HORTSCIENCE , VOL. 26(7), JULY 1991

HORTSCIENCE 26(7):887-890. 1991.

Bound versus Free Water in DormantApple Buds—A Theory forEndodormancyMiklos Faust and Dehua LiuFruit Laboratory, Beltsville Agricultural Research Center AgriculturalResearch Service, Beltsville, MD 20705

Merle M. MillardEnvironmental Chemistry Laboratory, Beltsville Agricultural ResearchCenter, Agricultural Research Service, Beltsville, MD 20705

G.W. StutteDepartment of Horticulture, University of Maryland, College Park,MD 20742

Additional index words. Malus domestica, chilling, magnetic resonance imaging,thidiazuron

Abstract. Intact apple (Malus domestica Borkh.) buds were examined by magneticresonance imaging (MRI). MRI did not excite water in unchilled apple buds and couldnot image it. When chilling was satisfied, images were produced. We interpret thisdifference to mean that water is in bound and/or structured form in dormant appleleaf buds before the chilling requirement is satisfied. Conversion of bound to free wateroccurred equally in the low-chilling-requirement cultivar Anna and the high-chilling-requirement cultivar Northern Spy only after 600 and 4000 hours of chilling, respec-tively. It appears that processes involved in satisfying chilling requirement are alsoconverting water in buds from bound to free form. Absence of free water in dormantbuds during the winter signifies endodormancy, whereas when the water is in freeform, buds are ecodormant. Thidiazuron, a dormancy-breaking agent, applied to par-tially chilled buds is instrumental in converting water to the free form within 24 hours.Summer-dormant buds contain free water, and they could be classified only as para-dormant. Based on proton profiles, ecodormant and paradormant buds cannot bedistinguished but endodormant buds can be readily identified.

Nondestructive imaging of plant tissues bymagnetic resonance imaging (MRI) is a use-ful method to assess the water status of cer-tain plant organs. Soft-tissue-contrast in MRIimaging is primarily due to differences inproton relaxation times. Relaxation charac-teristics are related to molecular motion.Theories on this subject were recently re-viewed (Wehrli, 1988). The two relaxationprocesses can be described by the longitu-dinal (Tl) and transverse (T2) relaxation timesand have been used to study water in planttissues (Chen et al,. 1978; Stout et al., 1978).

The average rate at which molecules reo-rient in a magnetic field is related to molec-ular size. Small molecules, such as water,reorient more rapidly and relax more slowlythan larger molecules, which reorient slowlyand relax much faster. Free water relaxesslowly (long T1 and T2 times) and gives asharply contrasting MR image, but, waterbound to or perturbed by macromolecules(called structured water) relaxes consider-ably faster (short T1 and T2 times) and doesnot show on the MR image (Fullerton, 1988).

Received for publication 17 Oct. 1990. The costof publishing this paper was defrayed in part bythe payment of page charges. Under postal regu-lations, this paper therefore must be hereby markedadvertisement solely to indicate this fact.

Water fractions may change in tissues as thetissue function changes. Thus, images ob-tainable from the tissues may also changeaccordingly.

Temperate-zone fruit trees, including ap-ple, require certain amounts of chilling be-fore they can resume growth. There aremodels that predict chilling requirement.However, little is known about the physio-logical and biochemical events that mark the

Fig. 1. Schematic illustration of imaging planeof apple buds. The two circles, transected bythe line A–B, correspond to the images of MRI.Occasionally, a graph is superimposed on theimage between A and B representing the rela-tive MR signal intensity for each pixel.

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Fig. 2. Magnetic resonance images of leaf buds of ‘Anna’ apple (A) after 200 h and (B) after 600 hof chilling. Note that (A) has only the stem section image and (B) shows both the the stem andbudsection images.

Fig. 3. Magnetic resonance images of leaf buds of ‘Northern Spy’ apple (A) after 1000 h and (B)after 4000 h of chilling. Similarly to ‘Anna’, (A) shows only the stem section image, whereas (B)has both stem and bud images.

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end of the chilling period (Faust, 1989). Langet al. (1985) proposed a unified terminologyfor dormancy. They distinguished betweenpara-, endo-, and ecodormancy dependingon factors regulating dormancy by physio-logical factors originating outside or insideof the affected structure, or by the environ-ment, respectively. Endodormancy in fruittree buds is thought to be released by chill-ing; thereafter, the buds are in ecodormancyuntil growth resumes (Faust, 1989). Para-dormant buds during summer dormancy canbe forced to grow by removing the terminalbud of the shoot. Such operation does notforce resumption of growth in buds that areendodormant. Resumption of growth also canbe forced by cytokinin-type compounds suchas benzyladenine in summer-dormant buds(Broom and Zimmerman, 1976) and by thi-diazuron (TDZ) in buds that may be char-acterized as paradormant (e.g. before chillingis initiated or during summer dormancy)(Steffens and Stutte, 1989, Wang et al.,

Here, we report the water status of applebuds, determined by MRI, during chilling,summer dormancy and forcing by TDZ.

‘Anna’, a low-chilling-requiring cultivar,and ‘Northern Spy’, a high-chilling-require-ment cultivar, were used for the studies.Shoots, grown during the summer, were cutlate in October and stored at 4C in a coldroom. Samples were removed after specifiedchilling hours had been imposed. Each shoot,after removal from the cold, was placed indistilled water for 20 to 24 h, at room tem-perature, before MRI analysis. Buds of ‘Anna’were treated with TDZ by painting a 100-ppm solution on the buds after removal fromstorage; these buds were kept at room tem-perature for 24 h, similar to untreated budsas described.

Summer-dormant buds to be imaged wereprepared on 25 July 1990, by removing theterminal buds in the orchard from some shootsand leaving others intact. Sample shoots ofboth types, without terminals and with setterminal buds, were collected 1 Aug. 1990.By sample collecting time, on shoots wherethe terminal bud had been removed, the toptwo lateral buds started to break. MRI wasperformed on buds below those breaking andbuds at comparable locations on shoots withterminal buds intact. MR images of replicatesamples were remarkably similar. Leaf budswere used for all imaging.

Shoots were placed in 10-mm MRI tubesfor imaging with a Bruker MSL, 400 MHz(9.4T) microimaging instrument (Broker Instr.Co., Billerica, Mass.). Conventional spinecho 2DFT images of matrix size 256 × 256were obtained with spatial resolution of 58.5× 58.5 × 720 µm. Method was similar tothat described by Kuhn (1990). In all im-ages, the TE was 14 ms and the TR was 514ms. First, images were taken with fewer beingaveraged for rapidity of examination. Thisprocedure resulted in excessive noise in theimages, but allowed rapid replication ofsamples. Then, selected samples were im-aged with more being averaged. Due to sig-nal noise considerations, between 100 and

HORTSCIENCE , VOL. 26(7), JULY 1991

Fig. 4. Magnetic resonance images of leaf buds of ‘Anna’ apple treated with 100 ppm TDZ after 200h of chilling. The image should be compared with Fig. 2A (untreated at the same chilling level) andFig. 2B (a fully chilled bud).

Fig. 5. Magnetic resonance image of summer-dormant buds of ‘Anna’ apple. (A) Image of lateralbud with intact terminal bud. (B) Image of lateral bud on a shoot where terminal bud was removed1 week before imaging.

512 averages were taken, resulting in dataacquisition times of up to 24 h. Image sliceswere taken perpendicular to the axis of theshoot about at the midpoint of the bud (Fig.1). Taking the image slice higher or lower

HORTSCIENCE , VOL. 26(7), JULY 1991

in the bud resulted in a smaller circle of thebud image. Hard copy of images was ob-tained using a Sony video graphic printerUP-811. Image of at least six buds were takenof each sampling date or bud type.

According to the proton profiles for bothcultivars, sufficient free water to produce animage was present only in the stem beforethe chilling requirement was satisfied (Figs.2A and 3A). Buds produced no image at thistime. Free water in the bud was readily ob-servable and created images in ‘Anna’ after600 h of chilling (Fig. 2B) and in ‘NorthernSpy’ after 4000 h of chilling (Fig. 3B). In-termediate stages before the chilling require-ment was satisfied showed only traces of freewater (images not presented). The moisturecontent of buds, determined by dry weight,was 49% before and 59% after chilling wassatisfied and the difference was statisticallynot significant. This result indicates that, inthe unchilled, endodormant buds, the wateris in a form that creates T1 or T2 times thatare too short to form an image. We inter-preted this result to mean that in endodor-mant buds, water is in bound or structuredform. This interpretation further implies thatendodormancy is a stage of bud developmentwhen water is in a bound or structured form.This situation contrasts with that of chilledbuds, in which sufficient quantity of wateris in free form to create images. So far, wedo not know, but do suspect from applicationof TDZ, that a substantial part, but not all,of the water is in a free form. Regardless ofthe portion of water that is in free form, chilledbuds differs greatly from endodormant budsas far as the form of water is concerned.

Chen et al. (1978), in winter wheat crowns,observed a significant decrease in relaxationtimes of T1 and T2 with nuclear magneticresonance (NMR) after 7 weeks of accli-mation. T2 time for tissue culture aggregatesof wheat was much longer than that in fullyhardy crowns (Stout et al. 1978). Stout etal. (1978) observed two populations of waterin ivy bark with NMR, one with short T1and T2 times (≈32% to 40% of the water)and another with long T1 and T2 times (≈60%to 68% of the water). They believed that thelarger quantity of water, which had the longerT2 time, was intracellular water, and the waterthat had short T2 time was associated withcell walls. We do not know the degree ofdormancy in wheat crowns or in ivy bark,but we believe that these studies indicate thatat least part of the water can be in bound/structured form in hardened and partiallydormant plant tissues–in contrast to thepresence of free water (long T2 time) in tis-sue culture aggregates, which represent ac-tively growing tissue.

The outside layer of bark, and an innerring, where the cambial layer and/or theyounger areas of xylem are, contain consid-erable free water even before chilling re-quirement of buds is satisfied. If the absenceof free water is considered as a criterion forendodormancy, as we advocate, tissues ofthe stem are not endodormant.

TDZ treatment changed the bound/struc-tured water-free water relationship within 24h in ‘Anna’, and the buds became visible onMRI images with a higher-intensity protonsignal than satisfying the chilling require-ment can produce (Fig. 4). Images of budswithout TDZ were the same as shown in Fig.

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2A. TDZ is not effective during endodor-mancy of other cultivars to induce growth(Steffens and Stutte, 1989) and needs to beapplied in late fall, before chilling is initi-ated, or to partially chilled buds when a sub-stantial part of the chilling requirement hasbeen satisfied. TDZ is also effective inbreaking summer dormancy of apples (Wanget al., 1986). From these limited experi-ences, it appears that TDZ is effective inparadormant buds to force growth or in budsthat are near to the end of their endodor-mancy. Because TDZ is effective after 200h of chilling (less than half the normal re-quirement) in ‘Anna’ buds in converting waterto free form, which is required for resump-tion of growth, it appears that ‘Anna’ budsare not in as deep dormancy as those of‘Northern Spy’, which require 80% or moreof their chilling requirement to be satisfiedbefore TDZ is able to break dormancy (Stef-fens and Stutte, 1989). Thus, we may belooking not only at a difference in length inendodormancy, but also a difference in thedepth of endodormancy. TDZ appears to bea useful tool to evaluate this aspect.

Summer dormant buds contain free water(Fig. 5 A and B). When the terminal bud isremoved and the top buds of the shoots areactivated, the proton profile of the still-dor-mant lateral bud (Fig. 5B) is somewhat moreintense than that of the lateral bud associatedwith an intact terminal bud of the shoot (Fig.5A). Nevertheless, based on proton profile,the dormant buds collected during the sum-mer contained free water and their protonprofile was definitely different from the budsthat were endodormant.

The xylem proton profile of shoots is muchless clear than similar profiles in endodor-mant or paradormant buds. Intensity of freewater of xylem of the the shoot with theterminal bud removed and the top two orthree buds activated (Fig. 5B) shows muchhigher free water concentration than the xy-lem of the shoot with the terminal bud andwith all the buds dormant (Fig. 5A). In fact,the xylem proton profile of shoots activatedto grow by removing the terminal bud (Fig.5B) is as strong as the xylem proton profileof stems on which buds were activated byTDZ (Fig. 4). This pattern contrasts with therelatively weak proton profile of the xylemof the chilled or unchilled dormant buds (Figs.2B and 3 A and B). However, occasionallywe had higher proton profile in the xylemeven in shoots with endodormant buds (Fig.2A). Although activation of buds either byTDZ or by removing terminals over para-dormant buds may increase free water in thexylem, we do not know the importance offree water in the xylem of endodormant orparadormant shoots without further research.

In considering winter buds, bound watersignifies endodormancy. As chilling is com-pleted, the buds have free water. They areready to grow, but they are still dormant be-cause the environment is not conducive togrowth. Such buds are classified ecodor-mant. Based on proton profile, summer-dor-mant (paradormant) buds are not differentfrom those in ecodormancy. Thus, based on

proton profiles, we cannot distinguish be-tween ecodormant and paradormant buds, butwe can definitely separate endodormant budsfrom the others.

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HORTSCIENCE , VOL. 26(7), JULY 1991