of plants to these two stresses in te- of VSP gene expression (CreeIman et al. 1992, Davis et al. 1993, Mason et al.

1988, Mason and Mullet 1990, Mason et al. 1992, Pena-Cortes et al. 1988, l!W.

Surowy and Boyer 1990).

VSP induction by water stress was reported by Mason and Mullet (1990).

They demonstrated that when older light-grown soybean plants were exposed to water stress, stem and leaf growth were inhibited and VSP mRNA increased in several plant parts. However, when the plants were rewatered, VSP mRNA readjusted toward prestress Ievels. It is well-hown that water stress induces ABA synthesis and many effects of water stress are due to an increase in ABA (Bensen et al. 1988, Harris et al. 1988). Since ABA was reported to have no effect on VSP gene expression in soybean (Anderson 1989,1991). it is not likely that water stress induces

VSP synthesis because of the increase of ABA. Creelman et al. (1990) found that soluble sugars increase in the hypocotyl zone of elongation of dark-grown seedlings during water deficits. They suggest that sugars mediate the effect on VSP expression under conditions of water stress-

Wounding seems to be a common inducer of VSP gene expression. Wound- induction of VSP gene expression has been studied in poplar (Davis et a1.1993), soybean (Mason and Mullet 1990), and potato (Pena-Cortes et al. 1988, 1989, 1991,

1992). Results have demonstrated that the accumulation of these proteins is not restricted to the wound site, but it is dso obsenred in non-wounded tissues (Mason et al. 1992, Berger et al. 1995). This indicates that upon wounding an inducing factor or wound hormone is released and is rapidly transported to other tissues of the plant.

The chemical nature of the signals involved in the wounding response process might be jasmonates (Creelman et al. 1992, Farmer et al. l992b, Parthier 1991, Pena-Cortes et al. 1992, Ryan 1992, Staswick et al. 1991). Creehet al. (1992) reported that

JAlMeJA increased in wounded tissues. Addition of MeJA to soybean suspension cultures increases the rnRNAs for three wound-responsive genes, indicating that

MeJA and JA also have a role in the mediation of several changes in gene expression associated with the plants' response to woundhg. Farmer et al. (1992b) reported that processes of induction of the two proteinase inhibitor proteins in leaves of tomato,

&alfa and tobacco by wounding and airborne methyl jasmonate appea.to be similar, and both may involve some common elements of a single signal transduction pathway. Moreover, the rates of synthesis of proteinase inhibitor proteins in response to methyl jasmonate are similar to the rates of synthesis induced by wounding

(Farmer and Ryan 1990, Graham et al. 1986). One profound insight into the signal pathway of jasmonates in the wounding response has been provided by Vick and

Zimmeman (1984). Jasrnonic acid in plants has been shown to originate fiom linolenic acid (Vick and Zimmerman 1984), wounding activates membrane-attaching lipases to release a polyunsaturated fatty acid (linolenic acid). This is then metabolized to jasmonate via the jasrnonic acid biosynthesis pathway. This mode1 is supported by the observation that two different physiological effects, synthesis of inducible protehase inhibitor and tendril coiling of Bryonia, are triggered not only by jasmonates but also by their precursors, in particular linolenic acid (Famer and

Ryan 1992a).

Pena-Cortes et al. (1989), on the other hand, strongly suggest that ABA plays a direct role in the induction of proteinase inhibitor II (PI II) genes in potato, tomato and tobacco upon wounding. They showed that directly spraying ABA onto leaves could induce the accumulation of PI II mRNA, and ABA-deficient mutants show a much Iesser induction of these genes upon wounding compared to wild-type plants. Moreover, of greater importance, clifferences in the PI II induction between wild-type and mutant plants are completely abolished by ABA application. In addition, upon

ABA spraying, the mutant potato plants show PI II expression as high as wild-type plants, demonstrating that a lack of wounding response of the PI II genes in the mutant plants is directly related to their low contents of endogenous ABA.

Supporthg evidence indicates that the endogenous ABA concentration increases 3- to 5-fold upon wounding and tbis elevation is not resbncted to the tissue that has been damaged directly but cm also be detected in the non-wounded systemically induced tissue (Pena-Cortes et al. 1991). On the other hand, PI II gene expression in potato and tomato leaves can be induced by JA (Pena-Cortes et al. 1992, Faxmer et al. 1992).

It is, therefore, considered that, in potato and tornato, ABA induces the expression of wound-responsive genes by activating JA biosynthesis. This results in higher endogenous JA and, in tum, this activates the expression of PI II and other wound- inducible genes (Pena-Cortes et al. 1995). Dammam et al. (1997) showed that, in addition to this effect, ABA may also play a more direct role in the activation of wound-inducible genes in potato leaves, and it involves a JA-independent pathway for ABA-induced gene expression. Since ABA is unable to induce VSP synthesis in soybean, whereas JA can (Anderson 1989, 1991, Mason and Mullet 1WO), it seems that besides the cornmon response mechanism, there is a separate response pathway for ABA and JA on VSP gene expression in different plant species. The role of nürogen and sucrose in VSP gene expression

Since VSP is a nitrogen storage form, it is Iogical to think that the nutrition of a plant would influence VSP gene expression. Several experiments had investigated the effects of nitrogen and soluble sugar supplernentations on the VSP gene expression in poplar, soybean, Arabidopsis thdima, and potato (Berger et al. 1995,

Coleman et al. 1994, Hattori et al. 1991, Johnson and Ryan 1990, Mason et al. 1992,

Sadka et al. 1994, Staswick et al. 1991, van Cleve and Apel 1993, Wenzler et al.

1989). The results show that nitrogen is not involved in signalling VSP gene expression, whereas sucrose has a direct role in vspB gene regulation in soybean

(Sadka et al. 1994), Arabidopsis rhaliana (Berger et al. 1995), and patatin and proteinase inhibitor II gene regulation in potato (Johnson and Ryan 1990, Pena-

Cortes 1992, Wenzler et al. 1989).

The fact that sugars increase in the hypocotyl during water deficit (Creelman et al. 1990) and that wound-induction of VSP mRNAs is greatly enhanced in illuminated plants, compared to dark-treated ones (Mason and Mullet 1990) point to a role for photosynthetic carbohydrate in the regulation of VSP expression. Mason et al. (1992) reported that in soybean, the triggers of VSP synthesis are CO-regulated by sucrose and MeJA. Increasing the sucrose concentration in the absence of MeJA had only a srnall effect on VspB expression in soybean suspension cell cultures and excised leaves. However, in the presence of MeJA, addition of sucrose resulted in a large enhancement of VspB expression. Similarly, Atwp mRNA accumulation in

Arabidopsis thliana, which encodes a protein homologous to VSPa and VSPP in soybean, is not induced significantly by sucrose alone (Berger et al. 1995). But in the presence of MeJA, increasing the sucrose concentration from 1% to 4% enhances

Ahrsp rnRNA levels five-fold. Although, Johnson and Ryan (1990), Wenzler et al.

(1989) and Hattori et al. (1991) did not mention the level of JA/M.eJA present in their materials, it is possible that the enhanced expression of the genes encoding patatin, proteinase inhibitor and sporamin by sucrose was actually the result of JA/MeJA and sucrose CO-stimulation.Since the materials used in their experiments were excised laves. petioles, stems, or explants, not the intact plants, cutting may have caused a wounding response, resulting in an increase in JAIMeJA in these tissues.

With respect to the signalling of VSP gene synthesis, the nature of the sucrose effect is unknown, but based on the above results there are two hypotheses. One is that sugar acts as a signal molecule in concert with JAMeJA to induce the expression of VSP genes. Another possibility is that JAMeJA is the sole signal molecule for induction of VSP transcription, and energy sources in the form of carbohydrate enhance this process.

In all of the above studies, the accumulation of storage protein was preceded by an increase in its corresponding mRNA. It seems that regulation of VSP gene expression is controlled at the transcriptional level. The other possible roles of VSP

As mentioned previousiy, many VSPs show enzymatic activities, or share homology with other known enzymes (Zhong et al. 1997); patatin is a lipid acyl hydrolase (Andrews et al. 1988). the VSPor, VSPP, and 94 kD VSPs in soybean leaves are acid phosphatases and lipoxygenase (DeWald et al. 1992, Tranbarger et al. 1991 ), and the VSP protehase inhibitor in potato tubers (Pena-Cortes et al. 1988).

This indicates that, besides the storage function, VSPs may have other biological roles, such as in pathogen resistance and fiost hardiness (Berger et al. 1995, Ehness et al. 1997, Kang and Titus 1987, Mason et al. 1992, Pomeroy and Siminovitch 1971,

Sauter et al. 1988, Sauter and Wellenkamp 1988, Wetzel et al. 1989% Wetzel and

Greenwood 1989).

Possible role of VSP in pathogen resistance

Ehness et al. (1997) reported that pathogen attack and wounding activate a cascade of defense responses and may also affect carbohydrate metabolism. They reported that fungal elicitors, chemical elicitors such as benzoic acid, and wounding repressed gene expression of Rubisco mRNA. Activation of a defense machinery requires energy and thus an induction of sink metabolism. Physiologicd studies indicate that both photosynthetic capacity and carbohydrate metabolisrn are dtered in response to pathogens (Tcsi et al. 1994, Wright et al. 1995). Since VSP synthesis is stimulated by wounding adorinsect attack (Berger et al. 1995, Davis et al. 1993,

Mason et al. 1992, Pena-Cortes et al. l988), these proteins may have defense roles.

This hypothesis is Mersupported by the fact that one of the VSPs in potato is a proteinase inhibitor and it can be induced by wounding or chewing insect attack

(Pena-Cortes et al. 1988). Moreover, recently, it has been demonstrated that transgenk rice plants harboring an introduced potato protehase inhibitor II gene show insect resistance (Duan et al. 1996).

Possible desof VSP infrost hardiness

Prior to winter, the leaves of deciduous trees and herbaceous perennial plants, which are vulnerable to freezing or drought, senesce and the nitrogen in the leaves is mobilized to parts of the plant capable of surviving the winter. Cytological studies of changes in the secondary phloem parenchyma cells of Robinia pseudoacacia during development of frost hardiness showed that the large central vacuole that is present in the summer is segrnented into separate vacuoles in the fall (Pomeroy and

Siminovitch 1971). These vacuoles are protein-storing organelles (Wetzel et al.

1989a). Thus it is thought that overwintering storage of nitrogen is related to frost hardiness (Kang and Titus 1987, Niki and Sakai 1981, Pomeroy and Siminovitch,

1971, Sauter et al. 1988, Sauter and WelIenkamp 1988, Siminovitch et al. 1968,

Wetzel et al. 1989%b, Wetzel and Greenwood 1989). Porneroy et al. (1970) reported a strong correlation between protein augmentation in Pinus bark and development of hsthardiness. In addition, they mentioned that free amino acid and carbohydrate levels did not correlate with hardiness. Changes in degree of winter hardiness has been also reported to be correlated with the appearance of specific storage proteins in the bark of Malus by Kang and Titus (1987). Sauter et al. (1988), Sauter and

Wellenkamp (1988) demonstrated that the accumulation of protein bodies in the ray cells of Salk and Populus matches the onset of winter dormancy in these species. In addition, the fact that genes related to cold-protection are dso expressed during the time of senescence (see review by Guy 1990, Hughes and DUM 1996) indicates a relationship between VSP and cold protection. Objectives

A previous study in our lahatory by Cyr and Bewley (1990a) demonstrateci that in dandelion root there is a predominant 18 kD protein, which shows seasonal changes. They suggested that the 18 kD protein acts as a vegetative storage protein.

Dandelion is a very common perennial weed. It is a rosette plant with very short underground stems and a long tap root Its growth is very extensive and mcult to control. It is so persistent because it has a strong root system that acts both as an overwintering and an asexual reproduction organ. During winter, all leaves above the ground are dead; however, the underground parts (short stem and long tap root) remain alive. When spring cornes new leaves grow out fiom the stems. Since the shsof dandelion are very srna11 and short, it is likely that the roots provide nutrients for this new leaf growth. The purpose of this study is to understand the overwintering strategy of dandelion and its available resources for spring leaf growth by determinuig the possible biological role of the 18 kD protein. 2. Purification of the 18 kD protein and the attempted isolation of a cDNA encoding the 18 kD rotei in 2.1 Introduction

A common way to better understand the biologicd function of a protein is to compare its amino acid sequence with that of other known proteins (Breiteneder et al. 1989, Schoning et al. 1996). Since there is no available sequencing method to determine the whole amino acid sequence of an intact protein, the full-length amino acid sequence of a protein is usually obtained by deduction from the full-length cDNA sequence encoding the protein (Breiteneder et al. 1989, Schoning et al. 1996).

Thus, to understand more about the 18 kD protein the first objective was to obtain the full-length cDNA encoding the 18 kD protein, and thereafter obtain the base

sequence and deduce the amino acid sequence.

For a protein, especially for those with no known function, as in the case of the 18 kD protein, achiewig its full-length cDNA sequence rquires several steps to be cornpleted. First, one or several fi-agments of amino acid sequence, such as the N- or C-terminal ends or one piece of interna1 amino acid sequence of the protein, have to be obtained. On the basis of the amino acid sequences, degenerate oligonucleotide probes are then made. The cDNA encoding the protein may then be isolated by screening a cDNA library using these degenerate oligonucleotide probes. The full- length cDNA sequence can then be determined by either the dideoxynucleotide method (Sambrook et al. 1989) or by using an automatic sequencer, such as 377

DNA sequencer (Perkin Elmer, AB1 Prism). There are two approaches to get partial sequenœs of proteins of interest. One is to use the microsequencing method (LeGendre et al. 1993). Proteins are fnst separateci by two-dimensional gel electrophoresis, such as isoelectric focussing (EF) followed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and are then electrophoretically transferred from the gel to a PVDF (polyvinylidene difluoride) membrane. The required proteins are then sequenced fiom the membrane by degradation fiom their N-terminus using the Edrnan reagent, phenyl isothiocyanate

(PITC) (Matsudaira 1993).

Protein sequencing from the PVDF membrane, however, is not always successful. Many eukaryotic proteins have blocked amino termini (Brown and

Roberts 1976, Brown 1979, Driessen et al. 1985). In this case, it is necessary to cleave proteins either enzymatically or chemically to obtain interna1 amho acid sequences. Prior to this, the proteins have to be recovered from a two-dimensional gel by electroelution (Stone and Williams 1993). This method, however, provides very low amounts of proteins (usually a few pg), and is not always successfd

(personal experiences).

The other approach to get partial sequences of a protein is to purify it by chromatography (Scopes 1994). Chromatography has been widely used in protein purScation, for it frequently gives the greatest increase in protein purity. The pdedprotein can then be either directly sequenced, or enzymatically or chemically digested for interna1 amino acid sequencing, if it is N-terminal-end blocked. In addition, high amomts of proteins can be obtauled by this method, and these can be used to produce the antibodies for locaiization studies.

This chapter describes the procedures used for obtaining partial amino acid

sequences of the 18 kD protein and the attempted isolation of a cDNA that encodes the 18 kD protein. Also demonstrated are the homologies between the partial

sequence of the 18 kD protein and the sequences of other known proteins. 2.2 Materials and Methods

2.2-1 Materiais

Dandelion roots were collected monthly on the campus of the University of

Guelph fiom May 1993 to May 1995. After being collected, roots were washed with

tap water and distilled water, then cut into srnail pieces (around 0.5 cm length),

fiozen with liquid nitrogen immediately, and stored at -80' C until use.

Protein purification by chromatography was conducted in the Dept. of Plant

Science, University of Adelaide, Australia Dandelion roots used for this experiment were collected fiom the roadside in the Adelaide Hills, South Australia, in June and

July 1996. Afier collection, the roots were washed and fiozen as described above.

2.2.2 Methods

2.2.2.1 Seasonal fluctuations of the 18 kD protein content in dandelion roots

2e2e2-1.1 Total soluble protein extraction from dandelion roots collected in

different months

Extraction was conducted according to the method of Cyr and Bewley

(1990a). Lyophilized dandelion roots 0.5 g obtained in six different months (January,

March, May, July, Septernber, November) were ground to a fine powder and extracted with 10 ml extraction buffer [O. 1 M sodium phosphate, 1OmM cysteine, 10

pl 10 rnM phenylmethyl-sulfonyl fluoride (PMSF)]. Before extraction, 0.5 g polyvinyl-polypyrrolidone (PVPP) was added to the buffer and dlowed to soak for

1 hour at 4' C. The PMSF was added just prior to extraction. Mer being centrifhged

(Eleckman, mode1 52-21) at 10,000 xg for 10 minutes, the supernatant was filtered with Miracloth,

There are many phenolic compounds present in dandelion root extracts which interfere with protein extraction and separation. Thus, a purification step to remove these phenolic compounds was conducted using phenol (Hwkman and Tanaka 1986).

To the crude extracts was added an equal volume of water-satwated phenol. This solution was vortexed for 2-5 minutes and centrifuged at 8,000 xg for 10 minutes at room temperature. The aqueous phase was discarded, but the interface between buffer and phenol was not disturbed. An equal volume of the extraction buffer was added to the phenol phase and the interface, the tube was vortexed and centnfuged, and the aqueous phase was again discarded as above. The protein within the phenol phase and the interface was precipitated overnight by adding 5 volumes 0.1 M ammonium acetate in methanol at -20' C. Pellets were washed in 5 ml 0.1 M ammonium acetate in methanol, and then 5 ml cold acetone, air dried, and dissolved in a protein loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol)

(modified fiom Laemmli 1970)-

2e2e2.12Detemination ofproiein quaniity Protein amounts were determhed by the BCA assay method according to the manufacturer's (Pierce) instructions. Bovine semalbumin (%SA) raflging fkom 100 pglml to 1,200 pg/ml was used as standard. Assay tubes were incubateci at 37' C for

30 minutes. Protein amounts were then detefailned at A562 nm using a DU44 spectrophotometer (Beckman).

2.2.2.1.3 Denaturing polyacrylurnide gel elecâophoresis (SDS-PAGE) of the total soluble protein

Forty pg protein from each extract were separated by SDS-PAGE (Laemmli

1970) with Bio-Rad mini Protean II dual slab apparatus. Separating gel: 15% T, 0.4%

C acrylamide, 0.375 M Tris-HC1, pH 8.8, 0.1% (wfv) SDS. Stacking gel: 2.2% T,

0.6% C acrylamide, 0.125 M Tris-HCI. pH 6.8,O. 1% (wlv) SDS. Gels were run with buffer (125 mM Tris base. 0.96 M glycine, 0.1% SDS, pH 8.3) at a constant 150 V for 1.5 hour, stained with Coomassie BriUiant Blue R in methano1:water:acetic acid

(5:4:1, v/v/v), and destained with methano1:water: acetic acid (25:160:16, v/v/v).

2.2.2.2 Determination of the number of subunits or isoforms of the 18 kD protein

Before being purified and sequenced, the number of subunits or isoforms of the 18 kD protein was detemiined. 2.2.2.2.1 Totd soluble protein extraction with buter containing or not coniaining the redzicing agent ~mercapioethanol(PME)

Lyophilized dandelion root (0.5 g) collected in December 1994 was ground to a hepowder in a mortar with pestle. This was extracted with 10 ml protein extraction buffer (0.1 M Tris-HC1, pH 7.0, 10 mM EDTA, 6 mM P-ME, 10 mM

Na,, 10 pl 10 rnM PMSF) or the same buffer without 6 rnM PME. The crude protein extracts were then pdedwith phenol as described in Section 2.2.2.1.1. The detedation of protein quantities were the same as described in Section 2.2.2.1.2.

Forty pg protein fiom each extraction were loaded onto an SDS-PAGE gel described in Section 2.2.2.1.3.

2.2.2.2.2 Two-dimensional elecaophoresik of the toial soluble protein of dandelion root

First dimensional gel electrophoresis involved using an isoelectric focussing

(IEF) gel. The method was modified fiom O'Farrell (1975). Second dimension electrophoresis used an SDS-PAGE gel (Laernmli 1970).

Lyophilized roots (0.5 g) collected in December 1994 were extracted with buffer (0.1 M sodium phosphate, pH 6.8,10 rnM cysteine, 3 mM PMSF). The extract was then precipitated by adding 4 volumes of cold acetone overnight at -20' C. The pellet was air dried and redissolved in 200 pl lysis buffer (9.5 M urea, 2% NP-40, 0.8% ampholines pH 5-7,0.8% ampholines pH 6-870-4%amphohes pH 3-10,5 4& vlv p-ME).

IEF electrophoresis: IEF tube gels (12 cm long x 1.5 mm inside diameter) were cast to contain 9.16 M ma,acrylamide (3.9%T75.4%C), 2% NP-40,2%ampholines

(0.8% pH 5-7,0.8% pH 6-8, 0.4% pH 3-10) (Bio-Rad), and to polymerize, TEMED

(0.08%) and AP (ammonium persulfate) (0.015%). Tubes were filled to within 1 cm of the top, and the gel solution overlaid with 50 pl 8M urea. After 1 hour the urea was replaced by 20 pl lysis buffer (same as above), and an additional overlay of water. These solutions were removed an hour later and new lysis baer added to the gel surface. The gels were pre-run in an IEF apparatus (Buchler Instnunents) with an anode solution of 0.01 M H3P04in the lower reservoir and a cathode solution of 0.02

M NaOH in the upper reservoir at 200 V for 20 minutes, 300 V for 20 minutes, and

400 V for 15 minutes. After the pre-run the upper gel surfaces were blotted dry.

Protein sample (50-100 pg, dissolveci in lysis buffer, total volume not in excess of 50 pl) was added, followed by 12 p1 sample overlay [8 M urea, 1% ampholines (0.4% pH 5-7,0.4% pH 6-8,0.2% pH 3-10)]. The gels were run at 400 V overnight, and then 800 V for 1 hour. Gels were extruded from the tubes for the second dimension separation. If the second dimension electrophoresis was not carried out immediately the gels were frozen within the tubes at -20°C. SDS-PAGE (conducted in a Bio-Rad Protean II slab ceil): SDS-PAGE gels (1.5 mm thick) were set up as separating gels only: 15%T, 0.4%C acrylamide, 0.375 M

Tris-HCI, pH 8.8,0.1% (w/v) SDS. The separating gels were poured almost to the top of the gel plates. The IEF tube gels were mounted onto the SDS-PAGE separating gel with 3% (w/v) agarose containing 0.1 92 M glycine, 0.025 M Tris-HCl, pH 8.3. Gels were run at a constant 70 V with ninning buffer: 0.1 92 M glycine, 0.025

M Tris, 0.1 % W/V SDS, pH 8.3 for 3-5 hours.

2.2.2.3 N-terminal-end sequencing of the 18 kD protein purified by two- dimensional electrophoresis

Two-dimensional electrophoresis as described above was used to purify the

18 kD protein for N-terminal end sequencing.

2.2.2.3.1 Eiectmp h oretic blotting for proiein sequencing:

Afier two-dimensional electrophoresis, the protein was transferred from the gel to PVDF (polyvinylidene difluoride) (Bio-Rad) membrane according to the method of LeGendre et al. (1993). Electrophoretic bloning was conducted in a Bio-

Rad Mini Tram-blot apparatus at a constant 10 V, 4' C, ovemight using transfer buffer (10 mM CAPS [3-(cyclohexylamine)-l-propanesulfonic acid] containing 10% methanol, 0.5 rnM DTT [DL-dithiothreitol], pH 11). AAer transfer, the PVDF membrane was washed with distilled water three times, 5 niinutes each and stained with 0.025% Coomassie Brilliant Blue R-250 dissolved in a 40% methanol solution for 5 minutes and destained with 50% methanol solution for 10-15 minutes or longer until the background was faint. The membrane was then air dned at room temperature on Kimwipe tissue papers and stored at 4' C before being sent out for sequencing.

The 18 kD protein on the PVDF membrane was sequenced in the Protein

Chemistry Core Facility, Howard Hughes Medical Institute, Columbia University,

New York, USA.

2.2.2.4 cDNA library construction

2.2.2.4.1 PolyA+ RNA isolation

Total polyA + RNAs were extracted and isolated according to the method of

Pramanik et al. (1993). Total RNA was extracted in one step fiom roots using extraction buffer [200 mM Tris-HCI, pH 8.5, 0.5 M NaCI, 35 rnM MgOAc, 0.5%

NP-40, 5 mM DTT, 200 pghd heparin, 0.2 M sucrose, 25 mM EGTA (ethylene glycol-bis (beta-arninoethyl ether) N,N,N', N'-tetraacetic acid)]. PolyA+ RNAs were then isolated fiom total RNAs by oligo d(T)-cellulose affinity chromatography

(modified fiom Sarnbrook et al. 1989).

Twenty ml extraction buffer were added to 5 g fiozen root powder (collected in December 1994). merthorough mwn& the slurry was centrifiiged at 10,000 xg in a centrifuge (Beckman, mode1 J2-2 1) at 4' C for 15 minutes. The supernatant was transferred to a new tube and 1/20 volume 5 M NaCl and 1/20 volume 10% SDS added. The tube was incubated at 65' C for 10-1 5 minutes, and then placed on ice immediately to rapidly cool the solution. Once the solution was cold, it was allowed to warm to room temperature and was then centrifuged at 3000 xg in a Hede refiigerated desktop centrifuge at 4' C for 5-10 minutes. The extract was kept on ice until ready to pass through an oligo (dT) column.

Oligo (dT)-cellulose type 7 (0.1-0.2 g per column, Pharmacia) was regenerated in 0.2 N NaOH for 5- 1 0 minutes, and the fine particles decanted off. The oligo (dT) cellulose was washed several times with water until the pH was 7, then packed in a sterile Dispocolumn (Bio-Rad, 0.9 cm diameter). The column was equilibrated with

20 ml binding buffer (0.5 M NaCl, 25 mM Tris-HCl, pH 7.5,0.5% SDS). The RNA extract was passed through the colurnn and collected in a sterile tube. Once the elution was finished, the extract was passed through the column a second tirne. The column was washed with binding bufTer until the OD, of the eluent was less than

0.05. Before eluting the polyA+ RNAs, the column was washed with at least 20 ml binding buffer without SDS.The poly A+RNAs were then eluted with sterile warm water (37* C). One ml water was added to the column three thes, and the eluent was collected as 3-5-drop fiactions in Eppendorf tubes and placed on ice immediately. To determine which hctions contained polyA+ RNAs, 5 pl solution korn each tube were dropped onto a plastic weigh-boat and 5 pl 2 pg/pl ethidium bromide added to each &op. These were then viewed under UV light. AU fiactions demonstrating a positive signal (red fluorescence) for RNA were combined. To the tube was added 0.11 volume 3 M NaOAc (pH 5.3) and 2.5 volumes 100% ethanol at -20' C. The tube coritents were mked and then placed at -20' C overnight to precipitate RNA. The pellet was washed with 75% ethanol, dried in a SpeedVac concentrator, and dissolved in 100 p1 sterile H,O. The amount and qudity of the polyA+ RNAs was determined on a DU-64spectrophotometer (Bechan) at 260 nm and 280 nrn.

2.2.2.4.2 cDNA librury construction ming Jgt II vecîor

A cDNA library was constructed using a senes of cDNA kits fiom Amersham: cDNA Synthesis System Plus, cDNA Rapid Adaptor Ligation Module, A-DNA In

Vitro Packaging Module, and cDNA Rapid Clonhg Module- Agtl 1 .

A. Fimt strand cDNA sythesis kit code RPN 125O:

Reaction mixtures: 50 pl and 20 pl total volume (solutions were added in order as follows) : 50 pl 20 pl

5 X fVst strand synthesis reaction buffer 10 pl 4 pl sodium pyrophosphate solution 2.5 pl 1 PI human placental ribonuclease inhibitor 2.5 pl 1 FI deoxynucleoside triphosphate mix 5 ~1 2 cil oligo dT primer 2.5 pl 1 cil

[C~~~PI-~CTP(ICN, 10 pCi/pl) 2 pl (20 pCi) total poly A+ RNA 12-5 ci1 (5 cl& 2-5 pl (1 ~g) sterile H,O 10 pl 5.5 pl

AMV reverse transcriptase 5 ci1 (20 U4g) Id.

U__------1---P-----U_ The 20 pl reaction rnix was used to determine the eficiency of the reverse transcription reaction. Both mixtures were incubated at 42OC for a minimum of 40

minutes.

B. Second stmnd cDNA sythesis fkit code RPN 12-56]:

Reaction mixture: 250 and 100 pl total volume:

250 pl

first strand cDNA reaction mix (above) 50 pl

second strand synthesis reaction buffer 93.5 p1

[C~~~PI-~CTP(ICN 1O pCi/pl) Elcoli ribonuclease H 5 ri1 (4 U) 1 pl

E. coli DNA polymerase 1 33 pl (1 15 U) 7 cil sterile H,O 68.5 pl 32.5 pl

The 100 pi mixture was again used to determine the efficiency of the transcription reaction. Both mixtures were incubated at 12' C for 60 minutes, 22' C

for 60 minutes and 70' C for 10 minutes. To the mix was then added 2 U T, DNA polymerase per pg of original mRNA and incubated at 37' C for 10 minutes. The

reaction was stopped by adding 4 pl 0.25 M EDTA (ethylenediaminetetraaceticacid),

pH 8.0 per 100 p1 of fmal reaction mix.

C. Pur@kation of double stranded cDNAs (accordhg to the Amersham

manufacturer 's instructio-) :

The double stranded cDNAs were purified by fust adding an equal volume of

phenol/chloroform, twice, and chloroform once. The removd of unincorporated

deoxynucleotide triphosphates fiom the reaction mDt and the recovery of cDNA was

achieved by adding an equal volume of 4 M ammonium acetate and twice the

combined volume of cold 100% ethanol. This mixture was chilled for 15 minutes on

ice and warmed to room temperature with gentle shakuig (to dissolve the unreacted

deoxynucleoside triphosphates precipitated during chilling). The tube was spun at top

speed in an Eppendorf centrifuge for 10 minutes. The supernatant was removed and the pellet washed once with 50 pl 2 M ammonium acetate, 100 pl cold 100% ethanol, centrifiged and dried. The pellet was again washed with 200 pl cold ethanol, centnfûged and dried. The cDNA pellet was then resuspended in 8.5 pl TE buffer (1 0 mM Tris-HCI, 1 mM EDTA, pH 8.0).

D.Analvsis ofthe qmthesis efficienotand the skes ofthe cDNAs accord in^ to the

Amersham manufacturer 's instructiom) : i) Synthesis efficiency:

The efficiencies of the reverse transcription and second strand transcription were determined by calculating the radioactivity incorporated into the synthesized cDNAs. Two pl aliquots of each reaction mixture containing the radioactivity were transferred to tubes containing 20 pl water. The solutions were mixed well and two aliquots of 2 pl of each solution were spotted onto two 2.4 cm discs A and B of

Whatrnan DE 81 paper, respectively. The two discs B, spotted with fist and second strand reactions were washed 6 times for 5 minutes in 0.5 M Na&PO,, and twice for

1 minute in water. Al1 filters were then thoroughly dried and radioactivity measured by liquid scintillation in an aqueous scintillation fluid. The percentage of input radioactivity incorporated into DNA was calculated by comparing the radioactivity counts between washed (B) and unwashed (A) fïiters. The mass of cDNA synthesized in the reaction was calculated based on isotope incorporation. ii) Size range of cDNAs:

The size range of the cDNAs synthesized was measured by allcaline agarose gel electrophoresis.

First and second strand reaction mixtures containing 10,000 cpm of radioactivity incorporated hto nucleic acid were added to 20 pl carrier DNA solution

(100 pg/ml salmon sperm DNA). One third of the combined volume (cDNA and carrier DNA) of 1 N NaOH was added to the each solution. The mixtures were incubated at 46' C for 30 minutes. The same volumes of 1 M HCl and 1 M Tris-HC1, pH 8.0, equal to that of NaOH were added to the mixhues, to which an equal volume of phenol/chloroform was also added. cDNAs were precipitated by adding 2.5 volumes of 100% ethanol ovemight at -20' C, dried and finally dissolved in 10 pl

alkaline agarose gel loading buffer (50 rnM NaOH, 1 rnM EDTA, 2.5% Ficol1400

(Pharmacia), 0.025% Bromophenol blue).

A 1.4% agarose gel was made in 50 rnM NaCl and 1 mM EDTA-When solid, the gel was covered by an excess of alkaline electrophoresis buffer (30 m.NaOH,

10 rnM EDTA) and incubated for at least 30 minutes prior to use.

The excess running buffer was rernoved and enough remained to just cover the gel to a depth 1 mm. Electrophoresis was carried out at voltages up to 7.5 Vfcm

until the dye had migrated out of the loading dot. A glas plate was placed directly

on top of the gel to prevent it from floating. Electrophoresis was continued until the dye marker had migrated 2/3 of the length of the gel. The gel was soaked for 30 minutes at room temperature in 7% trichloroacetic acid (two changes), and then mounted onto a glas plate and dried for 8 hours under many layers of Whatman

3MM paper, weighted with another glass plate. The dried gel was covered with

SaranWrap and exposed to X-ray film at -80' C for 7 days. The film was developed and the size of the cDNA was determined by cornparison with cDNA standards.

E. Ligation of aduptor to cDNA kit code RPN 1 7121:

Reaction mixture, 20 pl total volume:

cDNA (above) 8.5 pl

*L/K buffer 2 PI

enzyme enhancer 5

EcoR 1 adaptors 2.5 p1

T, DNA ligase 2 pl (2.5 U/p1)

------CC------*L/K buffer was provided with the kit. Its composition is not known. The mix was incubated at 16' C for 30 minutes. The reaction was stopped by adding 2 pl 0.25

M EDTA. STE buffer (10 mMTris-HCI pH 8.0,l mM EDTA-disodium salt, 50 mM

NaCl) was added to a final volume of 100 pl. j? Column ptirificatiudsize fiactionution Cf'adopted' cDNA:

The column was supplied with the Amersham 1-DNA in vitro packaging module (RPN 1712). The purification of adapted cDNA was conducted according to the given protocol.

Reaction mixture, 200 pl:

collected column sample

L/K buffer

T, polynucleotide kinase

steriled H,O

The mix was incubated at 370 C for 30 minutes and extracted with an equal volume of phenoVchloroform twice and chloroform twice. The cDNA was precipitated by adding 0.1 volume 3 M sodium acetate (pH 6.0) and 2.5 volumes cold 100% ethanol ovemight at -20' C. The pellet was washed with 75% ethanol, dried in a SpeedVac concentrator, and then resuspended in 20 p1 TE buffer at approximately 20 nglpl.

Ligation reaction mixtures (10 pl each): reaction 1 reaction 2 reaction 3 insert DNA blunt ended control DNA adapted cDNA adapted cDNA

2 pl (100 ng) 2 PI (40 W) 4 ~1(80ng) ngtii am 2 cil (1~) 2 cil (W 2 PI (1~)

* L-buffer 1 PI 1 VI 1 water 4 PI 4 PI 2 4

T, DNA ligase 1 pl (2.5 Ulpl) 1 pl 1 pl

__YI_------*L-buffer was provided with the kit. Its composition is not known. The mixtures were incubated at 16' C for 30 minutes.

1 In vitro ~acka- qf li~ationmktwes;

The whole of each 10 pl ligation mix was then packaged in vitro using the packaging extracts provided with Amersharn's 1-DNA in vitro packaging module

(R.PN 1717).

To each packaging reaction was added 10 y1 extract A and 15 pl extract B

(both extract A and B were provided with the kit). The mixtures were then incubated at 20' C for 2 hours. Mer incubation, 470 pl SM buffer (0.1 M NaCl, 50 rnM Tris-

HC1, pH 7.5,8 mM MgSO,, 0.01% gelath) were added. The packaged phage was stored at 2-8' C before use. J Prenuration of~hagevluting ce& /Rit code m717):

E. coli strain Y 1090 provided in the kit was used as phage plating cells. A single colony of Y 1090 hman LB plate (1% tryptone, 5% yeast extract, 1% NaCI, pH 7, 1.5% agar) containing 50 pg/d ampicillin was picked out and inoculated into

10 ml of MB (LB containing 0.4% maltose, 50 ampicillin, no agar), and then incubated at 37' C ovemight. One rdof the overnight culture was added to 50 mi prewarmed growth medium MB.The mixture was incubated at 37' C with vigorous shaking until the celIs had grown to an OD, of 0.5 (2.5 x 108celldml). The mixture was cooled on ice and centrifuged at 3000 x g for 10 min at 4' C. The cell pellet was resuspended in 15 ml ice-cold sterile 10 mM MgSO, and rnixed thoroughly. Cells were stored at 2-8' C before infection with phage.

K. Titratu&

Thirty pl of the final packaged phage mixture (fiom section 1) were added to

270 pl SM buffer. The solution was diluted 1O2 x, 10' x, 104 x, 16 x, and 106 x. One hundred pl of both Y 1090 plating cells (prepared as in section J) and each phage dilution were rnixed together to form a plating mixture. The mixture was incubated at 37' C for 15 minutes. To each plating mixture was added 4 ml liquid M-top (LB containing 0.4% maltose and 1.5% agar). The mixtures were immediately poured ont0 a LB plates containing 50 pg/ml ampicillin, which was spread with 100 pl of both 100 mM IPTG (isopropykhio-P-D-galactoside) and 2% (wk)X-gal(5-Bromo- khloro-3-indoyl-P-D-galactoside) one hour previously. Plates were incubated at 37O

C ovemight.

2.2.2.5 Screening of the cDNA library

The cDNA library was screened with the degenerate oligo probes derived 5om the sequence of 14 amino acids at the N-terminal end of one of the two major polypeptides. Methods used in this Section were modified f?om those in 'Molecular

Cloning' (Sarnbrook et al. 1989).

A. Pre~arationof re~lica-filters:

Plates (9 cm diameter) containing around 500 to 600 plaques were chosen for the filter lifting. In total, 20 plates were screened. They were incubated at 4' C for at least 1 hour before lifting. A filter disc (nitrocellulose membrane) was placed ont0 the surface of a plate and incubated for 1 minute. Both filter and plate were marked before peeling off the filter with blunt- ended forceps. A duplicate filter was placed on each plate, but the second filter was incubated 30 seconds longer than the first

one. The filters were then laid, plaque-side-up, for 7 minutes on Whatmarn 3 MM paper soaked with denaturing solution (0.5 M NaOH, 1.5 M NaCl). The filters were again transferred for 3 minutes ont0 another Whatman 3 MM paper soaked with neutralizing solution (0.1 M Tris-HC1, pH 7.5, 1.5 M NaCl). This step was repeated.

Finally the filters were rinsed with 2 X SSC (20X SSC: 3 M NaCI, 0.3 M sodium citrate, pH7.0 ), air dried on Whatman 3MM paper, and then baked at 80' C for one hour.

B. Oligomcleotide robe desipn:

Based on the fkt 14 amino acids of the protein N-temiinal end sequence, two oligonucleotide probes were designed:

The 14-amino-acid sequence of the N-terminal end:

AVAEFEITSSLSPS

AB5832 5'-GCT GTT GCT GAA TTT GAA ATT AC1 TC1 TC1 TTA-3'

AB5833 5'-GCT GTT GCT GAG TTC GAG ATT AC1 TC1 TC1 TTG-3'

Reaction mixture, 20 pl total volume:

4 pmol dephosphorylated oligomer 5 cl1

* 10 X buffer 2 ci1

[y" PIATP (ICN 10 pCi/pl) 5 cl1

T, polynucleotide kinase (Prornega) 2 U) * 10 X kinase buffer: 0.5 M Tris-HC1 pH 7.6, 0.1 M MgCl,, 50 mM DTT, 1 mM spemiidine, 1 mM EDTA.

The reaction mixture was incubated at 37' C for 60 minutes and inactivated at 65" C for 10 minutes.

D.Purification of Zabelled oligomer:

Sephadex G25 powder was soaked in TE (10 df Tris-HCl pH 8.0, 1 mM

EDTA) buffer ovemight. It was then packed to almost fil1 a sterile 15-cm-longglass

Pasteur pipet, which was blocked with glas fibre at the bottorn. The above labelling reaction mixture was then loaded onto the column, which was eluted with 100 pl TE buffer. Each 4-drop fiaction was collected as the radioactivity eluted off. The fiactions comprishg the fust peak of radioactivity were pooled.

E. Pre-kvbridization and hybridization:

The membranes were pre-hybridized at 42' C or 37OC for at least 3 hows in the following solution:

Final concentration

*20 x SSPE (or SSC) 5 x

2% Ficol1400 0.02%

2% PVP (polyvinylpyrrolidone) 0.02% 5% BSA (bovine senun albumin) 0.04 ml 0.02%

formamide 5 ml 50%

10% SDS 0.1 ml 0.1%

10 mgM salmon sperm DNA 250 pl 250 pg/ml

Sterile dm2O up to 10 ml

_------P------P------*20 x SSPE: 3 M NaCl, 0.117 M Na2PO4,0.02 EDTA, PH 7.4.20 x SSC:

3 M NaCl, 0.3 M sodium citrate, pH 7.0. The membranes were then hybridized with

probes at 42' C or 37' C overnight. The hybridization solution was the same composition as the above pre-hybridization solution, plus the labelled probes.

After overnight hybridization, the membranes were washed with 5 x SSPE (or

5 x SSC) at room temperature for 10 min, 5 x SSPE (or 5x SSC) + 0.1 % SDS at 42'

C or 37'~for 10 min, and 2 x SSPE (or 2 x SSC) at room temperature for 10 min.

The membranes were then wrapped with Saran wrap and exposed to a X-ray film

(Kodak) with an intensifjing screen at -80°C.

2.2.2.6 Protein purincation by chromatography

2m2m2m6mlDetennination of the optimal extraction buffer

This was detennined using dandelion roots collected in Guelph in December

1994. Seven 1 g lyophilized root preparations were ground to a fine powder in a

mortar with a pestie. The seven powders were then extracted with the seven extraction buffers Iisted below. The extraction methods and SDS-PAGE electrophoresis were conducted as describecl in Sections 2.2.2.1.1 and 2.2.2.1.3. The buffer extracting the highest amount of the 18 kD protein was chosen for the next purifcation ste~. A

Buffer 1: Mcnvaine (0.1 M citric acid, pH adjusted with 0.2 M Na&PO,)

pH 3.0;

Buffer 2: McIlvaine (sarne as above) pH 5.0;

Buffer 3: 0.1 M NaOAc, pH 4.5,10 mM EDTA,

6 mM P-mercaptoethanol (ME),10 mM NaN,, 3 mM PMSF;

Buffer 4:

10 mM NaN,, 3 mM PMSF;

Buffer 5: Buffer 3 + 2 M LiCl; pH 4.5

Buffer 6: Buffer 3 + 1 % Triton X- 100; pH 4.5

Buffer 7: 0.1 M sodium phosphate, pH 6.8, 10 rntM cysteine, 3 mM Ph

2.2.2.6.2 Total soluble protein extraction

Buffer 4 was chosen as the best extraction buffer for the 18 kD protein purification (see results section 2.3.1.5 A). Frozen, stored mots (250 g), previously collected from the roadside in the Adelaide HiUs, were extracted with 1 L of buffer

4. (The PMSF was added just before extraction). The slurry was shed with a stir bar for 3 hours at 4' C in a cold room, and then centrifuged at 10,000 xg for 30 minutes

at 4' C. The supernatant was filtered with Miracloth to remove the fine debris.

2-2.2.6-3Aûtein Punification

The following purifcation procedures were conducted according to the

methods of Hrmova and Fincher (1993, and personal communication).

i) Ammonium sulfate precipitafion

Proteins were collected as precipitates fiom the above crude protein extract at

five ammonium sulfate concentrations: O-20%, 204%,40-60%, 60-80%, 80-100%.

The amounts of ammonium sulfate added to each fraction were detenmined according

to Scopes (1994). This procedure was conducted in a cold room with constant

stirring. Ammonium sulfate was added slowly to the extract to avoid a steep increase

in concentration. Each addition took one to two hours. After reaching each

concentration, the solution was stirred for another 15 to 30 minutes, and then

cenmged at 10,000 xg for 10 minutes. Pellets were dissolved in 50-100 ml DE52

column buffer (20 mM Tris-HC1, pH 7.0,6 mM P-ME).

Two ml of solution were taken from each fiaction and protein was precipitated

for 30 minutes by adding 1/9th of the volume of the sample of 100% TCA (trichloroacetic acid) for a hal TCA concentration of 10%. Each pellet was dissolved in 50 pl protein loading buîîer (62.5 mM Tris-Ha, pH 6.8,2% SDS, 10% glycerol). Finpl were loaded onto an SDS-PAGE gel to determine which fiaction(s) contained the 18 kD protein. The SDS-PAGEwas conducteci as described in Section 2.2.2.1.3.

ii) Rotein sample preparation for anion exchange DE52 column pun'fication

Fractions containing the 18 kD protein were dialyzed in dialysis tubhg (pore size 6000 D) at 4' C in an 8 L bucket filed with buffer (20 mM Tris-HC1 pH 7.0,6 rnM P-ME, 10 mM Na&) oveniight to remove the ammonium sulfate since this interferes with the DE52 column pdication. The dialysis buffer was changed at least 4 times at Chour intervals. After dialysis, fractions were pooled and concentrated to a final volume of 40 ml in a 50-ml Amicon cell (Microcon, Amicon) with an Amicon membrane (3 kD cut-off, Amicon). The pH of the protein sample was checked More loading onto the DE 52 column to ensure it was the sarne as the buffer.

iii) Column preparation

Columns to be used for chromatography (Bio-Rad) were med with chromic acid and incubated ovemight before washing with large volumes of ddH20, and air ckyïng. The colurnns were fillecl with 5% dichloromethylsilane in n-hexane and inverted several times. The solution was poured out and the tubes were air dried fiom the bottom using plastic tubing connected to an air line. This step was repeated twice.

The column matrix was weighed and soaked in buffer. This matrix was mixed gently and thoroughly, and placed on ice for 15 minutes before the supernatant was discarded. This equilibration was repeated 4-5 times. The matrix was then slowly poured into the coated column. No air bubbles were allowed to form, nor was the ma& allowed dry. The column was washed with 10 bed-volumes of buffer before use. During the washing the elution rate and fraction size were set up.

iv) Anion exchange DE52 column pun'fication

The concentrated protein solution was loaded onto the prepared anion exchange DE52 (diethylaminoethyl cellulose, pre-swollen, Whatman) column (8 cm long and 1.5 cm diam.). The column was washed with master buffer (20 mM Tris-

HC1 pH 7.0, 6 mM P-ME), and the eluate was monitored at OD,, in a continuous flow ISCO UV reader. Once the W absorption basehe was stable, the column was eluted with elution buffer using a linear gradient of NaCl (50 ml each of O and 1 M

NaCl in master buffer). The flow rate was 0.5 dminute and the fraction size was 2 mVtube. Fipl of eluate were taken from each fraction, dried in a SpeedVac concenûator, and redissolved in 15 pl protein loading buffer. The presence of the 18 kD protein in the Factions was then determined by SDS-PAGE.

v) GelfiZtratrkm

Fractions containing the 18 kD protein were pooled and dialyzed against the gel filtration buffer (50 mM Tris-HC1, pH 7.0,6 mM PME, 10 mM NaNJ overnight.

The procedure was the same as described in Section 2.2.2.6.3 ii. Afier dialysis, the protein was concentrated to a final volume of 2 ml in a 10-ml Amicon cell with an

Arnicon membrane (3 kD cut-off). It was then Merpurified on a gel filtration column (Bio-Gel P-60, Bio-Rad, 90 cm long and 1.2 cm diam.). AAer loading, the column was eluted directly with 50 mM Tris-HCl, pH 7.0,6 rnM P-ME at a fiow rate of 0.9 dl0minutes, and the fiaction size collected was 0.9 &tube. The presence of the 18 kD protein in the fiactions was determined by SDS-PAGE.

vi) Chromatofocusing

Fractions containing the 18 kD protein were pooled and dialyzed against the chromatofocusing starting buffer: 25 rnM imidazole-HC1, pH 7.4. Protein was then loaded ont0 a chromatofocusing column @H7-5, Polybuffer exchanger: PBE 94,

Pharmacia LKB) (24 cm long and 0.9 cm diam.). Mer loading, the column was washed with starting buffer until the UV absorption baseline did not change. The column was then eluted with elution buffer: (Polybuffer 74, 1:6 dilution, pH 5.0). The flow rate was 1 mY3 minutes and fiaction size was 1 dtube. The presence of the 18 kD protein in the fiactions was checked by SDS-PAGE electrophoresis.

vii) GelfiIiration

The Polybuffer used for chrornatofocusing was removed fiom the protein since it interferes with N-temiinal end sequence determination. This was achieved by gel filtration, and the two 18 kD polypeptides were then Merpurified by a second gel filtration. The column size, master buffer, flow rate and fraction size were the same as described in Section 2.2.2.6.3 v.

viio N-terminal end sequencing

The two purified 18 kD polypeptides were then sequenced in the Department of Plant Science, University of Adelaide using a Hewlett-Packard G1005A protein sequencer (Hewlett Packard Company, Pa10 Alto, CA, USA) and the Hewlett-

Packard 3 .O sequencing routine, which is based on Edman degradation chemistry.

2.2.2.7 Cornparison of the N-terminal end sequence with other known proteins

Sequence cornparison between the N-terminal end sequence of the 18 kD polypeptide and known proteins was conducted using BLAST in GenBank frorn

World Wide Web sites (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-bI~t? 2.2.2.8 Northem blots using cDNAs encoding Mal d 1 and Bet v 1 allergen

proteins as probes

Results fiom the BLAST search indicated that the N-terminal end sequence

of the polypeptide with the higher pI value had homologies with allergen proteins,

Md d 1 fiom apple leaf and Bet v 1 fkom birch polien. Northem blots to detect

specific mRNAs encoding the 18 kD protein were performed using the cDNAs

encoding the Mal d 1 and Bet v 1 allergen proteins (Section 2.2.2.8.2) as probes.

2*2*2.8.1 Electrophoresis of RNA lhrough gels containittgformaldehyde (modz~ed from the methodr in 'Molecular Cloning ', Sambrook et al. 1989)

Gel D-aration

Agarose 0.2 g was melted by boiling in 12.4 mi ddH,O in a microwave. When

the agarose solution cooled to 60-55' C, 4 ml of 5 x MOPS buffer [0.1 M MOPS (3-

IN-morpholino] propanesulfonic acid), pH 7.0,40 mM sodium acetate, 5 mM EDTA, pH 8.0) and 3.6 ml of formaldehyde (37%) were added. The solution was mixed well

and poured.

Total ~olyA+ RNA sarnple pre~arationand electrophoresis For each gel, 2 pg (up to 4.5 pl) of polyA+ RNA isolated as described in

Section 2.2.2.4.1 were used. PolyA+ RNA was denahired by adding 2 pl 5 x MOPS,

3-5 pl formaldehyde, and 10 pl formamide and heated at 65' C for 10 minutes. Mer

heating, the mixture was cooled on ice immediately for 5 minutes. To it was then

added 2 pl sterile 6 x loading buffer (50% glycerol, 1 mM EDTA, pH 8.0, 0.25%

bromophenol blue, 0.25% xylene cyan01 FF) and loaded onto the above 1% agarose

gel. Electrophoresis was conducted at 60 V for about one hour. cDNAs encoding the

Mal d 1 and Bet v 1 were also loaded onto gels as positive controls.

RNA transfer fiom eei to membrane:

After electrophoresis, gels were washed in sterile water several times and

soaked in 20 x SSC for 45 minutes. Meanwhile, two pieces of nitrocellulose

membrane (GeneScreen hybridization transfer membrane, DuPont) were cut to the

size sarne as the gels, wet with sterile water and then soaked in 20 x SSC for at least

5 minutes. Two pieces of Whatman 3 MM paper were cut to the size of the two gels.

A plastic box was filled with 20 x SSC, covered with a clean glass plate, and

Whatman 3 MM filter paper wick was cut so that it covered the glas plate. The wick was wet completely with 20 x SSC and its ends were hanging into the 20 x SSC

\ solution. Gels were then placed together onto the wick with no air bubble present between the gels and wick. Membranes equilibrated with 20 x SSC were placed ont0 the gels and covered with the two pieces of Whatman 3 MM paper. Air bubbles between the gels and membranes and Whatman 3 MM were squeezed out. Areas around the gel were covered with Parafilm to avoid a shortcut flow of transfer buffer.

A stack of bloning paper was then placed onto the "sandwich". The RNA transfer fiom gel to membrane was conducted overnight.

Mer transfer, the membranes were washed with 6 x SSC for 5 minutes and air- dried on a Whatman 3 MM paper for at least 30 minutes. They were then baked at 80' C for two hous.

2-2-2.8.2 cDNA insert digestion and isolation from pikW1 75 plasntid

pMW175 plasmid containkg insert cDNAs encoding Mal d 1 and Bet v 1 were kindly provided by Dr. K. Hofbann-Sommergmber (Institut fur Genetik und

Allgemeine Biologie, Universitat Salzburg, Austria) (Breiteneder at al. 1989, Vanek- krebitz et al. 1995).

Insert dieestion:

The cDNA inserts were cut from their plasmid (pMW175) using restriction enzymes EcoR I and Nco 1( Boeh~gerMannheim). Both inserts were 480 bp long.

Digestion reactions (50 pl):

plasmid, containhg inserts 10 x EcoR 1 buffer 5 Pl

enzymes EcoR I and Nco 1 6 pl (3 pl each)

sterile dm20 up to 50 pl -- - The reactions were incubated at 37" C ovemight. The inserts were then

isolated by separation on 1% LMP (low melting point) agarose gel.

Insert isolation (modified fiom the method in 'Molecular cl on in^'. Sarnbrook et al.

1989)

LMP agarose 0.2 g (Gibco BRL) was heated until melted in 20 ml 1 x TAE

buffer (0.04 M Tris-acetate, 1 mM EDTA) and poured. Ten p1 of 6 x Ioading buffer

(0.25% bromophenol blue, 0.25% xylene cyan01 FF, 0.0 1 % ethidiurn bromide, 30%

glycerol in water) were added to 50 pl of the above digestion reactions. The reaction

mixtures were then loaded onto the gel which was nin at a constant 60 V for 1 hour

in lx TAE buffer. After ming, the region of the gel containing the cDNA inserts

was cut out with a razor blade under UV light. Gel slices were rnelted at 65' C for 5-8

minutes, and an equal volume of Tris-saturated phenol was added to each melted gel

solution. The mixtures were immediately vortexed for 2-3 minutes, and centrifuged

at full speed in a Eppendorf centrifuge for 5 minutes at 4' C. The aqueous fraction was collected and an equal volume of chloroform added. It was mDred and centrifbged as above. To every 100 pl liquid, 5 pl 5 M NaCl were added, and then 2 to 2.5 volumes of cold 100% ethanol. cDNA inserts were precipitated at -20' C overnight. The pellet was washed with 75% cold ethanol, dried in a SpeedVac concentrator, and dissolved in TE buf5er. The qymtity of cDNA were determined at

OD2m

2.2.2.8.3 cDNA probe Iabelling andpmi~~on

Probe labelling

cDNAs were labelled using a randody-primed DNA labelling kit (Boehringer

Mannheim). labelling mixtures (20 pl)

denatured cDNA 2 ~1(25ng)

dTTP+dGTP+dATP(O .S mM each) 3 pl (1 pl each)

[cr3'P]dCTP (ICN, 1 O pCi/pl) 5 PI

reaction mixture 2 PI

sterile ddH20 7 ~1

Klenow enzyme 1 pl(2unitdpl)

Robe purification with Sephadex G50 colum:

The cDNA probe purification was the same as in Section 2.2.2.5 D, except

-65- that the column was Sephadex G50 instead of G25. Also a sarne volume of dye

containhg 1% blue dextran and 1% orange G was added to the labelling mixture

before loading onto the column. The blue dye was used to indicate the location of the

labeiled probe since they CO-eluted.

2.2.2.8.4 Re-hybridizutihn and hybruiization of the membrane with the cDNA probes

Pre-hybridization and hybridization were conducted at 42' C. The method

used was the same as in Section 2.2.2.5 E, without formamide but using 20 x SSC.

After overnight hybridization, the membranes were washed with 2 x SSC +

1% SDS briefly at room temperature, at 42' C for 15 minutes, and 1 x SSC + 0.1 %

SDS at 42' C for another 15 minutes. The membranes were then wrapped in Saran

wrap and exposed to X-ray film (Kodak) with an intensifjmg screen at -80°C.

2.2.2.9 Western blot using antibody raised against Bet v 1 allergen protein

A western blot was also conducted according to the Bio-Rad manufacturer's

instructions to determine the homology between the 18 kD protein and Bet v 1.

Polyclonal and monoclonal antibodies raised against purified recombinant Bet v 1

allergen protein were also kindly provided by Dr. K. Hoffmann-Somrnergmber

(Section 2.2.2.8.2). The 18 kD protein extracted with buffer (0.1 M sodium phosphate pH 6.8, 6

rnM P-ME, 10 pl 10 mM PMSF in 10 ml buffer) as in Section 2.2.22.1 was used for

westem blots. Two 30 pg aliquots of total soluble protein fiom dandelion root were

loaded ont0 two acrylamide gels (15%T, 0.4%C). SDS-PAGE was conducted in the

same way as in Section 2.2.2.1.3. Mer electrophoresis, proteins were transferred

fkom the gel to nitrocellulose membranes (Bio-Rad) by electrophoresis. The

electrophoresis blotting was performed at a constant 10 V, 4' C overnight in buffer

(25 mM Tris-base, pH 8.3, 19 mM glycine, 20% methanol).

After transfer of protein, the membranes were blocked with 3% (w/v)

Carnation powdered miik in TBS (20 mM Tris-base, 500 mM NaCl, pH 7.5) for at

least 1 hour at room temperature with shaking, and washed in TTBS (0.05% [v/v]

Tween 20 in TBS) with gentle shaking twice for 5 minutes. Membranes were then treated with 1/1000 dilution of polyclonal rabbit anti-Bet v 1 antibodies or 1/10

dilution of monoclonal mouse anti-Bet v 1 antibody in 1% milk powder in TTBS

(antibody buffer) overnight at room temperature with gentle shaking. They were washed as above, and then reacted with a 1/2000 dilution of alkaline phosphatase

conjugated goat anti-rabbit IgG (Sigma) or rabbit anti-mouse IgG (Sigma) in antibody buser for 60 minutes at room temperature with shaking, washed as above, plus an additional wash for 5 minutes in TBS.Membranes were developed in colour- developing solution (100 pl solution A + 100 pl solution B in 10 ml carbonate buffer-see below) for 10 minutes in the dark, washed with deionized water, and dried in the dark between two pieces of Whatman 3 MM paper. Colour developing reagents (Bio-Rad): Solution A: 30 mg NBT (nitro-blue tetrazolium chlonde) in 1 ml

70% DMF WB-dimethylfomamide). Solution B: 15 mg BCIP (5-bromo-4chloro

3-indolyl phosphatephidine salt) in 1 ml 100% DMF. Carbonate buffed00 mM

NaHC03, 1 mM MgC1,.6H20, pH 9.8. 2.3 Resuits and Discussion

23.1 Rdts

2.3.1.1 Seasonul changes in the 18 RD protein in dandelion roots during the year

Fig. 2-1 demonstrates that there are two major proteins in dandelion roots, the

18 kD protein and one slightly lower in molecular mass, 16 kD. However, the 18 kD protein is more predominant. There are other proteins which are evident, such as those around 36 kD and 50 kD. Several proteins fluctuate during the year, including the 16 kD and 18 kDproteins, and the proteins around 36 kD and 50 kD,but only the change in the 18 kD protein is seasonal, even though this is not a large change. The amount of the protein is slightly higher in winter and fall than in the summer months

(May, July), when it is still predominant, however.

2.3.1.2 Subunit composition and number of LFofoms

Fig.2-2 indicates îhat there are slight differences between the protein profiles extracted by buffers with or without P-ME. A few proteins with high molecular masses, such as 32 k,,36 kD and 47 kD, were absent if the tissue was extracted using buffer containing P-ME, whereas several new proteins with low molecular masses at 24 kD, 20 kD and 13 kD were present. It is possible that these latter proteins are subunits of the 32 kD, 36 kD, and 47 kD proteins resulting from the disruption of disufide bonds between subunits by P-ME. However, the size of the 18 kD protein was the same whether or not the extraction buffer contained the reducing agent. It seems, therefore, that the 18 kD protein is not comprised of subunits connected by disulfide bonds. In lane 1 and 2 of Fig. 2-2, there is ody one predominant band (at 18 kD), instead of the two (at 18 kD and 16 kD) present in Fig.

2-1. This may be due to the different extraction buffers used for the two experiments.

The extraction buffer used for this experiment (Fig. 2-2) was Tris-HC1, pH 7.0, while

the buffer used for the data in Fig. 2-1 was sodium phosphate, pH 6.8. The 16 kD

protein was not extracted by Tris-HC1 buffer.

Two dimensional electrophoresis (Fig.2-3) demonstrates that there are more

than two 18 kD polypeptides, although there are only two major ones. Their pI values

are very close, between 5-6. Two proteins 19 kD and 16 kD, which are close to the

18 kD protein, are also evident. However, their pI values are quite different fiom that

of the 18 kD protein. The pI of 19 kD protein is around 6 and the pI of 16 kD protein

is around 5.

2.3. 1.3 N-tenninal-end sequence of the 18 kD protein punified by two-dimensional

electroph oresis

N-temiinal end sequencing of the two 18 kD polypeptide from the PVDF

membrane provided a 14-amino-acid sequence of one of the two major polypeptides

(polypeptide b indicated in Fig.2-3). The sequencer was not able to detect residues after the 14&amino acid , although the amount of protein was apparently sufficient.

This could be because of an intrachain disuIfide bond at that position that made it impossible to sequence the protein Mer.The N-terminal end of the other major polypeptide (a indicated in Fig. 2-3) was blocked and hence could not be sequenced.

A BLAST (Altschul et al. 1990) search of the GenBank through the Internet (World

Wide Web) failed to show homology between the 14-amino-acid sequence and any other sequenced proteins.

2.3.1.4 Screening the cDNA library

Screening the cDNA library with the degenerate oligonucleotide probes derived from the 14-amino-acid sequence was not successful. There was no hybridization signal even at low hybridization stringency (37' C).

The failure of the cDNA Library screening might be due to the improperly designed degenerate oligonucleotide probes derived from the 14 amino acids. The

Idamino-acid sequence at the N-terminal end was not long enough to provide sufficient information to design good probes, moreover, the sequence did not show

any homologies with other known proteins in the GenBank. Since no gene has been

isolated from dandelion there was no good reference that could be used to determine

genetic codon bias (Le. which base is most prevalent in the third wobble position) when designhg the oligonucleotide primers. To design good degenerate oligonucleotides and achieve more information about the homology between the 18 kD protein and other known proteins it was necessary to obtain a longer N-terminal end sequence or interna1 sequences. Thus, the more stringent purification methods (chromatography) were used to obtain a higher quantity and quality of the 18 kD protein.

2.3.1.5 Protein p ur~~cationby chromafogruphy

A) Determination of the optùnal buter

The amounts of total soluble protein extracted by 7 different buffers were determined and calculated. Buffers 1, 2, 3, 5 and 6 with low pH values (3-4.5) extracted much lesser arnounts of total protein than buffers 4 and 7 with higher pH values (6.7-7). The amounts of total proteins extracted by buffers 1, 2, 3, 5, and 6 were: 2 mg/,4 mg/,2 mg/,2.7 mg/,and 3 mg/l g dry root tissue, respectively, while buffers 4 and 7 extracted 10 mg/and 9 mg/l g dry root tissue. Furthemore, the SDS-

PAGE gel demonstrated that buffers 4 and 7 extracted proportionately more 18 kD protein than the other buffers (Fig. 24). Buffer 1,2,3,5 and 6 extracted more other proteins, such as the proteins around 13 kD (lane 1,3,5, and 6), 16 kD (lane 1,2,3,

5, and 6), and 21 kD (lane 2), and proteins between 35-66 kD (lane 1,2,3,5 and 6).

The i 8 kD protein is not the only predominant protein in these lanes since the contents of the proteins between 36-40 kD and at 16 kD are also quite hi& Cornparison of the profiles of the protein extracted by buffer 4 and buffer 7 shows that there is somewhat more 18 kD protein and less of other proteins extracted by buffer 4 than 7; buffer4extracted-protein was thus chosen for the next purification step.

B) Protein purjZcafr*onand the 18 kD proiein N-terminal end sequencing

Fig.2-5 shows that most 18 kD protein was precipitated in the 60430% ammonium sulfate fiaction, and a little in the 80-1 00% fiaction, with almost no 18 kD protein in fiactions 0-20%, 2040% and 40-60%. Thus, fkactions 60%-80% and

80%- 100% were taken for Merpurification by anion exchange chromatography

(DE52 column). After DE52 column separation the 18 kD protein was present in fiactions 6 to 24 (Fig.2-6). After hction 15, a lower molecular mass (16 kD) protein and a 2 1 kD protein were eluted. Fractions 6 to 15 were thus pooled for gel filtration chromatography which successfûlly separated the 18 kD protein fiom most other proteins, especially those with a higher molecular mas, such as the 28 kD, 35 kD,

43 and 44 kD proteins (Fig.2-7). During the gel filtration, the 18 kD protein started to elute out after fiaction 25 and was present mostly between fractions 28 and 36.

Fractions 28 to 38 were pooled for the chromatofocusing purification. During chromatofocusing two protein peaks were detected between fkactions 110 and 156

(Fig.2-8). SDS-PAGE shows that the two peaks were enriched in the 18 kD polypeptides and they were separated from most other proteins (Fig.2-9). The two peak fiactions were 124 and 140, the pH values of these two fractions were 5.56

(fraction 124) and 5.49 @action 140), which were taken as the pI values of the two polypeptides. Fractions 110 to 128 and 137 to 147 were pooled separately as crude

PI 5.56 polypeptide and PI 5.49 polypeptide, respectively. The two polypeptides were then pmed to near homogeneity on a second gel filtration column (Fig.2-10).

Cation exchange chromatography using carboxymethyl-cellulose (CM52,pre- swollen, Whatman) equilibrated in buffer (0.1 M NaOAc, pH 4.5,6 mM P-ME, 10 mM NaN,) was not useful in the isolation of the 18 kD protein since it did not bind to the column, and eluted with the wash buffer (0.1 M NaOAc, pH 4.5,6 mM P-ME).

This is most likely because the surface charge of the 18 kD protein is negative.

A 40-amino-acid sequence at the N-terminal end of the pI 5.56 polypeptide (b) was obtained (Fig. 2-1 1A). However, the N-terminal end of the pI 5.49 polypeptide

(a) was blocked and could not be sequenced.

2.3.I.6 Compatison of the 40-amino-acid sequence of the N-terminal end of the

18 kD protein with known proteins

A BLAST search in GenBank indicated that the 40-amino-acid sequence shares approximately 44% to 55% hornology with Bet v 1 fkom birch pollen and Mal d 1 from apple leaves (Breiteneder et al. 1989; Vanek-Krebitz et al. 1995), respectively, and several pathogenesis-related (PR) proteins (five were chosen to compare with the 40-amino-acid sequence of the 18 kD protein) (Fig. 2-1 1B).

2.3-1.7 Northem bloa using the cDWencoding the Mal d 1 and Bet v I aRèrgen proteins as probes

Although hybridization was conducted at mild suingency conditions (no formamide in the pre-hybridizartion and hybridization solutions, hybridization temperature 42' C, final washing solution 1 x SSC), there was no hybridization signal between the specific mRNA encoding the 18 kD protein and the cDNAs encoding either Mal d 1 or Bet v 1.

2-3.1.8 Western blots using polyclonal and monoclonal anidbodies mised aguinst

Bet v l allergen protein

The monoclonal antibody raised against purified recombinant Bet v 1 did not recognize the dandelion root 18 kD protein. However, polyclonal antibodies did cross-react with the 18 kD protein. Two proteins were detected at the 18 kD mark on western blots (Fig.2- 12). The amount of the total protein used for western blots was the same as that loaded onto the SDS-PAGE (Fig. 2-12 A). Cornparison of the proteins on SDS-PAGE gels and western blot indicated that the binding of the antibodies raised against the Bet v 1 with the 18 kD protein was very weak. In addition, the polyclonal antibodies recognized a 35 kD protein. 2.3.2 Discussion

2.3.2.1 Homologies of the I8 kD proiein with other known proteins

N-termind end sequencing of the 18 kD protein purïfied by chromatography provided a longer N-terminal end sequence (40 amino acids) than the protein obtained by two-dimensional electrophoresis and transferred to PVDF membrane.

The 40-amino-acid sequence showed homologies with allergen and PR proteins.

However, northem blots demonstrated that the homologies in the full length cDNA between the 18 kD protein and Mal d I and Bet v 1 were not very high, since there was no hybridîzation signal between the mRNA encoding the 18 kD protein and the cDNAs encoding the Mal d 1 and Bet v 1, even under rnild hybridization stringency conditions. Thus the idea of using these cDNAs as probes to isolate the cDNA encoding the 18 kD protein fiom a cDNA library was not feasible.

Monoclonal antibody raised against Bet v 1 did not recognize the 18 kD protein. Thus the 18 kD protein does not contain the specific epitope that the monoclonal antibody was raised against.

The fact that the polyclonal antibodies raised against Bet v 1 recognize both

18 kD polypeptides indicates that they are most likely isoforms.

Although the polyclonal antibodies cross-reacted with the 18 kD protein, the binding was not strong. The bands were extremely thin and faint compared to the bands which stained on acrylamide gels (Fig. 2-12). Moreover, the polyclonal antibodies were not specific to the 18 kD protein because they also cross-reacted with a 35 kD protein. Thus the polyclonal antibodies could not be used to screen a cDNA library either.

It was decided, therefore, that the cDNA encoding the 18 kD protein had to be isolated using degenerate prima derived fiom the 40-arnino-acid sequence of the

N-temiinal end to either screen the cDNA library or to conduct PCR amplification. Jan Mar May July Sept Nov

Fig. 2-1. Seasonal changes in the 18 kD protein of dandelion roots SDS-PAGE gel stained with Coomassie Brilliant Blue R-250; 40 pg of total soluble protein loaded in each lane. Fig. 2-2. SDS-PAGE gel of dandelion root proteins stained with Coomassie Brilliant Blue R-250 Total soluble proteins were extracted fkom dandelion roots collected in December 1994 (40 pg in each lane) 1. Protein extracted by buffer with B-ME. 2. Protein extracted by buffer without B-ME. + -- IEF 5 pI value 7

Fig. 2-3. Two dimensional electrophoresis of dandelion root protein (silver stained) Total protein was extracted from the root collected in December 1994; 80 pg total protein were loaded. Fust dimension: IEF. Second dimension: SDS-PAGE Arrow indicates the two major 18 kD polypeptides: a and b Fig. 2-4. Dandelion root proteins extracted with seven different buffers SDS-PAGE gel stained with Coomassie Brilliant Blue R-350; 40 pg of total soluble protein were loaded in each lane. 1. Buffer 1: McIlvaine pH 3 .O; 2. Buffer 2: MaIlvaine pH 5.0; 3. Buffer 3: 0.1 M NaOAc, PH 4.5, 10 mM EDTA, 6 mM PME, 10 rnM NaN,, 3 mM PMSF; 4. Buffer 4: 0.1 M Tris-HC1, pH 7.0, 10 mM EDTA, 6 mM P-ME, 10 mM Nd,, 3 mM PMSF; 5. Buffer 5: Buffer 3 + 2 M LiCl; 6. Buffer 6: Buffer 3 + 1% CWS+ 1% Triton X- 100; 7. Buffer 7: 0.1 M sodium phosphate, pH 6.8, 10 rnM cysteine, 3 mM PMSF. Fig. 2-5. 18 kD protein precipitated by five ammonium sulfate concentrations

SDS-PAGE gel stained with Coomassie Brilliant Blue R-25 0; Protein was loaded according to the volume, 10 pl each . Am SO,: 1.0-20%, 2.20-40%,3.40-60%, 4.60-80%, 5. 80-100% Fig. 2-6. SDS-PAGE gel of eluents after DE52 column chromatography (fraction 6 to 24) (stained with Coomassie Brilliant Blue R-250) 6-24: hction number; fraction 6 to 15 were pooled for the gel filtration purification. C. sample before DE52 column separation Fig. 2-7. SDS-PAGE gel of the eluents after gel filtration chromatography (fractions 1 to 48) (stained with Coomassie Brilliant Blue R-250) 1-48: fiaction number; fractions 28 to 3 8 were pooled for the chromatofocusing purification. 110 120 124 130 140 150 160 fiaction number Fig. 2-8. Absorbance at 280 nm of the eluate after chromatofocusing chromatography (fraction 110 to 156) (showing two protein peaks with peak fraction numbers of 124 and 140) Fig. 2-9. SDS-PAGE gel of the eluate after chrornatofocusing chromatography (fraction 110 to 156) (silver staining)

Fraction 1 10 to 128 and 137 to 147 were oooled into-. two - fractions for the final gel filtration p&ification. C. sample before chromatofocusing separation Fig. 2-10. SDS-PAGE gel of the isoforms of pI 5.49 (a) and pl 5.56 (b) after the final gel filtration chrornatography (silver staining) Fractions 6 to 16 in picture a were combined as isoform pI 5.49; Fractions 6 to 11 in picture b were combined as isoform p15.56; C. Sample before gel filtration separation A. The 40 aa of the N-terminal end of the pI 5.56 polypeptide AVAEFEITSSLSPSNIFKAFVIDFDTLAPKAEPETYKSK

B. Homologies of the 40 aa with known proteins 1. Allergen Mal d 1 protein - apple tree 18 kD 4 EFEITSSLSPSNIFKAE'VIDFDTIAPK

O .* . . .* . * ***** : : *.O O Mal d 1 1 ENEFTSEIPPSRLFKAFVLDADNLIPK

2. Allergen Bet v I - European white birch 18 kD 4 EFEITSSLSPSNIFKAFVIDFDTIAPK *. *. . .. **** : .-0 . Bet v 1 6 ETETTSVIPAARLFKAFILDGDNLFPK

3. Disease response resistance protein- PLrm sativum 18 kD 4 EFEITSSLsPSNIFKAFVIDFDTIAPK . *.** . ..*a ** : :: :: DRRP 7 EDEITSWP.PAILYKALVTDADTLTPK

4. PR-protein 2 (PVPR2) -PhmeofuswIgarir 18 kD 4 EFEITSSLSPSNIFKAE'VIDFDTIAPKA . . ** * : ::: ::: PVPR2 7 EDQTTSPVAPATLYKALVKDADTIVPKA

5. PR protein STH-2 1 SoIamun tuberom 18 kD 6 EITSSLSPSNIFKAFVIDFDTIAPK *. * * *** * : : . . STH-2 1 9 ETTTPVAPTRLFKALWDSDNLIPK

Fig. 2-11. Comparison of the 40 amino acid sequence with known proteins .- ,..,<' .. ,' ,' . - :. ...,. 7-._ --c , ..A::.; A. .-.,.,.. *,....- . .;.;,;J..,;-,;... . , ;j , '.>.-,--;,...: *.-.*-- -. . :!* 7. ;&*;.y: .."?... .--y .,x-;y,s; --.*. *,-- ..: .y,$w-$:;L--:.,: . . ..:&+<*Q-;: a-.; .,- )-s-.-,., .+rjr;." :2':: .i. +- ,. .r ' .-. - .. ...1. -.- . --.\< ;II-:' II. :@

Fig. 2-12. Western blot of the 18 kD protein using antibody raised against the Bet v 1 allergen protein from birch pollen A. Dandelion root protein profile on SDS-PAGE; stained with Coomssie Brilliant Blue R-250; 40 pg of total soluble protein were loaded. B. Western blot of the 18 kD protein recognized by the antibody raised against Bet v 1; 40 pg of total soluble protein were loaded. 3. Construction and characterization of the full-length cDNA sequence encoding the 18 kD protein and its genomic DNA-PCR products 3.1 Introduction

The polymerase chah reaction (PCR) is a simple, rapid and efficient procedure for in vitro enzymatic amplification of a specifc segment of DNA. It permits direct sequencing of nucleic acids without requiring the time-consuming and variably successful cloning procedure. It usually needs weeks to rnonths to prepare and screen a single cDNA library and to isolate and analyse candidate cDNAs. However, PCR takes only a few days to provide results. Thus, the PCR method was chosen to obtain the cDNA sequence encoding the 18 kD protein.

RACE (rapid amplification of cDNA ends)-PCR is a recent popular method for obtaining fuli-length gene sequences (Frohman 1990). In essence, the RACE protocol generates cDNAs by using PCR to amplify copies of the region between a single point in the transcrîpt and the 3' or 5' end (Frohman 1990, Frohrnan et al. 1988). In brief, for the 3'end. mRNA is reverse transcribed using a "hybrid" primer consisting of oligo

(dT) (17 residues) linked to a unique 17-base oligonucleotide (adaptor) primer.

Amplification is subsequently perfomed using the adapter primer, which binds to each cDNA at its 3'-end, and a primer specific or degenerate (as in the case of the 18 kD protein) to the gene of interest. For the 5' end, reverse transcription is performed using a gene-specifc- or oligo dT- (in this case) primer. A homopolyrner (poly A) is then appended by using a terminal tramferase to tail the first-stand reaction products.

Finally, amplification is accomplished using the hybnd primer previously descnbed and a second gene-specinc primer upstream of the fht one. The overlapping 3' and

5' end RACE products are combined to produce an intact filMength cDNA. The strategy is illustrated in more detail in Fig. 3-1.

The amino acid sequence determines the structure of a protein, and the structure, in tum, determines its fiinction (Darby and Creighton 1993, Zaidi and Smith

1996). Recently, due to the rapid development of the cornputer techniques, a few programs predicting the features and secondary structure of proteins based on amino acid sequence have been developed, such as PC/Gene@ and Gene Rumer@.

Information on the features and secondary structure of an unknown protein rnay provide useful data to allow its classification, and this was ceedout for the 18 kD protein.

Proteins with similar amino acid sequences may also have similar functions

(Schoning et al. 1996); thus comparison of the amino acid sequences of an unknown protein with the database rnay be informative in determinhg the function of the protein. A comparison was made of the sequence of the 18 kD protein with other known proteins using BLAST search in GenBank from World Wide Web sites.

Western blot analysis (Fig.2-12, Chapter 2) demonstrated that the two 18 kD polypeptides are most likely isoforms since the antibodies raised against the allergen protein, Bet v 1 fi0111birch pollen, recognized both. To Merdetermine if there are two isoforms, PCR amplification using the genomic DNA as templates was performed. Since the N-terminal end of the polypeptide with a lower pI value was blocked, and therefore its amino acid sequence could not be detennined, its full- length cDNA could not be obtained by RACE-PCR.PCR amplification of the genomic DNA was used instead in the expectation this would provide some information about its sequence. 3.2 Materials and Methods

3.2.1 Materiais

Dandelion roots used for total RNA extraction and mRNA isolation were

coliected on the campus of the University of Guelph in October, 1996. The roots were then washed, cut into small pieces, fiozen in liquid nitrogen, and stored at - 80' C until

use.

Young leaves for genomic DNA extraction were collected from the plants

growulg in a constant 20' C, 16 hours of light and 8 hours of dark regime in a growth chamber. Leaves were fiozen in liquid nitrogen kediately after collection and

stored at -80' C.

Analyses of the cDNA encoding the 18 kD protein, the products of genomic

DNA-PCR and their deduced amino acid sequences were accomplished using Align

Plus (Scientific and Education Software), Gene Runner (Hastings Software, Inc),

PC/Gene (Intelligenetics, Inc) .

3.2.2 Methods

3.2.2.1 Total RNA extraction

The method used for total RNA extraction was different fiom that outlined in

Section 2.2.2.4.1 in Chapter 2, and was modified fiom Jepson et al. (1991). Two g

fiozen roots were ground to fme powder in liquid nitrogen, and then extracted with 10 ml extraction buffer (400 mM NaCI, 50 mM Tris-HC1, pH 9.0, 1 % SDS, 5 mM

EDTA, 200 pg/d heparin, 10 mM Dm.An equal volume of phenoVchloroform was added to the extraction mixture. vortexed for 5 minutes, and then centrifuged at

10,000 x g at 4' C for 20 minutes. The aqueous phase was collected into a new clean

50 ml Sarstedt tube and an equal volume of chloroform added. The mixture was vorkxed and centrifugeci as above. The aqueous phase was collected in several 1.5 ml

Eppendod tubes, 113 volume 8 M LiCl added to each tube and mixed weU. Total

RNA was then precipitated ovemight at 4' C. The pellet was washed with 756 cold ethanol. dried in a SpeedVac concentrator for 2 minutes, and then dissolved in DEPC

(diethyl pyrocarbonate)-treated water. The quantity and quality of total RNA were detennined at OD 2601280 using a DU-64 spectrophotometer (Beckman).

33.23 Total poly A+ RNA isolation using an oiigo (dT)-cellulose col-

(modified from Sambrook et al. 1989)

Total poly A+ RNA was isolated using an oligo (dT)-cellulose column. This step was similar to that in Section 2.2.2.4.1. Oligo (dT)-cellulose type 7 (Pharmacia)

(0.2 g) was regenerated with 10-20 ml 0.1 M NaOH containing 5 mM EDTA. The column was then washed with buffer A (0.5 M LiCl, 20 mM Tris-HCI, pH 7.4,0.2%

SDS)until it was about pH 7.4. Total RNA was diluteci with 4 ml buffer A, heated to

65' C for 5 minutes, and cooled on ice to room temperature before being loaded onto the column. The eluate was collectecl in a sterile tube. Once the elution was fiaished, the eluate was passed through the column a second tirne. The column was washed with buffer A until the OD,of the eluent was less than 0.05. The column was washed with 20 ml beer B (0.2 M LiCl, 20 mM Tris-HCl, pH 7.4) containing 0.1% SDS. and then 20 ml buffer B without SDS. The poly A+ RNA was eluted with DEPC- treated water as 3-5-drop fractions in 1.5 ml Eppendorf tubes and placed on ice immediately.

RNA determination was the same as in section 2.2.2.4.1. Fractions which demonstrated a positive sigoal for poly A+ RNAs were combined, and a 1/20 volume of 8 M LiCl and 2 volumes of -20' C 100% ethanol added. The poly A+ RNA was then precipitated at -20' C oveniight. The pellet was washed with 75% cold ethanol, dried in a SpeedVac concentrator, and dissolved in 20 pl DEPC-treated water. The quantity and the quality of the polyA+ RNA were also checked at OD 260/280 using a DU-64spectrophotometer (Beckman).

3.2.23 Primer design for RACE-PCR

Fig. 2-1 1B indicates that a common motif m(V)]is present in both the N- terminal 40-arnino-acid sequence and the two allergen proteins (Mal d 1 and Bet v I).

A similar sequence (F)KAXV, around the same place, was also found in PR proteins

(Fig. 2- 11 B). Based on the 40-amino-acid sequence three degenerate primers were thus designed:

Primer 1: GCI GAGIA T'ITIC GAGIA ATI AC (encoding the N-terminal amino acids 2 to 8) A E F E 1 T

Primer 2: TTC AAG GCC TTï GTC (encoding the N-terminal amino acids 17 to 2 1) FKAFV

Primer 3: AAC/T AnTTC/T AAGIA GCI TTTIC (encoding the N-terminal amino acids 15 to 20) N 1 F K A F

3.2.2.4 3' end-RACE (rapid amplification of cDNA ends) PCR

3.2.2.4.1 Reverse transcription of poly A+ RNA

Reverse transcription was conducted using the RiBoClone cDNA synthesis systems AMV RT kit (Prornega).

In a stenle, Nase-free 1.5 ml Eppendorf tube, oligo dT+adaptor was added to the polyA+ RNA sarnple:

total polyA+ RNAs 3 ci1 (2 ci@

*ohgo dT + adaptor (Promega) 1 il1(1 W)

DEPC-treated H20 5 PI

*oligo dT + adaptor: StGATCCAGATCTCGAGAAGCC3'

The mixture was incubated at 70' C for 5 minutes and cooled on ice immediately. The following components were then added in the order shown: fmt strand 5X buffer 5

rRNasin ribonuclease inhibitor 25 U

sodium pyrophosphate, 40 mM 2.5 pl

AMV reverse transcriptase 15 U/pg RNA

nuclease-free H,O up to 25 pl

------UI__------p- ----CIIII__------U-L------The mixture was mixed gently, spun, and incubated at 42' C for one hour.

3.2.2.4.2. 3 ' end-RACE-PCR (modifedfrom Frohman 1990)

PCR cocktail (50 y 1):

10 x Taq buffer (Perkin Elmer) 5 Pl

DMSO (dirnethylsulfoxide, J.T.Baker) 5 Pl

dNTF's (3.75 mM each, Phamacia) 5 ci1

MgCI, (25 mM,Perkin Elmer) 4 Pl

*adaptor primer (25 pmoVp1) 1 Pl

* * gene-degenerate primer (25 pmoYp1) 3 ci1

cDNA (fmt strand) 1 ci1

AmpliTaq Gold (Perkio Elmer) 0.5 y1 (2.5 U)

sterile H,O 25.5 p1

* adaptor primer: 5'GCGGATCCAGATCTCGAGAAGCTT3'

* *gene-degenerate primers (Section 3.2.2.3) The PCR reaction was then conducted in a PCR machine (GeneAmp PCR

system 2400, Perkin Elmer) for 3 hours. PCR conditions:

30 cycles

94O C 94O C 42' C 72' C 72' C

10 minutes 30 seconds 40 seconds 1 minute 10 minutes

After the PCR reaction, 10 pl of each reaction mixture plus 5 pl of 6 x loading

buffer (0.25% brom~phenolblue, 0.2596 xylene cyan01 FF, 0.01 96 ethidium bromide,

30% glycerol in water) was loaded onto an 1% agarose gel. The gel was run as

descnbed in Section 2.2.2.8.2, and viewed under a UV light after ninning. The primer

producing the highest amount and most specific product(s) was used for the large

scale 3'-end-RACE PCR amplification.

3.2.2.4.3 PCR pruduct purification (modified from Davis et ai. 1986)

PCR products were isolated using an 1% agarose gel. Gel and sample preparation and electrophoresis were the same as in Section 2.2.2.8.2. A band with an

appropriate size of around 600 bp was cut out ftom the gel using a razor blade under

W light and placed in an Eppendorf tube. To this, 2-3 volumes of 6 M NaI were

added. The tube was incubated at 55' C for 5 minutes and 5 pl of silica suspension

(Sigma) added to the tube. The tube was then left on ice for 3 minutes for DNA to bind, vortexed and incubated for another 3 minutes on ice. This step was repeated 3 times. DNMsilica was precipitated by centrifbgation for 10 seconds at the maximum speed in an Eppendorf bench centrihge. The supernatant was discarded and the pellet washed 3 times in 200 pl washing solution (50% ethanol, 50% STE 1100 mM NaCl,

10 rnM Tris pH 7.5,l rnM EDTA]), dried using a SpeedVac concentrator, and 15 pl stenle water was added. The pellet was resuspended and incubated at 55' C for 5 minutes. The supernatant was collected dercentrifugation. The amount and size of the DNA was detemiined on a 1% agarose gel by cornparhg with a DNA mass ladder

(Gibco BRL) under W light.

3.2.2.5 5' end-RACE PCR (modified fiom Harvey and Dadison, 1991)

3.2.2. 5.1 Reverse transcription of polyA+ RNA:

Reverse transcription was conducted as above (section 3.2.2.4.1), but usuig oligo dT instead of oligo dT + adaptor as primer.

3.2.2.5.2 Purification of the reverse fianscripiun reaction mixture:

To the reaction mixture was added sterile water to 100 pl, and 10 pl 3 M sodium acetate (pH 5.2) and 60 pl isopropanol. The mixture was incubated on ice for

15-20 minutes. The pellet was recovered by centrifugation, washed with 75% ethanol, and resuspended in 100 pl sterile water. The precipitation and washing step were repeated once. This purified the first-strand cDNA fiom the excess nucleotides. 3.2.2.5.3 PolyadenyhtÏon ut the 5' end:

The cDNA pellet was resuspended in 21 pl sterile water, boiled for 2 minutes, rapidly cooled on ice, and then the following was added:

6 mM dATP (Pharmacia) 1

5 x tailing bufEer (Pharmacia) 6 pl

*TdT enzyme (Pharmacia) 2 pu15 U)

-CII_ *TdT enzyme: terminal deoqmucleotidyl tram ferase. The mixture was incubated at 370 C for 1 hour.

3.2.4.4 5 '-end RA CE-PCR:

PCR cocktail (50 pl):

10 x Taq buffer (Perkin Elmer)

DMSO (dimethylsul foxide, J.T.Baker)

(LNTPs (3.75 mM each, Pharmacia)

MgCl, (25 mM,Perkin Eher)

oligo dT + adaptor primer (20 pmol/yl)

*gene-specific primer 4 (20 pmolfpl)

cDNA (above)

AmpliTaq Gold (Perkin Elmer)

steriled H,O *gene-specific primer 4: 5' GTG CïT GTA TAC CGA GCC3' (encoding amino acid fiom 69th to 74th at N-terminal end)

The PCR was conducted under the same conditions as the 3'-end RACE-PCR.

The PCR product (450 bp) purification was also conducted as in Section 3 -2.2.4.3.

3.2.2.6 Cloning of the RACE-PCR products into pGEM-T Easy vector

Ligation of the RACE-PCRproducts (450 bp and 600 bp) into pGEM-T Easy vector and the transformation of E. coli 1 blue by the pGEM-T Easy vector were conducted according to the protocols described in the pGEM-T Easy vector system kit manual (Promega) .

3.2.2.6.1 Ligations of the RA CE-PCR products fo the pGEM-T Eas, vectoc

In two 0.5 ml tubes the ligation reactions were set up as below:

T, DNA ligase 10 x buffer 1 PI

pGME-T Easy vector 1 pl (50 ng)

PCR product 450 bp or 610 bp 2.5 pl (25 ng) or 1 p1(40 ng)

T, DNA ligase (3 Units/pl) 1

stenle H,O up to 10 pl

---Y- - -

The reactions were then mixed by pipetting and incubated at 4' C ovemight. 3.2.2. 6.2 Transfonn&ns using the pGEM- T Easy vector lïgution reactions

A) Pre~arationof cornDetent XL1 Blue ceUs

High efficiency competent cells (1 x 10' ch[colony forming units]/pg DNA) were used for transformations. The competent E. coli XL1 Blue cells were prepared by CaCl, method according to protocol (Sambrook, et al. 1989).

A single colony of XLl Blue celI from a stock plate was cultured overnight in

25 ml LB broth (1% w/v tryptone, 0.5% wlv yeast extract, 1% w/v NaCI, pH 7.0) containkg tetracyclin (50 pgM) at 37' C. One ml of this culture was then used to inoculate 25 ml SOB medium (2% w/v tryptone, 0.5% w/v yeast extract, 10 mM

NaCI, 2.5 mM KCI) and this was incubated at 37' C in a shaker until the OD,, was

0.55-0.66 (not exceeding 10' cells/rnl). The cells were transferred to sterile, ice-cold

50 ml polypropylene tubes and cooled by storing the cultures on ice for 10-15 minutes. The cultures were centrifuged at 3000 x g at 4' C for 10 minutes. The pellet was then resuspended in 1/3 (8.7 ml) original volume ice-cold 0.1 M CaCl,, centrifuged at 3000 x g at 4' C for 10 minutes, and gently resuspended in 1/12 of the original volume (2. lml) ice-cold 0.1 M CaCl,. Aliquots of 20pl were transferred to

1.5 ml Eppendorf hibes and stored at -80' C.

BI Transformation of com~tentXLl Blue ceUs by DGEM-T Easv vector containine;

RACE-PCRproducts The tubes containing the ligation reactions (Section 3.2.2.6.1) were spun to collect the contents at the bottom of the tube. Two pl of each Ligation reaction were added to new sterile 1.5 mlEppendorf tubes on ice. Fipl comptent XL1 Blue cells were transferred into each tube, they were gently flicked to mix the solution, and placed on ice for 20 min. After incubation, the cells were heat-shocked for 45-50 seconds in a water bath at 42' C, and Ilnmediately returned to ice for 2 minutes. SOC medium (2% wlv bactotryptone, 0.5% w/v yeast extract, 10 mM NaCl, 2.5 mM KCl,

10 mM MgCl, 20 mM glucose) at room temperature 950 pl was added to the tubes, which were incubated for 1.5 hours at 37' C with shaking (-150 rpm). One hundred pl of each transformation culture were plated onto duplicate antibiotic plates (LB plates containing 100 pg/rnl ampicillin and spread with 100 pl of both 100 mM IPTG and 2% X-Gd). The plates were incubated at 37' C overnight.

3.2.2.7 Plasmid preparation for sequencing (rnodified from the method in

'Molecular Cloning' , Sambrook et al. 1989)

3.2.2. 7. I Crude plùsmid extraction

White colonies were picked out and cultured individually in 20 ml LB containing 100 pg/ml ampicillui at 37' C overnight with vigorous shaking. The cultures were centrifuged at 4000 x g for 5 minutes. Pellets were suspended in 2 ml

STE buffer (0. l M NaCI, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The solution was transfmed into two 1.5 mi Eppendod tubes, and re-centrifbged at top speed in an

Eppendorf bench centrifuge for 5 minutes. Pellets were re-suspended in 200 p1 ice- cold solution 1(50 rnM glucose, 25 rnM Tris-CI, pH 8.0,10 mM EDTA) by vortexing.

To each tube 400 pl solution II (fieshly made, 0.2 N NaOH, 1% SDS) were added.

Tubes were inverted immediately 5 times and left on ice for 5 minutes. Another 300 pl solution III (fieshly made, mixture of 6 ml 3 M KOAc + 1.15 ml HOAc) were added to each tube. The tubes were inverted immediately 10 times, lefi on ice for 5 minutes, and then centrifuged as above for IO minutes at 4' C. Eight hundred p1 supernatant were transferred into clean tubes and 1 volume isopropanol was added.

The tubes were mixed well, incubated at room temperature for 10 minutes and centrifuged as above. The pellets were washed with 75% ethanol, vacuum dried and resuspended in 200 p1 TE (pH 8.0). RNAase A was added into each tube to a fmal concentration of 20 pghl and incubated at 37' C for 1 hour. The crude plasmid was

Merpurified with equal volumes of phenol/chloroform, and then chloroform. After purification, the plasmid was precipitated by adding 2.5 volumes of 100% ethanol and

0.1 volume of 3 M NaOAc pH 5.2. The plasmid was washed and dried, as described above, and dissolved in 50 pl TE buffer (pH 8.0).

3.2.2.7.2 Plusrnid purzjkatim

Cnide plasmids containhg RACE-PCR products were further purified by separation on a LMP (low melting point) agarose gel. The method was the same as in

Section 2.2.2.8.2.

Five clones with the 450 bp insert and 4 clones with the 600 bp insert were sent

out for sequencing after the plasmids were digested with EcoR 1 to check for the

presence of inserts. Sequencing was conducted with forward and backward Ml3

primers using an automatic sequencer (B10377)in DNA sequencing labs of both the

OMAFRA and the Department of Zoology, University of Guelph.

3.2.2.8 PCR ampiification ushg genomic DNA as templates

3.2.2.8.1 Genomic RNA isolation

The method for genomic DNA extraction in this section was modifed from

Ausubel et al. (1994). Five g frozen leaves were ground into fine powder in liquid

nitrogen and extracted with 25 ml extraction buffer (0.1 M Tris-HCI, pH 8.0,50 mM

EDTA, and 500 mM NaCl, 1% w/v Dm).The mixture was vortexed for 2 minutes,

and 1.7 ml 20% SDS was added. The solution was vortexed again and incubated at

65' C for 10-15 minutes. To the mixture 8.5 ml of freshly-made solution (a mixture of 60 ml 5 M KOAc, 11.5 ml glacial acetic acid and 28.5 ml sterile H,O) was added.

The mixture was incubated on ice for 20 minutes, then centrifugecl at 2000 x g for 20 minutes. The supernatant was fdtered through 8 layers of Miracloth. Fifteen ml isopropanol were added to the supernatant to precipitate the DNA and the solution incubated at -20° C for 30 minutes. DNA was spooled out with a glass rd, rinsed with

75% cold ethanol, dried in a SpeeaVac concentrator, and redissolved in 3 ml TE buffer. R,Nase A was added to the DNA solution to a final concentration of 20 pg/ml, which was then incubated at 370 C for 30 minutes. Five hundred pl 5 M NaCl and 400 pl prewarmed 10 x CTAB (cetyltrimethylammonium bromide) (700 mMNaCl 10% w/v CATB ) were added to the DNA solution and incubated at 65' C for 20 minutes.

The DNA was purified with an equal volume of pheno~chloroform,and then chloroform alone. DNA was precipitated by adding 2.5 volumes of 100% ethanol and incubated at -20' C for 30 minutes. Mer centrifûgation at top speed in an Eppendorf bench centrifuge for 10 minutes, DNA was washed with 75% ethanol, vacuum dried for'2 minutes, and redissolved in 200 pl stede ddH,O.

The quality and quantity of DNA was determined at OD260/280 using a spectrophotometer.

3.2.2.8.2 PCR amplifcation of the genomic DNA and its pproduct sequencing

Primer desimation

5 pairs of primers were designed for genomic DNA PCR (Fig. 3-2).

5' end primer: 3' end primer:

1. primer 3N & primer lC 2. primer 3N & primer4

3. primer 2 & primer4

4. primer 2 & prime 1C

5- Primer F3 & primer 3C primer 3N: corresponding to amino acids 1 to 8 primer 2: corresponding to amino acids 17 to 21 primer F3: corresponding to amino acids 28 to 35 primer 4: corresponding to amino acids 109 to 1 14 primer 3C: corresponding to amho acids 127 to 133 primer 1 C: corresponding to amino acids 150 to 156

PCR cocktail (50 ul):

10 x Taq buffer (Perkin Elmer)

DMSO (dimethylsulfoxide, J.T.Baker)

dNTPs (3.75 mM each, Pharmacia)

MgCl, (25 mM,Perkin Elmer)

5' end primer (25 pmoYp1)

3' end primer (25 pmoVp1)

genomic DNA (100 x dilution)

ArnpliTaq Gold (Perkin Elmer) sterile H,O 27.5 pl ----- The PCR reactions were then conducted under the following conditions:

30 cycles

94O C 94O C 5l0 C 72' C 72' C

10 minutes I minute lminute 2minutes 10minutes

PCR product detennination. de cation and seauencing

The PCR products in each reaction were detected on a 1% agarose gel as described in Section 3.2.2.4.2. Reactions which yielded products were repeated on a large scale. The PCR products were purifed by separation on a 1% LMP (low melting point) agarose gel. and recovery of PCR products was as described in Section 2.2.2.8.2.

The pmed products of genomic DNA PCR were then directly sequenced using primers which drive the PCR amplifcation, using an automatic sequencer in the

Department of Zoology, University of Guelph.

3*2*2.9Analysis of the cDNA encoding the 18 kD protein and its deduced amino acid sequence 3.2.2.9.1 ConstructÎon of the full-Iength rDNA sequence and its deduced amino ucid sequettce

Forward and backward sequence data of 4 clones of 3' end RACE-PCR products and 5 clones of 5' end RACE-PCRproducts were compared to each other using Align Plus and Gene Runner. This cornparison was to avoid the mistakes which were due to either mismatch by PCR amplifications or misreading in the sequencer.

The corrected sequences of the 3' and 5' end RACE-PCR products were then overlapped using Align Plus, and the fulClength cDNA sequence encoding the 18 kD protein was thus constmcted.

The arnino acid sequence of the 18 kD protein was deduced fkom the full- length cDNA sequence using Gene Runner.

3.2.2. 9.2 Some characteristics of the 18 kD protein

Characteristics of the 18 kD protein, such as arnino acid composition, charge density, signal peptide, eukaryotic secretory signal and organelle-targeting sequence prediction, site and signature detection, isoelectnc point, and hydropathy profile were analysed by PC/Gene and Gene Runner.

3.2.2.10 Cornparison of the cDNA eneoding the 18 kD protein and its deduced amino acid sequence with other known proteins DNA and protein sequence cornparison with other known proteins was conducted using BLAST search in GenBank. Sequences of 5 proteins with high homology to the 18 kD protein were retrieved fiom GenBank for Meranalysis.

3.2.2.11 Anaiysis of the PCR products amplified using genomic DNA as templates

Analysis of the PCR products amplified using genomic DNA as templates were also conducted using Align Plus and Gene Runner. As above (section 4.2.2.1.1) 3 forward and 3 backward sequences of each PCR product were compared to each other to correct the mismatch or misreading. The corrected sequences of the two products were then compared to each other and also compared with the full-length cDNA sequence. Cornparison of the deduced arnino acid sequences fiom the two genomic

DNA sequences and the full-length cDNA was also conducted. 3.3 Results and Discussion

3.3.1 Results

3.3.1.1 3'-end RACE PCR

Fig.3-3 shows that the 3'-end RACE PCR with degenerate primer 2 yielded the most product. There was only one PCR product (600 bp) fkom this reaction, and the product (lane 2) was brightest compared with the products fiom other reactions.

Reaction 1 produced two products, one of which had the same size as that in reaction

2: 600 bp. The other was 400 bp (lane 1). Primer 3 did not work well. There were also two products in reaction 3, but they were very faint. The size of one of the products was also 600 bp, the other was around 700 bp (lane 3). Primer 2 was thus chosen for conducting the large scale amplification of 3'-end RACE PCR.Lane 5 shows the 600 bp PCR product after purification.

3.3.1.2 Sr-endRACEPCR

5'-End RACE PCR was successfûl in amplification of the DNA of interest, and

Fig.3 -4 shows that atthough the products show a smear in lane 2, around 450 bp, there is a faint but clear band. After large scale amplification, a S'-end RACE PCR product of 450 bp was obtained derpurification (lane 4). 3-3.1.3 Cloning of the PCR products into pGEM-T Emy vector and sequencing

The RACE-PCR products were cloned into the pGEM-T Easy vector. After

being incubated with restriction enzyme EcoR 1, inserts of around size 450 bp were

detectable (Fig. 3-5). Sequences of the 3'end RACE PCR products in lanes 2-5 and

the 5'-end RACE PCR products in lanes 6-10 were obtained.

3.3.I.4 PCR ampüfications using genomic DNA as templates

Of the 5 pairs of primers used in PCR arnplincations, primers 3-N and 1C and

3-N and 4 did not yield any product (lane 1 and 2, Fig. 3-6). Primen 2 and 4 yielded

one product of - 1200 bp, primers 2 and 1C yielded two, one of -1 200 bp and the

other of 300 bp. The pair of F3 and 3C prirners also yielded two products, whose sizes

were very sirnilar, around 450 bp and 400 bp.

3.3.I.5 Thefia-length cDNA sequence encoding the 18 kDproteUl and its deduced amino acid sequence

The 744 bp full-length cDNA sequence obtained, together with the 156 amino

acid sequence deduced from it, are shown in Fig. 3-7. The cDNA has one open- reading fmme which extends from nucleotide 57 to 524. The 17 bp polyA tail is located 2 17 nucleotides downstream fiom the TGA translation terminal codon. The

220 bp 3' untranslated region contains a putative polyadenylation signal (AATAAA) starting at position 640. AT nucleotides are present at a much higher percentage

(63.57%) than that GC nucleotides (36.42%). The kt40 amino acids at the N- terminal end deduced from the nucleotides 57 to 176 c~rrespondexactly with the 40 amino acids at the N-terminal end determined directly fiom the purified polypeptide.

It is likely, therefore, that the cDNA encodes the 18 kD polypeptide of pI 5.56.

3.3.1.6AnaIysk of the 18 kDprotein

The amino acid composition of the 18 kD protein is given in Table 3-1. Three amino acids are absent fiom the proteh: Arg O, Gln (Q), and Trp (W). The most abundant amino acids are Ser (1 1.54%), Lys (10.26%), and Ile (9.62%). Polar and non-polar amino acids make up the highest percentages (53.2% and 46.89%, respectively) of the 18 kD protein. Acidic and basic amino acids only account for 26 and 23 out of 156 amino acids, respectively. Charge density data obtained fiom Gene

Rumer demonstrates that there are more negatively charged amino acids than positively charged (Fig 3-8). This Merconfirms the result obtained using cation exchange chromatography which suggested that the 18 kD protein is negatively charged on its surface (see results in Chapter 2). Gene Runner dso indicates that the pI value of the 18 kD polypeptide pI is 5.6 (Fig. 3-9) which is very close to 5.56 determined by measuring the pH value of the eluate fiaction fkom chromatofocusing.

The predicted rnolecular mass of the polypetide is 16.9 11 kD. There are two potential N-glycosylation sites (NXS) at tesidues 77 and 131,s protein kinase C phosphorylation sites at 36,39,5 1,65 and 142, and 3 casein kinase

II phosphorylation sites at 56,83, and 152.

Cornparison of the deduced amino acid sequence with the 40-amino-acid-N- terminal sequence obtained fiom the p&ed protein indicates that the 18 kD protein does not contain a signal peptide. The result fiom the PC/Gene program confmns this lack of a signal peptide. No part of the 18 kD protein sequence shows a minimum hydrophobie value of a signal peptide (Fig. 3- 10). Signal peptides are present in most

"secretory" proteins and they serve to initiate insertion into the endoplasmic retidum in eukaryotes (Heijne 1986). The PC/Gene program also indicated that there is no eukaryotic secretory signai sequence and nor any organelle-targeting sequences in the protein. The logical conclusion is that the 18 kD protein is a cytosolic protein.

3.3.1.7 Cornparison of the 18 kD protein with known proteins

The result from the BLAST search in GenBank, comparing the 18kD protein sequence with known protein sequences, is consistent with the result obtained by comparing only the 40-amino-acid N-terminal sequence (see Chapter 2). The 18 kD protein does show high homologies with a group of allergen and intracellular pathogenesis-related (PR) proteins. The order of the proteins demonstrating homology from high to low are: two parsley PR proteins, PRl-3 and PRl-1 (64% similady, 44% identity); allergen proteins from apricot fruit, pAPRI35 (62% simiiarity, 42% identity); allergen protein fiom cherry, PRUA 1 (6 156 sunilarity, 42% identity); allergen protein fiom apple, Md d 1 (61 % similarîty, 41% identity); allergen protein from hazel, Cor 1 (59% similarity, 42% identity); allergen protein from hornbearn, Car b 1 (63% similarity, 39% identity); allergen protein kom celery, API

G 1 (64% similarity, 40% identity); pathogen-induced and allergen protein fkom birch pollen, Bet v 1-Sc3 (59% similarïty, 41% identity), Bet v 1 (61% similarity, 38% identity).

Amino acid sequence alignments of the 18 kD protein to five homologous proteins: parsley PR protein (PR1-3), allergen proteins from apncot fniit (pAPRUS), cherry (PRUAl), apple (Mal d l), and bûch pollen (Bet v 1) are shown in Fig. 3-1 1.

There are three common blocks of amino acids present in all five proteins; two are short (FKAF and DGGS) and one is long (EGDGGVGTM). Sequence GXGGXG within the long common block EGDGGVGTIK is considered to be the conserved motif throughout all the members of the group (Hoffmann-Sommergruber et al. 1997).

It seems that the 18 kD protein is a member of this family.

3.3.1.8 The PCR products amplijied ushg genomic DNA as templates and their deduced amino adsequences

Cornparisons of the sequences of the 5 genomic DNA-PCRproducts with the cDNA sequence revealed that the 1200 bp and 300 bp PCR products were different in sequence fiom the cDNA obtained using 3'- and 5'- end RACE-PCRamplification.

However, sequences of products with sizes 450 bp and 400 bp corresponded to the cDNA sequence. The two products of the genomic DNA-PCR do not encode the same protein. Not only are the sizes of their introns different, but also the nucleotides in both intron and coding regions are slightly changed. The two introns and the two coding regions share 80.7% and 88% homology at the nucleotide level, respectively, and their deduced amino acid sequences (105 amino acids) show 85.7% identity.

The intron within the 450 bp product is 149 bp long, the other 83 bp long. The

450 bp product with long intron is designated as 1-PCR, the other was 2-PCR (Fig.3-

12). Both introns are inserted at the same position: at amino acid 59 between nucleotide 1 and 2 (starting at nucleotide 232), and are relatively AT rich (72.48% in

1-PCR and 79.52% in 2-PCR)compared with the coding regions (63.43% in 1-PCR and 65.39% in 2-PCR). The high AT content in the intron is a characteristic of plant introns (Goodall and Filipowicz 1989). The two exon-intron junctions were in accordance with the GT-AG nile, a consensus plant splice mle (Wolfe 1993).

3.3.1.9 Cornparison of the nucleotide and amino acid sequences of the two PCR products ccsing genomic DNA as templaks with the fuIZHength cDNA encoding the

18 kD prorein and its deduced nmino acid sequence Cornparisons of the DNAs and their deduced amino acid sequences of the two products of the genomic DNA-PCR with the full-Iength cDNA encoding the 18 kD protein and its deduced amino acid sequence are show in Figs. 3-12 and 3-13. Fig.

3-12 dernonstrates that the sequence of the coding region of 1-PCR is identical to the sequence of the cDNA obtained using 3'- and 5'-end RACE-PCR amplifications. Its deduced amino acid sequence (1 O6 amino acids) thus perfectly matches the amino acid sequence deduced fkom the cDNA (Fig. 3-1 3). Therefore, it can be concluded that 1-

PCR is a partial gene sequence encodùig the pI 5.56 18 kD isoform. It seems likely that 2-PCR encodes the other 18 kD isoform. 3.4 Discussion

Typicaily, storage proteins contain a signal peptide and are deposited in subcellular structures, the protein bodies (Bewby and Greenwood 1990). They are synthesized on the rough endoplasmic reticulum (RER) and during synthesis the signal peptide directs the insertion of the elongating polypeptide into the ER lumen.

When elongation and insertion are complete, dicot storage proteins are then transported via the Golgi apparatus to the vacuole, which fragments to fom protein bodies. However, the seed storage protein, urease which is present in many legume seeds (Bailey and Boulter 1971, Pernoflet and Mosse 1983) has been reported to be a cytosolic protein (Faye et al. 1986). Thus, although the 18 kD protein is cytosolic, it caanot be excluded as a storage protein.

The 18 kD protein shares a quite high homology (64-59% similarity, 44%-38% identity) with a group of allergen and intracelIu1a.r pathogenesis-related (PR)proteins.

In addition, it contains the common motif GXGGXG which is present in all proteins of this allergen and IPR group (Hoffmann-Sommergruber et al. 1997). No other allergen or IPR proteins have been reported in roots.

Allergen and IPR proteins are classifieci in similar groups of proteins based on the similarity of their amino acid sequences, which are highly conserved throughout many species (Breiteneder et al. 1989, 1992, 1995, Fristenslq et al. 1988, Swoboda et al. 1994, 1995b, Vanek-Krebitz et al. 1995) . The molecular masses of this group of proteins are quite similar, between 16.5-17 kD (Breiteneder et al. 1995). Their

cDNA and genomic DNA sequences have been determined during the last 10 yem,

and most proteins in this group have several isoforms (Breiteneder et al. 1993,

Swoboda et al. 1995a). AUergen proteins have been found in leaves, fruits, and pollen

of different trees and plants, such as alder (Breiteneder et al. 1992), apricot (Mbeguie-

A-Mbeguie et al. 1997), apple (Atkinson et al. 1996, Schoning et al. 1995, 1996,

Vanek-Krebitz et al. 1995), canot (Schoning et al. 1995), celery (Breiteneder et al.

1995, Schoning et al. 1995), cherry (Scheurer et al. 1996, Schoning et al. 1995),

European white birch (Breiteneder et al. 1989, Swoboda et al. 1995a). hazel

(Hoffmann-Sommerber 1996). IPR proteins have been reported to be induced in bean (Walter et al. 1990), parsley suspension cultures (Somssich et al. 1986, 1988), in pea (Fristensky et al. 1988) and in potato tuber discs (Matton and Brisson 1989) upon infection with bacterial or fungal pathogens, or after treatment with fungal elicitors. Among the allergen proteins, birch pollen allergen protein, Bet v 1, was the fust with a published cDNA-derived sequence from this subgroup of proteins

(Breiteneder et al. 1989). The parsley PR proteins were the fust PR proteins defmed as iniracellular PR proteins since they do not contain a signal peptide (Somssich et al.

1986).

Allergen proteins are so-named because they are capable of inducing allergen- specific IgE synthesis, therefore causing an allergie-reaction. Unlike PR proteins, Sommerpber et al. 1997). Recently, however, a subset of microbial pathogen- inducible Bet v 1-related mRNAs and proteins were found in birch cell suspension cultures (Swoboda et al. 1994). They were designated as Bet v 1-Sc1 , Sc2, and Sc3 and their amino acid sequences showed 70430% homology to the constitutively- expressed pollen allergen protein Bet v 1. This links the relationships between the allergen and PR proteins, and ailergen proteins may also have a defence fiction against pathogen attack.

The GXGGXG motif is a characteristic sequence (P-loop) of protein kinases, as well as other nucleotide binding proteins (Saraste et al. 1990). However, no member of this group of allergen and PRproteins has been shown to have kinase activity. Recently, a new type of ribonuclease fiom ginseng calli has been reported to have a sequence similarity with this group of allergen and IPR proteins (Moiseyev et al. 1994). Again, such activity has not been demonstrated for any of the members of this family of allergen and PRproteins. Although an increasing number of members of this gene family of allergen and IPR proteins has been isolated and sequenced, their fimction remains unknown.

Two partial gene sequences were obtained fiom PCR amplification using dandelion genomic DNA as templates. Sequence cornparison reveals that the two partial genes encode diffGent proteins. One sequence of the two partial genes corresponds to the cDNA encoding the 18 kD protein of pI 5.56. This Merc0dirn-s that there are two 18 kD protein isoforms present in dandelion roots.

The complete amino acid sequence of the isoform with pI 5.56 was obtained.

However, only a partial amino acid sequence (105 residues) was obtained for the other isofom. The two isoforms share 85.7% homology based on the 105 arnino acid sequence. Although it is most likely that the 2-PCR is the partial gene sequence encoding the 18 kD isoform with pl 5.49, this needs Merconfimation.

Both introns are inserted at the same position, at amino acid 59 between nucleotide 1 and 2 (or 60 if the fmt amino acid M is included, which is the case for other allergen and PR proteins, Hoffmann-Sommergmber et al. 1997) in the two partial gene sequences. Comparing the positions where introns are inserted with those in other allergen protein genes, a conserved pattern can be observed. Intron sequences are inserted within amino acid 62 between nucleotides 1 and 2 in Bet v 1 (Betzdaceae),

Cor a 1 (Corylaceae), Md d 1 (Rosaceae), pcPRl (Apiaceae) and PI49 (Fabaceue), arnino acid 57 between nucleotides 1 and 2 in STHZ. (Solanaceae) and amho acid 63 between nucleotides 1 and 2 in Aoprl (Liliaceae) (Hoffrilann-Sommergniber et al.

1997). Moreover, al1 genomic DNAs obtained encoding this group of proteins contain only one intron whose size ranges fiom 81-153 bp. It seems that there is a cornmon origin of intron insertion among these proteins. 3'-end RACE PCR 5'-end RACE PCR mRNA

reverse transcription reverse transcription (with primer: oligo dT + adaptor) (with primer: oligo dT) I cDNA 37i-TITITT+ adaptor 5'

removai of exce& oiigo dT prima and taiiing cDNA with dATP gene & &laPtor primer

PCR amplification with adaptor + oligo dT & gene primer 5'gene primer-!+ : : : : : : H++TT~+ adaptor 3'

5 ' adaptor + lTiTïT ---b 3'

j'adaptor + TïTTIT-; 3' 3': ------y-gene primer 5'

Fig. 3-1. Strategies for RACE-PCR ------Primer 3N------> -----primer 2---> 5 ' GCTGTCGCAGAGTfiGAGATCACT3 ' 5'TTCAAGGCCTTTGTT3' 1AVAEFEITSSLSPSNIFKAFVI 22 ------pr*r P3------> 5 ' GCACCTAAGGCTGAACCTGAAAC3 * 23DFDTIAPKAEPETYKSIKTIEGD45

<--- Primer 1C---- 3'GTGGGACTTCGAACAACS' 138LKKTFKAIETYVISHPEAC

Fig. 3-2. 5 pairs of primers used for genomic DNA-PCR Fig. 33. Products of 3'-end RACE PCR agarose gel stained with ethidium bromide 1. primer 1 + adaptor primer; 2. primer 2 + adaptor primer; 3. primer 3 + adaptor primer; 4. negative control; 5.3'-end RACE PCR product (600 bp) after purification Fig. 3-4. Products of S'-end RACE PCR agarose gel stained with ethidium bromide 1. S'-end RACE PCR amplification; 2. DNA mas ladder; 3. negative control; 4. S'-end RACE PCR product (450 bp) after purification. Fig. 3-5. RACE PCR products digested from pGEMT Easy vector with EcoR 1 agarose gel stained with ethidium bromide M: DNA mass ladder; 1,2,3,4, and 5: 3'-end RACE PCR products; 6,7, 8,9, and 10: S'-end RACE PCR products Fig. 3-6. Products of PCR amplification using genomic DNA as template (agarose gel stained with ethidium bromide) 1. primers 3N + 1C; 2. primers 3N + 4; 3. prhners 2 + 4; 4. primers 2 + 1C; 5. primers F3 + 3C; 6. positive control; 7. negative control. GGACAACTAAATCATTCAACTTCCTATATTTTTCTTTCACATMCMTCMTA

ATGGCTGTCGCAGAGTTTGAGATCACTTCTTCACTTTCTCCTTCCMTATTTTCMGGCCTTTGTTATT AVAEFEITSSLSPSNIFKAFVI

GATTTTGACACTATTGCACCTAAGGCTGAACCTGAAACTTACWTCCATmGACCATTGMGGCGAT DFDTIAPKAEPETYKSIKTIEGD

GGTGGTGTTGGRACCATCAAAAGCATTACATACAGCGATGGTGTCCCGTTCACMGCTC~GCACMG GGVGTIKSITYSDGVPFTSSKHK

GTTGATGCCATCGATTCAAACAACTTTAGTATCAGCTACACCATCTTTGAAGGTGATGTTTTAATGGGA VDAIDSNNFSISYTIFEGDVLMG

ATCATAGAGTCTGGTACTCATCATCTTRAOTTTTTACCTTCTGCTGATGGAGGCTCGGTATACWGCAC ISESGTHHLKFLPSADGGSVYKH

TCAATGGTGTTTRAATGCRRAOGTGACGCTAAGTTAACCGACGAAAATGTTAGCCTCATGAAAGMGGT SMVFKCKGDAKLTDENVSLMKEG

TTGAAAAAGACCTTTAAAGCGATTGAGACTTATGTCATTTCTCACCCTGMGCTTGTTGATTCATCTTG LKKTFKAIETYVISHPEAC

GATCCAATAAGTTTTGTTTCAACAAGACATOCTTAGTAGTGTGTACTCATMTTWCMGMTCGT TTTGTTCTTGTTTTCGAGTCCTTGCTTTCACCTAAATAAATGGCCATTTATGAGGTGGTACTTCGGTTT

Fig. 3-7. The cDNA scquencc encoding the 18 kD protein aiid its dediiced nmino acid sequence Kg. 3-8. pI value of the 18 kD protein amino acid number

Fig. 3-9. Charge density of the 18 kD protein 10 i 5 ;

-5 i -10 . -20 : hydrophilic

amino acid number Fig. 3-10. Hydropathy profile of the 18 kD protein Plot were made according to the method of Kyte and Doolittle using a window of nine residues. Negative values indicate hydrophilic regions. 18 kD PR1- 3 pAPRI3 5 PRUAl Maldl Betvl

18 kû PR1 - 3 pAPRI3 5 PRUAl Maldl Betvl

18 kD PR1 - 3 pAPRI3 5 PRUAl Maldl Betvl

Fig. 3-11. Cornparison of the 18 kD protein with other known proteins PR1-3 is the pathogenesis-related protein fiom parsley;PAU, PRUA 1, Mal d 1, and Bet v 1 are the allergen proteins fiom apricot hit, cherry, apple, and birch pollen. Amho acids which are same in al1 or most proteins are shaded. Common motifs present Li al1 proteins are underlined and bold.

18 k~ protein AVAEFEITSSLSPSNIFKAFVIDFDT~APKAEPETYKSIKTIEGDGG~GTIK

______d---_--______isoform 1 ______d---_--______APKAEPETYKS IKTIEGDGGVGTIK isoform 2 PKAEPETYKAVNI IQGDGGUGTIK **** 18 kD proterin S1TYSM;VPFTSSKHKVDAIDSNNFSISYTIFEGDVLMGIIESGTHHLKFLP isoform 1 SITYSDGVPFTSSKRKVDAIDSNNFSISYTIFEGDVLMLP isoform 2 SVTYSDGLPFTSSKHKVENVDTNNFSISYTIFEGDVLMGIIDAGTHHLKFLP * *** * **

Fig. 3-13. Cornparison of the amino acid sequences deduced from genomic DNA-PCR products with the 18 kD protein deduced from the fuli-length cDNA sequence Amino acids different £iom those deduced fiom the full-length cDNA sequence are in bold and marked with *. Table 3-1. Amino acid composition of the 18 kD protein

Amino acid

acidic amino acids basic amino acids non-polar polar 4. Effects of environmental perturbations on the expression of the gene encodina the 18 kD protein and ~roteh-svnthesis 4.1 Introduction

Although the 18 kD protein has homology with a number of allergen and IPR proteins, its function as a vegetative storage protein (VSP)still cannot be excluded.

A few studies have reported that VSPs can be induced by wounding (Berger et al.

1995, Davis et al. 1993, Mason and Mullet 1990, Mason et al. 1992, Pena-Cortes et al. l988), while wounding is also a common inducer of PR proteins (review of Bol et al. 1990, Sturm and Chrispeels 1990, Wamer et al. 1992). It seems that VSPs and PR proteins are somehow CO-relateci.The possible role of the 18 kD protein as a VSP was further studied in this and the next chapter.

As mentioned in the iiterature review, the major characteristics of a VSP are that it is preferentially synthesized during senescence and depleted during reactivation of meristem growth (Cyr and Bewley 1990b, Mussigmann and Ledoight 1989,

07Kennedyand Titus 1979). Although the amount of the 18 kD protein is high during the fall and winter and decreases during the spring and summer, these slight seasonal changes raise the question as to whether the 18 kD protein is a VSP. Determination of the seasonal expression patterns of the 18 kD protein gene at the mRNA level may provide more information about the tirne course of the synthesis and mobilization of the protein throughout the year, and help to assign a function. Furthemore, the spatial and temporal expression of the 18 kD protein gene during seedhg establishment and in the different parts of the mature vegetative plant may provide information about its role. Hence, the seasonal expression of the gene and its spatial and temporal expression during growth were determined. The number of genes encoding the 18 kD protein was also determined.

It is quite common that highly homologous proteins can be found in related species. Indeed, homologous VSPs, allergen and PRproteins have been detected in species belonging to the same family. For example, a cDNA probe encoduig the VSP in poplar hybridizes with the mRNA encoding the VSP in willow under high stringency hybridization conditions (Xu 1993), antibody raised to a 35 kD protein from cypress can react with proteins of VSPs extracted from the wood of redwood and yew (Hamis and Sauter 1991), and VSPs in soybea. and French bean share 658 and

71% identity (Rhee and Staswick 1992% b, Zhong et al. 1997). The major ailergen protein, Apig 1 in celery (Breiteneder et al. 1995), shares around 6 1% hornology with the PR proteins, PR1-3 and PR- 1, in parsley (Somssich et al. 1988). Both plants are rnembers of the Apiaceae family. Apple and cherry are members of the Rosaceae family, and their major ailergen proteins, Mal d 1 and PRUAl , share 75% identity

(Mbeguie-A-Mbeguie et al. 1997). A search for proteins sharing homologies with the dandelion 18 kD proteins in other members of Compositae family was conducted. 4.2 Materials and Methoàs

4.2.1 Materials

Dandelion roots were collected monthly on the campus of the University of

Guelph from May 1993 to May 1995. Roots of 8 plants of the Compositae were also collected on the University campus. Root samples were washed, cut into small pieces, and fiozen in liquid nitrogen and stored at -80' C as described in Chapter 2. Different organs of the dandelion were collected, fiozen and stored in the same way as above.

4.2.2 Methods

4.2.2.1 Seasonal, temporal, and spatial expression of the gene encoding the 18 kD protein in dandelion and detection of homologous proteins in the roots of other members of the Compositae

4.2.2.1.1 SDS-PAGE

Detemination of the temporal and spatial expression of the gene encoding the

18 kD protein in dandelion at the protein level, and its homology with proteins in other Compositae, was conducted with SDS-PAGE. Protein extraction, purifcation, quantitative determination, and electrophoresis were conducted as described in

Sections 2.2.2.1.1,2.2.2.1.2,2.2.2.1.3.

Different dandelion organs (0.1-0.5 g fresh weight), or the roots fiom plants from eight other species of Compositae (5 g khweight each) were ground to a fine powder in liquid nitrogen, and extracted with buffer (0.1 M sodium phosphate, lUmM cysteine, 10 10 mM PMSF) at a ratio of 1:5 sample : buffer. The cmde extracts were then purifed with phenol as in Section 2.2.2.1.1. Pellets were dissolved in a modifed Laemmli buffer (62.5 mM Tris-HC1, pH 6.8,2% SDS, 10% glycerol) and their protein quantities determined by the BCA assay method. Forty yg of each protein sample were loaded onto 15% polyacrylamide-SDS gels.

4.2.2.1.2 Northern blots

Determination of seasonal, temporal, and spatial expression of the gene encoding the 18 kD protein in dandelion at the mRNA level and its homologous genes in eight plants of the Compositae was conducted by northern blots. The methods used were similm to ihose described in Section 2.2.2.8.

Total RNA was extracted as described in Section 3.2.2.1. The RNA loading buffer used here was different from that in section 2.2.2.8: gel loading buffer (79 pl):

foimamide

5 x running buffer

formaldehyde (37%)

sterile glycerol

saturated bromophenol blue ethidium bromide 2 ci1

~~~~~~~~~~~~~~~C~U-______C_U______U______C_U______U______C_U______U______C_U______U______C_U______U______C_U______U_____----- ~~~~~~~~~~~~~~~C~U-______C_U______U______C_U______U______C_U______U______C_U______U______C_U______U______C_U______U_____----- P

To each 10 pg total RNA was added 10 pl gel loading buffer. The mixtures were heated at 65' C for 10 minutes and cooled on ice immediately for at least 5 minutes.

The 600 bp cDNA obtained by 3' end RACE-PCR was used as a probe for northern blots. Probe labelling and purification were the same as that in Section

2.2.2.8.3, except that the probe was labelled with [U~~P]~ATP.

Pre-hybridization and hybridization were conducted at 65' C for seasonal, temporal and spatial expression experiments, and at 42' C for detection of the homologous genes in other Compositae. The pre-hybridization and hybridization solutions were the same as in Section 2.2.2.8.4. The wash conditions for the former experiments were: 2 x SSC + 0.1 % SDS at room temperature for 5 minutes, 65' C for

30 minutes, and 0.1 x SSC+ 0.1 % SDS at 65' C for 30 minutes. However, the wash conditions for the latter experiment was: 2 x SSC a.18 SDS at room temperature for

5 minutes, at 42' C for 30 minutes, and 0.1 x SSC + 0.1% SDS at 42' C for 30 minu tes.

The membranes were wrapped with Saran wrap and exposed to an X-ray film

(Kodak) with an intensifjmg screen at -80' C. 4.2.2.2 Sonthern bIots

4.2.2.2. 1 Genomic DNA isolttfion

The latex present in all parts of the dandelion plant interfered with the quality of genomic DNA. Two DNA extraction kits (fkom Amersham and QIAGEN) and several other extraction methods including the one used in section 3.2.7.1 were tried but all failed. DNA extracted by these methods was not digested well by restriction enzymes: EcoR 1, Hind III, BamH 1, Nco 1, Xba 1. Eventually, the following method

(modified fiom Murray and Thompson 1980) was used .

Young leaves (2 g) were ground to a powder in liquid nitrogen, the powder

1yophiluRd overnight. The powder was placed into 1.5 ml Eppendorf tubes until less than half full with a spatula. Extraction buffer 500 pl (50 rnM Tris-HC1, pH 8.0,700 mM NaCl, 10 mM EDTA, 1% (w/v) CTAB, 1% B-mercaptoethanol) was added into each tube, and after mking with the powder, they were incubated at 56' C for 15 minutes and then cooled to room temperature only. To the tubes 500 pl of chloroform:arnylalcohol mixture 24: 1 (v:v) was added, mixed well, and centrifûged at top speed in an Eppendorf bench cenwge at room temperature for 5 minutes. The aqueous phases were transfened to new Eppendorf tubes, and to each tube 0.1 volume of 10% CTAB was added. The mixtures were then inverted gently and 1 volume chlorofoim:isoamylalcohol(24: 1) added. The mixtures were centrifuged at top speed in an Eppendorf bench centrifuge at room temperature for 5 minutes. The aqueous layers were transferred to new tubes, 1.2 volumes precipitation baer (50 mM Tri-

HCl, pH 8.0,10 mm EDTA, 1 % CTAB) were added, and the tubes innibated at room

temperature for at least 20 minutes. DNA-CïAB complex pellets were recovered by

centrifuging at top speed in an Eppendorf bench cenmgeat room temperature for

10 minutes. Each cornplex pellet was dissolved in 400 p11 M NaCl and the solutions

containing the DNA-CTAB combined. RNase A was added to the solution to the final

concentration of 20 p@, incubated at 37' C for 30 minutes, and the DNA was then

precipitated by adding 2-2.5 volumes of cold 100% ethano1 and incubating at -20' C

overnight. The pellet was centnfuged and washed with 75% ethanol, vacuum dried

and redissolved in 100 pl sterile ddHH,O. DNA quantity and quality were detennined

at 0D,60a8,with a spectrophotometer.

4.2.2.2.2 Genom ic DNA digestion with resfnktion envrnes

Genornic DNA (10 pg) was digested wiih restriction enzymes: EcoR 1, Hind

III, and Nco I.

digestion mixtures (30 pl):

10 x buffer

IO mM spermidine

1 mdml BSA

genornic DNA sterile ddH20 14.5 pl

enzyme 3 lil

----_CI ------

Digestion mixtures were incubated at 37' C overnight. After digestion 1 pl digestion solution was taken from each reaction, and the diggstion products separated on a 1% agarose gel.

4.2.2.2.3 Electrophoresis of digested genornic DNA

An agarose gel (0.8%) was prepared with 1 x TAE buffer. To each digestion mixture 10 pl of 6 x loading buffer (see Section 2.2.2.8.2) was added. A DNA mass ladder (Gibco BRL) was also loaded onto the gel as size rnarker, and the gel was run at 80 V for 2-3 hours in 1 x TAE buffer.

4.2.2.2.4 Denaturation of the DNA gel and hrznsfem*ngof the genomic DNA from gel to membrane

After electrophoresis the rnarker lane was cut off for ethidium bromide staining. The gel was cut to its final size and one corner was marked. It was then incubated in a denaturation buffer (500 mM NaOH, 1.5 M NaCl) for 30 minutes and

in a neutralization buffer (500 mM Tris-HCl, pH 7.2, 1.5 NaCl, 1 mM EDTA) for a

minimum of two washes (15 minutes each) at rwm temperature with gentle shaking. One piece of membrane (Genescreen, DuPont) was cut to the size of the gel and marked on one corner. The membrane was wetted with sterile ddH20 and soaked in

20 x SSC for at least 5 minutes. The transfer were conducted in the same way as described in Section 2.2.2.8.1.

After iransfening, the membrane was rinsed briefly with 6 x SSC, air drïed on a Whatman 3 MM paper for at lest 30 minutes, and then baked at 80' C for 2 hours.

4.2.2.2.5 cDNA probe labelling and purifiatioon

The cDNA probe used was the same as in Section 4.2.2.1.2 except that the 600 bp cDNA obtained by 3' end RACE-PCRwas randomly labelled with [a32P]d~TP+

[a"~]dcTPin order to get as strong a signal as possible.

4.2.2.2.6 Pre-hybtidizatim and hybridizafion of the membrane Whthe cDNA probe

Pre-hybridization and hybridization were conducted under the same conditions as in Section 2.2.2.8.4 at 65' C. The membrane was washed with 2 x SSC + 1% SDS briefly at room temperature, and 65' C for 30 minutes, and 0.5 x SSC + 0.1% SDS at

65' C for another 30 minutes. The membranes were then wrapped with Saran wrap and exposed to a X-ray film (Kodak, XOmat) with an intens-g screen at -80°C.

After one week exposure the same membrane was again washed with 0.1 x SSC + 0.1 % SDS at 65' C for 30 minutes, and then exposed to X-ray film (Kodak) at

-80' C again for an additional week. 4.3 Results and Discussion

43.1 Results

4.3.1.1 Seasonal changes in expression of the gene encoding the 18 kD protein

Fig. 4-1 shows that the amount of mRNA encoding the 18 kD protein underwent seasonal changes. The seasonal fluctuation was much greater than that at the protein level. The amount of the mRNA encoding the 18 kD protein was much lower during spring and summer than that during fdand winter. In May the expression of the gene was the lowest. Expression started to increase greatly during early faIl (September) and reached the highest peak in late fall (November). The expression decreased during winter (January, March). In March the specific mRNA decreased to almost the same amount as that in July.

4.3.1.2 Spatial expression of degene encoding the 18 kDprotein in other organs of dandelion

Total RNA and total soluble proteins were extracted from dry seed, peduncle, sepal, petal, ovary, mature Leaves, underground stems and roots.

Northem blots (Fig. 4-2) showed that positive hybridization signals were present only in the lanes of stem and root. Thus, the expression of the gene encoding the 18 kD protein is specifc to these organs.

Results from SDS-PAGE (Fig. 4-3) confirmed those obtaïned from the northem blot The predomhant 18 kD protein was present only in stems and roots. In the lane for dry seeds there was a high amount protein around 18 kD. However, when comparing its size with the 18 kD protein in stem and root closely, its molecular mass was found to be a little less than 18 kD, (about 17 kD). There were other major proteins present in dry seed, e.g. at 45 kD, 33 kD and 24 kD, with proteins of molecular mass around 45 kD king abundant. Predominant proteins other than 18 kD were also observed in the calyx (45 kD) and leaf (45 kD and 14 kD). Besides the 18 kD protein, two other proteins with molecular mass around 34 kD and 35 kD were also abundant in both stem and root.

4.3.1.3 Expression of the gene encoding the 18 kD protein during seedhg establishment

Total soluble proteins and total RNAs were extracted from dry seeds, one-day- germinated seeds, 5-day-old roots, 5-day-old stems, 5-day-old leaves, 15- day-old roots, and 30-day-old roots.

Northern blots showed that, except dry seeds (lane 1) and 5-day-old leaves

(iane 5)- mRNA encoding the 18 kD protein was present in al1 samples (Fig. 4-4).

Thus the expression of the gene encoding the 18 kD protein began very early, during seedling growth; it was expressed as soon as the radicle came out and was constitutively expressed thereafter in the root and stem. SDS-PAGE confirmed the presence of the 18 kD protein in 5day-old stem, 5-, 15-, 30-day-old root and its absence fkom the dry seed and 5-day-old leaf (Fig. 4-5). However, interestingly, there was no 18 kD band present in the extract fkom 1-day-germinated seed. It seems that the synthesis of the 18 kD protein did not start on the hrst day of germination, and that

there is a lag period between gene activation and protein synthesis. On the other hand,

the 18 kD protein was not a predominant protein in 5-day-old stems and roots.

Although the 18 kD protein was more evident in 15- and 30day-old roots, it was not

as abundant as in mature root extracts (Fig. 4-3).

4.3.1.4 Detection of homologous proteins in other Compositae

Total soluble proteins and total RNA were extracted from the roots of eight

Compositae: Cirsiurn arvense (Canada thistle), Cichorium in~bus(chicory),

Conyza canadensis (horse weed), Erigeron perennis (perennial fleabane), Lactuca scanola (wild lettuce), Rudbeckia triloba (three-lobed coneflower), Senecio vulgaris

(groundsel), and Sonchus anrensis (sow thistle).

The roots of eight species contained at least one predominant protein. The

molecular masses of some of them are very close to 18 kD, such as the 19 kD protein

in Canada thistle, 17 kD in chicory, 20 kD in three-lobed coneflower, and 18 kD and

19 kD in groundsel and sow thistle (Fig. 4-6). However, not one of them was

homologous to the dandelion 18 kD protein since the cDNA probe encoding the 18 kD protein did not recognize any message from these 8 plants (Fig. 4-7). This

indicated that the 18 kD protein is not a comrnon protein in the Cornpositae.

4.3.1.5 The number of gene copies encoding the 18 kû protein

Southern blots probed with the cDNA encoding the 18 kD protein showed

three major hybridization bands in all three lanes of genomic DNA that had been

digested with the restriction enzymes: Eco R 1, Hind III, and Nco 1, respectively, when

the membrane was washed wiîh 0.5 x SSC solution at 65' C (Fig. 4-8a). This suggests

that there are a minimum of three gene copies encoding the 18 kD protein. However,

if the membrane was washed with 0.1 x SSC at 65' C, only one hybridization band

was detected in each laue (Fig. 4-8b). Since there are two 18 kD isofonns, and both

share about 88% homology at the nucleotide level, it seems that the less strong

hybridization between the probe and the gene encoding the isofom 2 (PI 5.49) was

washed away under the high shingency washing conditions. This suggests that the

isoforrn with pI 5.56 may be encoded by one gene copy, and the other encoded by two

gene copies. There were also some faint hybridization signals present in dl three

lanes. Analysis of restriction enzyme sites using Gene Runner program indicated that

there are no restriction sites for the enzymes of EcoR 1, Hind III, and Nco 1 in the

sequences of the full-length cDNA and the genomic DNA-PCR products. It is most

likely, therefore, that the faint signals were caused by a low degree homology between the probe and other non-specific genes, instead of by hybridhtion between the probe and the partial gene sequences resulting fkom restriction enzyme digestion. 43.2 Discussion

Seasonal fluctuations in the expression of the gene encoding the 18 kD protein, as determined at the mRNA level, are considerable and are much pater than that seen at the protein level. During spring (May) and summer (July), the amount of the specific mRNA is very low, and lower in May than in July (Fig. 4-l), but the amount of the 18 kD protein is not low, with Little difference between the amount present in the roots in May and July (Fig.2-1). This might be due to the slow degradation of the protein in spring, suggesting the 18 kD protein is a very stable protein. In addition, during fall and winter the amount of the protein remains the same, though there is a big fluctuation in the amount of its mRNA between September and January. Synthesis of the 18 kD protein may not be controlled at the transcriptional level.

A similar pattern showing significant seasonal changes in the specific-mRNAs encoding bark storage proteins (BSP) has also been demonstrated in willow (Xu

1993). In willow bark, the amount of the BSP-mRNA is high during the faIl, reaches the highest amount in October and decreases gradually from November to December.

However, a linle BSP-mRNA is detectable in January, but no BSP-mRNA is present in February, March and in the spring and summer months (May and June). Moreover, unlike the 18 kD protein, the amount of the BSP in willow undergoes sipificant seasonal fluctuations as weU. The protein increases markedly in the fall (September) and remaines hi@ throughout the fa11 and winter (October, November, Decernber, January, February, March), and then decreases sharply during spring growth (May and

June). The seasonal changes at both the mRNA and protein level in willow bark are expected in that the protein encoded by this mRNA is a storage protein. It is preferentially synthesized during senescence, remains high when growth is retârded and is depleted during meristem reactivation. With respect to the 18 kD protein in dandelion, however, although the seasonal fluctuations of the amount of its mRNA are significant, its function as storage protein is questionable. Not only are the amounts of the 18 kD protein in spring and summer high (Fig. 2-l), but also, unlike the BSP-mRNA in willow bark, the expression of the gene encoding the 18 kD protein is still pronounced during the entire winter (January and March) after senescence has occurred. There must be other reasons for the synthesis of the 18 kD protein during winter, for example, for freezing protection.

The expression of the gene encoding the 18 kD protein is tissue specific, for it is expressed only in the roots and stems. In addition, the expression occurs very early, as soon as the radicle emerges from the seed, and is constitutively expressed thereafter. Protein synthesis is elevated during the seedling growth since the amount of the 18 kD protein increases proportionately in the protein profdes from 5-day-old roots to 15-day-old and 3û-day-old roots, and to mature roots (Fig. 4-5,4-3). AU these results indicate that the 18 kD protein has a function related to root and stem development. The lag penod behween protein synthesis and gene activation at the cornpietion of germination Merindicates that the synthesis of the 18 kD protein is not controlled at the transcriptional level.

Protein homologous to the 18 kD protein was not present in other members of

Compositae. Since none of the 8 plants belong to the same genus as dandelion, it is still possible that homologous proteins are present in those species which are more closely related to dandelion.

It has been reported that a 17 kD protein in the petennial weed, chicory, is a vegetative storage protein (Cyr and Bewley 1990a) and antigenically similar to the 18 kD protein in dandelion (Cyr and Bewley, pers. commun.). The antibodies raised against the dandelion 18 kD protein recognize the predodant protein in chicory, and vice versa. However, results from this study demonstrated that the homology at nucleotide level between the two genes is low, since no hybridization signals could be observed on northern blots (lane 2 in Fig. 4-7). Jan Mar May July Sept Nov

Fig. 4-1. Seasonal regdation of expression of the gene encoding the 18 kD protein top part: picture of an agarose gel stained with ethidium bromide showing equal loading of total RNA (10 ug each). bottom part: northern blot probed with cDNA encoding the pI 5 S6 isoform Fig. 4-2. Spatial expression of the gene encoding the 18 kD protein top part: pichue of total RNA on an agarose gel stained with ethidium bromide; bottom part: northem blot probed with cDNA encoding the pI 5.56 isoform. Fig. 4-3. Synthesis of the 18 kD protein in different parts of a dandelion SDS-PAGE gel stained with Coomassie Brilliant Blue R-250, 40 pg of total soluble protein were loaded in each lane. Fig. 4-4. Expression of the gene encoding the 18 kD protein during seedling establishment seed: dry seed; 1seed: one-day-germinated seed; Sleaf, Sroot, Sstem: 5-day-old led, root and stem; 1Sroot, 30root: 15-day-old and 30-day-old root. top part: pichire of total RNA agarose gel; bottom part: northem blot probed with cDNA encoding the pI 5.56 isoform. Fig. 4-5. Synthesis of the 18 kD protein during seedling establishment seed: dry seed; 1 seed: one-day-germinated seed; Sleaf, Sroot, Sstem: 5-day-old leaf, root and stem; 1 Sroot, 30root: 15-day-old and 30-day-old root. SDS-PAGE gel stained with Coomassie Brïlliant Blue R-250, 40 pg of total soluble protein were loaded in each lane. Fig. 4-6. SDS-PAGE of total soluble protein in roots of eight members of the Compositae gel was stained with Coomassie Brilliant Blue R-250, 40 pg of total soluble protein were loaded in each lane. C.dandelion; 1. Cirsium arveme; 2. Cichorium Nttybus; 3. Conyza cmudemis; 4. Erigeron perennis; 5. Lactuca scariola; 6.Rudbeckia triloba; 7.Senecio vulgaris; 8. Sonchus anensis. Fig. 4-7. Absence of the 18 kD protein fiom roots of other members of the Compositae top part: picture of total RNA on an agarose gel; bottom part: northem blot probed with cDNA encodhg the pI 5.56 isoform. C.dandelion; 1. Cirsium amerne; 2. Cichorium intybus; 3. Conyza canadensis; 4. Erigeron perennis; 5. Lacîuca scarida; 6. Rudbeckia triloba; 7.Senecio vulgaris; 8. Sonchus arvemis. 7126 bp- 5096 bp- 4072 bp- 3054 bp-

2036 bp- 1636 bp- 1018 bp-

Fig. 4-8. Southern blot of dandelion genomic DNA probed with the product of 3'-end RACE PCR a. membrane was washed with 0.5 x SSC, at 65' C; b. membrane was washed with 0.1 x SSC, at 65' C. 5. Effects of environmental perturbations on the expression of the gene encoding the 18 kD protein and protein synthesis 5.1. Introduction

Experiments conducted in this chapter were designed to find out which environmental factor may have an effect on the 18 kD protein gene expression.

Information obtained from these experiments may be critical in determining the possible role of this protein in dandelion roots.

The seasonal patterns of the 18 kD protein accumulation and decline suggest that seasonal environmental factors, such as photoperiod and temperature, are possible regdators of gene expression. Previous studies (Butler 1996, myself, not shown here), indicated that alte~gphotoperiod from long day to short day did not influence 18 kD protein synthesis or mobilization. In this Chapter, the effect of temperature on the gene expression and 18 kD protein synthesis was detemiined.

As mentioned in the Literature review, defoliation provides a good method to determine if a protein is a vegetative storage protein since defoliation leads to a

shortage of supply of curent photosynthate and the plant must acquire nutrients from

alternative sites to grow new leaves Oankwerts and Gordon 1989). This results in the decline of nitrogen reserves in the remaining parts of the plant to support new shoot

growth (Graber et al. 1927). Several proteins were identifed as VSPs by defoliation,

such as VSPs in the roots of alfalfa (Hendershot and Volenec 1993a). white clover

(Corre, et al. 1996), and le* spurge (Cyr and Bewley 1990~).

The environmental perturbations, drought and wounding are two common stresses that plants encounter. They are the common inducen of VSPs (Coleman et al. 1994, Creelman et al. 1992, Davis et al. 1993, Mason et al. 1988, Mason and

Mullet 1990, Mason et al. 1992, Pena-Cortes et al. 1988, 1991, Surowy and Boyer

1990). Their effects on the 18 kD protein synthesis were detected.

Sequence cornparison revealed that the 18 kD protein shares up to 44% homology with a number of intracellular pathogenesis-related (PR) proteins.

Synthesis of an IPR proîein in potato tubers has been reported to be induced by a. hornogenate of the fungus Phytophthora megaspenna (Matton and Brisson 1989).

The effect of this elicitor on 18 kD proîein synthesis in dandelion root discs was detennined. 5.2 Materiais and Methods

5.2.1 Materials

Plants used in this Chapter were grown fiom seeds collected fiom a single inflorescence of a dandelion growing on the campus of the University of Guelph.

Since dandelions reproduce apomictically (Asker and Jerling 1992), these plants were genetically identical.

Dandelion seeds were gerrninated on moist soi1 (a mixture of soil, sand and vermiculite) in a growth chamber at 20' C with a 16-hour photopenod. After three weeks the seedlings were transferred into 12-inch-diameter pots, 4 plants in each pot.

The plants were then grown in growth chambers at 20' C with a 16-hour photoperiod and 50% relative humidity for at ieast six months. At this age the root systems were large enough to provide sufficient materials for experimentation. The plants were watered twice a week dhgtheir growing period. Since the dandelions were growing so luxuriantly under the above conditions it was necessary to cut the foliage every two months. Any experirnental treatrnents were conducted at least one month after defoliation.

5.2.2. Methods

Plants were grown at two temperatures in growth chambers: 20' C and 5' C.

Both chambers were set for 16-hours of light, 8 hours of darkness and 50% relative 5.2.2.1 CoId shock treatments

Three pots of dandelions growing in the 20'~growth chamber were transferred to the 5' C growth chamber. Roots were collected at 36 hours, 72 hours and 5 days after transfer. Roots of plants growhg in the growth charnbers at constant 20' C and

5' C were collected as controls. Roots were washed, cut into smd pieces, fiozen with

Iiquid nitrogen, and stored at -80' C before extraction as described in the Matends and Methods section in Chapter 2.

5.2.2.2 Warm shock treatments

Opposite to cold shock experiment, three pots of dandelions growing in the 5'

C growth chamber were transferred to the 20' C growth chamber. Roots were colIected at 24 hours, 48 hours and 5 days after transfer. Roots used as controls were the same as above. Roots were washed cut into small pieces, frozen with liquid nitmgen, and stored at -80' C before extraction.

5.2.23 Defoliation treatments

Defoliation experiments were conducted on plants growing in both the 20' C and 5' C growth chambers. All leaves were cut off at the base of the petioles. Roots were collected at 24 hours, 48 hours and 6 days after defoliation at 20' C, or 36 hours,

72 hours and 6 days after defoliation at 5' C. Roots of non-defoiiated plants were collected as controls. Roots were washed, cut into small pieces, frozen with liquid nitrogen, and stored at -80' C before extraction.

5.2.2.4 Wounding treatments

Wounding experiments were also conducted under two conditions: 20' C and

5' C. Leaves were clipped around the edge with pliers, with care taken not to damage the main veins of the leaves. Roots were collected at 24 hours, 48 hours and 4 days after wounding at 20' C, or 36 hours, 72 hours and 4 days after wounding at 5' C.

Roots of non-wounded plants were collected as controls. Roots were washed, cut into small pieces, frozen with liquid nitrogen, and stored at -80' C before extraction.

5.2.2.5 Water stress treatment

This treatment was conducted in a 20' C growth chamber. Dandelions were watered thoroughly on the fmt day of the experiment and then water was withheld for up to 12 days. Roots were collected on the 2nd, 4th, 6th, 8th, 10th and 12th day after watering. On the 12th day the soi1 was dry and plants had started to wilt. Roots were washed, cut into small pieces, fiozen with liquid nitrogen, and stored at -80' C until extraction. 5.2.2.6 Fungal elicitor treatments

Roots of plants growing in 20' C and 9 C growth chambers were collected and washed with tap water, ddH20, and sterile H20several times, dried with a Kimwipe, cut into 0.5-cm-long pieces, and then incubated either in sterile water (4 g fresh weight root discd3 ml of sterile water) as control or in sterile water + fimgal elicitor

(homogenate of Phytophthora rnegasperma) (20: 1) at 20' C or 9 C. Homogenates of

Phytophthora megarpennu were kindly provided by Dr. Normand Brisson, University of Montreal, Canada. Root discs were collected at 8 hours, 24 hours, 48 hours and 4 days after incubation and stored at -80' C until use. Roots before incubation were used as control.

5.2.2.7 SDS-PAGE

Determination of the effects of the above environmental perhubations to either the synthesis or mobilization of the 18 kD protein were conducted with SDS-PAGE.

Root proteins were extracted, purified, quantified, and electrophoresed as described in Sections 2.2.2.1.1,2.2.2.1.2,2.2.2.1.3.

5.2.2.8 Northern bIots

Determination of the effects of above environmental perturbations on expression of the gene encoding the 18 kD protein at mRNA level was conducted using northern blots, as in Section 4.2.2.1.2. 5.3 Results and Discussion

53.1 Results

Before starhg all the experiments in this Chapter, total soluble proteins from tap roots and rniddle size laterai roots were extracted. The proportion of the 18 kD protein in total soluble protein profiles extracted from these two types of root were the same (data not shown). Thus results are not affected by the size of root materials.

53.1.1 Effect of cold shock on the gene expression and on 18 kD protein synthesis

Cold shock had a very evident effect on the gene expression at the mRNA level

(Fig. Ma). At 36 hours after being transferred from 20' C to 5' C, the amount of the specific-mRNA encoding the 18 kD protein in the root increased greatly and reached the same arnount as that in roots of control plants growing at 5' C, and rernained elevated for up to 5 days. However, the effect of cold shock on 18 kD protein synthesis was very small (Fig. 5- 1b), with possibly an increase in the quantity of the

18 kD protein seen in the 36 hour sample. At 72 hours, however, the arnount of the

18 kDprotein decreased to that seen in control roots and remained at that level until the 5th day.

Cold shock also had an effect on the quantity of other proteins. A 37 kD protein was present only in control roots growing at S0 C (Fig. 5-1b, lane C2). It seemed that the synthesis of this protein was also cold-induced, although its induction needed longer than 5 days. Cold shock suppressed the synthesis of a 35 kDprotein.

The arnount of the protein decreased at 36 hours and was hardly present at 72 hours or on the 5th day. However, this suppression was not permanent, for the 35 kD protein

was again present in control plants growing at 5' C (lane C2).Synthesis of a 36 kD

protein seemed not to be affected by cold shock, since no obvious changes were observed in its amount during the experiment.

53.1.2 Effects of warm shock on gene expression and 18 kD protein mobiüzation

As might be expected, warm shock showed the opposite effect to cold shock on the expression of the gene encoding the 18 kD protein (Fig. 5-2 a). The specific mRNA encoding the 18 kD protein decreased sharply 24 hours after king transfemed from 5' C to 20' C and maintained this low level during the rest of the experirnent.

After 48 hours the amount of the specific mRNA was almost the same as that in the roots of control plants growing at 20' C. However, the effect of warm shock on 18 kD protein mobilization did not show until the 5th day (Fig. 5-2 b), and the arnounts of the 18 kD protein were still high at 24 and 48 hours after transfer. The decrease on the

5th day was to almost the same amount as that in roots of control plants growing at 20'

C.

Coincident with the cold shock results, wam shock suppressed the synthesis of the 37 kD protein by the 5th day and hence the effect was quicker than the induction of synthesis by the cold temperature. Similar to cold shock, warm shock also suppressed the 35 kD protein synthesis. However, the suppression was transient also; the protein was again present in control plants growing at 20' C. The fact that both cold and warm shocks suppress 35 kD protein synthesis indicated that the metabolism of this protein was not related to the adaptions of plant to the temperature fluctuations.

The expression of the 36 kD protein was also not affected by warm shock.

53.13 Effects of the defoliation on the 18 kD protein expression

Defoliation at 20' c did not affect the 18 kD protein gene expression at either the mRNA or protein level (Fig. 5-3). The amounts of the specific mRNA and the 18 kD protein did not show obvious changes during the experiment (Fig. 5-3 a and b).

However, defoliation at 20' C affected some other protein accumulation. Three novel proteins at 20 kD, 15 kD and 14 kD were observed at 24 hours after defoliation.

Accumulation of the 22 kD protein was also stunulated at that tirne. The amounts of these proteins stayed at a high level up to the 6th day.

If defoliation was conducted at OC, its effects on gene expression and 18 kD protein synthesis were very obvious (Fig. 5-4). A large increase in the amount of the specific mRNA was observed at 36 hours &ter defoliation and this remained elevated at least until72 hours (Fig. 5-4 a). A decrease was observed on the 6th day when the amount of mRNA deched to a very low level, lower than in control plants. The same type of paaeni was also observed on the protein gel @g. 5-4 b). Accumulation of the

18 kD protein was stimulated at 36 hours and 72 hours after defoliation, but retumed to the control levels on the 6th day. Interestingly, the three new proteins highly induced, and the one protein up-regulated by defoliation at 20' C were not induced or up-regulated at 5' C. Instead, increases in the synthesis of proteins between 47 kD and

36 kD were obsewed.

53.1.4 Effects of wounding on gene expression and 18 kD protein synthesis

Wounding at 20' C did not have any effect on either 18 kD protein gene expression or protein synthesis itself (Fig. 5-5). No changes were observed in the amounts of either the specinc-mRNA on a northem blot (Fig. 5-5 a) nor of the 18 kD protein on an SDS-PAGE gel (Fig. 5-5 b) during the wounding experiment. Nor did it affect any other protein accumulation or mobilization (Fig. 5-5 b).

However, wounding at 5' C had signifcant effects on both 18 kD protein gene expression and protein synthesis (Fig 5-6). Both effects were very similar to that of defoliation at 5' C (Fig. 54). Fig. 5-6 shows that the amounts of the specifc-mRNA and the 18 kD protein increased at 36 hours and remained elevated at least until72 hours after wounding. They then decreased by the 4th day to levels similar to those of the control plants. There was an increase in 47 kD protein accumulation, but not in the proteins between 47 kD and 36 kD. 5.3.1.5 Effect of water stress on gene expression and 18 kD protein synthesis

Water stress did not have any effect on the 18 kD protein synthesis or its gene expression (Fig. 5-7). AIthough plants were not watered for up to 12 days in a 20' C growth chamber, there were no obvious changes in the amount of either the specific- mRNA or the 18 kD protein. Moreover, there were also no obvious changes in the arnounts of other proteins. The protein profiles extracted from roots collected on the

4th day, 6th day, 8th day, 10th day and 12th day of stress were very similar (Fig. 5-

7b).

5.3.1.6 Effects of the fimgal elicitor on gene expression and 18. kD protein synthesis

Northern blots and SDS-PAGE gels of root discs that were treated at 20' C and

5' C with fungal elicitor (homogenate of Phytophthora megaspema) showed this elicitor had no effects on 18 kD protein gene expression nor the synthesis of the protein (Fig. 5-8,s-9,s-10,and 5-1 1). Although the total RNA could not be extracted from root discs after 24 hours incubation in the control experiment, the expression patterns of the gene encoding the 18 kD protein in both control and elicitor treatments at 20' C during the first 24 hours incubation were the sarne (Fig. 5-8). The amount of the specfic-mRNA increased after 8 hours incubation and then decreased after 24 hours incubation. In the elicitor treatment, the amount of the specific-mRNA remained at a low level after 48 hours and 4 days incubation (Fig. 5-8 b). However, no changes were observed in the amounts of the 18 kD protein in either the control or elicitor treated roots (Fig. 5-9). The amount of the 18 kDprotein was low after 24 hours in the control roots, but this was due to a mistake in loading (Fig. 5-9 a). However, changes in other protein syntheses and mobilization were evident and similar in both control and elicitor treatments. The amounts of the proteins with molecular masses between

20 kD and 29 kD decreased after 8 hours incubation and were hardly detectable during the rest of the experiment, whereas a 16 kD protein accumulated after 8 hours of incubation and then declined after 48 hours and remained a low level on day 4.

The expression patterns of the gene encoding the 18 kD protein in both control and elicitor treatments at 5' C were also quite similar. Interestingly, after 8 hours incubation the amount of the specific-mRNA decreased instead of increasing as it did at 20' C, and it remained low up to 24 hours incubation (Fig. 5-10 a). It then increased to a sirnilar level to that in control roots after 48 hours incubation and stayed at the high level until the 4th day. Although the decreases at 8h and 24h in the elicitor- treated roots was not as obvious as in control roots, they were still observable (Fig. 5-

10 b). As in the 20' C treatment, the amount of the 18 kD protein did not show obvious changes in either control or elicitor treatments after 4 days of incubation (Fig.

5-1 1). 53.2 Discussion

AU the results of the northem blots presented in this Chapter were obtained fkom membranes washed using: O. 1 x SSC 0.1% SDS at 65' C for 30 minutes. The hybridization signals thus most likely represent the hybridization between the isoform pI 5.56 polypeptide and the probe. However, for some northem blots, such as defoliation and wounding at 20' C and 5' C, membranes were also washed with 0.5 x SSC and 0.1 x SSC and exposed to the X-ray films separately. The signal patterns were the same under al1 conditions, which indicates that the other isoform either undergoes the same changes as that of the pI 5.56 isoform, or it did not change in amount during the experiments.

Evidence obtained fkom the experiments in this Chapter further indicates that the 18 kD protein is not a vegetative storage protein. Defoliation did not stimulate the rnobilization of the 18 kD protein at 20' C but, rather. it caused an increase in protein accumulation at 5' C. Wounding was unable to induce the synthesis of the 18 kD protein at 20' C either, contrary to what has been obsewed for other VSPs in other plants (Coleman et al. 1994, Creelrnan et al. 1990, Davis et al. 1993, Farmer et al.

1992, Mason and Mullet 1990, Pena-Cortes et al. 1988, Surowy and Boyer 1990).

However, interestingly, the increase in 18 kD protein synthesis by wounding was observed at 5' C and the effects at both the mRNA and protein level were similar to those in the defoliation experiment. It seems that the plant recognizes defoliation as a wounding event However, defoliation dso has sorne unique effects. At 20' C, the - amounts of several novel proteins increase, but not at 5' C. This clifference might be due to slow metabolism at low temperature, and six days may not be long enough to show the effects of defoliation at 5' C. At 20' C, new leaves could be seen on the 6th day after defoliation, but new leaves had not corne out even at two weeks after defoliation at 5' C.

VSP amounts decline when growth is active, and it is logical to surmise that

VSPs should increase when growth is retarded. It is well known that water deficits inhibit plant growth (Salisbury and Ross 1992), and thus it is expected that water deficit stimulates VSP synthesis. Water stress has been reported to stimulate VSP synthesis in soybean leaves (Mason and Mullet 1990). However, no effect was detected on 18 kD protein synthesis even if the plants were not watered for up to 12 days. These results indicate that the 18 kD protein does not function in the same way as a traditional VSP.

There is no doubt that expression of the gene encoding the 18 kD protein is temperature-regulated. Results nom cold and wam shock experiments demonstrated that gene expression is up-regulated by low temperature (5' C) and inhibited by warm temperature (20' C). These results are coincident with the natural seasonal fluctuations in gene expression, Le. active during fall and winter and inactive during spring and summer. However, the responses to cold and warm shock at the protein level did not coincide with those at the specific-mRNA level. In the cold shock experiment, the elevated amount of the specinc-MA was detected by 36 hours and then remained high, while the increase of the 18 kD protein amount was transient, and was obsewed only in the 36 hour sample. In the warm shock experhent, a decrease in the amount of the 18 kD-protein- specific-rnRNA was observed 24 hours after king transferred from 5' C to 20' C, while the protein did not decline until the

5th day. This inconsistent response of the protein and its specific-&A was also observed during the seasonal changes, and merindicates that the gene expression is not controlled at the transcriptional level.

A lag period was also found between the decline in mRNA and its protein for the cold-responsive protein, CAP85, in spinach (Neven et al. 1993). One day after rehxrning from low temperature to warm conditions, the mRNA level was already low, but appreciable amounts of protein remained. Neven et al. (1993) suggested that slow degradation of CAP85 may provide protection for spinach in case frost retumed.

The same explanation may be also suitable for dandelion.

Low-temperature-responsive (LTR)genes have been reported to be controlled at both the transcriptional and post-transcriptional levels @unn et al. 1994, Hajela et al. 1990, Kirch, H-H. et al. 1997, Wolfraim et al. 1993). Even in one plant, some

LTR genes are transcriptionally regulated, while others are post-transcriptionally regulated (Dunn et al. 1994, Hajela et al. 1990). However, to date no LTR genes have been reported to be controkd at the transiational or post-translational level. Further experiments are needed to determine at which level the gene encoding the 18 kD protein is controlled.

The fact that both defoliation and wounding affected 18 kD protein gene expression and protein synthesis only at 5' C not at 20' C indicates that the effect is

actually related to the cold treatment The leaf may be the sensor of the plant to cold.

Defoliation and wounding of leaves at 5' C may stimulate the sensitivity of plants and

leaves to cold, and therefore stimulate gene expression and protein synthesis. It is

possible that the 18 kDprotein is related to cold protection.

There seerns to be a systemic signalling transduction pathway between the

roots and the leaves since the expression of the 18 kD protein in roots increased when

the leaves were wounded or when plants were defoliated at 5' C. Similar phenornena

have been reported for vegetative storage protein (VSP)synthesis in soybean leaves

and Arabidopsis thliana roots. Mason et al. (1992) reported that unwounded leaves

of soybean plants that were adjacent to wounded leaves showed increased Vsp

mRNA accumulation. They suggested that a soluble transported wound signal is

present in soybean leaves. Berger et al. (1995) also showed that the content of the

Atvsp mRNA in Arabidopsis thaliana increased in roots when rosette leaves were

wounded, indicating the existence of a transmissible wound factor. In the case of

dandelion, a transmissible cold receptor seerns to be present in leaves. Phytophthora megaspem is not a pathogen of potato and dandelion, but its elicitor induces IPR protein synthesis in potato tuber discs. It did not induce the 18 kD protein in dandelion root discs. Elicitors have been shown to mimic the effect of pathogen infection in different species (Chappell and Hahlbrock 1984, MacKintosh et al. 1994). It seems that the 18 kD protein is not a pathogen-inducible protein, although it is homologous to IPR proteins. This conclusion is Mersupported by the fact that wounding, a cornmon PR protein inducer, was unable to stimulate the

18 kD protein synthesis at 20' C.

hterestingly, contrary to the obsewation in the intact roots, the amount of the specific-mRNA encoding the 18 kD protein in root discs was up-regulated at 20' C and inhibited at 5' C after 8 hours incubation. This indicates that wounding caused by root cutîing has different effects from that of leaf wounding on gene expression and the former wounding must be much more complex than the latter. The different responses of the gene in root discs to warm (20' C) and cold (5' C) temperature further indicates that 18 kD protein gene expression is temperature-regulated.

Moreover, the responses of the root discs to cutting at the 18 kD protein level was also inconsistent with its mRNA; the amount of 18 kD protein remained the same during the entire experiment. This again indicates that the 18 kD protein amount is not controlled by the amount of transcripts. Fig. 5-1. Effect of cold shock (20' C to 5' C) on expression of the gene and 18 kD protein synthesis a: top part: picture of RNA on an agarose gel showing equal loading of total RNA ( 10 pg each); bottom part: northem blot probed with cDNA encoding the pI 5.56 isoform; b: SDS-PAGE gel stained with Coomassie Bdliant Blue R-250; 40 pg of total soluble protein were loaded in each lane. C 1 : control plants growing at 20' C; C2: control plants growing at 5' C; 36h, 72h, and 5D: hours and days after transfer. arrows indicate the 35 kD and 37 kD proteins. Fig. 5-2. Effect of warm shock (5' C to 20' C) on expression of the gene and 18 kD protein mobilization a: top part: picture of RNA on an agarose gel showing equal loading of total RNA (1 0 pg each); bottom part: northem blot probed with cDNA encoding the pI 5S6 isoform; b: SDS-PAGE gel stained with Coomassie Bdliant Blue R-250; 40 pg of total soluble protein were loaded in each lane. C 1 : control plants growing at SOC; C2: control plants growing at 20' C; 24h, 48h, and 5D: hours and days afier transfer. Arrows indicate the 35 kD and 37 kD proteins. Fig. 5-3. Effect of defoliation at 20' C on expression of the gene and 18 kD protein synthesis a: top part: picture of RNA on an agarose gel showing equal loading of total RNA ( 1 0 pg each); bottom part: northem blot probed with cDNA encoding the pI 5.56 isoform; b: SDS-PAGE gel stained with Coomassie Brilliant Blue R-250; 40 pg of total soluble protein were loaded in each lane. C: control, plants were not defoliated; 24h, 48h, and 6D: hours and days after defoliation. Fig. 5-4. Effect of defoliation at 5' C on expression of the gene and 18 kD protein synthesis a: top part: picture of RNA on an agarose gel showing equal loading of total RNA ( 10 pg each); bottom part: northern blot probed with cDNA encoding the pI 5.56 isoform; b: SDS-PAGE gel stained with Coomassie Brilliant Blue R-250; 40 pg of total soluble protein were loaded in each lane. C: control, plants were not defoliated; 36h, 72h, and 6D: hours and days after defoliation. Fig. 5-5. Effect of wounding at 20' C on expression of the gene and 18 kD protein synthesis a: top part: picture of RNA on an agarose gel showing equal loading of total RNA ( 10 pg each); bottom part: northem blot probed with cDNA encoding the pI 5.56 isoform; b: SDS-PAGE gel stained with Coomassie Bnlliant Blue R-250; 40 pg of total soluble protein were loaded in each lane. C: control, plants were not wounded; 24h, 48h, and 4D: hours and days after wounding Fig. 5-6. Effect of wounding at 5' C on expression of the gene and 18 kD protein synthesis a: top part: picture of RNA on an agarose gel showing equal loading of total RNA (1 0 pg each); bottom part: northem blot probed with cDNA encoding the pI 5.56 isoform; b: SDS-PAGE gel stained with Coomassie Brilliant Blue R-250; 40 pg of total soluble protein were loaded in each lane. C: control, plants were not wounded; 36h, 72h, and 4D: hours and days after wounding Fig. 5-7. Effect of water stress at 20' C on expression of the gene and 18 kD protein synthesis a: top part: picture of RNA on an agarose gel showing equal loading of total RNA ( 10 pg each); bottom part: northern blot probed with cDNA encoding the pI 5.56 isoform. b: SDS-PAGE gel stained with Coornassie Brilliant Blue R-250; 40 pg of total soluble protein were loaded in each lane; 2D, 4D, 6D, 8D, 10D, and 12D: days out of water.

Fig. 5-9. Effect of elicitor (homogenate of Phytophthora megasperma) on 18 kD protein synthesis at 20' C SDS-PAGE gel stained with Coomassie Bdliant Blue R-350; 40 pg of total soluble protein were loaded in each lane. a. control, without elicitor; b. with elicitor; C . before incubation; 24h, 48h, and 4D: hours and days afier incubation. Fig. 5-10. Effect of elicitor (homogenate of Phytophthora megasperma) on 18 kD protein gene expression at 5' C a. control, without elicitor; b. with elicitor; top part: picture of RNA on an agarose gel showing equal loading of total RNA ( 10 pg each); bottom part: northem blot probed with cDNA encoding the pI 5.56 isoform. C. before incubation; 24h, 48h, and 4D: hours and days after incubation. Fig. 5-11. Effect of elicitor (homogenate of Phytophthora megasgerma) on 18 kD protein synthesis at 5' C SDS-PAGE gel stained with Coomassie Bdliant Blue R-250; 40 pg of total soluble protein were loaded in each lane. a. control, without elicitor; b. with elicitor; C. before incubation; 24h, 48h, and 4D: hours and days after incubation. 6. General discussions The physical environment in which organisms grow is not constant, and

fluctuations occur during a year. The only way that perennid plants can cope with changeable environmental conditions is by undergohg physiological, structural and biochemical adjustments. One common unfavourable environmental condition that

temperate species encounter is freezing. The strategies that plants use to avoid

freezing damage involve genetic programme alterations, that is, they suppress the

synthesis of some constitutive cellular proteins and synthesize specific proteins which

relate to cold-acclimation (review by Guy 1990, Hughes and Dunn 1996).

A number of genes encoding low-temperature-responsive (LTR)proteins have

been isolated fkom a variety of different species that undergo &dg stress (Boothe

et al. 1997, Cattivelli and Bartels 1990, Hajela et al. 1990, Houde et al. 1992, Kirch

et al. 1997, Neven et al. 1993, Schaffer and Fischer 1990, WoIfraim et al. 1993,

Zhang et al. 1993). Expression of these genes is induced or enhanced by low

temperature. Although no functions of these LTR proteins have been elucidated, the

levels of expression of these LTR proteins have been found to positively correlated

with fieezing tolerance (Guy et al. 1992, Houde et al. 1992, Mohapatra et al. 1989,).

Dandelion, as a persistent perennial weed, inevitably encounters freezing

stress during overwintering. Results from this study indicate that the expression of

the gene encoding the predominant 18 kD protein in roots is seasonally changeable.

Expression is the highest in the late fall when the temperature starts to decline to around zero and remains at a relatively high level even in January when the temperature is below zero. The 18 kD protein gene then decreases in expression when spnng cornes. Cold shock (20' C to 5' C) and warm shock (5' C to 20' C) experiments confii that the expression of the gene is up-regulated by cold temperature (5' C) and dom-regdatecl by wmtemperature (2$ C). Moreover, the fact that defoliation and wounding have effects on gene expression and 18 kD protein synthesis only at 5' C indicate that the effects are modulated by cold, instead of defoliation or wounding. It is possible that defoliation and wounding rnay increase the sensitivity of the plant to cold due to damage to the leaves. All these results together indicate that the 18 kD protein is a low-temperature-responsive (LTR) protein and that it may have a role in cold protection.

A common feature among many (Lm)proteins is their stability upon boiling in aqueous solution (Boothe et al. 1995, Lin et al. 1990, Lin and Thomashow, 1992%

Neven et al. 1993). This property reflects that these proteins are extremely hydrophilic which is also characteristic of many dehydration-induced proteins. A major component of freezing tolerance may be to tolerate dehydration (Steponkus

1984, Yelenosky and Guy 1989), which is caused by the presence of ice in the extracellular spaces (Guy 1990). Indeed, many LTR genes which have been investigated are also responsive to drought (Cattivelli and Bartels 1992, Guy et al.

1992, Hajela et al. 1990, Neven et al. 1993, Kurkela and Franck 1990, Thomashow 1993). Furthemore, many studies have demonstrated that a mild drought stress can increase fieezing tolerance (Cloutier and Andrews 1984, Cloutier and Siminovitch

1982, Guy et al. 1992, Siminovitch and Cloutier 1982). This indicates that there is a comected mechanism involving both fieezing- and dehydration-tolerance (Neven et al. 1993). However, results from this study indicate that the expression of the gene encoding the 18 kD protein in dandelion roots is not drought-inducible. Evidence fiom the research of Neven et al. (1993) and Urao et al. (1994) support the possibility that drought-stress and fieezing-stress can operate by two different mechanisms.

Although the LTR protein in spinach, CAP85, is both low-temperature and water stress responsive, its accumulation at low temperature is likely not caused by water stress since the water potential did not decrease. Urao et al. (1994) reported that two genes that encode Ca2+-dependentprotein kinases in Arabidopsk thaliana, are induced by drought and high-salt stresses, but not by low-temperature stress or heat stress. All these results indicate that besides the common response mechanisms to stress, a different and unique response mechanism is present in both low temperature and water stress.

Unlike most LTR proteins, the 18 kD protein does not show strong characteristics of dehydration-tolerance-related proteins (DTR), such as very high hydrophilicity, having repeat motifs of serine, or lysine-rïch repeats (Neven et al.

1993). However, it did exhibit above 55% hydrophilicity and was misshg the amino acid Trp (W), which is generally absent from DTR proteins. It is rich in serine

(1 1.54%) and lysine (10.26%), though they were not present as serine clusters or lysine-rich repeats as in DTR proteins. Similarly, an LTR protein in alfalfa, pSM2075

(Luo et al. 1991), which is similar to the dehydrin B18 in barley (Close et al. 1989)- does not have either the serine cluster nor the lysine-rich repeat and is glycine-rich.

The possibility that the 18 kD protein is a DTR protein cannot be excluded, even though it is not drought-inducible. At least it has similar physical propemes.

Iden-g the location of a protein in cells is often essential in establishing its biological role. Several studies have determined that the subcellular localization of LTR proteins is in soluble fractions of the ceU, especially in the cytoplasm (Boothe et al. 1997, de Beus et al. 1997, Houde et al. 1995, Lin and Thornashow 1992b,

Neven, et al. 1993). Using both light and electron microscopy, and biochemical fractionation, Houde et al. (1995) indicated that the LTR proteins in wheat, the

WCS 120 protein fdy,are highly present both in the cytoplasm and nucleus of cens in wheat leaves but none of the farnily members are found in the celI walls or in other organelles. They suggested that these proteins are involved in a general protective role by surrounding vital cellular proteins and protecting thern from unfolding or aggregating during freezhg or dehydration. Their cryoprotection assays demonstrated that the WC120 proteins protected lactate dehydrogenase against denaturation upon freezing. A similar explmation was proposed for the cryoprotective role of BSA in protecting enzyme preparations during storage at low temperature (Tamiya et al.

1985). Other LTR proteins localized in cytoplasm are the BN28 in Brnrsica napus

(Boothe et al. 1997, de Beus et al. 1997) and the CAP85 protein in spinach (Neven et al. 1993). A drought-induced protein (Close et al. 1993) is also localized in the

cytoplasm of ceus. In common with îhese LTR proteins, the 18 kD protein was

identified as a cytosolic protein by comparing its deduced amino acid sequence from

the full-length cDNA with the N-terminalend sequence obtained directly from the pdied polypeptide and by the PCfGene program. This characteristic of the 18 kD protein plus the high amount which is present in cells allows the speculation that the

18 kD protein plays a protective role during fkeezing.

Aithough the precise roles of LTR proteins have not been elucidated, a few proposed functions of these proteins have been reported, including as an anrifreeze

(Hon et al. 1994, 1995, Kurkela and Frank 1990), in membrane stabilization (Hincha et al. 1990), water binding (McCubbin et la. 1985, Roberts et al. 1993), and cryoprotection of proteins (Kamoka and Oeda 1994, Lin and Thornashow 1992%

Houde et al. 1995). With respect to the 18 kD protein, a previous study indicated that the 18 kD protein is not an antifreeze protein (Butler 1996). Houde et al. (1995) suggested that the cryoprotective efTect of the WCS120 protein family is due to its abundance during cold acclirnation. The 18 kD protein is very predominant during fall and winter and it may have a similar role to these proteins. However, this needs Werconfirmation.

On the other hand, results fiom this studies indicate that the 18 kD protein shares up to 44% homology with a group of allergen and PRproteins and it contains the common motif which is present in all the members of this group. This indicates that the 18 kD protein is a member of this family. Although the 18 kD protein seems not to be pathogen-inducible, other IPR proteins which show homology with the 18 kD protein are pathogen inducible (Somssich et al. 1988). Hence, the 18 kD protein may also have a role in pathogen defence. A simila-result was demonstrated in winter rye. Hon et al. (1995) reported that proteins accumulating during cold acclimation in winter rye were identified having both antifreeze activity and a similarity to three classes of PR proteins, endochitinases, endo-pl ,3-glucanases, and thaumatin-like proteins. They suggested that these proteins play a dual role in freezing tolerance and disease resistance in ovemintering rye. A positive CO- relationship between cold-acclimation and fimgal-resistance has also been demonstrated in a number of keeezing-tolerant grasses. (Tronsmo 1984, 1985, 1993,

Tronsmo et al. 1993). For example, in perennial grasses, timothy (Phleum pratense) and cocksfoot (Dactylis glomew), the development of their freezing tolerance during cold acclimation is positively correlated with theU achievement of resistaoce to the snow molds Typhula ishikunensis and Fusarium nivale (Tronsmo, 1984,

1993). PR proteins can be induced by low temperature, e.g. the acidic PR proteins and a basic endochitinase in barley (Tronsmon et al. 1993), and a PR protein, osmotin, in potato (Zhu et al. 1993). It is thus possible that the 18 kD protein plays roles in both hzing tolerance and disease resistance.

The expression of the gene encoding the 18 kD protein is tissue specifc, it is constitutively expressed only in the stems and roots, and is expressed as soon as the radicle cornes out from the seed. In addition, the amount of the 18 kD protein proportionately increases during seedling development; it seems that the 18 kD protein is also related to the development of the roots and stems. It needs to be clarified whether the increase in the 18 kD protein during seedling development may increase the tolerance and resistance abilities of the roots and stems to freezing and diseases. LTR proteins have been reported to be highly expressed in vascular bundles and their bordering parenchyrna cells in wheat seedlings (Houde et al. 1995). Tanino and McKersie (1984) have shown that the vascular tissues in cold-acclimated winter wheat are very sensitive to freezing stress. The high amount of the LTR protein in these tissues suggests a cold protective role, and may help to maintain metabolite transport in vascular tissues at low temperatures. During seedling establishment, the vascular tissues are developed and expanded, and it is possible that the 18 kD protein is localized in vascular tissues of the stems and roots in dandelion and the increased amount of the 18 kD protein may due to the increased growth of vascular tissues.

Detemination of the localization of the 18 kD protein in roots and stems will help cl- these hypoîheses.

Results from this study indicate that the 18 kD protein is not likely to be a

VSP. First, the amount of the 18 kD protein is still high during spring and summer when growth is most active and a lot of nutrients are required. Northern blots indicate that the amount of the specifc-mRNA is very low in May, suggesting that this high amount of the protein during spring is due to slow degradation of the protein. This property does not fit the characteristics of a VSP. Secondly, defoliation, which is a good method to determine the VSP function, cannot stimulate the mobilization of the 18 kD protein. Other environmental perturbations, water stress and wounding, which have been reported to induce synthesis of several VSPs in many plants, are unable to stimulate gene expression in dandelion either. Moreover, the protein is not deposited into a common VSP subcellular site, protein bodies; it is cytosolic. Ali these observations together indicate that the 18 kD protein does not function as a VSP.

Fuîure studies

Although the results from this study indicate that the 18 kD protein is low- temperature-responsive and may relate to cold protection, this needs to be further confirmed. Experiments, such as cryoprotection assays, deterrnination of the role of the 18 kD protein in membrane stabilization and water binding, and the tissue localization of the 18 kD protein in stems and roots of dandelion, will be very valuable in investigating its possible role in the cold protection. The gene promoter characterizes gene regdation and expression, and promoter analysis should be done to help to determine the role of the protein.

This study also indicates that there seems to be a transmissible cotd factor present in Ieaves that trigger 18 kD protein synthesis in the dandelion plant. Further experiments can be done to detemine the nature of the signal involved in the cold response. It has been suggested that the phytohormone abscisic acid (BA) is involved in the low-temperature response process. Several researchers have indicated that the amount of ABA increases when plants were subjected to low temperature

(Chen et al. 1983, Guy and Haskell 1989) and application of ABA induces synthesis of some LTR proteins (Chen et al. 1983, Chen and Gusta 1983). ABA may participate in the signahg of LTR gene expression. However, not all genes regulated by low temperature are regulated by exogenous ABA, and some are expressed in mutants with defects in ABA signal transduction (Gilmour and

Thornashow 1991, Nordin et al. 1991). It seems that there are two responsive mechanisms that plants can exhibit to low temperatures: ABA-dependent and ABA- independent responses. It has been also proposed that calcium functions as a second messenger in response to chilling (Knight et al. 1991) and cold acclimation @hg and

Pickard 1993, Monroy et al. 1993). A transient change in the amount of cytosolic calcium was obsewed in response to cold shock (Knight et al. 1991) and the modulation of calcium charnels was activated by low temperature (Ding and Pickard * 1993). Results fiom Monroy et al. (1993) and Monroy and Dhindsa (1995) provided more simcant evidences for the involvement of calcium as a second messenger during cold acclimation. They showed that low temperature induced the influx of ceU wall calcium into the cytosol and calcium chelators, calcium channel blockers, and inhibitors of calcium-dependent protein kinases (CDPKs) prevent cold acclimation.

It seems that calcium rnay also play an essential role in low-temperature signal transduction leading to cold acchation. It will be exciting to fmd out which of the above signal paîhways is involved in the cold response in dandelion.

The 18 kD protein seems not be controlled solely at the transcriptional level.

Further experirnents are needed to determine at which level the expression of the 18 kD protein is controlled. Knockout of 18 kD protein synthesis in a transgenic plant by transforming the plant with the antisense to the gene will provide the most direct evidence of its function of the protein.

This work has provided information of the 18 kD protein and its gene, and provides a framework for f-wther studies to elucidate the role of this abundant component of the stems and roots of the perennial weed, dandelion. References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, J. (1990) Basic alignment search tool. J. Mol. Biol. 215: 403-410.

Anderson, J. M. (1991) Jasmonic acid-dependent increase in vegetative storage protein in soybean tissue cultures. J. Plant Growth Regul. 10: 5-10.

Anderson, J. M. (1989) Membrane-derived fatty acids as precursors to second messengers. In: Second messengers in plant growth and development. Boss, W. F. and Morre, D. J. eds., A. R. Liss, New York. 18 1-2 12.

Anderson, J. M., Spilatro, S. R., Klauer, S. F., and Franceschi, V. R. (1989)Jasmonic acid-dependent increase in the level of vegetative storage proteins in soybean. Plant Sci. 62: 45-52.

Andrews, D. L., Beames, B., Summers, M. D., and Park, W. D.(1988) Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovhs vector. Biochem. J. 252: 199-206.

Asker, S. E., and Jerling, L. (1992)Apomixis in plants. CRC Press, Boca Raton, 23 1- 234.

Atkinson, R. G., Perry, J., Matsui, T., Ross, G. S., and Macrae E. A. (1996) A stress-, pathogenesis,-, and allergen-related cDNA in apple fniit is also ripening-related. New Zealand J. Crop Hom. Sci. 24: 103-107.

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Preparation and analysis of DNA. In: Current protocols in molecula.biology. John Wiley & Sons, Inc. USA.

Bailey, C J., and Boulter, D. (1971) Urease, a typical seed protien of the Leguminosae. In: The chemotaxonomy of the Leguminosae. Harbome, J. B., Boulter, D., and Turner, B. L., eds. Academic Press, New York London. 485-502.

Barber, L. D., Joern, B. C., Volenec, J. J., and Cunningham, S. M. (1996) Supplemental nitrogen effects on alfalfa regrowth and nitrogen mobilization from roots. Crop Sci. 36: 1217-1223. Bensen, R., Boyer, J., and Mullef R. (1988) Water deficit-induced changes in abscisic acid, growth, polysomes, and translatable RNA in soybem hypocotyls. Plant Physiol. 88: 289-294.

Berger, S., Bell, E., Sadka, A., and MuIlet, J. E. (1995) Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, Light and phosphate. Plant Mol. Biol. 27: 933-942.

Bewley J. D., and Black, M. (1994) Mobilization of stored seed reserves. In: Seeds: Physiology of development and germination. Second edition, Plenum Press, New York, London. 293-343.

Bewley, J. D., and Greenwood, J. S. (1990) Protein storage and utilization in seeds. In: Plant physiology, biochernistry and molecular biology. First edition, Dennis, D. T., AND Turpin, D. H. Longman Group UK Limiteci. London. First edition. 456-469.

Bol, J. F., Linthorsf H. J. M., and Cornelissen, B. J. C. (1990)Plant pathogenesis- related protehs induced by virus infection. Annu. Rev. Phytopath. 28: 11 3- 138.

Boothe, J. G., de Beus, M. D., and Johnson-Flanagan, A. M. (1995) Expression of a low-temperature-induced protein in Brassica napus. Plant Physiol. 108: 795-803.

Boothe, J. G., Sonnichsen, F. D., de Beus, M. D., and Johnson-Flanagan, A. M. (1997) Purifkation, characterization, and structural analysis of a plant Iow- temperature-induced protein. Plant Physiol. 113: 367-376.

Bowler, C., Montagu, M. V., and Inze, D. (1992)Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:83-116.

Breiteneder, H., Ferreira, F., Hoffmann-Sommergruber, K., Ebner C., Breitenbach, M., Rumpold, H., Kraft, D., and Scheiner, 0. (1993)Four recombinant isoforms of Cor a 1, the major allergen of hazel pollen, show different IgE-binding properties. Eur. J. Biochem.. 212: 355-362.

Breiteneder, H., Ferreira, F., Reikerstorfer, A., Duchene, M., Valenta, R., Hoffmann- Sommergmber, K., Ebner, C., Breitenbach, M., Kraft, D., and Scheiner, 0. (1992) Complementary cloning and expression in Escherichia coli of Aln g 1, the major allergen in pollen of alder (Alnzuglutinosa). J. Allergy. Clin. Irnmunol. 90:909-917. Breiteneder, H., Hohann-Sommergmber, K., OTRiordain,G., Susani, M., Ahom, H., Ebner, C., Kraft, D., and Scheiner, 0.(1995) Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and stnichiral relationships to a group of 17 kD hee pollen allergens. Eur. J. Biochem. 233: 484-489.

Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraff., D., Rumpold, H., Scheiner, O., and Breitenbach, M (1989) The gene coding for the major birch pollen allergen Bet v 1, is highly homologous to a pea disease resistance response gene. EMBO J. 8:1935-1938.

Brue, V., Guerbette, F., Kader, J. C., Kamangara, G. C., Svenson, B., and von Wettetein-Knowles, P. (1989) A lOkD barley basic protein transfers phosphatidylcholine from liposomes to mitochondria. Carlsberg Res. Commun. 4: 8 1-84.

Brown, J. L. (1979) A cornparison of the turnover of amino-terminally acetylated and nonacetylated rnouse L-cell proteins. J. Biol. Chem. 254: 1447.

Brown, J. L., and Roberts, W.K.(1976) Evidence that approximately eighty percent of the soluble proteins from Ehrlich ascites cells are amino-terminally acetylated. J. Biol. Chem. 25 1: 1009.

Butler, S. M. (1996) An investigation of an 18 kD polypeptide in the subterranean organs of dandelion (Tararacurn oficinale Weber). MSc thesis. Department of Botany, University of Guelph.

Cattivellli, L., and Bartels, D. (1990) Molecular cloning and characterization of cold- regulated genes in barley. Plant Physiol. 93: 1504-15 10.

Cattivelli, L., and Bartels, D. (1992) Biochemistry and molecular biology of cold- inducible enzymes and proteins in higher plants. In: Inducible plant proteins. Wray, J. L. ed, Cambridge Univ. Press, Cambridge, UK. 267-288.

Chandler, P., and Robertson, M. (1994) Gene expression regulated by abscisic acid and a related wound-induced gene are differentially induced by nitrogen. Plant Physiol. 106: 21 1-215.

Chappell, J. and Hahlbrock, K. (1984) Transcription of plant defence genes in response to W light or fimgal elicitor. Nature 3 11 : 76-78. Chen, H. H., and Gusta, L. V. (1983) Abscisic acid-induced freezing resistance in cultured plant cells. Plant Physiol. 73: 71-75.

Chen, H. H., Li, P. H., and Brenner, M. L. (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71: 362-365.

Clausen, S., and Apel. K. (1991) Seasonal changes in the concentration of the major storage protein and its mRNA in xylem ray cells of poplar trais. Plant Mol. Biol. 17: 669-678.

Cloutier, Y., and Andrews, C. J. (1984) Efficiency of cold hardiness induction by desiccation stress in four winter cereals. Plant Physiol. 76: 595-598.

Cloutier, Y., and Siminovitch, D. (1982) Correlation between cold- and drought- induced fiost hardiness in winter wheat and rye varieties. Plant Physiol. 69: 256-258.

Close, T. J., Fenton, R. D., Yang, A., Asghar, R., DeMason, D. A., Crone, D. E., Meyer, N. C., and Moonan, F. (1993) Dehydrin: The protein. In: Plant responses to cellular dehydration during environmental stress. Current Topics in Plant Physiol. 10:104-118.

Close, T. J., Kortt, A. A., and Chandler, P. M. (1989) A cDNA-based cornparison of dehydration-induced proteins (dehydrins) in barley and corn. Plant Mol. B iol. 13: 95- 108-

Coleman, G. D., Banados, M. P., and Chen, T. H. H. (1994) Poplar bark storage protein and a related wound-induced gene are differentially induced by nitrogen. Plant Physiol. 106: 2 11-215.

Coleman, G. D., Chen, T. H. H.,Ernst, S. G., and Fuchigami, L. (1991) Photoperiod control of poplar bark storage protein accumulation. Plant Physiol. 96: 686-692.

Coleman, G. D., Chen, T. H. H., and Fuchigami, L. H. (1992) Complementary DNA cloning of poplar bark storage protein and control of its expression by photoperiod. Plant Physiol. 98: 687-693.

Coleman, G. D., Englert, J. M., Chen, T. H. H., and Fuchigami, L. H. (1993) Ph ysiological and environmental requirements for poplar (Populus deltoides) bark storage protein degradation. Plant Physiol. 102: 53-59. Corre, N. Bouchart, V., Oq,A., and Boucaud, J. (1996) Mobilization of nitrogen reserves during regrowth of defoliated Tnfolium repens L. and identification of potential vegetative storage prokins. J. Exp. Bot. 47: 11 1 1 - 11 18.

Creelman, R A., Mason, M. S., Bensen, R. J., Boyer, J. S., and Mullet, J. E. (1990) Water deficit and abscisic acid cause differential inhibition of shoot versus root growth in soybean seedhgs: analysis of growth, sugar accumulation, and gene expression. Plant Physiol. 92: 205- 214.

Creelman, R. A., Tierney, M. L., and Mullet, J. E. (1992) Jasmonic acidhethyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression. Proc. Natl. Acad. Sci. USA. 89: 4938-4941.

Culvenor, R. A., and Simpson, R. J. (1991) MobWtion of nitrogen in swards of Tnfolilun subtermeum L. during regrowth after defoliation. New Phytol. 117: 8 1- 90.

Cunningham, S. M., and Volenec, J. J. (1996) Purification and characterization of vegetative storage proteins from alfalfa (Medicago sativa L.) taproots. J. Plant Physiol. 147: 625-632.

Cyr, D. R., and Bewley J. D. (1990a) Proteins in the roots of the perennial weeds chicory (Chicorium intybus L.) and dandelion (Tararacwn ofinale Waber) are associated with overwintering. Planta 182: 370-374.

Cyr, D. R., and Bewley, J. D. (1990b) Annual rhythmicity of nitrogen reserves in the roots of pemicious perennial weeds. In: Importance of root to shoot communication in the response to environmental stress. Davies, W. J., and Jeffcoat, B., eds, British Society for Plant Growth Regulation Monograph 21: 353-368.

Cyr, D. R., and Bewley, J. D. (1990~)Seasonal variation in nitrogen storage reserves in the roots of lem spurge (Euphorbia esula) and responses to decapitation and defoliation. Physiol. Plant. 78: 361-366.

Dankwerts, J. E., and Gordon, A. J. (1989) Long-term partitionbg, storage and remobilization of I4c assimilated by Trfolium repens (cv. Blanca). Ann. Bot. 64: 533-544.

Dammam, C., Rojo, E., and Sanchez-Serrano, J. J. (1997) Abscisic acid and jasmonic acid activate wound-inducible genes in potato through separate, organ- specific signal transduction pathways. Plant J. 11 : 773-782.

Darby, N. J., and Creighton, T. E. (1993) Protein structure. IRL Press at Oxford University Press.

Dathe, W., Ronsch, H., Preis, A., Schade, W., Sembdner, G., and Schreiber, K. (1981) Endogenous plant hormones of the broad bean, Vicia faba L. (-)-jasmonic acid, a plant growth inhibitor in pericarp. Planta 153: 530-535.

Davis, J. G., Dibner, M.D., and Battey, J.F. (1986) Methods in molecular biology. Elsevier Science Publishing 123- 125.

Davis, J. G., Egelkrout, E. E., Coleman, G. D-, Chen, T. H. H., Haissig, B. E., Riemenschneider, and Gordon, M. P. (1993) A family of wound-induced genes in Populus shares common features with genes encoding vegetative storage proteins. Plant Mol. Biol. 23: 135-143. de Beus, M. D. Boothe, J. G., and Johnson-Flanagan, A. M. (1997) Temporal and spatial expression of BN28, a low-temperature-induced protein in Brassica napus. Cm. J. Bot. 78: 28-35.

DeWald Da, Mason, H. S., and Mullet, J. E. (1992) The soybean vegetative storage proteins Vspa and Vsppare acid phosphatases active on polyphosphates. J. Biol. Chem. 267: 15958-15964.

Ding, J. P., and Pickard, B.G. (1993) Modulation of mechanosensitive calcium- selective cation charnels by temperature. Plant J. 3: 713-720.

Driessen, H. P. De Jong, W. W. Tesser, G. I., and Bloemendal, B. (1985) Cnt. Rev. Biochem. 18: 28 1-325.

Duan, X., Li, X., Xue, Q., Abo-El-Saad, M., Xu, Dm, and Wu, R (1996) Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biochem. 14: 494-498.

DUM, M. A., Goddard, N. J., Zhang, L., Pearce, R. S., and Hughes M. A. (1994) Low-temperature-responsive barley genes have different control mechanisms. Plant Mol. Biol. 24: 879-888.

Dunn, M. A., Moms, A., Jack, P. L., and Hughes, M. A. (1992) A low temperature- responsive translation elongation factor 1a from barley (Hordewn vulgare L.). Plant Mol. Biol. 23: 22 1-225.

Ehness, R., Ecker, M., Godt, D. E., and Roitsch, T. (1997) Glucose and stress independently regulate source and sink metabolism and defence mechanisrns via signal transduction pathways involving protein phosphorylation. Plant Cell9: 1825- 1841.

Enon, J. L., Ballo, B., May, L., Bussell, J., Fox, T., Thomas, S. R. (1991) Isolation and characterization of a tomato acid phosphatase cornplementary DNA associated with nematode resistance. Plant Physiol. 97: 1462-1469.

Farmer, E. E., and Ryan C. A. (1990) Interplant communication: airborne methyl jasrnonate induces synthesis of proteinase inhibitors in plant leaves. Proc. Natl. Acad. Sci. USA 87: 77 13-77 16.

Farmer, E. E., and Ryan, C. A. (1992a) Octadecanoid precursors of jasmonic acid activity the synthesis of wound-inducible proteinase inhibitors. Plant Cell. 4: 129- 134.

Farmer, E. E., and Ryan, C. A. (1992b) Reguiation of expression of proteinase inhibitor genes by methyl jasmonate and jasmonic acid. Plant Physiol. 98: 995- 1002.

Faye, L., Greenwood, J. S., and Chrispeels, M. J. (1986) Urease in jack-bean (Canavalia ensiformis (L.) DC) seeds is a cytosolic protein. Planta 168: 579-585.

Franceschi, V. R. (1986) Temporary storage and its role in partitioning arnong sinks. In: Phloem transport. Cronshaw, J. M., Lucas, W. J., and Giaquinta, R. T., eds. Alan R. Liss, New York 399-409.

Franceschi, V. R., and Giaquinta, R. T. (1983a) The paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation. 1. Ultrastructure and histochemistry during vegetative development. Planta 157: 41 1-42 1.

Franceschi, V. R. and Giaquinta, R. T. (1983b) The paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation. IJ. Structure, metabolic and cornpartmental changes during reproductive growth. Planta 157: 422-43 1.

Franceschi, V. R., Wittenbach, V. A., and Giaquinta, R. T. (1983) Paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation. Plant Physiol. 72: 586-589.

Fristensky, B., Horovitz, D., and Hadwiger, L. A. (1988) cDNA sequences for pea disease resistance response genes. Plant Mol. Biol. 11 : 7 13-7 15.

Frohman, M.A (1990) RACE: Rapid amplification of cDNA ends. PCR protocols: A Guide to Methods and Amplification. Innis, M. A., eds, Academic Press, San Diego. 28-38.

Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full- length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85:8998-9002.

Gilmour, S. J. and Thomashow, M. F. (199 1) Cold acclimation and cold-regulated gene expression in abscisic acid mutants of Arabidopsis thaliana. Plant. Mol. Biol. 17: 1233-1240.

Gilmore, S. J., Amis, N. N., and Thomashow, M. F. (1992) cDNA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana. Plant Mol. Biol. 18: 13-21.

Goodall, G. J., and Filipowicz, W. (1989) The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell58: 473-483.

Graber. L. F., Nelson, N. T., Luekel, W. A., and Albert, W. B. (1927) Organic food reserves in relation to growth of alfalfa and other perennial herbaceous plants. Research Bulletin 80, Univ. Wisconsin, Madison.

Graham, J. S., Hall, G. Pearce, G., and Ryan, C. A. (1986) Regulation of synthesis of proteinase inhibitors 1 and II mRNAs in leaves of wounded tomato plants. Planta 169: 399-405.

Greenwood, J. S., Demmers, C., and Wetzel, S. (1990) Seasonal dependent formation of protein-storage vacuoles in the inner bark tissues of Salk microstachya. Can. J. Bot. 68: 1747-1755.

Greenwood, J. S., Stinissen, H. M. M. Peurnans, W. J., and Chnspeels, M. J. (1986) Sambucus nigra aggluiinin is located in protein bodies in the phloem parenchyrna of the bark. Planta 167: 275-278. Guy, C. L. (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. AMU. Rev. Plant Physiol. Plant Mol. Biol. 41 : 187-223.

Guy, C. L., and Haskell, D. (1989) PreIiminary characterization of high molecular mass proteins associated with cold acclimation in spinach. Plant Physiol. Biochem. 27: 777-784.

Guy, C. Haskell, D., Never, L., Klein, P., and Smelser, C. (1992) Hydration-state- responsive proteins link cold and drought stress in spinach. Planta 188: 265-270.

Hajela, R. K., Horvath, D. P., Gilmour, S. J., and Thornashow, M. F. (1990) Molecular cloning and expression of cor (cold-regulated) genes in Arabidopsis thaliana. Plant Physiol. 93: 1246-1252.

Hm,U., and Sauter, J. J. (199 1) Storage proteins in the wood of Taxodiaceae and of Ta~us.J. Plant Physiol. 138: 497-499,

Harley, C. P., Regeimbal, L. O., and Moon, H. H. (1958) The role of nitrogen reserves in new growth of apple and the transport of P from roots to leaves during early-spring growth. Proc. Am. Soc. Hort. Sci. 72: 57-63.

Harris, M. J., Outlaw, W. H., Mertens, R., and Weiler, E. W. (1988) Water stress induced changes in the abscisic acid content of guard cells and other cells of Vicia faba L. leaves as determined by enzyme-amplified immunoassay. Proc. Natl. Acad. Sci. USA. 85: 2584-2588.

Harvery, P. J., and Boulter, D. (1983) Isolation and characterization of the storage protein of yam tubers (Dioscorea rotundata). Phytochem. 22: 1687-1693 -

Harvey, R.J. and Darlison, G (199 1) Random-primed cDNA synthesis facilitates the isolation of multiple S'tDNA ends by RACE.Nucl. Acids Res. 19: 4002

Hattori, T., Nakagawa, T., Maeshima, M., Nakamura, K., and Asahi, T. (1985) Molecular cloning and nucleotide sequence of cDNA for sporamin, the major soluble protein of sweet potato tuberous roots. Plant. Mol. Biol. Int. I. Fund. Res. Genet. Eng. 5: 3 13-320.

Hattori, T., Fukumoto, H., Nakagawa, S., and Nakamura, K. (199 1) Sucrose-induced expression of genes encoding for the tuberous root storage proteins, sporamin, of sweet potato in leaves and petioles. Plant Cell Physiol. 32: 79-86. Heijne, G. von (1986) A new method for predicting signal sequence cleavage sites. Nucl. Acids. Res. 14: 4683-4690.

Heilmeier, H., Schulze, E. D., and Whale, D. M. (1986) Carbon and nitrogen partitionhg in the biennial monocarp. Arctium tomentosum. Mill. Oecologia 70: 466- 474.

Hendershot, K. L., and Volenec, J. J. (1993a) Taproot nitrogen accumulation and use in overwinte~galfalfa (Medicago sotivu L.). J. Plant Physiol. 141 : 68-74.

Hendershot, K. L., and Volenec, J. J. (1993b) Nitmgen pools in taproots of Medicago sativu L. after defoliation. J. Plant Physiol. 141: 129-135.

Hem,E. M., Hankins, C. N., and Shannon, L. M. (1988) Bark and leaf lectins of Sophora japonica are sequestered in protein-storage vacuoles. Plant Physiol. 86: 1027-1031.

Hincha, D. K., Heber, U., and Schmitt, J. M. (1990) Proteins fiom frost-hardy leaves protect thylakoids against mechanical fkeeze-thaw damage in vitro. Planta 180: 416- 419.

Hofmiann-Sommergmber, K. (1996) Corylus avellana gene for major allergen Cor a 1 (clone CAGC 1 1). GenBank No. 272440.

Hoffmann-Sornrnergruber, K., Vanek-Krebitz, M., Radauer, C ., Wen, J., Ferreira, F.. Scheiner, O., and Breiteneder, H. (1997) Genomic characterization of members of the Bet v 1 farnily: genes coding for allergens and pathogenesis-related protein share intron positions. Gene 197: 91-100.

Hon, W-C,Griffith, M., Chong, P., and Yang, D. S. C (1994) Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.) leaves. Plant Physiol. 104: 97 1-980.

Hon, WC., Griffith, M., Mipan,, A., Kwok, Y. C., and Yang, D. S. C. (1995) Antifieeze proteins in winter rye are similar to pathogenesis-related proteins. Plant Physiol. 109: 879-889.

Houde, M., Daniel, C., Lachapelle, M., Nard, F., Laliberte, S., and Sarhan, F. (1995) Immmolocalization of -hg-tolemce-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues. Plant J. 8: 583-593. Houde, M., Dhindsa, R. S., and Sarhan, F. (1992) A molecular marker to select for freezing tolerance in Gramineae. Mol. Gen. Genet. 234: 43-48.

Hrmova, M., and Fincher, G. B. (1993) Purification and properties of three (1+3)-P- D-glucanase isoenzymes fiom young leaves of barley (Hordeum vulgare). Biochem. J. 289: 453-461.

Huang, J-F., Bantroch, D. J., Greenwood, J. S., and Staswick, P. E. (1991) Methyl jasmonate treatment eliminates ceIl-specinc expression of vegetative storage protein genes in soybean leaves. Plant Physiol. 97: 15 12- 1520.

Hughes, M. A., and Dunn M. A. (1996) The molecular biology of plant acclimation to low temperature. J. Exp. Bot. 47: 291-305.

Hughes, M. A., Dunn, M. A., Pearce, R. S., White, A. J., and Zhang, L. (1992) An abscisic-acid-responsive, low temperature barley gene has homology with a maize phospholipid transfer protein. Plant Ceil Environ. 15:861-865.

Hurknian. W.J., and Tanaka, C. K. (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 8 1:802-806.

Jepson, 1, Bray, J., Jenkins, G., Schuch, W., and Edwards. K. (1991) A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue. Plant Mol. Biol. Rep. 9: 131-1 38.

Johnson, R. J., and Ryan, C. A. (1990) Wound-inducible potato inhibitor II genes: enhancement of expression by sucrose. Plant Mol. Biol. 14: 527-536.

Kang, S. M., and Tihis, J. S. (1980) Qualitative and quantitative changes in nitrogenous compounds in senescing leaf and bark tissues of the apple. Physiol. Plant. 50: 285-290.

Kang, S. M., and Titus, J. S. (1987) Specific proteins may determine maximum cold resistance in apple shoots. J. Hort. Sci. 62: 28 1-285.

Kamoka, T., and Oeda, K. (1994) and characterization of COR85- oligomeric complex fiom cold-acclimated spinach. Plant Cell Physiol. 35: 60 1-61 1.

Keeney, D. R. (1980) Rediction of soi1 nitrogen availability in forest ecosystems: A literature review. For. Sci. 26: 159- 17 1. Keil, M., Sanchez-Serrano, J., and WiImitzer, L. (1989) Both wound-inducible and tuber-specific expression are mediated by the promoter os a single member of the proteinase inhibitor II gene famiry. EMBO J. 8: 1323-1330.

Kinz T. H., Ourry, A., Boucaud, J., and Lemaire, G. (1991) Changes in source-sink rehti011ship for nitmgen during regrowth of lucane (MediCago sativa L.) following removal of shoots. Aust. J. Plant Physiol. 18: 593602.

Kirch. H-H., Berhi, J., GlacPnski, H., Snlamim. F., and Gebhardt. (1997) Structural organization, expression and promoter activity of a cold-stress-inducible gene of potato (Solanum tuberosm L.) Plant Mol. Biol. 33: 897-909.

Knighf M.R., Campe& A. K,Smith, S. M., and Trewavas, A. J. (1991) Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352: 524-526.

Kurkela, S., and Franck, M. (1990) Cloning and characfensation of a cold- and AB A- inducible Arabidopsis gene. Plant Mol. Biol. 15: 137-144.

Laemmli UK (1970)Cleavage of structural proteins during the assembly of the head of bactenophage T,- Nature 227:680-685.

Langheinrich, U., and Tischner, R. (1991) Vegetaîive storage proteins in poplar: induction and characterization of a 32- and a 36-kiiodalton polypeptide. Plant Physiol. 97: 1017-1025.

Larsen, J. N. (1996) Pollen ailergen Car b 1. GenBank No. 280168.

Lefewe, J., Bigot, J., and Boucaud, J. (1991) Origin of folk niîrogen and changes in free amino acid composition and content of leaves, stubble and mots of peremial ryegrass during regrowth afkr defoliation. J. Exp. Bot. 42: 89-95.

LeGendre, N., Mdeld, M., Weiss, A., and Matsudaira, P. (1993) Purification of proteins and peptides by SDS-PAGE. In: A practical guide to protein and peptide purification for microsequencing . Matsudaira, P., ed, Second edition, Academic Press, San Diego, CA 77-78.

Lin, C., Guo, W. W., Everson, E., and Thomashow, M. F. (1990) Cold acclimation in Arabidopsis and wheat. A response associated with expression of rdated genes encoding 'boihg-stable' polypeptides. Plant Physiol. 94: 1078- 1083. Lin, C., and Thomashow, M. F. (1992a) A cold-regdateci Arabidopsis gene encodes a polypeptide having potent cryoprotective activity. Biochem. Biophys. Res. Commun. 183: 1103-1 108.

Lin, C., and Thornashow, M. F. (1992b) DNA sequence analysis of a complimentary DNA for cold-regdateci Arabidopsis gene cor 15 and characterization of the COR15 polypeptide. Plant Physiol. 99: 519-525.

Luo, M., Lin, L., Hill, R. D., and Mohapatra, S. S. (1991) Primary structure of an environmental stress and abscisic acid-inducible alfalfa protein. Plant Physiol. 17: 1267- 1269.

MacKintosh, C., Lyon, G. D., and MacKintosh, R. W. (1994) Protein phosphatase inhibitors activates anti-fungai defence responses of soybean cotyledons and cell cultures. Plant J. 11 : 53-62.

Maeshima, M., Sasaki, T., and Asahi, T (1984) Characterization of major proteins in sweet potato tuberous roots. Phytochem. 24: 1899-1902.

Mason, H. S., DeWald, D. B., Creelrnan, R. A., and Mullet, J. E. (1992) Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars. Plant Physiol. 98: 859-867.

Mason, H. S., Guerrero, F. D., Boyer, J. C., and Mullet, I. E. (1988) Proteins homologous to leaf glycoproteins are abundant in stems of dark-grown soybean seeding. Analysis of proteins and cDNAs. Plant Mol. Biol. 1 1: 845-856.

Mason, H. S., and Mullet, J. E. (1990) Expression of two vegetative storage protein genes during development and in response to water deficit, wounding and jasmonic acid. Plant Cell2: 569-579.

Matsudaira, P. (1993) Introduction. Ln: A practical guide to protein and peptide purification for microsequencing. Matsudaira, P., ed, Second edition, Academic Press, San Diego, CA 1- 13.

Matton, D. P., and Brisson, N. (1989) Cloning, expression, and sequence conservation of pathogenesis-relateci gene transcripts of potato. Mol. Plant-Microbe hteract. 2: 325-33 1.

Mbeguie-A-Mbeguie, D., Gomez, R-M.,and Fils-Lycaon (1997) Sequence of an allergen-, stress-, and pathogenesis-related protein fkom aprïcot fimit (Accession No. U93 165). Gene expression during fruit ripening. (PGR97- 180) Plant Physiol. 115:1730.

McCubbin, W. D., Kay, C. M., and Lane, B. G. (1985) Hydrodynamic and optical properties of the wheat gem Eni protein. Can. J. Biochem. Cell Biol. 63: 803-81 1.

Millard, P., and Neilsen, G.H. (1989) The influence of nitrogen supply on the uptake and remobilization of storage N for the seasonal growth of apple trees. Am. Bot. 63: 301-309.

Mohapatra, S. S., WoIfraim, L. Poole, R. J., and Dhindsa, R. S. (1989) Molecular cloning and relation to freezing tolerance of cold-acclimation-specific genes of alfalfa. Plant Physiol. 89: 375-380.

Moiseyev, G. P., Beintema, J. J., Fedoreyeva, L. I., and Yakovlev, G. 1. (1994) High sequence similarity between a ribonuclease from ginseng calluses and funguselicited protein from parsley indicated that intracellular pathogenesis-related proteins are ribonucleases. Planta 193: 470-472.

Monroy, A. F., and Dhindsa, R. S. (1995) Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25' C. Plant Cell 7: 321-331.

Monroy, A. F., Sarhan, F., and Dhindsa, R. S. (1993) Cold-induced changes in freezing tolerance, protein phosphorylation, and gene expression: Evidence for a role of calcium. Plant Physiol. 102: 122% 1235.

Murray, M. G., and Thompson, W. F. (1980) Rapid isolation of high-molecular- weight plant DNA. Nucl. Acids Res. 8: 4321-4325.

Mussigmann, C., and Ledoigt, G (1989) Major storage proteins in Jerusalem artichoke tubers. Plant Physiol. Biochern. 27: 8 1-86.

Neven, L. G., Haskell, D. W., Hofig, A., Li, Q-B., and Guy, C. L. (1993) Characterization of a spinach gene responsive to low temperature and water stress. Plant Mol, Biol. 2 1: 29 1-305.

Niki, T. and Sakai, A. (1981) Ultrastructural changes related to frost hardiness in the cortical parenchyma cells fkom mulberry twigs. Plant CeU Physiol. 22: 17 1 - 183.

Nsimba-Lubaki, M., and Peumans, W. J. (1986) Seasonal fluctuations of lectins in barks of elderberry (Sambucus nigra) and black locust (Robiniapseudoucacia). Plant PhysioI. 80: 747-75 1.

Nordin, K., Heino, P., and Palva, E. T. (1991) Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L) Heynh. Plant Mol. Biol. 16: 1061-1071.

O'Farrell P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J Biol. Chem. 250: 4007-402 1.

O'Kennedy, B. T., Hemerty, M. J., and Titus, J. S. (1975) Changes in the nitrogen reserves of apple shoot bark. Physiol. Plant. 45: 419-424.

O'Kennedy, B.T., and Titus, J. S. (1979) Isolation and mobilization of storage proteins from apple shoot bark. Physiol. Plant. 45: 419-424.

Oland, K. (1959) Nitrogenous reserves of apple trees. Physiol. Plant 12: 594-648.

Ourry, A., Bigot, J., and Boucaud, J. (1989) Protein mobilization from stubble and roots, and proteolytic activities during pst-clipping re-growth of perenaial ryegrass. J. Plant Physiol. 134: 298-303.

Ourry, A., Boucaud, J., and Salette, J. (1988) Nitrogen rnobilization from stubble and roots during re-growth of ryegrass. J. Exp. Bot. 39: 803-809.

Ourry, A., Kim, T. M.,and Boucaud, J. (1994) Nitrogen reserve mobilization during regrowth of Medicago sativa L. Relationships between availability and regrowth yield. Plant Physiol. 105: 83 1-837.

Paiva, E., Lister, K. M., and Park, W.D. (1983) Induction and accumulation of major tuber proteins of potato in stems and petioles. Plant Physiol. 7 1 : 16 1 - 168.

Parthier, B. (1990) Jasmonate: Hormonal regulator or stress factors in leaf senescence? J. Plant Growth Reg. 9: 1-7.

Parthier, B. (1991) Jasmonate, new regdators of plant growth and development: Many facts and few hypotheses on their actions. Bot. Acta. 104: 446-454. Pena-Cortes, H., Fisahn, J., and Willmitzer, L. (1995) Signal involved in wound- induced proteinase inhibitor II gene expression in tomato and potato plants. Roc. Natl. Acad. Sci. USA 92: 4106-41 13.

Pena-Cortes, H., Liu, X., Sanchez-Serrano, J. J., Schmid, R., and Wïtzer, L. (1992)Factors affecthg gene expression of patatin and proteinase-inhibitor41 gene families in detached potato leaves. Planta 186: 495-502.

Pena-Cortes, H., Sanchez-Serrano, J. J., Mertens, R., Willmitzer, L., and Prat, S. (1989)Abscisic acid is involved in the wound induced expression of the proteinase inhibitor II gene in potato and tomato. Proc. Natl. Acad. Sci. USA. 86:9851-9855.

Pena-Cortes, H., Sanchez-Serrano, J. J., Rocha-Sosa, M. M., and Willmitzer, L. (1988)Systemic induction of proteinase inhibitor II gene expression in potato plants by wounding. Planta 174: 84-89.

Pena-Cortes, H., Willmitzer, L., and Sanchez-Serrano, J. J. (1991) Abscisic acid mediates wound induction but not developmental-specific expression of the proteinase inhibitor II gene family. Plant Cell3: 963-972.

Pemollet. and Mosse. (1983) Structure and location of legume and cereal seed storage proteins. In: Seed proteins. Daussant, J.. Mosse, J., and Vaughan, J., eds, Academic Press, New York, London 155-19 1.

Peumans, W. J., Nsirnba-Lubaki, M., Broekaert, W. F., van Damme, and Els, J. M. (1986) Are bark lectins of elderberry (Sumbucus nigra) and black Locust (Robinia pseudoacacia) storage proteins? In: Molecular biology of seed storage proteins and lectins. Plant. Physiol. 53-63.

Polacco, J. C., Thomas, A., L., and Bledsoe, P. J. (1982)A soybean seed urease-nuU produces urease in cell culhue. Plant Physiol. 69: 1233- 1240.

Pomeroy, M. K., and Siminovitch, D. (1971) Seasonal cytological changes in secondary phloem parenchyma cells in Robinia pseudoacacia in relation to cold hardiness. Can. J. Bot. 49: 787-795.

Porneroy, M. K., Suninovitch, D., and Wightman, F. (1970) Seasonal biochemical changes in the living bark and needles of red pine (Pinus resinosa) in relation to adaption to freezing. Can. J. Bot. 48: 953-967. Pramanik, S.K., Reynolds, T. L., MacIsaac, S. A., and Bewley, J. D. (1993) Rapid and efficient purifcation of seed messenger RNA without pheno1:chloroform extraction. Seed Sci. Res, 3: 137-139.

Rapp, W. D., Lilley, G. G., and Nielsen, N. C. (1990) Characterization of soybean vegetative storage proteins and genes. Theor. Appl. Genet. 79: 785-792.

Rhee, Y., and Staswick, P. E. (1992a) Nucleotide sequence of a soybean vegetative storage protein vspA gene. Plant Physiol. 98: 792-793.

Rhee, Y., and Staswick, P. E. (1992b) Nucleotide sequence of a soybean vegetative storage protein vspB gene. Plant Physiol. 98: 794-795.

Roberts, J. K., Disirnone, N. A., Lingle, W. L., and Dure, L. III (1993) Cellular concentrations and uniformity of cell-type accumulation of two Lea proteins in Cotton embryos. Plant Cell5: 769-780.

Ryan, C. (1992) The search for the proteinase inhibitor-inducing factor, PIIF. Plant Mol. Biol. 19: 123-133.

Ryan, C., and An, G. (1988) Molecular biology of wound-inducible proteinase inhibitors in plants. Plant Cell Environ. 11: 345-349.

Ryan, C., Pearce, G., and Kunkel, R. (1976) Variability in the concentration of three heat stable proteinase inhibitor proteins in potato tubers. Am. Potato J. 53: 443-455.

Sadka, A. DeWald, D. B., May, G. D., Park, W. D., and Mullet, J. E. (1994) Phosphate modulates transcription of soybean VspB and other sugar-inducible genes. Plant CeU 6: 737-749.

Salisbury, F. B. and Ross, C. W. (1992) Stress physiology. In: Plant physiology. 4th ed. Wadsworth, Belmont, CA 575-600.

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual. Second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Sanchez-Serrano, J. J., Amati, S., Keil, M., Pena-Cortes, H., Prat, S., Recknagel, C., and Willmitzer, L. (1990) Stress responses and disease resistance. The molecular and cellular biology of the potato. Vayda, M. E. and Park, W. D. eds, C.A.B., International, Wallingford, U. K. 57-69.

Saraste, M., Sibbald, P., and Wittinghofer, A ((1990) The P-loop a common motif in ATP- and GTP-binding proteins. TIBS 90:430-434.

Sauter, J. J,. and van Cleve, B. (1990) Biochemical, immunochemical, and ultrastructural snidies of protein storage in poplar (Populus x canadensis 'robusta ') wood. Planta 183: 92-100.

Sauter, J. J., and van Cleve, B. (1989) Immunochemical localization of a willow storage protein with a poplar storage protein antibody. Protoplasma 149: 175- 177.

Sauter, J. J., van Cleve, B., and Apel, K. (1988) Protein bodies in ray cells of Populus x canadensis Moench 'robusta'. Planta 173: 3 1-34.

Sauter, J. J., and Wellenkamp, S. (1988) Protein storing vacuoles in ray cells of willow wood (Salk carea L.). IAWA (Int. Assoc. Wood Anat.) Bull. 9: 59-65.

Schaffer, M. A. and Fischer, R. L. (1990) Transcriptional activation by heat and cold of a thiol protease gene in tomato. Plant Physiol. 93: 1486- 1491.

Scheurer, S. Metzner, K., Haustein, D., and Vieths, S. (1996) Prunus avium cherry- allergen PRUA 1 mRNA, complete cds. GenBank No. U66076.

Schmelzer, E., Kruger-Lebus, S., and Hahlbrock, K (1989) Temporal and spatial patterns of gene expression around sites of attempted fungal infection in parsley leaves. Plant Cell 1: 993-1001 .

Schoning, B., Vieths, S., Petersen, A., and Baltes, W. (1995) Identification and characterization of allergens related to Bet v 1, the major birch pollen allergen, in apple, cherry, celery and canot by two-dimensional immunobloning and N-terminal mcrosequencing. J. Sci. Food Agric. 65: 43 1-440.

Schoning, B., Ziegler, W. H., Vieths, S., and Baltes, W. (1996) Apple allergy: The cDNA sequence of the major allergen of apple, determineci by perforîning PCR with a primer based on the N-terminal amino acid sequence, is highly hornologous to the sequence of the major birch pollen allergen. J. Sci. Food. Agric. 71: 475-482.

Scopes, R. K. (1994) Protein purification. Springer-Verlag, New York 346-348. Sennerby-Forsse, L. (1986) Seasonal variation in the ultrastructure of the cambium in young stems of willow (Salk viminalis) in relation to phenology. Physiol. Plant. 67:529-537.

Siminovitch, D., and Cloutier, Y. (1982) Twenty-four-hour induction of freezing and drought tolerance in plumules of winter rye seedlings by desiccation stress at room temperature in the dark. Plant Physiol. 69: 250-255.

Siminovitch, D., Rheaume, B., Pomeroy, K., and Lepage, M. (1968) Phospholipid, protein and nucleic acid increases in protoplasm and membrane structures associated with development of extreme fieezing resistance in black locust tree cells. Cryobiol. 5: 202-225.

Somssich, 1. E., Schmelzer, E., Bollma~,J., and Hahlbrock, K. (1986) Rapid activation by fungal elicitor of genes encoding 'pathogenesis-related' proteins in cultured parsley cells. Proc. Natl. Acad. Sci. USA 83:2427-2430.

Somssich, 1. E., Schmelzer, E., Kawalleck, P., and Hahlbrock, K. (1988) Gene structure and in situ transcript localization of pathogenesis-related protein 1 in parsley. Mol. Gen. Genet. 213:93-98.

Spilatro, S. R., and Anderson, J. M. (1989) Characterization of a soybean leaf protein that is related to the seed lectin and is increased with pod removal. Plant Physiol. 90:1387- 1393.

Staswick, P. E. (1989) Developmental regulation and the influence of plant sinks on vegetative storage protein gene expression in soybean leaves. Plant Physiol. 89: 309- 3 15.

Staswick, P. E. (1990) Novel regulation of vegetative storage protein genes. Plant CeU 2: 1-6.

Staswick. P. E. (1994) Storage proteins of vegetative plant tissue. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 303-322.

Staswick, P. E., Huang, J-F., and Rhee, Y (1991) Nitrogen and methyl jasrnonate induction of soybean vegetative storage protein genes. Plant Physiol. 96: 130-136.

Steponkus, P. L. (1984) Role of the plasma membrane in freezing injury and cold

-224- acclimation. AMU. Rev. Plant Physiol. 35: 543-584.

Stone, K. L., and Williams, K. R. (1993) Enzymatic digestion of proteins and HPLC peptide isolation. In: A practical guide to protein and peptide purification for rnicrosequencing. Matsudaira, P., ed, Second edition, Academic Press, San Diego, CA 43-69.

Sturm A., and Chrispeels, M. J. (1990) cDNA cloning of carrot extracellular P- hctosidase and its expression in response to wounding and bacterial infection. Plant Cell2: 1107-1 119.

Surowy, T. K., and Boyer, J. S. (1990) Low water potentials affect expression of genes encoding vegetative storage proteins and plasma membrane ATPase in soybean. Plant Mol. Biol. 16:25 1-262.

Swoboda, L, Jilek, A., Ferreira, F., Engel, E., Hoffmann-Sornmerpber, K., Scheine, O., Krafe, D., Brieiteneder, H., Pittenauer, E., Schmid, E., Vicente, O., Heberle-Bors, E., Ahom,H., and Breitenbach, M. (1995a) Isoforms of Bet v, the major birch pollen allergen, analyseci by liquid chromatography, rnass spectrometry, and cDNA cloning. J. Biol. Chem. 270: 2607-26 13.

Swoboda, I., Scheiner, O., Kraff D., Breitenbach, M., Heberle-Bors, E., and Vicente, 0. (1994) A birch gene family encoding pollen allergens and pathogenesis-related proteins. Biochem. Biophys. Acta. 1219: 457-464.

Swoboda, I., Scheiner, O, Heberle-Bors, E., and Vicente, O (1995b) cDNA cloning and characterization of three genes in the Bet v 1 gene family that encode pathogenesis-related proteins. Plant Ce11 Environ. 18: 865-874.

Tamiya, T., Okahashi, N., Sakuma, R., Aoyarna, T., Akahane, T., and Matsumoto, J. J. (1985) Freeze denaturation of enzymes and its prevention with additives. Cryobiol. 22: 446-456.

Tanino, K. K., and McKersie, B. D. (1984) Injury within the crown of winter wheat seedlings after freezing and icing stresses. Can. J. Bot. 63: 432-436.

Taylor, B. K., and May, L. H. (1967) The nitrogen nutrition of the peach tree. II. Storage and mobilization of nitrogen in young trees. Aust J. Biol. Sci. 20: 389-4 11.

Taylor, B. K., van den Ende, B., and Canterford, R. L. (1975) Effects of rate and timing of Ritrogen applications on the performance and chernical composition of young pear trees cv. Williams Bon Chretien. J. Hort. Sci. 50: 29-40.

Tecsi, L. L, Made, A. J., Smith, A. M., and Leegood, R. C. (1994) Complex, localized changes in CO2 assimilation and starch content associated with the susceptible interaction between cucumber mosaic virus and a cucurbit hostoPlant J. 5: 837-847.

Thomashow, M. F. (1993) Gene induced during cold acchation in higher plants. In: Advances in low temperature biology. Steponkus, P. L. ed, JAI press, London, 2: 183-21 O.

Todd, L. (1991) The role and the regdation of storage proteins in the bark of temperature tree species. MSc thesis. Department of Botany, University of Guelph.

Tranbarger, T. J., Franceschi, V. R. Hildebrand, D. F., and Grimes, H. D. (1991) The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles. Plant Cell3: 973-987.

Tromp, J. (1983) Nutrient reserves in roots of fniit trees, in particular carbohydraies and nitrogen. Plant Soil. 73: 410-413.

Tromp, J., and Ovaa, J. C. (1973) Spring mobilization of protein nitrogen in apple bark. Physiol. Plant. 29: 1-5.

Tromp, J., and Ovaa, J. C. (1979) Uptake and distribution of nitrogen in young apple trees afier application of nitrate or ammonium, with special reference to asparagine and arginine. Physiol. Plant. 45: 23-28.

Tronsmo, A. M. (1984) Predisposing effects of low temperature on resistance to winter stress factors in grasses. Acta Agric. Scand. 34: 210-220.

Tronsmo, A. M. (1985) Inducecl resistance to biotic stress factors in grasses by frost hardening. In: Plant production in the north. Kaurin, A., Junttila, O., and Nilsen, J. eds. Norwegian University Press, Tromso, Norway 127- 133.

Tronsmo, A. M. (1993) Resistance to winter stress factors in half-subfamilies of Dactylis glomeruta, tested in a controlled environment. Acta Agric. Scand. 43: 89-96.

Tronsmo, A. M., Gregersen, P., Hjeljord, L., Sandal, T., Bryngelsson, T., and Collinge, D. B. (1993) Cold-induced disease resistance. In: Mechanisms of plant defence responses. Fritig, B., and Legrand, M., eds, Kluwer Academic, The Netherlands 369.

Truernk E., Schmid, J., Epple, P., IlIig. J., and Sauer, N. (1996) The sink-specific and stress-regdateci Arabidopsis STP4 gene: enhanced expression of a gene encoding a monosaccharide transporter by wounding, elicitors and pathogen challenge. Plant Cell8: 2169-2182.

Urao, T., Katagiri, T., Mizoguchi, T., Yamaguchi-Shinozaki, K., Hayashida, N.. and Shinozaki, K. (1994) Two genes that encode Ca2+-dependent protein kinases are induced by drought and hi&-sdt stresses in Arabidopsis thaliana. Mol. Gen. Genet. 244: 331-340.

Van Cleve, B., and Apel, K. (1993) Induction by nitrogen and low temperature of storage protein synthesis in poplar trees exposed to long days. Planta 189: 157- 160.

Van Cleve, B., Clausen, S., and Sauter, J. J. (1988) Immunochernical localkation of a storage protein in poplar wood. J. Plant Physiol. 133: 371-374.

Vanek-Krebitz, M., Hoffmann-Sommergruber, K., and Breiteneder, H. ( 1995) Cloning and sequencing of Mal d 1, the major allergen from apple (Malus domestica), and its immunological relationship to Bet v 1, the major birch pollen. Biochem. Biophys. Res. Comrn. 214538-551.

Vick, B. A., and Zimmerrnan, D. C. (1984) Biosynthesis of jasmonic acid by several plant species. Plant Physiol. 75: 458-46 1.

Vierling, E. (1991) The roles of heat shock proteins in plants. AMU. Rev. Plant Physiol. Plant Mol. Biol. 42: 579-620.

Walter, M. H., Liu, J. W., Grand, C., Lamb, C. J., and Hess, D. (1990) Bean pathogenesis-related (PR) proteins deduced from elicitor-induced transcripts are members of a ubiquitous new class of consewed PR proteins hcluding pouen allergens. Mol. Gen. Genet. 222: 353-360.

Wamer, S. A. J., Scott, R., and Draper, J. (1992) Characterisation of a wound- induced trmscript fiom the monocot asparagus that shares similarity with a class of intracellular pathogenesis-related (PR) proteins. Plant Mol. Biol. 19: 555-56 1. Weidhase, R A., Le-, J., Ktamell, H. M., Sembdner, G., and Parthier, B. (1987) Degradation of ribulose-1,5-bisphosphate carboxylase and chlorophyll in senescing barley leaf segments ûiggered by jasmonate, by jasmonic acid methylester, and counteraction by cytokinin. Plant Physiol. 69: 161-1 66.

Wenzler, H., Mignery, A., Fisher, L., and Park, W. (1989) Analysis of a chimeric class-1 patatin-GUS gene in transgenic potato plants: High level expression in tubers and sucrose-inducible expression in cultured leaf and stem explants. Plant Mol. Biol. 12: 41-50.

Wetzel, S., Demmers, C., and Greenwood, J. S. (199 1) Protein-storing vacuoles in inner bark and leaves of softwoods. Trees 5: 196-202.

Wetzel, S., Demmers, C., and Greenwood, J. S. (1989a) Spherical organelles, analogous to seed protein bodies, fluctuate seasonally in parenchymatous cells of hardwoods. Can. J. Bot. 67: 3439-3445.

Wetzel, S. Demmers, C., and Greenwood, I. S. (1989b) Seasonally fluctuahg bark proteins are a potential form of nitrogen storage in three temperate hardwoods. Planta 178: 275-28 1.

Wetzel, S., and Greenwood, J. S. (1989) Protein as a potential nitrogen storage compound in bark and leaves of several softwoods. Trees. 3: 149-153.

Wetzel, S., and Greenwood, J. S. (1991) The 32-kilodalton vegetative storage protein of Salk microstachya Turz. Characterization and immunolocalization. Plant Phsiol. 97: 771-777.

Williamson, V. M., and Colwell, G. (1991) Acid phosphatase-1 frorn nematode resistant tornato. Plant Physiol. 97: 139- 146.

Wittenbach, V. A. (1982) Effect of pod removal on leaf senescence in soybeans. Plant Physiol. 70: 1544- 1548.

Wittenbach, V. A. (1983a) Effect of pod removal on leaf photosynthesis and soluble protein composition of field-grown soybeans. Plant Physiol. 73: 121- 124.

Wittenbach. V. A. (1983b) hirification and characterization of a soybean leaf storage glycoprotein. Plant Physiol. 73: 125-129. Wolfe S. T. (1993) Molecular and cellular biology. Wadsworth ,Belmont, California. CA 592-594.

WolfkÛm, L. A., Langis, R., Tyson, H., and Dhindsa, R. S. (1993) cDNA sequence, expression, and transcript stability of a cold acclimation-specific gene, casl& of alfalfa (Medicago falcuta) ceIls. Plant Ph ysiol. 10 1: 1275- 1282.

Wright, D. P., Baldwin, B. C., Shephard, M. C., and Scholes, J. D. (1995) Source- sink relationships in wheat leaves infected wiîh powdery mildew. 1. Alterrations in carbohydrate metabolism. Physiol. Mol. Plant Pathol. 47: 237-253.

Xu, X. Y. (1993) Seasonal changes in BSP (bark storage protein) specific mRNA in willow (Salix alba L.) MSc thesis. Departrnent of Botany, University of Guelph.

Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, bw-temperature, or high-salt-stress. Plant Cell6: 25 1-264.

Yelenoslq, G., and Guy, C. L. (1989) Freezing tolerance of citrus, spinach, and petunia leaf tissue. Osmotic adjustment and sensitivity to freeze induced cellular dehydration. Plant Physiol. 89: 444-45 1.

Zaidi, 2. H., and Smith, D. L. (1996) Protein structure-function relationship. Plenum Press, New York.

Zhang, L., Dunn, M. A., Pearce, R. S., and Hughes, M. A. (1993) Analysis of organ specificity of a low temperature responsive gene family in rye (Secale cereale L.) J. EXP.Bot. 44: 1787-1793.

Zhong, P-Y.,Tanaka, T., Yamauchi., DmT and Minamikawa, T. (1997) A 28- kilodalton pod storage protein of French bean plants. Plant Physiol. 113: 479-485.

Zhu, B.. Chen, T. H. H., and Li, P. H. (1993) Expression of an ABA-responsive osmotin-like gene during the induction of fieezing tolermce in Solanum commersonii. PIant Mol. Biol. 2 1: 729-735. l MAGE EVALUATION TEST TARGET (QA-3)

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