development, distribution, andcharacteristics ofintrinsic, … · development, distribution,...

Plant Physiol. (1988) 88, 915-9220032-0889/88/88/0915/08/$0 1.00/0

Development, Distribution, and Characteristics of Intrinsic,Nonbacterial Ice Nuclei in Prunus Wood1

Received for publication April 5, 1988 and in revised form June 24, 1988

DENNIS C. GROSS*, EDWARD L. PROEBSTING, JR., AND HEATHER MACCRINDLE-ZIMMERMANDepartment ofPlant Pathology, Washington State University, Pullman, Washington 99164-6430; andIrrigated Agricultural Research and Extension Center, Washington State University,Prosser, Washington 99350

ABSTRACT

Ice nuclei active at approximately -2°C and intrinsic to woody tissuesof Prunus spp. were shown to have properties distinct from bacterial icenuclei. Soaking 5-centimeter peach stem sections in water for 4 hourslowered the mean ice nucleation temperature to below -4°C, nearly 2°Clower than stems inoculated with ice nucleation-active Pseudomonassyringae strain B301D. Ice nucleation activity in peach was fully restoredby air-drying woody stem sections for a few hours. The ice nuclei inwoody tissue were inactivated between 40 and 50°C, but unaffected bytreatment with bacterial ice nucleation inhibitors (i.e. NaOCI, tartaricacid, Triton XQS-20), sulfhydryl reagents (i.e. p-hydroxymercuriben-zoate and iodine) and Pronase. Ice nuclei could not be dislodged fromstems by sonication and were shown to be equally distributed in peachbud and internodal stem tissue on a per unit mass basis; outer and innerstem tissues were also indistinguishable in ice nucleation activity. Devel-opment of ice nuclei in immature peach and sweet cherry stems did notoccur until midsummer and their formation was essentially complete bylate August. Once formed the ice nuclei intrinsic to woody stems werestable and unaffected by seasonal changes in growth. The apparentphysiological function of the ice nuclei is discussed in relation to super-cooling and mechanisms of cold hardiness in Prunus spp.

Frost tender plants avoid intracellular ice formation and con-comitant lethal injury because of the capacity of water in plantcells to supercool. Although pure water supercools to approxi-mately -40°C before homogeneous ice nucleation occurs, icegenerally forms at higher temperatures because exogenous icenuclei order water molecules into a configuration conducive togrowth of ice crystals (23, 29). An ice nucleus is active at adiscrete threshold temperature, and only a few substances, pri-marily crystalline forms of organic compounds, show ice nuclea-tion activity at temperatures above -5°C (14). Accordingly, waterwithin the tissues of annual plants, such as beans, tomatoes andmaize, has the capacity to supercool to temperatures below -5°Cbecause these tissues lack intrinsic ice nuclei active at highfreezing temperatures (23). Appreciable supercooling, however,does not occur in most woody plants such as Prunus spp., whichbegin freezing around -2°C apparently due to one or more typesof constitutive ice nuclei within woody tissue (9, 17). Accord-ingly, water contained in dormant flower primordia supercools

'This report is based, in part, on research conducted and supported asa part of SAES Western Regional Research Project W-130. Plant Pa-thology New Series No. 0006, College of Agriculture and Home Econom-ics, Research Center, Washington State University, Pullman 99164-6240.

to extremely low freezing temperatures by mechanisms thatpromote the formation of a barrier impervious to penetration byextracellular ice (6, 12, 28). Prunus flowers and other vegetativetissues, in contrast, are highly susceptible to frost injury becausethey are not protected by such a natural barrier to ice propagationinitiated by intrinsic ice nuclei within woody stem tissues (17,27). Consequently, the potential for frost protection from con-trolling INA2 bacteria on flowers would possibly be achieved ifwood-associated ice nuclei could be inactivated to permit super-cooling of frost-sensitive tissues.A few species of phytobacteria, including some strains of

Pseudomonas syringae and Erwinia herbicola, generally composethe only source of ice nuclei on herbaceous plants active attemperatures above -5°C (22, 23). Consequently, supercoolingwas limited by the presence of epiphytic INA bacteria, and frostinjury was a function of the number of INA bacteria and theirice nucleation frequency (22, 23). Woody plants also commonlyharbor high populations of INA bacteria. For example, popula-tions of INA P. syringae sometimes exceeded 106 CFU/g offlowers from deciduous fruit trees and composed over 90% ofthe total bacterial population (13, 15). Populations of epiphyticINA bacteria were dynamic, fluctuating widely in response tochanging environmental conditions. Accordingly, populations ofINA bacteria were infrequently detected on trees grown in aridparts of the Pacific Northwest (15). Despite undetectable popu-lations of INA bacteria, flowers attached to woody stems frozeat approximately -2°C (3, 4, 7, 17, 26, 27). This observationsuggested that woody tissues contained intrinsic ice nuclei activewithin the same temperature range as most INA bacteria. Sucha phenomenon prompted considerable debate concerning thepossible existence of INA bacteria in internal stem tissues or thepresence of INA remnants of dead bacterial cells (2, 3, 18, 27).It was especially difficult to rationalize the physiological advan-tage imparted by indigenous ice nuclei that interfered with thesupercooling of flowers or fruitlets and accentuated frost injury.There is growing evidence that the ice nucleation activity

associated with woody stem tissues of peach is nonbacterial inorigin. First, independent studies by Ashworth et al. (7) andGross et al. (17) noted that peach stems were INA beginning atabout -2°C regardless of whether INA bacteria were detected ornot. Furthermore, suppression of INA bacteria in orchards witheither bactericides or antagonistic bacteria failed to elicit signifi-cant frost protection (27). Second, bacterial ice nuclei did notremain active for extended periods in nature based on seasonalmonitorings of activity (13, 15). Anderson and Ashworth (2),moreover, observed that bacteria killed with streptomycin rapidlyand irreversibly lost ice nucleation activity upon exposure to

2Abbreviations: INA, ice nucleation-active; MNT, mean ice nucleationtemperature; CFU, colony-forming units.

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temperatures above 20'C, indicating that dead cells of INAbacteria were unlikely to maintain appreciable activity beyond afew hours under most orchard conditions. Finally, Ashworth andDavis (8) treated peach shoots with inhibitors of bacterial icenucleation (i.e. chemicals that quickly inactivate bacterial icenuclei [22, 23]) and showed no effect on the ice nucleationtemperature of shoots. Autoclaved shoots, in contrast, super-cooled to approximately -4°C, which was 1.5 to 2.0°C lowerthan the ice nucleation temperature of untreated peach shoots.Only the preliminary report of Haefele and Lindow (18) countersthe existence of an ice nucleus intrinsic to woody tissue. Theyobserved that the mean supercooling temperature of untreatedpear twigs was -2.4°C, whereas those treated with benzethoniumhydroxide and copper sulfate had mean supercooling tempera-tures of -4.2 and -4.9°C, respectively; extensive washing of peartwigs also reduced the mean supercooling temperature to -3.8°C.These results indicated that the ice nuclei associated with dor-mant pear twigs were superficial and not inherent to pear tissue.The general characteristics of the ice nuclei intrinsic to woody

tissues of peach are described in this study. Tests of variousenvironmental effects (e.g. soaking, drying, temperature) andchemicals (e.g. bacterial ice nucleation inhibitors, sulfhydrylreagents, Pronase) on stability were used to show that the wood-associated ice nuclei are nonbacterial in origin. In addition, thelocation of high levels of intrinsic ice nucleation activity withinpeach stems was determined, and its seasonal development mon-itored in both peach and sweet cherry stems. The importance ofintrinsic ice nuclei is also discussed in relation to cold hardinessand physiological processes associated with supercooling inPrunus.

MATERIALS AND METHODS

Plant Material. Peach (Prunus persica [L.] Batsch.) cultivar'Redhaven' and sweet cherry (Prunus avium L.) cultivar'Bing,'grown at the Irrigated Agriculture Research and Extension Cen-ter, Prosser, WA, were used as the source of Prunus stem tissue.To protect peach trees from contamination by indigenous INAbacteria, trees were sprayed with a mixture of the bactericidesstreptomycin sulfate(150 ppm; Agristrep 17, Pfizer ChemicalCo.) and oxytetracycline (100 ppm; Mycoshield, Pfizer ChemicalCo.) at approximately 3-week intervals from prebloom to thesmall fruit stage of development. Ice nucleation experimentswere conducted on peach stems collected from February (fulldormancy) to May (small fruitlet stage of development) 1987.The only exceptions were peach and sweet cherry samples col-lected between May and October 1986, which were used to studythe development of wood-associated ice nuclei in maturingstems. Stems were stored in plastic bags at4°C until use.

Prior to MNT analysis, peach or sweet cherry stems were

routinely cut into 5-cm sections. Foliage, when present, was

removed with a razor blade and discarded. In experiments relat-ing mass to MNT, buds and fruitlets were excised at their stembase with a razor blade. Samples consisted of buds, fruitlets, or

1-cm internodal stem sections frozen in increments of 1, 5, and10 per test tube.The tomato (Lycopersicon esculentum Mill.) cultivar 'Castle-

martII,' grown in the greenhouse 4 to 5 weeks from seed, was

used as the source of tomato stems. All foliage was removedfrom stems prior to cutting into 1-cm stem sections; preweighedstem sections were frozen in increments of 1, 5, and 10 per testtube.Assay for Epiphytic INA Bacteria. Plate assays for enumerat-

ing populations of INA and total bacteria on peach and sweetcherry samples were done as previously described (15). Stemswere cut to 5-cm lengths and foliage, if present, was excised anddiscarded. Two 20-stem samples per orchard were processedwithin 24 h of collection for enumeration of bacterial opula-

tions. The minimum detection level of INA bacteria was approx-imately cell/g fresh weight of stem tissue.INA Bacterium and Inoculation. The highly active ice nucleat-

ing strain B301D of Pseudomonas syringae pv syringae, whichexpresses one -5°C ice nucleus per 3 x 102 cells (16), was usedto inoculate peach stems. The bacterium was grown on King'smedium B (20) agar for 2 d at 20°C, which is conducive for highexpression of ice nucleation activity (15). Cells were suspendedin sterile deionized water to a concentration of to 3 x 108

CFU/mL. Peach stems were inoculated by immersion for 2 minin a freshly prepared bacterial suspension; noninoculated stemswere immersed for 2 min in sterile deionized water. Stems were

blotted on paper towels for 5 min and then processed for MNTmeasurements.

Determination of MNT. The test tube freezing method (17)was used to determine the MNT of stem, bud and fruitletsamples. Samples were immersed in deionized water (24mL) inautoclaved test tubes (40 tubes per treatment), and equilibratedat-1.5C in a refrigerated water bath (EX-400 bath and EN-850refrigeration unit with ±0.007°C accuracy according to the man-

ufacturer [Neslab Instruments, Inc., Portsmouth, NH 03801]).Frozen tubes were counted after a 20-min incubation. Thetemperature was then lowered by a1°C increment, equilibratedfor 20 min, and the number of newly frozen tubes counted. Thisprotocol was repeated until all 40 tubes per analysis had frozen.The MNT was calculated as the mean temperature at which iceformation occurred per 40 tubes. The MNT was determined induplicate for all treatments and the values then averaged; eachreplicate within a treatment was frozen in a separate water bath.Temperature and Sonication Treatments. The effect of high

temperatures on the MNT of 5-cm peach stem sections was

tested by sealing 40 stem sections in a plastic bagfilled withdeionized water (100mL). All air was removed from the bagprior to sealing it with heat. The bags were submerged in a

constant temperature water bath adjusted to either 25, 40, 50, or85°C; two 40-stem bags were prepared for each temperature.After a 60-min incubation, the stems were removed from thesealed bags and air-dried at22.5°C (±1C) for 4 h prior todetermination of MNT.

Peach stems sealed in water-filled bags as described above, butnot heat treated, were sonicated for 60min using a standardbenchtop ultrasonic cleaner. Ice was added at appropriate inter-vals throughout the treatment period to maintain the tempera-ture61

1 OC. Control stems sealed in water-filled bags were incu-bated at4°C for 60 mmn without sonication. After incubation,stem samples were rinsed three times with deionized water(4°C)and air-dried as described above.

Chemical Treatments. The chemicals and the concentrationsused to treat 5-cm peach stem sections are listed in Table I.Tartaric acid, Triton XQS-20, iodoacetamide, and p-hydroxy-mercuribenzoate were purchased from the Sigma Chemical Co.,NaOCI and guanidine HCI (99% [w/w]) were purchased fromthe Aldrich Chemical Co., Inc., and Pronase (type XIV) waspurchased from Calbiochem. The iodine was dissolved in a buffercontaining 10 mm MgCl2, 24 mm KI, and 50 mm borate (pH8.0) (25). lodoacetamide and p-hydroxymercuribenzoate were

dissolved in 50 mm Tris (pH 7.2) (21). Pronase was prepared in10 MM MgCl2, 0.1% (v/v) Triton XQS-20, and 50 mm Tris (pH7.5). Stem sections were treated by immersing them for 60minat22.5°C (±+1C in a solution (100 mL/40 stem sections) of a

given chemical. Two replicates of 40-stem sections were preparedfor each treatment; controls included peach stems treated withbuffers minus the sulfhydryl reagents or Pronase. After chemicaltreatment, the stems were blotted on paper towels and air-driedat22.5° C (±

1°C) for 4 h prior to determination of MNT.

916GROSS ET AL.



NONBACTERIAL ICE NUCLEI INTRINSIC TO PRUNUS WOOD

Table I. Effect of Various Chemicals on theMNT ofPeach StemsMature peach stems (5-cm long) were treated by immersing them for 60 min at 22.5°C (± 1C) in a solution

(100 mL/40 stem sections) of a particular chemical. After treatment, the stems were blotted on paper towelsand air-dried at 22.5°C (± 1C) for 4 h prior to determination ofMNT. Two replicates of 40-stem sections wereprepared for each treatment.

Chemical Concentration MNTa b SE

Bacterial Ice Nucleation InhibitorsWater (control) -2.21 a ±0.13Triton XQS-20 0.1% (v/v) -2.49 ab ±0.16Tartaric acid 0.2 M -2.69 abc ±0.16NaOCl 0.5% (v/v) -2.78 bc ±0.28Na2CO3 0.1 M -3.04 cd ±0.04ZnSO4 + urea 0.05 M + 0.5 M -3.43 de ±0.03CuSO4 50 mM -3.79 ef ±0.09Na2CO3 + urea 0.1 M + 1 M -4.07 fg ±0.07H3PO4 2.0% (v/v) -4.22 fg ±0.27Guanidine HCI 1 M -6.23 j +0.23

Acids, Bases, and Organic SolventsH2SO4 2 N -6.90 k ±0.10KOH 1 N -5.92 ij ±0.24Ethanol 95% (v/v) -5.16 h ±0.08n-Butanol Concentrated -5.48 hi ±0.08Chloroform Concentrated -5.81 ij ±0.13

Sulfhydryl Reagents and Pronasep-Hydroxymercuribenzoate 1 mM -2.40 ab ±0.15Iodine 0.02 mm -2.47 ab ±0.07Iodine 0.005 mm -2.83 bc ±0.03lodoacetamide 50 mM -4.37 g ±0.09Pronase 30 U/mL -2.53 ab ±0.0LSD 0.05 0.43LSD 0.01 0.58

a Each MNT value is the average MNT oftwo replicates, 40 stem sections per replicate. b Values followedby the same letter do not differ at the 5% level of significance according to Duncan's multiple range test.

RESULTS

Bacterial Populations Recovered from Prunus Stems. Popula-tions of total bacteria from peach and sweet cherry stem sectionsusually ranged between 10' and 103 CFU/g fresh weight. Popu-lations tended to be greater in the spring and summer monthsthan during winter dormancy. In addition, sweet cherry stemsusually yielded about 10-fold higher populations of total bacteriaper gram than peach stems sprayed with antibiotics at regularintervals in the spring.INA bacteria were not recovered from any ofthe stem samples

used in these studies of the wood-associated ice nucleus. Non-INA fluorescent pseudomonads were occasionally isolated at lowpopulations of generally less than 10 CFU/g.

Effects of Water and Temperature on the MNT of PeachStems. The MNT of 5-cm peach stem sections was approxi-mately -2.5°C regardless of whether they were either noninocu-lated or inoculated with a high population of INA strain B30ID(Fig. 1; Table II). However, the activity of bacterial ice nucleiwas clearly differentiated from that of intrinsic, wood-associatedice nuclei by prolonged soaking of stems in water. After only 4h of soaking, noninoculated stems exhibited an MNT of -4.3°Cwhich was 1.8°C lower than that of stems inoculated with bac-terial ice nuclei (Fig. 1). Further soaking for 8 h or longer loweredthe MNT of noninoculated stems only slightly to a minimumbetween -4.4 and -4.8°C, which was in sharp contrast to nosignificant change in the MNT of inoculated stems even after 24h of soaking. Measurements of presoaking and postsoakingweights of peach stems showed approximately 20% water uptakefollowing 24 h of soaking.The inactivation of wood-associated ice nuclei by prolonged

C)0

z

0~o-e INOCULATED_-. NONINOCULATED

- 1

-2-K

-3-

-4-

6 4 A 12 16 20

TIME (HOURS)FIG. 1. Effect of time of soaking in deionized water on the MNT of

mature peach stems inoculated or noninoculated with INA P. syringaeB301D. Peach stem sections 5-cm in length were dipped for 2 min ineither a bacterial cell suspension (1-3 x 1O' CFU/mL) or sterile deionizedwater prior to soaking at 4°C in test tubes containing deionized water(one stem section per tube). Each MNT value is the average MNT oftwo replicates, 40 stem sections per replicate. The SE for the MNT valuesranged from 0 to +0. 14°C.

917

24




Table II. Reversibility ofthe Effect ofSoaking on the MNT ofPeach StemsMature peach stems (5-cm long) were either soaked 24 h in deionized water at 4°C or incubated without

soaking for 24 h at 4°C. Prior to determining the MNT in part A, half of the soaked and nonsoaked stemswere inoculated with INA P. syringae B30ID by dipping in a cell suspension (1-3 x 108 CFU/mL) for 2 min;the remaining stems were dipped in sterile water. After determinations of MNTs in part A, stem sections wereair-dried for 4 h at 22.5°C (±1C). In part B, the soaking and nonsoaking conditions were reversed for theinoculated and noninoculated stems used in part A. Each MNT value is the average MNT of two replicates,40 stem sections per replicate.

Part A Part B

Condition Inoculateda MNT SE Condition Inoculateda MNT SE

°C °cNonsoaked + -2.52 ±0.04 Soaked + -2.99 ±0.01Nonsoaked - -2.19 ±0.21 Soaked - -4.68 ±0.05Soaked + -2.59 ±0.31 Nonsoaked + -2.76 ±0.18Soaked - -4.60 ±0.27 Nonsoaked - -2.73 ±0.20

LSD 0.05 0.91 LSD 0.05 0.53LSD 0.01 1.51 LSD 0.01 0.88

a Inoculated, +; noninoculated, -.

soaking in water was not permanent and could be readily reversedby air-drying (Table II). For example, in part A noninoculatedpeach stem sections soaked for 24 h in water had an MNTapproximately 2°C lower than nonsoaked, noninoculated stems.When the soaking or nonsoaking conditions were reversed inpart B for these same peach stems (after prior air-drying for 4 h),theMNT was correspondingly reversed by about 2°C. In contrast,inoculated stems either soaked or nonsoaked in water showedno significant difference in MNT and were similar in MNT tothe noninoculated, nonsoaked stems.

Inactivation of wood-associated ice nuclei caused by extendedsoaking in water was fully reversed by simply drying the peachstems for only 4 h at 22.5°C (± 1C) (Fig. 2). However, extensivedrying for 16 h or longer lowered the MNT to slightly below-3°C, which was nearly 1°C lower than the MNT for stems driedfor 4 or 8 h. Stems which had not been previously soaked butnevertheless dried for the same lengths of time also showed asimilarly marked lowering of the MNT after 16 h. Furthermore,stems dried for 16 h showed extensive shriveling not seen in thecase of stems dried 4 or 8 h. Attempts to reverse the partialinactivation of the wood-associated ice nuclei caused by drying

0-

_-1

C) -2-0

-3-/

-4-

-5-0 4 8 12 16

TIME (HOURS)FIG. 2. Effect of time of drying at 22.5°C (±1°C) on the MNT of

mature peach stems (5-cm long) previously soaked for 24 h in deionizedwater. Each MNT value is the average MNT of two replicates, 40 stemsections per replicate. The SE for the MNT values ranged from ±0.05° to±0.1 30C. The average MNT ofpeach stems prior to soaking was -2.27°Cwith an SE of ±0.24°C.

for 16 h were unsuccessful. Stems dried for 16 h and thensuccessively soaked in water for 24 h and air-dried for 4 hcontinued to freeze at an MNT below -3C.At temperatures of 40°C or less the wood-associated ice nuclei

were highly stable, exhibiting an MNT of around -2.5°C (Fig.3). Activity of the wood-associated ice nuclei appeared to be losttotally after exposure to a temperature between 40 and 50C.The MNT of peach stems exposed to 50°C for 60 min droppedto -6.3°C, a value about 3.5°C lower than the MNT of stemsexposed to 40°C. Exposure of stems to higher temperatures didnot cause additional losses in ice nucleation activity.

Distribution of Wood-Associated Ice Nuclei in Peach Stems.Extensive exposure of peach stems to sonication demonstratedthat the ice nucleation activity was intrinsic to stem tissues andnot loosely associated with the surface, as generally observedwith INA bacteria (22). Stems sonicated for 60 min (followed bythorough rinsing and air-drying for 4 h) had an average MNT of-2.2°C, a value identical to the MNT of stems merely soaked inwater for 60 min and air-dried (data not presented). Maceratesof stem tissues did not yield a detectable population of INA

u I

-1-

-2-C)0 -3-

z -4-

-5-

-6-

-720 30 40 50 60 70 80 90

TEMPERATURE (0C)FIG. 3. Effect of prolonged exposure to temperatures ranging from

25 to 85°C on the MNT of mature peach stem sections. Peach stems (5-cm long) were sealed in plastic bags containing 100 mL of deionizedwater, and then submerged and incubated in a constant temperaturewater bath for 60 min. Stem sections were air-dried at 22.5°C (± 1C) for4 h prior to determinations of MNTs. Each MNT value is the averageMNT of two replicates, 40 stem sections per replicate. The SE for theMNT values ranged from ±0.09 to ±0.20°C.

.

918 GROSS ET AL.

P-1




bacteria, indicating that INA bacteria did not reside within theintercellular spaces or vessels which are impervious to the effectsof sonication. Decreased MNTs for stems were observed only ifthe water within the ultrasonic chamber was not strictlyprevented from accumulating heat during operation.The possible localization of ice nuclei in either the inner wood

or outer bark tissues was analyzed in freezing tests. Stem seg-ments, from which all buds were earlier removed with a razorblade, were cut longitudinally to the wood, and the bark tissueswere separated from the inner wood by peeling. The respectiveaverage fresh weights of the bark and wood samples were 0.23and 0.24 g/5-cm stem section. The average MNT for the barkand wood samples when combined in test tubes was -2.2°C. Incontrast, the average MNTs of the bark and wood samples whenanalyzed separately were -2.6 and -2.8°C, respectively, indicat-ing that the ice nuclei were about equally distributed in the outerand inner tissues of peach stems. The higher MNT of combinedtissue samples as compared to the MNT of either the outer orinner tissues alone was attributed to the doubling of total massper test tube.Comparisons ofpeach bud to internodal stem tissues collected

prior to bloom showed them to be essentially equivalent ininherent ice nucleation activity on a mass basis (Fig. 4). Bud andinternodal stem tissues exhibited a linear relationship betweenthe MNT and the log of sample mass with no appreciabledifference in line slope between the two tissues, which indicatesuniform distribution of ice nuclei within these tissues. Internodalstems collected in the young fruitlet stage of development wereunchanged in ice nucleation activity per unit mass relative tostems collected prior to bloom. However, the MNT of fruitletswas approximately 1°C per unit mass lower than postbloominternodal stem, prebloom internodal stem, and prebloom budtissues. Therefore, equivalent levels of ice nucleation activitywere observed for fruitlet tissues only when their mass wasincreased three- to four-fold relative to internodal stem or pre-bloom tissues. The slope of the line for fruitlets was neverthelessunchanged from the prebloom stage of development. This indi-cated that, although the ice nuclei occurred in lower numbers

z%

z_2

0.0* II

-2.0-

-4.0-

-6.0-

-8.0 -

-1.O 0.0 1.

per unit mass as fruitlets grew, their distribution was relativelyuniform within the fruitlet sample.The average MNT of tomato stems was over 4°C lower per

unit mass than the MNT of peach stems (Fig. 4). For example,the MNT for 0.66 g of tomato stem tissue was -7.0°C, demon-strating the inherent ability oftomato vegetative tissues to super-cool to relatively low temperatures.

Seasonal Development of Wood-Associated Ice Nuclei. Thedevelopment of wood-associated ice nuclei in peach and sweetcherry stems followed a similar seasonal pattern (Fig. 5). Youngstem shoots early in the growing season showed no appreciableintrinsic ice nucleation activity. For example, immature stemsin May expressed an MNT below -4°C, which was nearly 2°Clower than the MNT of mature 1-year-old stem sections. Thewood-associated ice nuclei developed in stems during the mid-summer period of growth as evidenced by the sharp increase inthe MNT of stems collected in July. By the end of August thewood-associated ice nuclei appeared to be fully developed sincethe MNT of stems from the current season was less than 0.5°Clower than the MNT of 1-year-old stems. The 0.5°C differenceapparently was due to the larger mass for 1-year-old stem sectionswhich averaged about 40% larger than stem sections from thecurrent season. For example, sweet cherry stem sections fromthe current and previous seasons, respectively, averaged 0.95 and1.64 g when collected in October. Extrapolation of MNTs fromthe log of these tissue masses using the linear relationship shownfor peach stems in Figure 4, confirmed the observed differencein MNTs of about 0.5°C.The monthly average MNT values for 1-year-old stem sections

from both peach and sweet cherry were relatively consistentduring the 6-month period, rarely dipping below the -2 to -3°CMNT range. Exceptions were 1-year-old stem sections collectedfrom sweet cherry in July and peach in September. Explanationsfor such fluctuations in MNT are unknown but may partiallyreflect exposure to hot daytime summer temperatures at Prosser,WA.

Stability of Wood-Associated Ice Nuclei to Chemicals. Of thebacterial ice nucleation inhibitors tested, the wood-associated icenuclei were highly sensitive to guanidine HCl, yielding an averageMNT of-6.2°C, and somewhat less sensitive to H3PO4, Na2CO3,Na2CO3 plus urea, CuSO4, and ZnSO4 plus urea (Table I). Peachstem sections treated with sodium hypochlorite, tartaric acid, or

01%0

LU]

zLLiOfLXIL-

I aU-.0

LOG [ FRESH WEIGHT (g) ]FIG. 4. Relationship between sample fresh weight and MNT for peach

stems, buds and fruitlets, and tomato stems. Peach bud (0) (y = -2.12+ 1.92x, R2 = 1.00) and subtending stem (-) (y = -1.91 + 2.2 1x, R2 =0.99) samples were collected on March 10 (first pink developmentalstage); the peach fruitlet (O) (y = -3.17 + 2.06x, R2 = 0.99) andsubtending stem (-) (y = -2.15 + 1.58x, R2 = 1.00) samples werecollected on April 29. Tomato internodal stem (A) (y =-6.66 + 1.38x,R2= 0.99) samples were obtained from 4- to 5-week-old plants. All stemswere cut into 1-cm sections of internodal tissue and tested in incrementsof 1, 5, and 10 sections per tube. Buds and fruitlets were also dividedinto increments of 1, 5, and 10 per tube. Each MNT value is the averageMNT of two replicates, 40 tubes per replicate.

z

MAY JUNE JULY AUG SEPT OCTMONTH

FIG. 5. Seasonal differences between the MNT of newly developingwood and the MNT of mature wood from the previous growing season.Samples were collected once during the last 10 d of each month. Peachand sweet cherry stem samples were excised of all foliage and cut into 5-cm sections prior to MNT measurements. Each MNT value used tocalculate differences between newly developing stems and 1-year-oldstems was the average MNT of two replicates, 40 stem sections perreplicate.

919

-2.0




Triton XQS-20 were largely unchanged in MNT relative to thatof the water control.Exposure to a strong acid (H2SO4) or base (KOH) as well as

various solvents (ethanol, n-butanol, and chloroform) fully in-activated the wood-associated ice nuclei since the MNTs of 5-cm peach stem sections were below -5°C (Table I). In contrast,only iodoacetamide of the sulfhydryl reagents tested had anappreciable effect, lowering the MNT of peach stem sections toapproximately -4.4°C. Pronase had no effect on the MNT.

DISCUSSION

Although several studies (3-5, 7, 8, 17, 26, 27) providedevidence that woody stem tissues of peach contained intrinsicice nuclei active at approximately -2°C, there was no cleardifferentiation between their activity and that of INA bacteria.Evidence from this study clearly indicates that there are nonbac-terial ice nuclei associated with woody tissues: (a) INA bacteriawere not recovered either externally or internally from stemsamples used in these tests; (b) soaking of peach stem sections inwater for only about 4 h inactivated intrinsic ice nuclei, whereasthere was no effect on the activity of bacterial ice nuclei addedto stems; (c) activity of intrinsic ice nuclei was regained by dryingstem sections for about 4 h; (d) intrinsic ice nuclei were inacti-vated by exposure to temperatures between 40 and 50°C, whereasbacterial ice nuclei are reported to lose activity at around 33°C(2); (e) sonication of peach stems for 60 min did not affect icenucleation activity associated with woody stems, whereas soni-cation is reported to remove 60 to 95% of all bacteria, includingINA Pseudomonas syringae, from leaves as compared to totalcounts from macerated tissue ( 19); (f) some bacterial ice nuclea-tion inhibitors (i.e. tartaric acid and Triton XQS-20) had noeffect on the activity of the ice nuclei intrinsic to peach stems;(g) the sulfhydryl reagents, iodine and p-hydroxymercuriben-zoate, had no effect on activity of intrinsic ice nuclei, whereasthey are reported to inactivate cell-associated bacterial ice nuclei(21, 25); and (h) Pronase did not inactivate intrinsic ice nuclei,whereas bacterial ice nuclei are highly sensitive to proteolyticactivity (23, 25).

Differences in methodology apparently are responsible for thediscrepancy between our results and those ofHaefele and Lindow(18) regarding the source of ice nucleation in woody tissue. The1.5°C lower mean supercooling temperature observed for ex-

tensively washed pear twigs (18) probably reflects prolongedsoaking without a drying period before ice nucleation measure-ments. Although our soaking experiments used peach stems,similar supercooling responses were observed for pear, sweetcherry, and apple stems after prolonged soaking in water (ourunpublished observations). Furthermore, it was not surprisingthat many of the bacterial ice nucleation inhibitors originallydescribed by Lindow (22) also inhibited the ice nuclei in peachwood, since their effects are relatively nonspecific. For example,the mean supercooling temperature of -4.9°C reported for peartwigs treated with copper sulfate (18) is similar to the lowerMNTobserved for peach stems treated with copper sulfate in this study(Table I). In contrast, 0.5% NaOCl, 0.2 M tartaric acid, and 0. 1%Triton XQS-20 failed to lower the MNT of peach stem segmentseven though at these concentrations they have been reported togive significant control of frost injury to immature pear fruit inorchards at -3°C (22) and to field-grown leaves of maize andtomatoes exposed to -4°C (24). The relative contribution ofINAbacteria and wood-associated ice nuclei to frost injury due tomild radiative frosts of -3°C in fruit trees has been discussed(17, 27).

In contrast to our results, Ashworth and Davis (8) reportedthat copper sulfate and phosphoric acid had no effect on the icenucleation temperature of peach stems. In some ice nucleationanalyses, peach internode tissue was cut into 1-cm sections and

then placed in test tubes containing 1 ml of water, eight sectionsper tube. Untreated stem sections or stem sections briefly rinsedin water surprisingly showed no activity at temperatures above-4°C. In our tests, copper sulfate and phosphoric acid reducedthe MNT of peach stem sections to approximately -4°C, anMNT 1.5 to 2.0°C lower than water-treated peach stems (TableI). Apparently, peach internodal segments in the Ashworth andDavis (8) study had somehow lost appreciable activity associatedwith the intrinsic ice nuclei during experimental manipulations;cutting of stems into 1-cm sections was not responsible based onour analyses of effects of sample mass on MNT (Fig. 4). In othertests (8), peach shoots were similarly treated with these chemicalsand the initial ice nucleation temperature was measured bydifferential thermal analysis. Copper sulfate and phosphoric acidagain were reported not to affect the ice nucleation temperatureof shoots, as judged by comparisons to untreated field-grownshoots which froze at -2.4°C. Although the ice nucleation tem-perature reported for untreated peach shoots is consistent withour MNT data, there is no obvious explanation for the conflictingresults for copper sulfate and phosphoric acid on activity ofintrinsic ice nuclei.Ashworth et al. (7) later tested the effects of various chemicals,

including chloroform, ethanol, KOH, and H2SO4, on ice nuclea-tion activity associated with ground peach wood. Ground woodtreated with H2SO4 had the lowest ice nucleation temperature of-7.3°C while all other treatments, including untreated groundtissue, froze at approximately -5.5°C. Although these tempera-tures are in good agreement with the MNT values observed for5-cm peach stem sections similarly treated with the same chem-icals (Table I), the grinding process itself causes complete loss inactivity at -5°C (5). These results further illustrate the impor-tance of proper handling of stem tissue in ice nucleation studiesto ensure that activity is not lost due to auxiliary factors.

Analysis of peach stem and flower tissues by Andrews et al.(5) indicated that intrinsic ice nuclei were concentrated in pedi-cels, although the MNT measured for pedicels was below -5°C.Ice nuclei active above -3°C were found in this study to be aboutequally distributed in peach bud and internodal tissues per unitmass (Fig. 4). Moreover, there was no measurable difference inice nucleation activity between the inner and outer tissues frommature stems. Because tomatoes lack an intrinsic ice nucleusactive above -5°C, the MNT for tomato stems was about 4°Clower per unit mass as compared to peach wood (Fig. 4); similarresults for tomato were reported by Anderson and Ashworth (1).Consequently, the freezing behavior of peach flowers, fruit, andleaves more closely resembles that oftomato stems per unit masssince they also lack an intrinsic ice nucleus active above -5°C(17).Ashworth et al. (10) noted that the relationship between sample

mass and ice nucleation temperature for all plant tissues followeda modified singular hypothesis (30) for ice nucleation. In thismodel, endogenous ice nuclei exhibit threshold activity within aspecific temperature range, although the occurrence of an icenucleation event is stochastic since its probability is dependenton sample mass and length of exposure (1 1). Accordingly, icenucleation increases exponentially as temperatures are decreased,as observed in Figure 4, until temperatures exceed the range inwhich the ice nucleus is active. The upper temperature limit foractivity of intrinsic ice nuclei in peach is approximately -1.5°Cbased on our freezing analysis by the test tube method. INAbacteria, however, follow the singular hypothesis (29) wherebythe ice nucleation temperature is dictated by the most active INAbacterial cell within a sample population. Therefore, plant sam-ples harboring bacterial ice nuclei active at warm freezing tem-peratures are frozen independently of sample mass and do notsupercool below -2.5°C.The ice nuclei inherent to Prunus wood and active above -3°C

920 GROSS ET AL.




apparently are not formed in growing stems until midseason, butare completely formed by late August when stems are almostmature and no longer increase appreciably in mass. Ashworth etal. (7) also observed that young peach stems froze at lowertemperatures than stems from the previous year, although theynoted a more gradual increase in ice nucleation activity duringthe summer growth period. Once formed, the ice nuclei arerelatively constant in activity per unit mass and not influencedby seasonal growth cycles (8; Figs. 4 and 5).The chemical nature of the ice nucleus (or nuclei) in Prunus

wood remains to be determined, although evidence suggests that,unlike bacterial ice nuclei (23, 31), it is not a protein. This isbased partly on its insensitivity to Pronase and to the sulfhydrylreagents, p-hydroxymercuribenzoate and iodine, which quicklyinactivate bacterial ice nuclei (21, 25). In addition, the effect ofsoaking stems in water indicates that the ice nucleating sub-stance(s) undergoes conformational changes upon hydrationwhich are not conducive to ice crystal formation; however,soaking had no effect on activity of the proteinaceous bacterialice nucleus (Fig. 1; Table II). A number of organic compoundsincluding some amino acids, steroids, and terpenes are activeabove -5°C, but only in crystalline form (14). Accordingly,recovery of full ice nucleation activity by air-drying soaked stemsmay be due to recrystallization of an organic constituent ofwood. The ice nuclei cannot be leached from woody tissue withwater, although the ice nucleating substance(s) may have beensolubilized or denatured by treatment with ethanol or otherorganic solvents to cause loss of activity (Table I).Treatment of peach stems with guanidine, a general protein

denaturant, gave the only indication that the ice nucleus (ornuclei) might contain a protein component. Guanidine reducedthe MNT of 5-cm stem segments to below -6°C, which wasmore than 2C lower than stems treated with any of the otherice nucleation inhibitors; only the strong acid or base treatmentswere approximately equivalent in reducing the MNT of stems(Table I). In contrast, the related protein denaturant, urea,showed only weak activity in similar tests. Stems treated with 1

M guanidine for just 2 min had an MNT of about -4°C, prompt-ing interest in testing guanidine on whole peach branches in anorchard. However, a 60 min exposure to 0.1 M guanidine was

ineffective in lowering the MNT of peach stem sections. Appli-cation of 0.1 M guanidine to peach branches also proved to bephytotoxic.The occurrence of ice nuclei intrinsic to Prunus wood suggests

a possible role in cold hardiness by initiating extracellular iceformation at specific sites in tissues (12). Quamme (28) observedthat extracellular ice formation was initiated at warm freezingtemperatures in the bud scales of peach, causing a redistributionof water within dormant flower bud tissues toward the ice crystalsink. It was speculated (6, 28) that a dry region eventually formedat warm freezing temperatures between the primordium and theice nucleated bud scales and axis. This creates a barrier topropagation of ice crystals into primordia and thereby permittingdeep supercooling of intracellular water to between -15 and-25C. Extracellular ice formation at temperatures near 0°C isadvantageous because it starts the redistribution of water withinbuds soon after exposure to freezing temperatures. Moreover,fewer and larger ice crystals are formed at warm freezing tem-peratures. Ice nucleation at lower temperatures promotes rapidand fatal growth of small ice crystals throughout the bud. Con-sequently, the presence of endogenous ice nuclei in bud scalesand wood active at a threshold temperature between -1 and-2°C appears to be advantageous to the deep supercoolingprocess since bud resistance progressively increases as watermigrates to external sites of ice formation. Ashworth and Davis(9) measured the initial ice nucleation temperature of severalwoody plant species in the field and noted that all froze above

-2°C, which suggested that intrinsic ice nuclei active near 0°Care widely distributed in woody plants.The presence of ice nuclei active at about -2°C significantly

promoted survival of deacclimating peach and sweet cherry budsexposed to temperatures of -8 to -10°C as compared to budsfree of such ice nuclei (4, 17). Ice nucleation at temperaturesnear -10°C apparently resulted in the rapid formation of smallice crystals which spread to the embryo (6, 12). At about bloom,however, a transition from a frost-tolerant to a frost-sensitivephase was observed in which ice formation at -2°C was injuriousto floral organs. These studies (4, 17) demonstrated that inacti-vation of ice nuclei in wood after anthesis would be beneficial tosurvival since supercooling would occur in flower or fruitlettissues. The challenge now is to establish environmental condi-tions in orchards or find a suitable chemical which would tem-porarily inactivate intrinsic ice nuclei without deleterious sideeffects to trees.Although we have substantial evidence that ice nucleation

activity intrinsic to peach wood is nonbacterial in origin, addi-tional work is needed to define the chemical nature of the icenucleating substance(s) and the mechanism by which it triggersice formation. Our studies nevertheless describe the generalenvironmental and chemical parameters affecting aw";ri -of theice nuclei, which need to be considered in future .nocho'micaland physiological studies. Furthermore, the apparent physiolog-ical function of the ice nuclei in promoting cold hardiness ofwoody plants illustrates the importance of supercooling andendogenously controlled ice nucleation during dormancy anddeacclimation.

LITERATURE CITElD

1. ANDERSON JA, EN ASHWORTH 1985 Ice nucleation in tomato plants. J AmSoc Hortic Sci 110: 291-296

2. ANDERSON JA, EN ASHWORTH 1986 The effects of streptomycin, desiccation,and UV radiation on ice nucleation by Pseudomonas viridiflava. PlantPhysiol 80: 956-960

3. ANDERSON JA, EN ASHWORTH, GA DAVIS 1987 Nonbacterial ice nucleationin peach shoots. J Am Soc Hortic Sci 112: 215-218

4. ANDREWS PK, EL PROEBSTING, DC GRoss 1983 Differential thermal analysisand freezing injury of deacclimating peach and sweet cherry reproductiveorgans. J Am Soc Hortic Sci 108: 755-759

5. ANDREWS PK, EL PROEBSTING JR, DC GROSS 1986 Ice nucleation andsupercooling in freeze-sensitive peach and sweet cherry tissues. J Am SocHortic Sci 11 1: 232-236

6. ASHWORTH, EN 1982 Properties of peach flower buds which facilitate super-cooling. Plant Physiol 70: 1475-1479

7. ASHWORTH EN, JA ANDERSON, GA DAVIS 1985 Properties of ice nucleiassociated with peach trees. J Am Soc Hortic Sci 1 10: 287-291

8. ASHWORTH EN, GA DAVIS 1984 Ice nucleation within peach trees. J Am SocHortic Sci 109: 198-201

9. ASHWORTH EN, GA DAVIS 1986 Ice formation in woody plants under fieldconditions. HortScience 21: 1233-1234

10. ASHWORTH EN, GA DAVIS, JA ANDERSON 1985 Factors affecting ice nucleationin plant tissues. Plant Physiol 79: 1033-1037

11. BiGG EK 1953 The supercooling of water. Proc Phys Soc B66: 688-69412. BURKE MJ, LV GUSTA, HA QUAMME, CJ WEISER 1976 Freezing and injury in

plants. Annu Rev Plant Physiol 27: 507-52813. CODY YS, DC GROSS, EL PROEBSTING JR, RA SPorrs 1987 Suppression of ice

nucleation-active Pseudomonas syringae by antagonistic bacteria in fruit treeorchards and evaluations of frost control. Phytopathology 77: 1036-1044

14. FUKUTA N 1966 Experimental studies of organic ice nuclei. J Atmos Sci 23:191-196

15. GROSs DC, YS CODY, EL PROEBSTING JR, GK RADAMAKER, RA SpoTrS 1983Distribution, population dynamics, and characteristics of ice nucleation-active bacteria in deciduous fruit tree orchards. Appl Environ Microbiol 46:1370-1379

16. GRoss DC, YS CODY, EL PROEBSTING JR, GK RADAMAKER, RA Spom 1984Ecotypes and pathogenicity of ice-nucleation-active Pseudomonas syringaeisolated from deciduous fruit tree orchards. Phytopathology 74: 241-248

17. GROSS DC, EL PROEBSTING JR, PK ANDREWS 1984 The effects of ice nuclea-tion-active bactmria on temperatures of ice nucleation and freeze injury ofPrunus flower buds at various stages of development. J Am Soc Hortic Sci109: 375-380

18. HAEFELE DM, SE LINDOW 1982 Localization and quantification of ice nucleiand ice nucleation active bacteria associated with dormant and growing peartissue (abstr). Photopathology 72: 946

921



922 GROSS ET AL.

19. HAEFELE DM, SE LINDOW 1987 Flagellar motility confers epiphytic fitnessadvantages upon Pseudomonas syringae. Appl Environ Microbiol 53: 2528-2533

20. KING EO, MK WARD, DE RANEY 1954 Two simple media for the demonstra-tion of pyocyanin and fluorescin. J Lab Clin Med 44: 301-307

21. KOZLOFF LM, MA SCHOFIELD, M LUTE 1983 Ice nucleating activity of Pseu-domonas syringae and Erwinia herbicola. J Bacteriol 153: 222-231

22. LINDOW SE 1983a Methods of preventing frost injury caused by epiphytic ice-nucleation-active bacteria. Plant Dis 67: 327-333

23. LINDOW SE 1983b The role of bacterial ice nucleation in frost injury to plants.Annu Rev Phytopathol 21: 363-384

24. LINDOW SE, DC ARNY, WR BARCHET, CD UPPER 1978 Control of frostdamage to plants in the field with bacterial ice nucleation inhibitors (abstr).Phytopathol News 12: 138

25. PHELPS P, TH GIDDINGS, M PROCHODA, R FALL 1986 Release of cell-free icenuclei by Erwinia herbicola. J Bacteriol 167: 496-502


26. PROEBSTING JR EL, PK ANDREWS, D GROSS 1982 Supercooling young devel-oping fruit and floral buds in deciduous orchards. HortScience 17: 67-68

27. PROEBSTING EL JR, DC GROSs 1988 Field evaluations of frost injury todeciduous fruit trees as influenced by ice nucleation-active Pseudomonassyringae. J Am Soc Hortic Sci 113: 498-506

28. QUAMME HA 1978 Mechanism of supercooling in overwintering peach flowerbuds. J Am Soc Hortic Sci 103: 57-61

29. VALI G 1971 Quantitative evaluation of experimental results on the heteroge-neous freezing nucleation of supercooled liquids. J Atmos Sci 28: 402-409

30. VALI G, EJ STANSBURY 1966 Time-dependent characteristics of the heteroge-neous nucleation of ice. Can J Phys 44: 477-502

31. WOLBER PK, CA DEININGER, MW SOUTHWORTH, J VANDEKERCKHOVE, MVAN MONTAGU, GJ WARREN 1986 Identification and purification of abacterial ice-nucleation protein. Proc Natl Acad Sci USA 83: 7256-7260



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