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Western immunoblot analyses showed that small heat shock proteins (smHSPs) are low or undetectable in the peel of ‘Fuji’, ‘Jonagold’, ‘Criterion’, ‘Gala’, and ‘Delicious’ apples [(Malus sylvestris (L.) Mill var. domestica (Borkh.) Mansf.] growing shaded within the tree canopy (shade apples), but are high in apples growing exposed to direct sunlight (sun apples). ‘Fuji’, ‘Jonagold’, and ‘Gala’ sun apples sampled biweekly between 1 July and 21 Oct. 1997 were highest in content of smHSPs on 31 July, 13 Aug., and 10 Sept., corresponding to some of the warmest periods of the sampling period. The smHSPs started to disappear first in ‘Gala’, the earliest maturing cultivar, and last in ‘Fuji’, the latest maturing cultivar indicating that maturity might play a role in regulating smHSP accumulation. In sun apple fruit left on trees for 60 to 120 days beyond commercial maturity and exposed to field temperatures as low as –4 C, a 71.7 ku (u = unified atomic mass unit) polypeptide was detected with a polyclonal antiwheat (Triticum aestivum L.) HSP70 in the peel and cortex of all five cultivars. While no smHSPs were detected in these apples, three smHSPs, as detected by antibodies against pea (Pisum sativum L.) cytosolic HSP18.1, could be induced in the same fruit 24 hours after heating to 45 C for 4 hours. In ‘Fuji’ shade apples heated at 40 C, smHSP accumulation was detected after the second hour of a 4-hour heat treatment and continued to increase over the next 48 hours at 22 C. Levels of HSP70 did not change in ‘Fuji’ shade apples heated at 45 C for 2, 4, or 6 hours, but smHSPs became detectable immediately after each of these heat treatments and further increased over the next 24 hours at 22 C. Accumulation of smHSPs was maximal in the 4-hour heat treatment. After a 4-hour heat treatment at 45 C, smHSPs increased during the next 48 hours at 22 C and then declined by 72 hours. Using two-dimensional electrophoretic analysis, as many as 17 proteins ranging from 15 to 29 ku were found to accumulate in the peel 48 hours after a 4-hour heat treatment. Thus, apples can respond rapidly to high temperature stress, even at advanced stages of maturity, by synthesizing smHSPs, which likely play an important role in protecting cellular biochemical processes during these periods of stress. nonheat-stressed vegetative tissue, but can increase 2000-fold (to over 1% of total protein) upon heat stress (DeRocher et al., 1991; Waters et al., 1996). In vitro, several smHSPs protect cellular constituents and metabolic functions. For example, HSP18.1, a Class I cytosolic smHSP in pea (Pisum sativum), forms oligomers of 12 subunits with molecular weights between 200 to 240 ku (Lee et al., 1995) and can bind denatured enzymes. Thus, smHSPs help by preventing aggregation and promoting refolding back to functional enzymes (Jakob et al., 1993; Lee et al., 1995; 1997). This and other evidence suggest that smHSPs play an important role in adaptation of plants to high-temperature stress, but the precise mechanism of their functioning is still unclear (Härndahl et al., 1999). Our major goal is to understand the cause(s) of sunburn of apples (Malus sylvestris var. domestica), and to determine how fruit acclimate to survive conditions of high temperature and solar irradiation. We (L.E. Schrader and J. Zhang, unpublished data) have found that sunburn occurs in apples only when the fruit surface temperature exceeds 45 C, and that fruit not acclimated to stressful environmental conditions are more susceptible to sunburn. For example, shaded fruit that are suddenly exposed to sunlight, by pruning or the shifting of a branch laden with fruit, will sunburn at a peel temperature 1 to 4 C lower than will fruit that are acclimated by daily exposure to full sunlight (L.E. Schrader, unpublished data). Also, fruit are more likely to sunburn if rapidly changing weather conditions cause a sharp increase in air temperature than if air temperature increases graduReceived for publication 27 Dec. 2000. Accepted for publication 27 Apr. 2001. We gratefully acknowledge financial support of the Washington State Tree Fruit Research Commission. We also thank Elizabeth Vierling at the University of Arizona for generous donations of anti-HSP18.1 antibodies from pea and antiwheat HSP70 antibodies. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. Former research associate. Current address: Indian River Research and Education Center, University of Florida, 2199 S. Rock Rd., Ft. Pierce FL 34945-3138; e-mail: mrit@gnv.ifas.ufl.edu. Corresponding author; e-mail: schrader@wsu.edu. Many organisms accumulate heat shock proteins (HSPs) in response to high nonlethal temperatures (Vierling, 1991). Several classes of HSPs exist and the composition of HSPs varies among organisms. The HSP70s are highly conserved among prokaryotes and eukaryotes and their expression is both constitutive and heatinduced (Lindquist and Craig, 1988; Vierling, 1991). The HSP70s act to prevent aggregation of unfolded proteins (Hendrick and Hartl, 1995). Gatenby and Viitanen (1994) and Hartl (1996) have described the constitutive presence of HSP70s and their role as molecular chaperones. The small heat-shock proteins (smHSPs) are between ≈15 to 30 ku (u = unified atomic mass unit) and are less conserved in nature (Waters et al., 1996) than are the HSP70s. While the predominant HSPs of many eukaryotes are the HSP70s, the smHSPs predominate in plant species during heat stress (Waters et al., 1996). Furthermore, while some HSP70s are constitutively expressed (Perdue et al., 1998), smHSPs are rarely expressed in ally over several days. Acclimation occurs in the field and helps protect fruit from sunburn, but the factors responsible for this induced tolerance are not known. We hypothesized that HSPs are induced in peel of sunexposed fruit and play a role in acclimating apples to near-lethal temperatures. One objective of the present study was to characterize HSP accumulation in the peel of apples from several cultivars and maturity stages using fruit growing shaded within the tree canopy (shade apples) or exposed to direct sunlight (sun apples). A second objective was to examine induction of HSPs under controlled environments to characterize the influence of temperature and duration of heat shock (i.e., length of incubation at a given temperature) on the rise and fall of HSP70 and smHSPs. Ferguson et al. (1998) reported increased expression of HSP mRNA and radiolabeled protein in apple fruit cortex (≥5 mm below the skin of the fruit) associated with high daily temperatures. Because sunburn occurs in the apple peel, we studied proteins in the peel (epidermal and subepidermal tissue up to ≈1 mm from the surface) using antibodies specific to certain HSPs. Preliminary reports of our work on HSPs in apple fruit were published previously (Kochhar et al., 2000; Ritenour et al., 1998). Materials and Methods SAMPLE COLLECTION. During the 1997 growing season, ‘Jonagold’, ‘Criterion’, ‘Gala’, and ‘Delicious’ apples were harvested from 10-year-old trees trained to a slender spindle at the Washington State University Tree Fruit Research and Extension Center (TFREC), Wenatchee, Wash. ‘Fuji’ apples were collected from 11-year-old trees trained to a central leader and located ≈8 km north of the TFREC. From these orchards, apples growing exposed to direct sunlight between at least 10 AM and 2 PM (sun) and growing shaded within the tree canopy (shade) were harvested and transported to the laboratory in plastic bags for treatments and/or HSP analysis. Fruit that were shaded in the canopy were chosen for comparison to sun fruit rather than using the shaded side of sun fruit because the back side of sun fruit was warmed by heat dissipated from the exposed side, and also intercepted some scattered and reflected irradiation. Unless otherwise stated, heat treatments and samples for HSP analysis were taken on the day of harvest. All experiments included at least three replicates (one fruit per replicate) and replicates gave similar results except where noted. For HSP analysis, 1 g of apple peel was excised with a peeler from the most heated area of sun apples (area receiving direct sunlight during the hottest part of the day) or from the most shaded area of shade apples. The thin tissue segments were immediately frozen using dry ice, freeze-dried, and then stored at –34 C until HSPs were extracted. Apples were used from the same orchards in 1999 except that ‘Fuji’ apples were harvested from 3-year-old trees trained to a central leader at the TFREC. To determine whether HSPs were still present in apples 60 to 120 d beyond commercial maturity and if HSPs could still be heat-induced in these apples, sun apples of the five cultivars were harvested 22 Dec. 1999 from the same trees used in 1997. Tissue samples were excised, frozen, and stored as before. Apples harvested at maturity and placed in cold storage (4 C) were compared to those left in the field. HEAT TREATMENTS. ‘Fuji’ shade apples were heat treated in a temperature-controlled water bath to the desired temperature by placing apples inside 3.8-L, Ziploc freezer bags (DowBrands L.P., Indianapolis, Ind.) (0.069-mm-thick plastic) and submerging the apples (four per bag) below water level. The bags were braced so that they remained open at the top for gas exchange. Using copper-constantan thermocouples (0.254-mm-diameter wire) inserted just beneath the peel, peel temperatures were monitored during incubation in the water bath. In a 40 C bath, peel temperature increased from room temperature (22 C) to 38 C in 45 s, and then increased asymptotically to 40 C during the next 20 min. In the 45 C bath, peel temperature increased from room temperature to 41 C in 72 s, and then increased asymptotically to 45 C during the next 32 min. After heat treatments

Characterization of Heat Shock Protein Expression in Apple Peel under Field and Laboratory Conditions