Proc. Natl. Acad. Sci. USAVol. 92, pp. 7148-7152, August 1995Plant Biology

Sequence analysis of a mannitol dehydrogenase cDNA from plantsreveals a function for the pathogenesis-related protein ELI3

(salt stress/carbohydrate metabolism/polyols/celery/salicylate)

JOHN D. WILLIAMSON*t, JoHAN M. H. STOOP*, MARA 0. MASSEL*, MARK A. CONKLINGt, AND D. MASON PHARR*Departments of *Horticultural Science and tGenetics, North Carolina State University, Raleigh, NC 27695-7609

Communicated by Charles S. Levings III, North Carolina State University, Raleigh, NC, May 10, 1995

ABSTRACT Mannitol is the most abundant sugar alcoholin nature, occurring in bacteria, fungi, lichens, and manyspecies of vascular plants. Celery (Apium graveolens L.), aplant that forms mannitol photosynthetically, has high pho-tosynthetic rates thought to result from intrinsic differencesin the biosynthesis of hexitols vs. sugars. Celery also exhibitshigh salt tolerance due to the function of mannitol as anosmoprotectant. A mannitol catabolic enzyme that oxidizesmannitol to mannose (mannitol dehydrogenase, MTD) hasbeen identified. In celery plants, MTD activity and tissuemannitol concentration are inversely related. MTD providesthe initial step by which translocated mannitol is committedto central metabolism and, by regulating mannitol pool size,is important in regulating salt tolerance at the cellular level.We have now isolated, sequenced, and characterized a MtdcDNA from celery. Analyses showed that Mtd RNA was moreabundant in cells grown on mannitol and less abundant insalt-stressed cells. A protein database search revealed that thepreviously described ELL3 pathogenesis-related proteins fromparsley and Arabidopsis are MTDs. Treatment of celery cellswith salicylic acid resulted in increased MTD activity andRNA. Increased MTD activity results in an increased abilityto utilize mannitol. Among other effects, this may provide anadditional source of carbon and energy for response to patho-gen attack. These responses ofthe primary enzyme controllingmannitol pool size reflect the importance of mannitol metab-olism in plant responses to divergent types of environmentalstress.

Mannitol is a six-carbon noncyclic sugar alcohol found indiverse organisms ranging from bacteria to higher plants.Mannitol is present in more than 100 species of higher plants,where it can be a significant portion of the soluble carbohy-drate (1-3). For instance, celery (Apium graveolens) translo-cates up to 50% of its photoassimilate as mannitol, with theremainder being sucrose (4). Both translocated carbohydratesare assimilated during growth of nonphotosynthetic hetero-trophic (i.e., sink) tissues. Other postulated roles for mannitolinclude carbon storage, free radical scavenging, and osmopro-tection (4-7).The use of mannitol as a photoassimilate and translocated

carbohydrate is reported to be advantageous to the plant inseveral ways. Celery, a C3 plant, has carbon fixation ratesequivalent to those ofmany C4 plants (8). This may result fromboth increased NADP/NADPH turnover compared to plantsthat exclusively form sugars and from the additional cytosolicsink for photosynthetically fixed CO2 provided by mannitolsynthesis (7, 9, 10). In addition to the increased carbon fixationthat accompanies mannitol biosynthesis, the initial step ofmannitol utilization generates NADH, thus giving a higher netATP yield than the catabolism of an equal amount of sucrose(7). Finally, mannitol-producing plants also exhibit a high

degree of salt tolerance due to the function of mannitol as anosmoregulator and compatible solute (6, 11, 12). Celery plantsgrown in hydroponic culture with a salinity equivalent to 30%that of sea water show dry weight gains equal to plants grownat normal nutrient levels (12). In addition, tobacco that wasgenetically engineered to synthesize mannitol through theintroduction of the Escherichia coli NAD-dependent mannitol-1-phosphate dehydrogenase acquired significant salt tolerance(6).

Metabolite pool sizes in plants are usually determined byrelative rates of synthesis and utilization. The isolation andcharacterization of a plant NAD-dependent mannitol dehydro-genase (MTD), the enzyme responsible for the oxidation ofmannitol to mannose in celery, was reported by our laboratory(13). MTD is a monomeric mannitol:mannose 1-oxidoreductasewith a molecular mass of "40 kDa (13, 14). In celery plants, theexpression ofMTD is highly regulated. MTD activity is highest inyoung actively growing root tips, is also high in young rapidlygrowing (sink) leaves, but is not detected in mature photosyn-thetic (source) leaves. Extractable MTD activity from differenttissues is inversely correlated with mannitol concentration (13).Additional evidence that mannitol oxidation serves as a startingpoint for the entry of carbon into metabolism is that celery tissuesalso contain high hexokinase and phosphomannose isomeraseactivity (15). These three enzymes provide a pathway for theconversion of mannitol to fructose 6-phosphate for entry intocentral metabolism.

Recent characterization of mannitol biosynthetic and uti-lizing enzymes in plants suggests that mannitol pool size isregulated in response to salt stress primarily at the level ofutilization or turnover (11, 12). Salt-stressed celery plantsaccumulate mannitol throughout the plant. This is due pri-marily to the down-regulation ofMTD in sink tissues, resultingin decreased mannitol utilization and increased pool size (12).In celery cell suspension cultures, MTD activity is also stronglyinfluenced by carbon source and is highest in mannitol-growncells (16). This, however, does not appear to be simplesubstrate regulation, because in intact plants tissue mannitolconcentration is inversely related to MTD activity. We haveascertained, in fact, that in addition to salt, the presence ofsugars may also down-regulate MTD expression (17). Forexample, transfer of cells to medium totally lacking carbohy-drates is accompanied by an initial increase in MTD activitycomparable to that seen on transfer to mannitol. In addition,celery cultures containing mannose plus mannitol, or sucroseplus mannitol, have lower MTD activity than cultures withmannitol alone (17). This suggests that sugars suppress MTDexpression. This is consistent with Obaton's report (18) that inflowering celery, mannitol decreased only after stored sugarsfell below 1% of dry weight.

Given the evident potential for osmoprotection, increasedphotosynthesis, and more efficient sink metabolism provided

Abbreviations: MTD, NAD-dependent mannitol dehydrogenase; PR,pathogenesis related; SA, salicyclic acid.TTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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by mannitol metabolism, the engineering of plants with bothbiosynthetic and catabolic enzymes is a compelling goal.Toward this end we isolated, sequenced, and characterized amannitol dehydrogenase cDNA§ (Mtd) from celery cells. Byusing this cDNA as a probe, we analyzed the Mtd gene responseto salt, carbon source, and, because of its homology to thepathogenesis-related (PR) protein ELI3, its predicted re-sponse to salicyclic acid (SA).

MATERIALS AND METHODSPlant Tissue Growth and Treatment. Celery cell suspension

cultures used in these studies were maintained in MS medium(19) with mannitol (180 mM), mannose (180 mM), or sucrose(90 mM) as the sole carbon source. Relative growth rates forcells maintained on these carbon sources are similar (16);however, MTD activity is highest for cells grown in mannitol.After 14 days, cells were transferred to fresh medium contain-ing the same carbon sources and grown for 3 or 4 days asindicated prior to further treatment or harvest. Sucrose-growncells used in salicylate response experiments were supple-mented with either salicylic acid (to 1 mM from a 100 mMstock in distilled H20) or an equal volume of distilled H20 andgrown for an additional 24 h. At the conclusion of the varioustreatments, cells were harvested by filtration, washed withdistilled H20, and frozen in liquid nitrogen. Frozen tissue wasground to a fine powder in a mortar and pestle in liquidnitrogen and stored at -80°C.

Protein and Enzyme Assays. Protein extractions and MTDactivity assays were performed as described (16). Proteinconcentrations were determined by the method of Bradford(20). Protein blot analyses of MTD in E. coli cell extractscontaining equal amounts of protein were performed asdescribed (21) by using polyclonal antisera raised againstgel-purified MTD (14).

Library Construction and Clone Isolation. Poly(A)+ RNAisolated from mannitol-grown cell suspension cultures 3 daysafter subculturing was used for construction of a directionalcDNA expression library in A-ZIPLOX (GIBCO/BRL).Phage in the unamplified primary library containing cDNAencoding MTD were detected as described (22) by usingpolyclonal antisera raised against gel-purified MTD (14)."Mannitol-specific" subtractive probes were also prepared asdescribed (21) and used to identify phage containing cDNAsfor sequences more abundant in mannitol- vs. mannose-growncells. Phage giving strong positive signals in either screen wereplaque-purified and excised as plasmids by using the E. coliexcision host DH12S (23). Resulting colonies were gridded onnitrocellulose disks soaked with isopropyl 13-D-thiogalactosideand incubated on LB agar and appropriate antibiotics. Colo-nies containing cDNA encoding MTD were verified by usingMTD polyclonal antisera as described (21).DNA and Protein Sequence Analysis. Deletion subclones of

clone p5-4, containing a putative near full-length Mtd cDNA,were made by using the Erase-a-Base kit (Promega). In additionto vector universal and reverse sequencing primers, internaloligonucleotide primers were used to facilitate sequencing. Oli-gonucleotides were synthesized and clones were sequenced at theIowa State University Nucleic Acid Facility. Sequence analysiswas performed using PC/GENE (IntelliGenetics), GenBank On-line Service, and at the National Center for BiotechnologyInformation by using the BLAST network service (24).

Nucleic Acid Manipulations. DNA colony blots were pre-pared as described (21). For genomic hybridization analysis,total genomic DNA was isolated from celery by the method ofDellaporta et al. (25) and digested with restriction enzymes.

§The sequence reported in this paper has been deposited in theGenBank data base (accession no. U24561).

Plasmid DNA was isolated from single colonies by using analkaline lysis miniprep (21) and digested with restrictionenzymes. For both plasmid and genomic DNA blot analysis,restriction fragments were separated by electrophoresis on Trisacetate/EDTA/agarose gels and blotted onto nitrocellulose.For analysis of MTD transcript accumulation, total RNA wasisolated from celery cells in suspension culture as described(26), and poly(A)+ RNA was isolated from total RNA by usingoligo(dT)-cellulose chromatography (5 Prime -> 3 Prime,Inc.). Both total and poly(A)+ RNA were separated ondenaturing 1.2% agarose/formaldehyde gels and transferredonto nitrocellulose. Resulting blots (both DNA and RNA)were hybridized overnight at 65°C as described (21) with32P-labeled 1.3-kbp Not I-Sal I insert of clone p5-4 containingthe entire Mtd cDNA (Fig. 1). Hybridizing bands and colonies

AF H

0c)V4

H H HH H H

H H H H HH H 13 a 4H

to.-94

I I-

200bp

BCCTCTCCTATTTCATTAAACAATCTCAAATTTTTATTTTGACAATGGCGAA

M A lC

ATCGTCAGAAATTGAACACCCTGTCAAGGCTTTTGTCTGGCCTGCAAGGaS S I B H P V A F W A A R D

pep 1ACACTACTaaTCTCCTTTCTCCGTTTAAGTTTTCCAGAAGGGCAACAGGT

T T O L L S P F K F S R R A T 0

GAGAAGGATGTGAGGCTCAAAGTTCTaTTTTGTGCAGTTTGTCATTCTGABK D V R L K V L F C a V C H S D

pop 2TCATCACATGATCCATAATAACTGGGCTTCACCACGTATCCTATCGTTCH H M I H N N W a * T T Y P I V P

CTGGGCACGAAATTGTTGGTGTOGTGACTGAAGTTGGGAGCAAAGTGGAAG H E I V G V V T B V G S X V E

Xho IIAAAGTCAAGGTCGGAGATAATGTTGGAATTGGGTGCTTAGOTTGATCTGK V X V G D N V G I G C L V G S C

HFHU

H

Q q

-A /

IHC

so3

10020

15037

20054

25071

30088

350105

TCGTTCATGTGAAAGTTOCTGCGACAACAGGGAGAGTCACTGTGAAA.ATA 400R S C B S C C D N R B S H C S N T 122

CAATAGATACCTACGGTTCTATATATTTTGATGOAACCATGACACACGGA 450I D T Y G S I Y P D G T 2 T H 0 139

Dal IGOGTALTTCCGATACTATGGTTTGCGGACGAACATTTCATTCTTCGATGC 50o00 V S D T M V A D 1 H F I L R W P 156

,bAAGAATTTGCCACTCGATTCTOGTGCTCCTCTATTGTGTGCCGGGATCA 550K N L P L D S 0 A P L L C A G I T 173

CAACTTATAGTCCCCTGAAATACTATGGACTCGACAAGCCTGGTACTAAG 600T Y S P L K Y Y C L D K P G T K 190

ATTGOTGTTGTAGGCTTAGGTGGGCTAGGCCATGTAGCTGTGAAGATOGC 650I 0 V V G L G G L G H V A V K M A 207Hind III

AAAAGCTTGGTGCACAGGTTACGGTAATAGATATTTCTGAAAGCAAAA 700K A F 0 A Q V T V I D I S E S K R 224

GGAAGGAAGCATTGQAAAAACTCGGTGCTGATTCTTTCTTGTTAAATAGT 750K K A L E K L G A D S F L L N 5 241

SSt I Cla IGACCAGGAACAAATGAAGGGCGCCACTACCTCACTTGATGGAATTATCA 80o0D Q E Q M K G A R S S L D G I I D 258

.TACTGTACCTGTGAATCACCCTCTTGCTCCACTGTTTGATCTATTAAA0C 850T V P V N H P L A P L F D L L X P 275

Hind IIICTAATCGAAA2=GTTCATGOTTGGTGCACCTGAAAAGCCCTTTGAGCTG 900N G K L V M V 0 A P E K P F E L 292

HindIIICCAGTGTTCTCTTTGCTTAAGGGGAGAA&AGZCTTGGAGGCACTATTAA 950P V F S L L X 0 R K L L 0 0 T I N 309

p-p3TGGTGGGATAAAGGAAACACAAGAAAT0CTTGATTTTGCAGCAAAGCACA 1000G G I T Q E M L D F AA XK N 326

Pvu IIACATAA;A&O ATGTTGAAGTTATTCCTATGGACTATGTGAACACCGCA 1050

I T A D V E V I P M D Y V N T A 343

ATGGAGAGACTTGTGAAGTCAGATGTTCGATACAGATTTGTCATCGACAT 1100M E R L V K S D V R Y R F V I D I 360

Ave IITGCTAATACGATOA92AOGAAGAAAGTTTGOGOOCCTAGAGACACCOGO 1150A N T H R T E E S L G A 365

TCTTTAAATCOACTACATATCTCTACAGAKATG0OCTACCAATGCGTCCA 1200TATTTOTGTACCAGACTTGGGCATAAATCATTTTTATGTATTTTATTTAT 1250CTTTTTCTCTTTTT 1298

FIG. 1. Mtd cDNA. (A) Partial restriction map of the cDNA clonep5-4. An arrow indicates reading frame and orientation of the MTDcoding region, and a bar indicates the scale. The entire Not I-Sal Ifragment was used as a probe in subsequent analyses. (B) Nucleotideand deduced amino acid sequences of the Mtd cDNA. Selectedrestriction enzyme cut sites are indicated, and peptide sequencesconfirmed by direct amino acid sequencing of purified MTD (14) aredouble underlined.

W.M., i I 1'"444-

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were visualized by autoradiography.

RESULTSIsolation of a Mtd cDNA. Celery cells grown on mannitol as

the sole carbon source have substantially higher MTD activitythan cells grown on mannose as the sole carbon source (16).Because MTD activity reaches maximal levels between 3 and4 days after subculturing, poly(A)+ RNA from 3-day-subcultured mannitol-grown cells was used for construction ofa directional cDNA expression library. Phage containingMTD-encoding cDNA were detected in the isopropyl ,B-D-thiogalactoside-induced unamplified primary library by usingantisera raised against gel-purified MTD (14). Of 3 x 105plaques screened, -1% were antibody-positive. Mannitol-specific subtractive probes were also prepared and used toidentify phage in the library containing sequences more abun-dant in mannitol vs. mannose grown cells. Of 15 stronglyantibody-positive clones and 29 mannitol-enhanced subtrac-tive clones selected at random, all antibody-positive clones and8 of the subtractive clones cross-hybridized.

Clones containing the largest putative Mtd cDNA insertswere identified by restriction analysis. The 1.3-kbp putativefull-length clone p5-4 was sequenced and found to contain asingle open reading frame of 1095 nt, encoding a peptide of 365amino acids with a predicted molecular mass of 39.7 kDa (Fig.1). Molecular mass of purified celery MTD was previouslydetermined to be -40 kDa, and sequences of three trypsin-generated peptides had been determined (14). These were (i)AFGWAAR, (ii) VLF(C/S)GVCHSDHHMIHNNWGF(manually terminated), and (iii) LLGGTINGGIK The doubleamino acid obtained in the fourth position of peptide 2suggests the presence of two nearly identical Mtd alleles, thecodon difference between cysteine and serine being a single G

ELI3EL13-2ELI3-1MTm

ELI3ELI3-2EL13-1MTD

ELI3'ELI3-2ELI3-1 WIMTD I

ELI3ELI3-2ELI3-1MTD

EhI3ELI3-2ELI3-1MTD

EL13ELI3-2ELI3-1Mm

C change. The molecular mass and sequence of the pre-dicted cDNA translation product of clone p5-4 correspond tothose of the purified MTD protein (Fig. 1B), confirming itsidentity as an authentic Mtd sequence. DNA blot analyses oftotal genomic DNA from celery suspension cells using theentire Not I-Sal I insert (Fig. 1A) of clone p5-4 as a probeshowed a simple hybridization pattern consistent with one toa few copies of the Mtd gene per haploid genome (data notshown).ELI3 PR Proteins Are MTDs. The deduced amino acid

sequence of clone p5-4 was compared with protein sequenceson file in the data bases by using a BLAST search (24). Thisshowed that the Mtd sequence encodes consensus zinc andNAD/NADH binding domains characteristic of many dehy-drogenases (Fig. 2). The deduced MTD peptide sequence wasalso found to be essentially identical to those of the ELI3 PRproteins from parsley and Arabidopsis (27). These amino acidsequences have an overall 83% identity with an additional 10%similarity.Accumulation of Mtd Transcript in Response to Carbon

Source, NaCl, and SA in Celery Cells in Suspension Culture.MTD activity increases in mannitol-grown celery cells insuspension culture for 3-4 days after transfer to fresh medium(16) and remains high until limited by depletion of mediumcomponents. No comparable increase in MTD activity is seenin similarly treated cultures grown in medium containingmannose or sucrose as the sole carbon source. Further, celerysuspension cells and celery roots grown in increasing salinityshow a dose-dependent decrease in MTD activity (7, 12). Foranalyses here, celery suspension cells were transferred to freshmedium containing mannitol, mannose, sucrose, or sucrose/0.3 M NaCl. Total proteins were extracted from cells 4 daysafter transfer, and MTD activity was determined (16) (Fig. 3).Differences in MTD activity were as reported (16, 17), with

1Zn binding

UPNVm I *3 I I

I Y I M~ z-r''

n

I

;A

FIG. 2. Amino acid comparison of MTD and the ELI3 PR proteins. The deduced amino acid sequence of NAD-dependent MTD from Apiumgraveolens was aligned with those of ELI3 from parsley (Petroselinum crispum) (fragment) and ELI3-1 and ELI3-2 from Arabidopsis thaliana (27).Identical amino acids are indicated by a black background, similar amino acids (neutral substitutions) are indicated by a gray background, and adash indicates a space added to preserve alignment. The zinc and NAD/NADH binding motifs are indicated by bracketed labels. Peptide sequencesconfirmed by amino acid sequencing of purified MTD (14) are double underlined. Identity is -83% with an additional 10% similarity. Similaritygroups used were A,G,S,T; I,L,V,M; F,Y,W; D,E,N,Q; and K,R,H.

-- F -_3 ID

F -z :ess 3e:M-3ERRSlDR3 3 NT

H I LV _ V| _

I ; 33 SWS'^:V F

*_ .. .3 *3 - I-11:z:vs: ZS..I

-P ESXE Gb:S:e"sR:XE3 M-CS'SE MIX:1e!= I s ; F̂ ,|.|-§b X@" :X Es:sS@":BW :' e .e EI

Proc. Natl. Acad. Sci. USA 92 (1995)

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1.4

E 1.2

45-.8

0.6

2 0.2 ...0

Mt Ms Suc NaCI

Mtd (pA+)

Mtd (tot)

FIG. 3. Effect of carbon source and NaCl on MTD activity and MtdRNA accumulation in celery cells. Celery cell suspension cultures weretransferred to fresh medium containing mannitol (Mt), marnose (Ms),sucrose (Suc), or sucrose/0.3 M NaCl (NaCl). Cultures were incubatedfor 3 days and harvested. Total proteins were extracted and MTDactivity was determined. One unit (U) of MTD activity is defined as1 ,umol/h. Total (tot) and poly(A)+ (pA+) RNA was extracted fromcells from each treatment and relative amounts ofMtd transcript weredetermined by RNA blot analysis.

activity being highest in mannitol-grown cells, much lower insucrose- and mannose-grown cells, and lowest in cells grown insucrose/0.3 M NaCl. To see whether transcript accumulationmirrors these differences, relative amounts of Mtd transcriptwere assessed by RNA blot analysis. Total and poly(A)+ RNAblots were probed with a 32P-labeled 1.3-kbp Not I-Sal Ifragment (Fig. 1) containing the entire Mtd cDNA insert.Differences in amounts ofMtd transcript paralleled differencesin MTD activity (Fig. 3).

Identification ofMTD as a PR protein also led us to examineits response to SA. Celery cells in suspension culture grown for24 h in sucrose-containing medium supplemented with 1 mMSA had almost 20-fold higher MTD activity than samples fromcells grown in unsupplemented cultures (Fig. 4). RNA blot

E

.5

1.0

0.8

0.6

0.4

0.2

0-SA +SA

Mtd

FIG. 4. Differential MTD activity andMtd transcript accumulationin the presence and absence of exogenous SA. Celery cell suspensioncultures were transferred to fresh sucrose-containing medium andgrown for 72 h. Replicate cultures were then supplemented with SA (to1 mM) (+SA) or with an equal volume of water (- SA) and incubatedfor an additional 24 h. Total proteins were extracted and MTD activitywas determined. One unit (U) ofMTD activity is defined as 1 ,umol/h.Total RNA was extracted from cells from each treatment and relativeamounts of Mtd transcript (Mtd) were determined by RNA blotanalysis.

analyses of total RNA showed that this difference was paral-leled by a corresponding change in Mtd RNA accumulation.

DISCUSSIONPrevious work in this laboratory demonstrated (13) the exis-tence of a NAD-dependent mannitol dehydrogenase in celerythat catalyzes the conversion of mannitol to mannose. Afterphosphorylation by hexokinase and subsequent isomerizationby phosphomannose isomerase, mannose ultimately enterscentral metabolism as fructose 6-phosphate. MTD appears tocontrol mannitol pool size by regulating mannitol turnover orutilization in response to various stimuli. For instance, MTDis highly expressed in heterotrophic (sink) tissues of wholeplants and in cell suspension cultures in the presence of itssubstrate mannitol. However, in the presence of sugars and/orNaCi (7, 16, 17), MTD expression is repressed. The derepres-sion of MTD expression in sugar-depleted sink tissues allowsincreased utilization of mannitol in central metabolism. Con-versely, the negative regulation ofMTD expression in responseto NaCl leads to higher mannitol concentrations and maycontribute to salt tolerance (7, 12).Comparison of molecular mass and partial amino acid

sequence of purified MTD protein with those deduced fromthe cDNA sequence indicates that p5-4 is an authentic Mtdclone (Fig. 2). The transcript size revealed by RNA blotanalysis (-1.3 kbp) was comparable to the cloned insert size(1298 bp), further indicating that the clone is nearly full length.RNA blot analyses of both total and poly(A)+ RNA showedthat changes in the amount of Mtd transcript in celery cells insuspension culture paralleled changes in MTD activity mod-ulated both by carbon source and NaCl (Fig. 3). Thus, thereported changes in MTD activity are regulated, at least inpart, at the level of RNA accumulation.

Subsequent analysis of the deduced MTD protein sequenceindicated that MTD, and hence mannitol, also may be involvedin plant responses to pathogen attack. A BLAST search of theprotein databases revealed that a group of PR proteins ofpreviously unknown function, the ELI3 proteins of parsley andArabidopsis (27), are in fact MTDs. In leaves ofArabidopsis, thepathogen-induced expression of these genes was proposed tobe critical in resistance to Pseudomonas and is dependent onthe presence of a wild-type copy of the R-gene resistance locusRPM1 (27). Various combinations of susceptible and resistantArabidopsis and virulent and avirulent pathovars of Pseudo-monas syringae were evaluated. In all cases, incompatibleinteractions (resistant host or avirulent pathogen) were char-acterized by a more rapid accumulation ofMtd RNA than werecompatible interactions (susceptible host). SA mediates anumber of plant resistance responses to pathogen attack.These responses range from the direct suppression of enzymeactivity, as for catalase (28), to the increased accumulation ofspecific PR proteins (29). Not surprisingly, therefore, we wereable to demonstrate that both MTD activity and Mtd RNAdramatically increase in celery suspension cells treated withSA. Although, to our knowledge, neither parsley nor Arabi-dopsis had previously been reported to make mannitol, wefound (14) in separate studies that parsley contains substantialamounts of mannitol, MTD protein, and regulated MTDactivity.

Plant responses to pathogen attack are complex, and whilea number of specific responses have been delineated, thefunctions of many PR proteins remain obscure. Our worksupports the observation that a number of PR proteins may beproteins with well-defined metabolic roles (e.g., ,3-glucanase;ref. 30) that may also be activated during pathogen attack.While not specifically defining the role of MTD in pathogenresponse, our findings suggest several possibilities. As detailedabove, MTD expression is repressed in the presence of sugars.The SA derepression of MTD expression in the presence of

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sucrose allows access to mannitol as an added carbon andenergy source. This might be advantageous in tissues under-going induction of defense mechanisms by allowing access toadditional energy and carbon to support the increased demandfor both inherent in many pathogen responses (e.g., cell walland phytoalexin synthesis). Alternatively, lower mannitol intissues may affect the invading pathogen directly. Becausemannitol is an antioxidant, its removal, like the inactivation ofcatalase (28), might enhance the pathogen-induced oxidativeburst reported by several groups (31). Further, as a number ofplant pathogens (especially fungi) can utilize mannitol as acarbon and energy source, its removal may retard pathogeningress.The identification of MTD as a PR protein opens various

lines of inquiry into the possible biological basis for thisunexpected relationship. Thus, with our previous findings (7,12, 14, 17), these resplts suggest that mannitol metabolismplays an important role in plant responses to diverse biotic andabiotic stresses.

We thank Dr. M. Ehrenshaft for many valuable discussions and Mr.M. Williams for photographic support. This research was supported inpart by U.S. Department of Agriculture Grant 9302250 to D.M.P. andM.A.C.

1. Bieleski, R. L. (1982) in Encyclopedia of Plant Physiology: NewSeries, eds. Loewus, F. A. & Tanner, W. (Springer, New York),Vol. 13A, pp. 158-192.

2. Lewis, D. H. (1984) Storage Carbohydrates in Vascular Plants, ed.Lewis, D. H. (Cambridge Univ. Press, Cambridge, U.K.), pp.143-184.

3. Thompson, M. R., Douglas, T. J., Obata-Sasamoto, H. & Thorpe,T. A. (1986) Physiol. Plant. 67, 365-369.

4. Loescher, W. H., Tyson, R. H., Everard, J. D., Redgwell, R. J. &Bieleski, R. L. (1992) Plant Physiol. 98, 1396-1402.

5. Smirnoff, N. & Cumbes, Q. J. (1989) Phytochemistry 28, 1057-1089.

6. Tarczynski, M. C., Jensen, R. G. & Bohnert, H. J. (1993) Science259, 508-510.

7. Pharr, D. M., Stoop, J. M. H., Williamson, J. D., Studer Feusi,M. E., Massel, M. 0. & Conkling, M. A. (1995) HortScience, inpress.

8. Loescher, W. H. (1987) Physiol. Plant. 70, 553-557.9. Rumpho, M. E., Edwards, G. E. & Loescher, W. H. (1983) Plant

Physiol. 73, 869-873.10. Fox, T. C., Kennedy, R. A. & Loescher, W. H. (1986) Plant

Physiol. 82, 307-311.11. Everard, J. D., Gucci, R., Kann, S. C., Flore, J. A. & Loescher,

W. H. (1994) Plant Physiol. 106, 281-292.12. Stoop, J. M. H. & Pharr, D. M. (1994) Plant Physiol. 106, 503-

511.13. Stoop, J. M. H. & Pharr, D. M. (1992) Arch. Biochem. Biophys.

298, 612-619.14. Stoop, J. M. H., Williamson, J. D., Conkling, M. A. & Pharr,

D. M. (1995) Plant Physiol. 108, 1219-1225.15. Stoop, J. M. H. & Pharr, D. M. (1994) J. Am. Soc. Hort. Sci. 119,

237-242.16. Stoop, J. M. H. & Pharr, D. M. (1993) Plant Physiol. 103, 1001-

1008.17. Pharr, D. M., Stoop, J. M. H., Studer Feusi, M. E., Williamson,

J. D., Massel, M. 0. & Conkling, M. A. (1995) in Carbon Parti-tioning and Source-Sink Interactions in Plants, eds. Madore, M. &Lucas, W. (Am. Soc. Plant Physiol., Rockville, MD), in press.

18. Obaton, M. F. (1929) Rev. Gen. Bot. 41, 622-633.19. Murashige, T. & Skoog, F. (1962) Physiol. Plant. 15, 473-497.20. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.21. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press,Plainview, NY), 2nd Ed.

22. Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA80, 1194-1198.

23. Lin, J.-J., Jessee, J. & Bloom, F. (1992) Focus 14, 98-101.24. Altschul, S. F., Gish, W., Miller, W., Meyers, E. W. & Lipman,

D. J. (1990) J. Mol. Biol. 215, 403-410.25. Dellaporta, S. L., Wood, J. & Hicks, J. B. (1983) Plant Mol. Biol.

Rep. 1, 19-21.26. Williamson, J. D., Quatrano, R. S. & Cuming, A. C. (1985) Eur.

J. Biochem. 152, 501-507.27. Kiedrowski, S., Kawalleck, P., Hahlbrock, K., Somssich, I. E. &

Dangl, J. L. (1992) EMBO J. 11, 4677-4684.28. Chen, Z., Silva, H. & Klessig, D. F. (1993) Science 262, 1883-

1886.29. Yalpani, N. & Raskin, I. (1993) Trends Microbiol. 1, 88-92.30. Kauffmann, S., Legrand, M., Geoffroy, P. & Fritig, B. (1987)

EMBO J. 6, 3209-3212.31. Sutherland, M. W. (1991) Physiol. Mol. Plant Pathol. 38, 79-93.

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