Transcripts Accumulating during Cold Storage of Potato ...Transcripts Accumulating during Cold...

Plant Physiol. (1994) 104: 445-452

Transcripts Accumulating during Cold Storage of Potato (Solanum tuberosum 1.) Tubers Are Sequence Related to

Stress-Responsive Genes'

Joachim van Berkel, Francesco Salamini, and Christiane Gebhardt*

Max-Planck-lnstitut für Züchtungsforschung, Carl-von-Linné-Weg 1 O, D-50829 Koln, Germany

During the adaptation of plants to low temperature, changes in gene expression can be induced in a variety of tissues. Low- temperature-regulated gene expression was studied in cold-stored potato (Solanum tuberosum 1.) tubers by two-dimensional electrophoresis of in vitro translation products. As a response to cold treatment, the relative amount of mRNA encoding at least 26 polypeptides changed. By differential screening of a cDNA library, 16 clones corresponding to cold-inducible transcripts were isolated. lhey were classified into four non-cross-hybridizing groups. RNA hybridizations using representative clones from each group revealed different temporal accumulation patterns for the cold- inducible transcripts. mRNAs homologous to the cDNA clones were first detectable after 1 to 3 d of cold treatment, and the highest level of expression was reached after 3 to 7 d. Transcripts corresponding to cDNA clones C113 and C119 were transiently expressed, whereas the steady-state level remained high for cDNA clones C17 and Cl2l during the cold storage period of 4 weeks. lhe DNA sequences of two cDNA clones, C17 and C119, have been determined. lhe polypeptide predicted from the DNA sequence of C119 is sequence related to small heat-shock proteins from other plant species. lhe deduced protein sequence of C17 exhibits strong homology to the dehydrin/RAB group of dehydration stress- and abscisic acid-inducible polypeptides and to cold-induced proteins from Arabidopsis and spinach.

Higher or lower than optimal temperatures exert stress on plants and induce changes in cellular metabolism leading to adaptation (Sachs and Ho, 1986; Guy, 1990). Severa1 tem- perate plants increase their freezing tolerance ("cold harden- ing") when exposed to low, nonfreezing temperatures. In storage organs of many such species, sugars accumulate in response to the cold treatment, a phenomenon known for a long time as "cold sweetening" (Miiller-Thurgau, 1882). Mechanisms and regulation of the biochemical processes leading to cold sweetening are in large part still unclear. What is known is that chilling alters the lipid composition of the cell membranes and increases the enzymic activity of specific steps in different metabolic pathways related to carbohydrate and amino acid metabolism (Guy, 1990; Thomashow, 1990). This implies that a shift to high or low temperatures can

' Supported by European Chips and Snacks Association Research Ltd. and the European Community under European Collaborative Linkage of Agriculture and Industry through Research contract No. AGRE 0001.

* Corresponding author; fax 49-221-5062-413. 445

induce differential gene expression, as first observed and extensively investigated in the case of heat shock (reviewed for plants by Sachs and Ho, 1986).

The number of studies describing cold-induced variations in mRNA populations is steadily increasing (reviewed by Guy, 1990, and Thomashow, 1990). Moreover, a number of cDNA clones encoding cold-regulated transcripts have been isolated from aerial tissue of alfalfa, Arabidopsis, barley, spinach, tomato, and wheat (for review see Cattivelli and Bartels, 1992, and refs. therein). Among the few cold-induced genes for which a possible function has been proposed is C14 of tomato, a gene encoding a protein closely related to thiol proteases (Schaffer and Fischer, 1988).

Low-temperature-regulated gene expression in cold-stored potato (Solanum tuberosum L.) tubers is the subject of this paper. Cold-induced metabolic changes leading to the accumulation of SUC, Glc, and Fru, resulting from starch-sugar interconversion, are typically observed in harvested potato tubers (Isherwood, 1973). This sweetening during long-term conservation of tubers at low temperature is negatively cor- related with the quality of processed products (Schwimmer et al., 1957). The characterization and the study of cold- inducible genes expressed in potato tubers and their promoters is, therefore, part of a strategy to suppress cold- temperature sweetening by manipulating the expression of certain genes at low temperature.

MATERIALS AND METHODS

Plant Material and Chilling Conditions

Tubers of field-grown potato plants (Solanum tuberosum ssp. tuberosum L. cv Saturna) were stored after harvest for 4 to 6 weeks at 20°C and then kept at 4OC between 1 and 28 d. Tuber samples were removed from storage at 4OC after 1, 3, 7, 10, 14, 21, and 28 d. After 28 d at 4OC the tubers were shifted back to 20°C for 1 and 7 d. Control tubers were stored continuously at 20°C for the whole period. Sampled, tubers were peeled, cut in small pieces, immediately frozen in liquid N1, and stored at -8OOC.

RNA lsolation

For the extraction of total RNA the method described by Schroder and Schroder (1982) and Schroder et al. (1988) was

Abbreviation: RFLP, restriction fragment length polymorphism.

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446 van Berkel et al. Plant Physiol. Vol. 104, 1994

used with slight modifications. Fifty grams of frozen tuber material were ground to a fine powder in a Waring blender with liquid NZ and dispersed in 50 mL of prewarmed (65OC) 0.4 M Tris-HC1 (pH %O), 0.1 M NaCl, 0.04 M EDTA, 5% (w/v) SDS, 0.1% (v/v) 2-mercaptoethanol. Following two pheno1:chloroform (1:1, v/v) extractions, macromolecular contaminants were removed by centrifugation after precipi- tation at room temperature by ethanol added to a final concentration of 20% (v/v). Nucleic acids were ethanol pre- cipitated and collected by centrifugation. The nucleic acid pellet was washed twice at 4OC with 3 M Na-acetate (pH 6.0) and dissolved in sterile high-purity water. Poly(A)+ RNA was isolated by affinity chromatography on oligo(dT)-cellulose (Maniatis et al., 1982).

In Vitro Translation of Poly(A)+ RNA

In vitro translation of polyadenylated tuber RNA was performed for 90 min at 3OoC using a rabbit reticulocyte lysate (N 90; Amersham, Braunschweig, Germany). Transla- tion mixtures contained 80% (v/v) reticulocyte lysate, 2 pg of poly(A)+ RNA, and 3.02 X 105 Bq [35S]Met (Amersham; 3 X 1OX3 Bq mmol-’) in a total volume of 23 pL. For two-dimensional electrophoresis, 23 mg of urea and 1 volume of sample buffer (9.5 M urea, 2% LKB ampholine [pH 3.5-101, 5% [v/v] 2-mercaptoethanol, 2% [v/v] Nonidet P-40) were added, and the mixture was loaded onto IEF tube gels.

Two-Dimensional Electrophoresis

For the first dimension, proteins were separated on 14-cm- long IEF tube gels (1.5 mm diameter) as described by Bartels et al. (1988). In the second dimension, the focused proteins were separated on 7.5 to 15% gradient polyacrylamide gels overlaid with a 4% polyacrylamide stacking gel (Laemmli, 1970). The gels were fixed in 6% TCA, fluorographed according to the method of Bonner and Laskey (1974), and exposed to Kodak X-Omat x-ray film. Methylated I4C-proteins (Amersham) were used as molecular mass markers for SDS gel electrophoresis.

Construction and Screening of a cDNA Library

A cDNA library was prepared in the X ZAP I1 vector (Short et al., 1988) from 6 pg of poly(A)+ RNA isolated from potato tubers exposed to cold stress at 4OC for 7 d. cDNA synthesis and in vitro packaging were camed out using the ZAP-cDNA synthesis kit (Stratagene, Heidelberg, Germany) according to the manufacturer’s instructions. About 25,000 plaque-form- ing units were differentially screened by in situ plaque hybridization (Maniatis et al., 1982) on Hybond-N nylon filters (Amersham) using two poly(A)+ RNA probes end labeled with 32P according to the method of Bartels and Thompson (1983). The first probe was poly(A)+ RNA isolated from cold- stressed tubers (7 d at 4OC), and the second probe was poly(A)+ RNA from tubers kept continuously at 20OC. Hy- bridization was performed overnight at 65OC in 6X SSC (1X SSC = 0.15 M NaCl and 0.015 M Na-citrate [pH 7.0]), 5X Denhardt’s solution, 1% (w/v) Gly, 0.5% (w/v) SDS, 50 pg mL-’ of denatured salmon sperm DNA, 5 pg mL-’ of poly(A).

Clones hybridizing only to RNA from cold-stressed tubers were selected and rescreened. In vivo excision of the Blue- script SK phagemid from the X ZAP 11 vector was camed out as described by the manufacturer (Amersham).

Northern Blot Analysis

Total RNA (60 pg) from each point of the cold treatment program was separated on 1% agarose gels containing formaldehyde (Maniatis et al., 1982; formaldehyde concentration 0.66 M according to that recommended by Davis et al., 1986) and blotted onto Hybond-N nylon filters (Amersham). The filters were hybridized to [c~-~~P]dCTP-labeled cDNA probes obtained by random primer labeling (Feinberg and Vogel- stein, 1984). Hybridizations were performed ovemight at 42OC in 50% (v/v) formamide, 5X SSC, 5X Denhardt’s solution, 0.5% (w/v) SDS, 20 pg mL-’ of denatured salmon sperm DNA. After hybridization the filters were washed with severa1 buffer changes of 0.5X or 0.1X SSC, 0.1% (w/v) SDS at 65OC. For subsequent hybridizations of the nylon membranes to different DNA sequences, previous probes were removed by incubating the membranes for 15 min in 0.1% (w/v) SDS at 75OC.

Southern Blot and Segregation Analysis

Southern blots of genomic DNA of potato were prepared as described by Gebhardt et al. (1989). Segregation analysis and mapping of cDNA clones were performed according to the method of Gebhardt et al. (1991).

DNA Sequencing and Data Analysis

The DNA sequence of the cDNA clones was detennined in the Bluescript vector on both strands by dideoxynucleotide sequencing with the T7 polymerase kit (Pharmacia, Freiburg, Germany) and sequence-specific oligonucleotide primers (Sanger et al., 1977).

Nucleic acid and protein sequence data were analyzed using the UWGCG program library of the University of Wisconsin Genetic Computer Group (Devereux et al., 1984).

RESULTS

Low Temperature lnduces Changes in the Translatable Tuber mRNA Population

The effect of cold treatment on gene expression in potato tubers was studied by two-dimensional electrophoresis of in vitro translation products. Poly(A)+ RNA was isolated from potato tubers (cv Satuma) stored at 4OC for 3, 7, and 14 d and from unstressed tubers stored at 2OoC for 14 d as the control. The RNAs were translated in vitro in the presence of [35S]Met in a rabbit reticulocyte lysate system; labeled translation products were analyzed by two-dimensional electrophoresis, followed by fluorography.

The protein patterns revealed by two-dimensional electrophoresis consisted of more than 250 spots. Whereas the leve1 of most of the translation products remained unchanged during cold treatment, reproducible changes due to cold storage were observed for at least 26 polypeptides. Figure 1,



Cold-Regulated cDNAs from Potato Tubers 447

© e IEF

AMr X 10'3

7d 4°C

Control

92.5—

69 —

30 — »•

14.3- »

BMr X 10"3

92.5—

69 — «

46 —

30 — «•

14.3-

cMr X ID'3

92.5—

69 —

46 —

30 —

14.3— »o«

Figure 1. Two-dimensional separation (IEF/SDS-PACE) of labeledin vitro translation products of polyfA)* RNA isolated from potatotubers. A, Fluorograph of the products of poly(A)+ RNA from tubersstored for 7 d at 4°C. B, Fluorograph of the products of poly(A)+

RNA from control tubers kept at 20°C. Polypeptide products forwhich changes were observed reproducibly during cold treatmentof potato tubers are indicated by squares. C, Schematic drawing ofthe translation product patterns. Solid spots indicate translationproducts increasing during cold treatment. Spots are numberedaccording to the group to which they have been assigned based oninduction kinetics (see text).

IEF

A and B, shows the patterns of in vitro synthesized proteinsderived from poly(A)+ RNA of potato tubers exposed to a 7-d cold treatment and from poly(A)+ RNA of unstressedtubers, respectively. In the figure, spots representing majorchanges are shown in boxes. The induction kinetics of cold-regulated mRNAs was studied by comparing the translationproducts obtained at different times of the cold treatment.According to the changes observed, the polypeptide productswere assigned to five different groups. In the schematicrepresentation of the two-dimensional profiles (Fig. 1C), theinduction behavior of the polypeptide products can be in-ferred based on the group number attached to the spots. Thepolypeptides of group 1 increased rapidly to a high, constantlevel within 3 d after transfer of the tubers to 4°C. Group 2proteins did not increase before 1 week of cold storage. ThemRNAs coding for group 3 proteins accumulated continu-ously during the period assayed. Group 4 polypeptides weretransiently induced within the 1st week of cold treatment.Except for those of group 4, all cold-induced polypeptideswere also detectable in low amounts in the translation prod-ucts of control RNA prepared from unstressed tubers. Thegroup 5 polypeptides decreased or disappeared after the shiftto 4°C.

Based on temperature-dependent mRNA kinetics, tubersstored for 7 d at 4°C were chosen as source materialof poly(A)+ RNA for the construction of a cDNA library: atthis stage nearly all detectable cold-induced proteins werepresent.

Isolation of cDNA Clones EncodingCold-Induced Transcripts

About 25,000 recombinant phages were differentiallyscreened by in situ plaque hybridization using as differentialprobes 32P-end-labeled poly(A)+ RNA from cold-stored tub-ers and from unstressed control tubers. Based on reproducibledifferential hybridizations, 16 cDNA clones putatively cor-responding to cold-regulated transcripts were selected. Thesize of the cDNA inserts ranged from 500 to 1300 bp. ThecDNA clones were classified into four groups by cross-hy-bridization experiments with Southern blots of the restrictionenzyme-digested plasmids (data not shown). Representativeclones of each of the four groups (CI7, CI13, CI19, CI21)were further characterized by northern analysis.

Expression of Cold-Induced Transcripts

By RNA gel blot hybridization experiments with total RNA,we confirmed that the four selected cDNA clones (CI7, CI13,CI19, CI21) were homologous to cold-inducible transcripts(Fig. 2). No hybridization signal was detected with RNA fromcontrol tubers stored at 20°C (0 d, 4°C). The size of thetranscripts was 1.0 to 1.1 kb for CI7 and CI19, 1.2 kb forCI13, and 0.8 to 0.9 kb for CI21.

The time course of the accumulation of cold-regulatedtranscripts was investigated using total RNA from tubers thathad been stored for 1 to 28 d at 4°C and RNA from tubersthat were shifted back to 20°C after 28 d of cold storage.Transcripts homologous to the selected cDNA clones werefirst detectable after 1 to 3 d of treatment (Fig. 2). Maximum




CI7

CI13

CI19

CI21

4324

0 1 3 7 1428 28281 7

days, 4 °Cdays, 20 °C

Figure 2. Northern gel blot hybridizations of four selected cold-induced cDNA clones to total RNA (60 fig per lane) isolated frompotato tubers stored at 4°C and 20°C for the number of daysindicated. The transcripts hybridizing to CI7 and CM 9 are 1.0 to 1.1kb long. CI13 detects a transcript of 1.2 kb, and CI21 detects atranscript of 0.8 to 0.9 kb. Bottom panel, As a control to ensure thatequal amounts of RNA were loaded in each lane, the same filter asabove was hybridized with the potato clone 4324 coding for rRNA.

ative of a single or low copy number of the correspondinggenes (data not shown). Several RFLP alleles were present inthe germplasm pool. Segregation analysis allocated all majorgenomic restriction fragments to a single genetic locus, con-firming, therefore, the single-copy nature of the genes codingfor CI7 and CI19 (data not shown). CI7 was linked withoutrecombination to marker loci GP75 and GP165 on potatochromosome IV. CI19 was mapped to the chromosome seg-ment between marker loci GP198(a) and CP137(c) on chro-mosome XI (Gebhardt et al., 1991).

Genomic restriction patterns obtained with CI13 and CI21

CI71 TATAATTTCAATATTCAAACTTTCACTTTACGTTAAAaTTCTTCATAATTTGTTTaATTG 60

61 AAAAAAAAGAAGAACAAAlEHBGCTGATCACTACGAACAGAAGAAGCCATCAGTTGAAGA 1201 M A D Q Y E Q N K P S V E E 1 4

121 GACTGTTGGTGCCAAOOTGGAGGCTAGTGATCGTCGTGTGTTTGATTTCATTGTGAGTAA 1801 5 T V G A N V E A S D R G V F D F I V S K 3 4

181 AAGAGAGGAAAAACCAACTCATGCTCATGAAGAAGACGCAATTTCATCTGAGTTTTGTGA 2403 5 K E E K P S H A H E E E A I S S E F C E 5 4

241 GAAAGTTAAAGTAAGTGAAGAAGAAGAACACAAGGAGCAAAAGAAAGAAGACAAGAAACT 5005 5 K V K V S E E E E H K E E K K E E K K L 7 4

}01 TCATGCATOAAGTAGCACCTCTAGTAGCTCGAGTGACGAGGAGGAAGAAATTCGAGACCA 36075 H R S S S S S S S S S D E E E E I C E D 94

361 TGGACAGATAACCAAGAAGAAGAAAAAGAAGGGATTGAAGGAAAAGATTAAGGAAAAAAT 4209 5 G Q I T K K K K K K C L K E K I K E K I 1 1 4

421 ATGTGGTGAioAOAAGGAACAAGTGAAAAGACAGGATAGCTCAGTTCCAGTTGAGAAATA 4801 1 5 S G D H K E E V K T E D T S V P V E K Y 1 3 4

481 CGAGGAAACAGAGGAGAAAAAAGGATTTCTAGAAAAAATTAAGGAGAAATTGCCAGGTGG 5401 3 5 E E T E E K K C F L E K I K E K L P C G 1 5 4

541 AGGAGATAAGAAGAGGaAaGAAGTGGCGaCGCGAOGAGCACCACCACCOGCTaCGGTGGA 6001 5 5 G H K K T E E V A A P P P P P P A A V E 1 7 4

601 aCATGAGCCCGACGGAAAGGAGAAAAAGCGATTTTTGGACAAAATTAAGGAGAAATTACC 6601 7 5 H E A E G K E K K G F L D K I K E K L P 1 9 4

661 AGGATAGGACTCAAAGAGTGAAGAGOAAAAGCAGAAGCAAAAACACSZEATTAAATGAAT 7201 9 5 G Y H S K T E E E K E K E K D - 2 0 9

721 GTTTATTTGTTGATGTTTTTTACTTTTCCCATGTTATGATTaTGTTTCTGTGTTCCTTTG 780

781 GTTATTAAGTGTTTTTATTTCCTTCACTTTGTTTTTTaGATTTTGAACCATTTTGTTTAG 840

841 TACTGTATTTGATATTTCTGTATGTAAGTGCATATATATTGTTTTGTATATGTGCCTTAT 900

901 GTGTATGAACTTTGATGATATGGATTTGTAAAATGCAGTTGATTTTAAAAAAAAAAAAAA 960

961 AAAAAAAAAAA 971

induction was reached after 3 d for clones CI7 and CI19 andafter 7 d for CI13 and CI21. The mRNA species correspond-ing to clones CI13 and CI19 were transiently expressed,whereas the steady-state level of the transcripts correspond-ing to clones CI7 and CI21 remained stable during the wholeperiod of cold storage. The amount of all transcripts decreasedstrongly after the tubers were shifted back to 20°C.

The presence of equal amounts of total RNA on the north-ern blots was confirmed by control hybridizations using asprobe a clone coding for rRNA of potato (Landsmann andUhrig, 1985).

Location of Cold-lnducible Genes on the Potato RFLPLinkage Map

The inserts of cDNA clones CI7, CI13, CI19, and CI21were used to assay on Southern blots the DNA polymorphismpresent in a germplasm pool of diploid potato breeding linesand to determine the position of the corresponding genes onthe potato RFLP linkage map (Gebhardt et al., 1991). Germ-plasm survey Southern filters probed with CI7 or CI19showed low complexity of genomic fragment patterns indie -

CI191 GAAGATOATCAAAAGTACATAGTATAG'ItJDEAGGGTCATCAGCAAATTAACATTGCTCAT 601 M R V I S K L T L L I 1 1

61 CATTTCTATTGCTTCGATTTTCCAGGTATCATCACTAGCCGCAGATCCGTCATCACTTCT 1201 2 I S I G C I F Q V S S L C A D A S S L L 3 1

121 ACCATTAATCTTACAGCAAATGATTaGCAGCAATCCTCCTAATACATTTCTTCATCCATT 1803 2 P L I L D Q M I C S N P A N T F L D P F 5 1

181 GAAAGTTTTGGAACAAATACCATTGGGATTAGAAAAGAGAGAGGAGAGTACTCTGCCGCT 2405 2 K V L E Q I P F G L E N R E E T T L P L 7 1

241 TTOTATTGCCAGACTAGACTGGAAAGAGACCGCGGAGGGCCACGTGATAAGOATAGACGT 3007 2 S I A R V D W K E T A E G H V I S I D V 9 1

301 ACCACCATTGAAGAAAGATCATATAAAOATAGAOATTGAAGAAAAGAGAGTGTTGAGAGT 3609 2 P G L K K D D I K I E I E E N S V L E V 1 1 1

361 GAGGGGAGACCGGAAGAAAGAAGAAGAGAAAAATCATOAGCACAATGATTGGCATTGTGT 4201 1 2 S G E R K K E E E K N D E Q N H U H C V 1 3 1

421 TGAAACCACCTACCaCAACTTCTGCAOACAGTTCCGTTTaCCGCAOAATGCTGATATTGA 4801 3 2 E R S Y G K F U H Q F R L P E N A D I D 1 5 1

481 TACAATGAAAGCTAAGCTTGAAAATGGGOTGCTTACTATTTGTTTTGCTAAATTaTCTCC 5401 5 2 T M K A K L E N C V L T I S F A K L S A 1 7 1

541 TCATAGAATTAAAGGTCCTAAAGTTGTGTCTATTGAGAGGAAACAAGAAGCAAAAGAGTC 6001 7 2 D R I K G P K V V S I E S K Q E G K E S 1 9 1

601 CTCTGTTAGAGAACAGCTTBfflTTTTATGAGTTACTGTGTTTTCTOTICATOATGTCCTG 660192 S V R E E L • 197

661 CTGTGCTTTAATTTGATGTTTTCTAGTATTTTGOTATCTGATATGTGATAAAAGATGCTT 720

721 AAATGTAATCTTATTTTGAAATATTATATATATTTTTAATTTGAOAAAAAAAAAAAAAAA 780

781 ATAAAAAAAAAAAAAAAAAAAAA 803

Figure 3. Nucleotide sequences and deduced amino acid se-quences of the cDNA clones CI7 and Cl 19. The putative translationstart codons and the stop codons are marked.



Cold-Regulated cDNAs from Potato Tubers 449

A 1

CI19 SIG FQV SSLGA

S F RR.SNV pea 22.7 M : z a F F L g I L A ADFPL alf 18.2 ......................... ...... soy 18.5C ............................ ...... N F RR.NNV

ara 17.4 ............................ ...... S F RR.TNV tom 66 ............................ ...... R I R R S S P

51 100 . . . . : : ::::m pB CI19 pea 22.7 . . . . alf 18.2 VWD P NS ALSASFP... RENS soy 18.5C VWD P NT LSSASFPEFS RESS tom 66 VFD P ST NSG ....... . € O S ara 17.4 VWD F GL TNAPA..... KDVA

101 150 CI19 pea 22.7 alf 18.2 soy 18.5C tom 66 ara 17.4

151 200 CI19 SK pea 22.7 EE alf 18.2 1s

tom 66 1s ara 17.4 1s

soy 18.5C 1s

c119

alf 18.2 G ............ sov 18.5C G ............ to; 66 ara 17.4

B C17 pea dehyd barley DE maize D3 rice 16C ara RAB18 tom TAS14 cott D11

C17 pea dehyd barley DE maize D 3 rice 16C ara RAB18 tom TAS14 cott Dll

C17 pea dehyd barley D8 maize D3 rice 16C ara RABl8 tom TAS14 cott D11

C17 pea dehyd barley 08 maize 03 rice 16C ara RAB18 tom TAS14 cott D11

C17 pea dehyd barley D8 maize D3 rice 16C ara RABl8 tom TAS14 cott D11

C17 pea dehyd barley D8 maize D3 rice 16C ara RABl8 tom TAS14 cott Dll

G... ......... G ............

1 5 0 .................... ..MADQYEQN KPSVEETVGA NVEASDRGVF .................... ..MAEENQNK YEETTSATNS ETEIKDRGVF ................................. MEY..QG QHGH.ATDKV ................................. MEYGQQG QHGHGATGHV ................................. ME.NYQG QHGYGA.DRV

MAQYGNQDQM RKTDEYGNHV QE. ....... ..TGVYQGTG TGGMMGGTG. .................................. MAHFQN QYSAPEVTQT

MASYQNRPGG QATDEYGNPI QQQYDEYGNP MGGGGYGTGG GGGATGGQGY

51 100

EEYGQPV.AG ......... H GGFTG..GPT GTHGAAGVGG AQLQATRDGH DVYRNPV.AG .QYGGGATAP GGGHGVMGMG GHHAGAG. .. GQFQPVKEEH

DFIVS..KRE EKPSHAHEEE AISSEFCEKV KV ........ SEEEEHKEEK DFLGG..KKK DEEHKPAQDE AIAHEFGHKV TLYEAPSETK VEEEGEKKHT

DQYGNPV.GG VEHGTGGMRH GTGTGGMGQL GEHGGAGMGG GQFQPAREEH

GTGGQGY.GS GGQGYGTGGQ GYGTGTGTEG FGTGGGARHH GQEQLHKESG ..TGGMM.GG TGGEYGT..Q GMGTGT..,. ...... HHHE GQQQLRR ... DAYGNPTRRT DEYGNPIPTQ ETGRGILGIG GHHHGGHHG. .......... 101 150

.......... 151 200

...................... ............ ..HATATTGG ........................................... HHDQSGQ ........................................... QHEG ... ........................................... NKEHQSQ

201 250 AAPPPPFPAA VEHEAEGK.. TTPPPVVAAH VPTETTATTT IHE A ........ G EYAGTHGTE. .AT QH... ..... AYGQQGHTGG AYAT..GTE. .GT H ......... T ...... GAG TGTGVHGAEY GNT H ......... AQAMGGMGSG YDAGGYGGEH H.. GR ........ .......... .EYGQTTGE. ... H ......... ATSTTTFGQG PTYHQHHREE RSD LWSAISY...

... ;;:;i;;a

E.... 261 . DHHKDETTS H ........... ........... ............ ........... ........... ...........

Figure 4. A, Comparison of the amino acid sequences of the predicted CI19 protein and small heat-shock proteins from pea (Helm et al., 1993), alfalfa (alf; Gyorgyey et al., 1991), soybean (soy; Raschke et al., 1988), tomato (tom; Fray et al., 1990), and Arabidop-

were also polymorphic and more complex, suggesting the existence of more than one copy in the potato genome. Segregation analysis revealed two genetic loci for C12 1, both on chromosome IV. One was in the chromosome segment bordered by the marker loci GP220(a) and GP172 and the other was in the segment bordered by the loci TG62 and GP35(d). One locus was identified for CI13 on chromosome V between marker loci GP28(b) and GP198(c) (Gebhardt et al., 1991).

DNA Sequence Analysis of cDNA Clones C17 and C119

The DNA sequences of clones CI7 and CI19, representative of a stably induced and a transiently expressed transcript, respectively, have been determined. Both sequences contain open reading frames coding for small proteins of 23.7 (CI7) and 22.3 kD ((319). The nucleotide sequences and the deduced amino acid sequences are shown in Figure 3.

The cDNA clone CI19 is 803 bp long and contains an open reading frame encoding a polypeptide of 197 amino acid residues. The predicted CI19 protein is sequence related to small heat-shock proteins from pea (Helm et al., 1993), soybean (Schoffl et al., 1984; Raschke et al., 1988), Arabidop- sis (Helm and Vierling, 1989; Takahashi and Komeda, 1989), wheat (McElwain and Spiker, 1989), tomato (Fray et al., 1990), and alfalfa (Gyorgyey et al., 1991). The regions of amino acid homology (in total about 45% identity and 65% similarity) were found dispersed throughout the whole sequence. The best homology (55% amino acid identity and 67% similarity) was found when C119 was compared with a family of small heat-shock proteins localized in the endomembrane system of heat-shocked pea seedlings (Helm et al., 1993). CI19 also shares with this type of protein the N- terminal and C-terminal extensions as compared to other small heat-shock proteins. Helm et al. (1993) showed that the N-terminal extension of the pea proteins codes for a signal peptide. A comparison of CI19 with PsHSP22.7 (pea 22.7) from pea and a selection of four other heat-shock proteins from alfalfa, soybean, tomato, and Arabidopsis is shown in Figure 4A.

cDNA clone CI7 has a length of 971 bp, and the protein deduced from its open reading frame is composed of 209 amino acids. The predicted C17 protein is extremely hydro- philic because of its high content (52%) of charged amino acids (it contains, for example, 48 Glu and 40 Lys residues). The deduced C17 protein sequence exhibits strong homology in characteristic regions to cold-induced proteins from Ara- bidopsis (rabl8; Lang and Palva, 1992, COR47; Gilmour et al., 1992) and spinach (CAP85; Neven et al., 1993). Similar

sis (ara; Takahashi and Komeda, 1989). Regions of homology (identity and similarity) are indicated in black. B, Comparison of the amino acid sequences of the predicted C17 protein and of members of the dehydrin/RAB/LEA family of proteins: pea dehydrin (dehyd) cognate (M. Robertson and P.M. Chandler, unpublished data; CenBank accession No. S25121), barley D8 and maize D3 (Close et al., 19891, rice RAB 16C (Yamaguchi-Shinozaki et al., 1989), Arabidopsis RAB 18 (Lang and Palva, 1992), tomato TAS 14 (Codoy et al., 19901, and cotton (cott) LEA D11 (Baker et al., 1988). The characteristic sequence motifs conserved among the different proteins are shown in black boxes.




homologies were also detected between C17 and several dehydration- and ABA-induced proteins from different plant species, e.g. dehydrins in barley and maize (Close et al., 1989), LEA (late embryogenesis abundant) proteins in cotton (Baker et al., 1988), RAB (responsive to ABA) proteins in rice and maize (Yamaguchi-Shinozaki et al., 1989; Vilardell et al., 1990), and desiccation-induced proteins in Craterostigma plantagineum (Piatkowski et al., 1990). The characteristic sequence features of the C17 protein are a cluster of nine contiguous Ser residues and three repeats of a Lys- and Glu- rich motif that are highly conserved between the above- mentioned proteins. Unlike the CI7 polypeptide, most of the related proteins contain only two copies of the Lys-rich motif. Figure 4B shows the amino acid sequence comparison between C17 and a selection of seven related polypeptide sequences from different species.

DISCUSSION

Regulation of gene expression in response to cold stress has been studied until now in leaves, shoots, and roots of a number of plant species (Cattivelli and Bartels, 1992) but not in plant storage organs such as potato tubers. Here, different regulatory elements might be involved in adaptation to low temperatures and long-term preservation of viability in the cold. Promoters that can be activated by shifting tubers to low temperatures and deactivated by retuming them to nor- mal temperatures would be useful tools for the temperature- regulated expression of truns-genes in tubers. As a first step toward the identification of such cold-inducible promoters, we studied the modification of transcript levels upon low- temperature treatment in potato tubers. At least 22 transcripts encoding translation products ranging in size from 10 to 60 kD increased in their steady-state levels (Fig. 1). The majority of the translatable mRNAs present in untreated tubers remained, however, detectable at a similar level after cold treatment. Such a behavior has been reported for other plant species exposed to cold stress (Meza-Basso et al., 1986; Mo- hapatra et al., 1987; Cattivelli and Bartels, 1989) and is quite different from the typical plant response to heat shock (Sachs and Ho, 1986; Guy, 1990).

Different accumulation kinetics have been observed: a first group of translation products reached a plateau within 3 d after a strong increase; others did not increase before 1 week in cold storage (group 2 proteins) or accumulated continuously during 2 weeks (group 3 proteins). A fourth group of proteins appeared only transiently within 2 weeks. Because the first RNA sample was taken 3 d after the start of the cold treatment, it is possible that some mRNAs coding for proteins of group 1 had started to accumulate even earlier. An increase of cold-inducible translation products as early as within 6 h was described, for instance, in barley coleoptiles (Cattiv'elli and Bartels, 1989) and within 24 h in Arabidopsis plantlets subjected to low-temperature treatment (Kurkela et al., 1988). On the other hand, a delayed component of the cold-stress response, as observed in the group 2 proteins of potato tubers, has also been found in spinach leaves 8 d after starting the cold treatment (Guy et al., 1985) and in plantlets of Solanum commersonii after up to 14 d (Tseng and Li, 1990). The induction pattems we have obtained with cold-stressed tub-

ers of S. tuberosum are comparable to the cold-induced alterations in the pattem of in vitro translation products of RNA from stem-cultured plantlets of the tuber-bearing wild species S. commersonii (Tseng and Li, 1990). Most of the changes in the tubers appeared to have occurred or to have been initiated after 7 d in cold storage. This stage was, therefore, chosen for cDNA cloning of cold-induced transcripts.

Sixteen cDNA clones were isolated by differential screening and classified into four non-cross-hybridizing groups. The finding of several independent clones for each group indicates that they correspond to transcripts of relatively high abundance in the mRNA population from which the library was constructed. A large proportion of the more than 20 cold-induced transcripts that had been identified by their translation products was not detected by the differential screening method used. The difference between the relative abundance of those transcripts in the two mRNA populations used for differentially screening the cDNA library might not have been sufficiently large to allow detection. RNA hybridizations using representative cDNA clones of each group as probes revealed that the homologous transcripts accumulated to a high level in potato tubers in response to the cold. They were not detectable in the mRNA population of unstressed tubers. Transcripts homologous to cDNA clones C17 and CI19 reached their highest level of expression after 3 d and those corresponding to clones CI13 and CI21 after 7 d (Fig. 2). The transcript levels of C17 and CI21 remained high during the cold storage period of 4 weeks and quickly decreased after the tubers were retumed to 2OOC.

In contrast, the level of transcripts corresponding to CI13 and CI19 decreased during prolonged cold storage. Based on their expression kinetics, clone CI7 may code for a group 1, CI21 for a group 3, and CI13 for a group 4 polypeptide. cDNA clones corresponding to group 2 polypeptides were not recovered. CI19 showed an expression pattem not clearly compatible with any of the protein groups described by the in vitro translation experiments. Both the stable expression in the cold and the decline of the mRNA level after an increase in the temperature have been described for cold- induced cDNA clones from several species (Schaffer and Fischer, 1988; Mohapatra et al., 1989; Cattivelli and Bartels, 1990; Hajela et al., 1990; Kurkela and Franck, 1990). Tran- sient expression of cold-induced transcripts, as observed for CI13 and CI19, was reported only by Neven et al. (1992) for spinach. The transient expression pattem may be an indica- tion that the gene product functions in the process of cold adaption, whereas a stable expression would be expected for a gene product required for maintaining the adapted state of the tissue.

Results of northem analysis with the C17, CI13, CI19, and CI21 cDNAs indicate that cold stress induces transcriptional activation of the corresponding genes or the selective stabilization of these mRNAs. Hajela et al. (1990) showed by nuclear run-on transcription experiments that three cold- regulated transcripts of Arabidopsis were controlled primarily at the posttranscriptional level, whereas a fourth one was regulated largely at the transcriptional level. A temperature- regulated thiol-protease gene in tomato was found to be controlled at the transcriptional level (Schaffer and Fischer, 1990). The two possibilities are currently being tested by the



Cold-Regulated cDNAs from Potato Tubers 45 1

functional analysis of the promoters of two of the cold- induced genes described in this paper.

The polypeptide predicted from clone CI19 had consider- able homology to small heat-shock proteins (Schoffl et al., 1984; Raschke et al., 1988; Helm and Vierling, 1989; Mc- Elwain and Spiker, 1989; Takahashi and Komeda, 1989; Fray et al., 1990; Gyorgyey et al., 1991; Helm et al., 1993). The closest resemblance, including the presence of a signal peptide, was found between CI19 and a family of small heat- shock proteins of pea shown to be localized in the endomembrane system (Helm et al., 1993). This sequence homology suggests that CI19 may be resident in the same cellular compartment in the tuber. The same or a similar motif as the sequence NDELK at the carboxyl terminus of the pea protein PsHSP22.7, interpreted as being an ER retention signal (Helm et al., 1993), was, however, not observed in CI19. It is known that, in addition to heat shock, osmotic stress, ABA, or water stress can also induce the synthesis of specific heat-shock proteins (Czamecka et al., 1984; Heikkila et al., 1984). Whether CI19 responds to forms of stress other than cold temperature has not been tested so far. Neven et al. (1992) have also shown that a protein homologous to 70-kD heat- shock proteins increases transiently in spinach seedlings upon cold stress. The induction of similar proteins at both low and high temperatures suggests that the plant response to low temperature may be, in part, functionally similar to the response to high temperature.

cDNA clone C17 encodes a polypeptide homologous to other cold-induced cDNAs of Arubidopsis (Gilmour et al., 1992; Lang and Palva, 1992) and spinach (Neven et al., 1993) as well as a group of proteins induced under dehydration stress like the dehydrins and RAB proteins. The homology was, however, restricted mainly to certain characteristic regions of the polypeptide. A cluster of Ser residues and/or a repeat of a Lys-rich motif, which is present in three copies in C17, are well conserved in a11 of the proteins cited (Fig. 4B). The Lys-rich motif has the potential to form amphiphilic helices, which have been proposed to have a function in the stabilization of desiccation-sensitive sites in the cell (Dure et al., 1989). ABA and desiccation can induce freezing tolerance at noninductive temperatures, and both drought and cold impose osmotic stress on the cell (Guy, 1990, and refs. therein). At the molecular level, genes induced by water stress and ABA are also induced by cold stress in barley, rice, and spinach (Hahn and Walbot, 1989; Cattivelli and Bartels, 1992). In contrast, genes induced by cold temperatures can respond to water stress and ABA (Hajela et al., 1990; Kurkela and Franck, 1990; Nordin et al., 1991). The cited findings suggest that common steps may exist in the pathways leading to protein induction in response to different types of environmental stress. Whether this is also the case in cold adaptation of potato tubers remains to be shown. To test the response of our cold-induced transcripts to other forms of stress, potato plants transformed with promoter/reporter gene fusion con- structs will be studied. Our present data add evidence to the idea that plants respond, in part, to various forms of stress with the synthesis of structurally and functionally related molecules.

Received June 24, 1993; accepted October 11, 1993. Copyright Clearance Center: 0032-0889/94/104/0445/08.

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