Platu, Cell and Envirotitnent (1985) 8, 333-^338

Extraorgan freezing in wintering flower buds ofCornus officinaiis Sieb. et Zucc.

MASAYA ISHIKAWA & AKIRA SAKAI The Institute of Low Temperature Science, Hokkaido University,Sapporo 060, Japan

Received 11 Februaty 1985; accepted for publication 28 February 1985

Abstract. Extraorgan freezing as a tnechanism forincteasing cold hardiness was shown using flowerbuds of Cornus offieinalis Sieb, et Zucc, Differentialthermal analysis (DTA) revealed that florets in flowerbuds of C. offieinalis owed their cold hatdiness todeep supercooling and also that slower cooling ratesiticreased the supercooling ability of florets.During slow stepwise cooling (5°C h ' ' ) , the watercontent of florets decteased and that of scales(involucral bracts) incteased, which lesulted inaccumulation of ice within the scales. This was tnoreextensive in early winter and early spring buds thanmid-winter ones. Flower buds with silicone oil in thespace between florets and scales also showed asimilar decrease in water content of florets atid anincrease in that of scales. This indicated that watertnigration from the florets to the scales probablytook place by way of the peduncles and thereceptacle, possibly through their vascular traces,and not directly frotn the surface of the florets to theice sink in (he fortn of vapour. Possible mechanismsof extraorgan freezing are postulated along with thisfinding,

Kev-wcnds: Cornus offieinalis Sieb. et Zucc. (Cornaeeae); differen-liai thermal analysis: cold hardiness: extraorgan freezing: flowerbuds; freezing injury: ice nucleation: supercooling: va,sculartissues: water relations.

Introduction

11 is well known that the flower buds of sotnewintering temperate woody species deep-supercool asa mcchanistn for cold hatdiness (George, Burke &Weiser, 1974; Gtahatn & Mullin, 1976; Quatntne,1978; Rajashekar & Burke, 1978; Proebsting &Sakai, 1979; Sakai, 1979; Ishikawa & Sakai, 1981,1982), It is also known that slower cooling rates orstorage at subfreezing tetnperatutes increases thesupercooling ability of florets in sotne of these flowerbuds (Ishikawa & Sakai, 1981; Burke & Stushnofl",1979), In cold hatdy Rhododendron flower buds,changes in supetcooling ability were attributed towater tnigration frotn flotets to scales which tnay actas an ice sink during the slow freezing (Graham &

Correspondence: Masaya Ishikawa, Crop Development Centre,t^Jniversity of Saskatchewan, Saskatoon, Saskatchewan, CanadaS7N OWO.

Mullin, 1976; Ishikawa & Sakai, 1981), Thisphenotnenon—slow dehydration of the supercooledorgan as a whole and completnentary fortnation ofice in sotne other tissues at naturally occurringcooling tates—was defined as 'extraorgan freezing'by Ishikawa & Sakai (1982), Extraotgan freezitig ischaracterized by the presence of a barrier at theorgan or tissue level against ice propagation into thesupctcooled organ frotn already frozen tissues. Thebarrier tnay also serve as a path for waterttanslocation frotn the supercooled organ or tissuesto the ice sink if it is penneable to water and not toice. However, in angiosperm woody plant flowerbuds, neither the tissue acting as the barrier nor thepath for the water tnigration has been wellcharacterized. Here we teport on the details ofextraotgan freezing in flower buds of Cornusoffieinalis. The evidence suggests that the pedunclesand the receptacle are involved in water transloca-tion frotn the florets to the scales, possibly via theirvascular ttaces, during slow freezing.

Materials and methods

Twigs with flower buds were collected frotn thenorthern side of a single tree (about 5 tn tall), Cornusoffieinali.s Sieb, ct Zucc, in the Botanical Garden ofHokkaido Univetsity on 16 Decetnber 1980, 18Januaty and 3 April 1981,

DilTerential thcnnal analysis (DTA) was carriedout as described befote (Ishikawa & Sakai, 1981),Exothertns were detected with 0,2 tntn Cu-constantan thertnocouples, atnplified 40 titnes andrecorded on potentiometric recorders. An excisedtwig (about 1 ctn) with a flower bud was attached tothe thermocouple and wtapped with alutniniutn foil.The samples were cooled at the rate of 2,5 to 9 Ch~ ' . Eight to ten flower buds were used for eachDTA, In sotne experiments, to check the correlationof exothertns to itijuty of florets, cooling was stoppedduring the course of DTA, buds were rewarmed, andincubated in a polyethylene bag at rootn tetnperaturefor a week to evaluate itijury of the florets visually.

To study the effect of cold tt eattnent or precoolingon exotherm temperatures and the water content offlower buds, 15 cm twig segments with flower budswere enclosed in polyethylene bags containing a

333

334 M. ISHIKAWA & A. SAKAI

Involucralbracts Florets

Twig

Figure 1, A sketeh of the longitudinal section of a C. oflieinalisflower bud. Broken lines indieate vaseular traees. Arroviis a. h ande point out where the bud was exeised from the twig for DTA. Inusual DTA experiments, buds were exeised at line it. *Spaee wheresilieone oil was inserted.

small atnount of snow to prevent desiccation andcooled at 5 C decretnents daily (0 to —15 C).Subsequently, the flower buds in the frozen statewere excised at each temperature and usually ninebuds were used for water content determination.Eight buds were cooled further to —45 C andexothertn temperatures were recorded. The othereight buds were carefully observed with a binocularmicroscope to find the location of ice. To determinethe water content of the frozen buds, each bud wasdissected into its cotnponents in cold rootns at —5 to— 15 C. Each part was then wrapped withaluminium foil and put into small tightly fittingpolyethylene bags in order to minimize tissue waterloss during rewanning. After the bags had beenbrought to room temperature, tissue weight wasmeasured. In sorne experitnents silicone oil wasinserted with a syringe into the space between theflorets and the scales without datnaging thetn(Fig. I). They were then cooled in the satne manneras described above and used for DTA and watercontent determination. Water content was expressedas a percentage of dry weight, which was detertninedafter 24 h of oven-drying at 70 to 80 °C.

Results and discussion

Low temperature exottierms (LTE) and infiiry toftorets

A wintering flower bud of C. offieinalis has about 20to 25 florets surrounded by four involucral bracts orscales (Fig, 1). A typical DTA profile of a llower bud

(a )

\

(b )

HTE

AJLTE

(c 1

^ f

0°C Stored9°C/H

0°C Stored5°C/'H

k-9°C Stored

i

-10 -15 - 2 0 - 2 5

Temperature (°C)

- 3 0

Figure 2, Typieal DTA profiles of C. oflicitudis llower buds on 3April, eooled at dilTerent rates. When eooled at 9 C h " ' , sealesand Horets froze together as indieated by the large exother'rn (a),(b) When the llower bud was cooled at 5"C h " ' . seales IVo/.e frrst.as shown by IITE. and the small spikes (LTE) between —8 and— 18"C indieated lethal fr-eezing of llorx-ts. (e) The bud waspreeooled to —9"C in 2 d (the scales had already frozen: thus, noHTE appeared), then eooled further at 5"C h " ' without rhawing.See legend to Fig. .̂ Ibr the meaning of the arrow.

collected on 3 April and cooled at 5 C h ' is shownin Fig. 2b. As shown in the case of Cornus ftorida L.Rower buds (Sakai, 1979), the nutnetous spike-likeLTE represented the fteezing of supercooledindividual florets, which resulted in their death,pr-obably due to intracellular ice formation. On theother hand, the high temperature exothertn (HTE)represented the non-lethal extracellular freezing ofthe scales and other tissues. When a (lower bud wasrewarmed at LTE yielding tetnpetatures during thecourse of DTA (for example, at the arrowedtetnperature in Fig. 2c), the nutnber of florets injuredcoincided with the nutnber of spikes (LTE) produced(Fig. 3c). No itijury was detected in the other tissuesof the flower bud, including involucral bracts(scales), peduncles, receptacle, and adjacent twigtissues. These data indicate that Cornus florets owedtheir cold hardiness mechanistn to supercooling, andthat the nucleation of the supercooled Horets resultedin their death, as dernonstr'ated in Rtiododendron(Graham & MuUin, 1976; George el at., 1974;Ishikawa & Sakai, 1981, 1982) and Pruniis(Quatiime, 1978) flower buds.

EXTRAORGAN FREEZING IN CORNUS FLOWER BUDS 335

Figure 3. (a), (b) Cross-seetions of Cornus llower buds on 3 April, eooled to — 9 °C in 2 d. Iee erystals (1) formed in tbe seales,that is, involuetal btaets (B) while the llorets (F) were in a dehydtated state, (b) Cross-seetion of 0.8 mm thieknessphotogtaphed with polarized light. Eaeh iee erystal was reeognized by the dilTetent eolour. (e) Florets of a C. offieinalis (lowerbud whieh was taken out of the freezer at - 2 2 " C in the midst of the LTE emerging tetnpetature range after prefreezing to- 9 ° C in 2 d (an example of a DTA protile of a bud treated in this way is shown in Fig. 2e; the arrow indieates thetemperature at whieh eooling was stopped and the bud was tewartned), then rewartned and itieubated for a week at roomtetnperature. tnvolueral braets (seales) wete stripped olT. Greenish yellow florets were alive (L), and yellowish brown oneswere dead (K). Magniiieation x 10.

Effect of cooting rates and preeooting on ETE andwater eontent of flower bud eomponents

When flower buds (3 April) were cooled at 9°C h~ ' ,LTE appeared at vety high tetnpetatures and werenot separated from the HTE (Fig. 2a). In eontrast,when the bud was precooled at about 5"C d~' to— 9"C and subsequently subjected to DTA, LTE(killing temperature of florets) were shifted to lowertetnperatures and the LTE size was reduced (Fig. 2c).

To elucidate the cause of the increase insupercooling ability of flotets during precooling to— 9 °C, the flower buds wet e cooled slepwise (5 Cd" ' ) and the water content of flower bud parts andlocalization of ice were deteitnined at each subzerotemperature. As shown in Fig. 4, during the stepwisepreeooling to —15 C, the water content of theflorets greatly decreased and that of the scalesincreased in a concomitant fashion. There were nolarge changes in the water contents of other tissues

and of the whole flower bud. These ehanges in watercontent of florets and scales during precooling weremore extensive in early winder (16 Decetnber) andearly spring (3 April), when the initial water contentof florets was high, than in flower buds of tnidwinter(18 January) (Fig. 4).

The incteased water content in the involucralbracts (scales) can be attriliuted to the accutnulalionof ice crystals within them (Fig. 3a, b). Because ofthe ice segregation, a large air space had developedwithin the scales and they looked like empty bagswhen observed in a thawed state. Early winter budswhich had not experienced frosts in the held did nothave a distinct air space such as seen in tnidwinterbuds and arlilicially lYozen buds.

These observations pennil one to conclude thatduring the slow cooling of Cornus flower buds, thescales freeze lirst and that water tnigrates frotn theflorets to the scales whete ice is formed; that is, theflorets undergo extraorgan freezing during the slow

336 M. ISHIKAWA & A. SAKAI

2 4 0

2 0 0

160

S 120

80

4 0

t6 0«c. O—O Scat*

A—A Ftortf

*•—A Bud axis

D—D Twig

Jan.

- 5 - t o - t 5

3 Aprit

0 - 5 - t o - t 5

Ttmperafure ( °C )

-5 - t o - t 5

Figure 4. Changes in water eontent of flower bud parts of C. offieinalis during cooling to — I5°C using daily decrements of5"C. Flower buds were eolleeted at three times during the eold season.

cooling in a sitnilar manner to cold hardyRhododendron flower buds (Ishikawa & Sakai, 1981).The decreased water content of the florets seems toresult in the enhanced supercooling ability of florets(Fig. 2c), as indicated with Rhododendron flowerprimordia (Ishikawa & Sakai, 1981).

Involvement of the hasat part of flower buds, possihlvvaseutar ti.i.iues, in the extraorgan freezing

We attempted to detertnine the pathway for watermigration between the florets and the scales, Siliconeoil was inserted with a syringe into the space betweenthe florets and the scales without datnaging them toprevent direct transloeation of water as vapour fromthe surfaee of the florets to the scales (Fig, 1). Evenin this case, during cooling to — 9°C, the floret watercontent decreased and that of the scales increased tothe same degree as in intact control buds (Fig. 5).This indicates that water in the floret may nottranslocate directly from the surface of the florets tothe scales in the fortn of vapour, but mainly throughthe peduncles and the reccptable. Even though thepossibility of direct water Iranslocation as vapourfrom the florets to the ice sink in flower buds in situcannot be denied, the path by way of the pedunclesand the receptacle alone seems to translocate thefloret water successfully and accotnplish extraorgan'freezing.

Longitudinal sections of flower buds of Cornusrevealed that florets, peduncles, the reeeptacle andscales were interconnected by vascular traces andthat there were no distinct gaps or special tissues toprevent ice propagation into the florets (data notshown). Water transloeation may occur eitherthrough these vaseular traces or the intercellularspaces between the parenchymal cells surroundingthem.

Involvement of the basal part of the flower bud in

supercooling of florets was suggested by anotherexperiment. At the cooling rate of 2 to 3"C h ',florets in a winter bud (18 ,Ianuaty) excised al line a(Fig. 1) supercooled normally (LTE: —19 lo— 23 C). (In the previous experiments of DTA, theflower buds used were all excised frotn the line a.) Incontrast, in the buds cut at line h, LTE were shiftedto higher temperatuies (LTE: - 5 to - 2 0 X ) , andwhen excised at line e, they did not deep-supercooland froze with other lissues (LTE: -^6 to — 7 C).This may indicate the necessity of an 'intact" twig

Scate Floret Axil Twig Tatat

Figure 5, Changes in water eontent of C. offieittatis flower budparts on .1 April during eooling frotn 0 to —9"C in 2 d. (D)Water eontent of llower buds parts at OC (the mean of flowerbuds wilh and wilhoul silicone oil); (W) water content at - 9 ' C(without silieone oil); (m) water eontent at —9 C (with silieoneoil in the buds). The water eontent tneasutetnents wete done justbefore cooling at 0 C and also at — 9"C with ftozen flower buds.

EXTRAORGAN FREEZING IN CORNUS FLOWER BUDS 337

adjacent to the flower bud in the supercooling of thellorets.

In the extraorgan freezing of conifer buds (Ahies,Pieea. Lari.x) and hydrated lettuce seeds, it has beenshown that the ctown tissue (Sakai, 1983), and theendosperm with its cuticle layer (Ishikawa & Sakai,1982; Keefe & Moore, 1981), respectively, are thebarriers against ice penetration into the supercooledotgans. The water in these supercooled organsfreezes on the opposite side of the barriers, having icectystals segregated. In contrast, in flower buds ofRhododettdron (Ishikawa & Sakai, 1981), Prtwtis(Ashworth, 1982), and Cornus offtcinatis (the presentstudy), no special tissues or gaps have beenrecognized as the barrier against ice intrusion at thebasal part of the flower pritnordia. Nevertheless, theimportance of intactness of the bud axis inpreventing ice penetration into the supercooledflower primordia has been detnonstrated by Quamnie(1978) and Ashwotth (1982). In the case of C.offtcinatis, it sectiis that vascular traces interconnect-ing the Horets atid the scales, together with theintercellular spaces atnong the parenchytnal cellssurrounding the vascular tissues, may not only be abarrier against ice propagation into the florets frotnthe already frozen tissues, but may also serve as thepath for water migration frotn the florets to thescales during the slow freezing.

Questions of how the pathway can be permeableto water and not to ice and what the driving force forthe water migration is, remain unsolved. However,this can be postulated as follows. As indicated inFig. 2b, the scales froze lirst in spite of their lowerwater content compared to the florets (Fig, 4). Thiscould be due to the presence of ice nucleatingbacteria (Lindow et at., 1978), to the ptescnce ofintrinsic substances of high ice nucleating abilitywithin the scale tissues, or to the tetnperaturegradient fortned between scales and florets during theprocess of cooling. On the other hand, it iseonceivable that flotets tnay lack the bacteria orsubstances of high ice nucleating activity to avoidtheir spontaneous freezing. Once the freezing inscales starts, ice tnay not propagate in florets andinstead ice segregates within scales if the tetnperatureof the water soutce (floiets) is beyond their fteezingpoint, atid/or if there is any barrier between thewater souree and the iee aecutnulation site. Theformer possibility tnay hold true at the early stagesof freezing in seales. However, as the tetnperaturegoes down further, the floret water may be in asupercooled state because the water flow frotn thesource to the sink is not rapid enough to remove allthe freezable water at that temperature frotn florets(Ishikawa & Sakai, 1981), Therefore, the ptesence ofa barrier seetns essential and could be localized at thebase of the scales, possibly in vascular tissues. Onespeculation of such a barrier comes from thesitnilarity of extraorgan freezing to frost-heaving ofsoil and ice needle formation on the soil surface; it is

known that in soil with a grain size of 2 to 5 pm,frost-heaving results very easily, but if the grain sizeis tnore than 50 //m, it is difficult for frost-heaving tooccur (Beskow, 1935). The capillary diatneter ofvascular tissues seetned to be at tnost 20 to 30 /nn inCornus flower buds (data not shown) and appearedcotnparable to the case of frost-heaving. Anotherpossible type of barrier is deposition of a substancewhich prevents ice crystal growth in vascular tissuesor elsewhere.

The driving force for transloeation of water inthe extraorgan freezing of Cornus flower buds tnaybe attributed to the chemical potential diflerencebetween liquid water and ice in the first place, as inthe case of extracellular freezitig (Levitt, 1980),Subsequently, ice formation in the scales may alsolower the water potential of cells in the scales andadjacent tissues, which fortns a water potentialgradient between the scales and the florets. Thisgradient could also be a driving force for watertnigration. When the water potential diflerencebeeotnes stnaller as the water content of floretsdecreases during extraorgan freezing, the watertransloeation rate tnay be further reduced. When thefloret water content is teadily low, as in ,Ianuarybuds (Fig. 4), the water potential gradient betweenflorets and scales during extraorgan freezing tnightbe stnaller, which results in lower exttaorgan icesegregation. Further research to check the validity ofthe hypothesis is going on.

In sutntnary, the flower buds of C. offieinatis wereshown to undergo extraotgan IVeezing during slowfreezing. The involucral btaets (scales) were an icesink. The extraorgan freezing tnay be a tnechanistnto inctease the supercooling ability (cold hatdiness)of the florets in response to prolonged subfreezingtemperatutes, especially in early winter and earlyspring when the floret water content is relativelyhigh. These features were essentially similar to theextraorgan freezing iti cold hardy Rhcnhdendrcmflower buds (Ishikawa & Sakai, 1981), but tnoretypically shown in the case of Cornus flower buds.T'aking advantage of the simple structure of theflower buds of C. offieinalis (a eluster of floretssurrounded by four scales), we injected silicone oilitiside the flower buds to ptevent direet water vapourtranslocation frotn the florets to the scales. Thisexperitnent indicated that the water tnigration duringthe extraorgan freezing may take place through thepeduncles and the receptacle, possibly through theirvascular traces.

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