Download Growth-induced Microcracking and Repair Mechanisms of Fruit

Proceedings of the SEM Annual Conference
June 1-4, 2009 Albuquerque New Mexico USA
©2009 Society for Experimental Mechanics Inc.
Growth-Induced Microcracking and Repair Mechanisms of Fruit Cuticles
Eric A. Curry, Plant Physiologist
USDA, Agricultural Research Service, Tree Fruit Research Laboratory, 1104 N. Western Ave.,
Wenatchee, Washington 98801 [email protected]
ABSTRACT
The apple cuticle is an amazing tissue system that is both dynamic and environmentally responsive. Not only
does it attenuate excessive solar radiation and deter pathological attack, it presents an effective barrier against
both water loss due to desiccation stress and water uptake during periods of standing surface moisture. From the
moment the flower bud unfolds, exposing tissue to water vapor deficit stress, the pro-cuticle begins covering the
tissue surface with an overlapping network of lipid-based microtubules produced by and connected to epidermal
cells. Ambient temperature and water vapor partial pressure sensed by these cells help modulate microtubule
composition. As microtubules elongate distally, they aggregate to form planar microcrystalline wax platelets,
which co-polymerize to form an organic solvent-insoluble, semi-permeable cutin matrix. Fruit growth, induced by
collective fruit cell enlargement, necessitate stretching and shearing of the cutin matrix, causing microtubules to
snap, thereby creating fresh elongating distal ends. The processes of microtubule elongation, aggregation,
crystallization and polymerization function concurrently as long as the fruit continues to enlarge. This
simultaneous “stretching, shearing and stitching” mechanism allows fragile underlying cells to expand and divide
during ambient conditions of very low relative humidity and temperatures up to 40 °C. Scanning electron
microscopy has provided a window into this amazing living polymer.
Introduction
Like most aerial plant structures, apple fruit are covered with a cuticle that protects them against desiccation,
contamination and other external stressors [1-4]. Generally, this cuticular layer is made up of epicuticular wax
(non-polar organic solvent-extractable wax) embedded within a matrix of solvent-insoluble cutin composed of
covalently cross-linked hydroxy and hydroxyl-epoxy fatty acids, all produced by epidermal cells [5-6]. Epicuticular
wax is composed primarily of very-long-chain aliphatic aldehydes, primary, secondary and tertiary alcohols,
alkanes, ketones and esters derived from saturated very-long-chain fatty acids with chain lengths between 24-34
carbons [7-8]. Other components may include terpenoids, flavanoids, sterols and β-diketones and their
derivatives [9]. Epicuticular wax, the basis of cuticle development, is biosynthesized as soon as epidermal cells of
developing meristem sense desiccation pressure. Wax production continues throughout storage [10-11] until the
cells of origin are depleted of substrate, are biochemically inhibited or become necrotic.
Ultrastructural analysis of the plant cuticle surface using scanning electron microscopy (SEM) has shown
epicuticular wax crystal-like aggregates of varying shape on numerous plant species [12-13], the structure and
form of which are largely determined by their unique chemical composition [14]. Although such epicuticular wax
crystalline structures have been reported on apple fruit [15-18] some have questioned whether their appearance
is artifactual [19] due either to the methods of preparation for SEM examination, or because of the conditions
-6
under which samples are viewed (partial pressures ranging up to 10 Pa).
Apple fruit of different cultivars vary in surface wax morphology and chemical composition during development, as
well as during and after cold-storage and subsequent shelf-life [20]. The characteristics and composition of apple
cuticular wax also change in response to environmental stresses such as rain acidity [21], temperature [22-23]
and radiation [24]. Differences in cuticular absorption of both ultraviolet and visible light have been reported in
shaded vs. sun-exposed portions of certain apple cultivars [25]. Because function is linked to structure which is
based on composition, these environmental cues can potentially alter the way in which the cuticle performs.
External physiological disorders related to dysfunctions or aberrations in the development of the peel (cuticle,
epidermis and hypodermis) are often linked to climatic conditions at a specific developmental stage during the
growing season. These may include frost ring, russet, stain, sunburn, cracking, splitting, flecking, lenticel
marking, sunscald and storage scald [26-27]. Thus, understanding the development of peel tissues and their
biochemical responses to environmental conditions would contribute to our knowledge of the etiology of these
disorders as well as to the development of solutions to prevent or minimize their incidence. This study was
undertaken to investigate the mechanism of cuticle growth under normal ambient conditions, as a preliminary
means of learning of conditions that may alter its composition, structure and function.
Materials and Methods
Apple tissue sample collection
Beginning 1 week after anthesis, and approximately every 2-3 weeks thereafter, two apples from each of three
separate trees were sampled from a uniform planting of mature 'Golden Delicious'/EMLA 106 trees in Malaga,
WA. On the first sampling date, young fruitlets about 8 mm in diameter from the interior canopy were carefully
excised with a razor blade, and placed upright in small vials with the pedicel base immersed in 1 ml distilled H2O.
The sun-exposed side was marked with a dot of ink from a permanent marker. Samples were placed in a cooler
held at 10 °C and transported (20 min) to the laboratory for further preparation. Fruit sampled throughout the
growing season were treated similarly, except that no vial of water was used when the fruit diameter was greater
than 25 mm. The last fruits sampled were no less than 80 mm in diameter, and were removed in September, at
least 135 days after anthesis but before internal ethylene concentrations exceeded 1 µl·l-1 (i.e., before initiation of
ripening). Pesticides were applied sparingly in the orchard and all samples were harvested at least 14 days after
topical application of any compound. All apples were free of any obvious surface disorder or marking, such as
might be caused by russet, sunburn, insects, pathogens, dirt or mechanical injury.
Sample preparation for scanning electron microscopy
Most of the tissue sample preparation was accomplished within 1 hour of fruit excision. An untouched portion of
cuticle approximately 3 mm in diameter and 0.5 mm thick was shaved by hand from the fruit, opposite the sunexposed side, using a 0.012 mm thick, double-edge stainless steel razor that had been rinsed with acetone and
air-dried to remove any residual oil. The shaved cuticle section was fixed to a 12 mm aluminum stub using
double-sided carbon tape, by pressing only the edge of the entire section onto the tape using a pair of fine-tipped
tweezers under a stereo-microscope. The stub was placed in a small, glass vacuum desiccator containing
4
packaged silica gel and kept at 10 °C and 1.3 x 10 Pa for 30 min.
For comparison with air-dried samples, similarly sampled peel tissue was freeze-dried by first placing the shaved
cuticle onto the surface of a clean aluminum block (9 x 7 x 5 cm) resting in, and temperature-equilibrated with
liquid N2. After all samples had been placed on the aluminum block, the entire “block + sample” assembly was
transferred to a small, glass vacuum desiccator connected to a freeze-drier where it remained at 100 Pa for 18 h.
Freeze-dried samples were removed and, under a stereo-microscope, affixed to a 12 mm aluminum stub using
double-sided carbon tape by pressing only on opposite edges of the tissue using a stainless steel microprobe.
The stub was placed in a glass vacuum desiccator containing packaged silica gel and kept at 10 °C and 1.3 x 104
Pa until further preparation.
Platinum sputter-coating and post-coating treatments were identical for air-dried and freeze-dried samples.
Mounted tissue was coated with platinum using a Desk II cold sputter coater (Denton Vacuum Inc., Morristown,
NJ, USA) fitted with a tilting omni-rotating head. With the sample 47 mm from the platinum target, a coating
thickness of approximately 20 nm was achieved after 75 s at 40 mA and 2.6 Pa. Coated samples were returned
to the vacuum desiccator and held under low vacuum at 1.3 x10-4 Pa and 10 °C until examined microscopically
using a Tescan Vega II LS SEM (Tescan, s.r.o., Brno, CZ) equipped with both secondary and back-scattered
electron detectors. Unless stated otherwise, images were obtained at 10 kV and 7.5 x 10-3 Pa.
Results and Discussion
Microcracking of the fruit cuticle surface of ‘Golden Delicious’ was observed on the earliest through the latest
apples sampled, with the main differences being both depth and width of the cracks, as cuticle thickness
increases with time after anthesis (data not shown). In this report, only images from samples taken from mature
fruit will be shown.
On mature fruit at harvest, before ripening, normal microcracking is shown in the image series in Figure 1 (A-D).
A
B
1
2
C
D
3
2
1
Figure 1. Epicuticular microcracking of mature ‘Golden Delicious’ apple peel at harvest before
initiation of fruit ripening. View fields of 1 mm (A) and 0.5 mm (B) indicate extent and depth of
concurrent cracking and self-repair. Enlargement of inset A1 (C) and A2 (D) show epicuticular
cracking at different stages (arrows). Extracuticular structure in C is a desiccated trichome.
Maximum width of the microcracking is fairly constant (20-25 µm) suggesting relatively uniform activity of cell
growth (division of the epidermal cells plus enlargement of fruit cortex cells) underlying the cuticular complex.
Figures 1C and 1D have view fields of 100 µm to show superficial stages of cuticle growth (C) and relative depth
(D) of cuticular complex. Visible stages of cuticular microcracking as shown by the arrows in Figure 1C are: 1)
stretching of co-polymerized wax crystals to produce flattened, tangential “bridges”; 2) shearing of the stretched
structures previously described, both on the surface as well as of those wax platelet aggregates subtending the
surface “bridges”, thereby exposing microtubular “growing” ends; and 3) extension of wax microtubules and
microtubular aggregates from both sides of the separated surface to fill-in the crack and re-establish a cuticle
depth commensurate with the surrounding tissue. The following figure (Fig 2.) illustrates these processes
(stretching, shearing and stitching) together the separated cuticle (steps 1- 3, above) at the surface and deeper
within the cuticle.
1
3
2
Figure 2. Epicuticular microcracking of mature ‘Golden Delicious’ apple peel at harvest before
initiation of fruit ripening. Arrows indicate stage of microcracking: 1) stretching of wax platelet
aggregates; 2) shearing and/or separation of wax platelet aggregates; and 3) elongation and coaggregation of platelets from both sides to fill-in or “stitch” together the crack. Angle of SEM
image is 15° from vertical.
Importantly, the processes (stages 1-3) of cuticle microcracking—stretch, shear and stitch—occur as long as the
fruit is undergoing enlargement. This is a normal process and the modus operandi of cuticle growth.
Interestingly, the thickness of the cuticle is a function of the genetic predisposition of the cultivar as well as the
environment in which the fruit is growing. Cuticle thickness increases rapidly through spring as temperature
increases and water vapor pressure decreases. Once the cells beneath the cuticle are protected from excessive
water movement in either direction (into or out of the fruit) the optimum cuticle thickness is maintained. Figure 3
shows a cross section of ‘Golden Delicious’ peel sampled at harvest and cut with a razor to show the epidermal
cells embedded within the cuticle. Epidermal cell size is roughly maintained after about 30 days of initial growth.
A
B
Figure 3. Cross section of ‘Golden Delicious’ apple peel at harvest. Angle of SEM image (A) is
45° from vertical to show surface microcracking in relation to epidermal cells as well as size of
epidermal cells in relation to subtending hypodermal cells that have stopped dividing and are
enlarging. Image (B) is and enlargement of the white dotted inset in (A) (0° from vertical).
Arrows indicate depth of the surface cracking.
Figure 3 shows the relationship of epidermal cells and surface microcracking. Groups of dividing cells are shown
enlarging (A) and, therefore, causing the upheaval of the cuticle resulting in microcracking. It is important to note
that all tissues distal to the epidermal cell membrane must undergo concomitant growth including the cell wall, the
insoluble cutin matrix and the epicuticular wax platelet aggregates. The enlargement of the inset in A indicates
that, although relatively deep, the microcracking never reaches the epidermal cell was surface. Indeed, in the
event this happens due to rapidity of fruit enlargement or extreme stress, often a secondary protective layer is
produced (suberin) which often appears as a darkened, rough portion of the peel (russet).
Conclusions
Cuticle thickness increases with time after anthesis to a thickness governed by plant genetics, cultural
management and environmental condition. For example, some apple cultivars such as ‘Cox’s Orange pippin’ are
grown predominantly in cooler, more humid environments and often develop an expected russet over much of the
fruit surface. In other parts of the world, such as in the arid, high desert of eastern Washington, such cultivars as
‘Delicious’ and ‘Gala’ are usually russet free, and considered unmarketable if cuticular russet exceeds 1-2
percent. Indeed, the causative factors for physiological (non-pathological) surface incongruences responsible for
market loss, and consequent development of remedial treatments underlie research investigating the nature of
apple cuticle growth and development.
Literature Cited
[1] Bell, HP. The protective layers of the apple. CANADIAN JOURNAL OF RESEARCH, 15C: 391-402, 1937.
[2] Halloway, PJ. Structure and histochemistry of plant cuticular membranes: An overview. In: THE PLANT
CUTICLE. (Cutler, D. F., Alvin, K. L. and Price, C. E., Eds.), Academic Press, London, 1-32, 1982.
[3] Lendzian, KJ. and Kerstiens, G. Sorption and transport of gases and vapors in plant cuticles. REVIEWS OF
ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY, 121: 65-128, 1991.
[4] Jenks, MA., Joly, RJ., Peters, PJ., Rich, PJ., Axtell, JD. and Ashworth EN. Chemically induced cuticle
mutation affecting epidermal conductance to water vapor and disease susceptibility in Sorghum bicolor (L.).
PLANT PHYSIOLOGY, 10: 1239-1245, 1994.
[5] Post-Beitenmuller, D. Biochemistry and molecular biology of wax production in plants. ANNUAL REVIEWS
OF PLANT PHYSIOLOGY AND PLANT MOLECULAR BIOLOGY, 47: 405-430, 1996.
[6] Jetter, R. and Schäffer, S. Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes
of the epicuticular wax film during leaf development. PLANT PHYSIOLOGY, 126: 1725-1737, 2001.
[7] Walton, TJ. In: METHODS IN PLANT BIOCHEMISTRY: LIPIDS, MEMBRANES AND ASPECTS OF
PHOTOBIOLOGY. (Harwood, JL. and Bowyer, JR., Eds.), Academic Press, 4: 105-158, 1990.
[8] Petracek, PD. and Bukovac, MJ. Rheological properties of enzymatically isolated tomato fruit cuticle. PLANT
PHYSIOLOGY, 109: 675-679, 1995.
[9] von Wettstein-Knowles, P. In: WAXES: CHEMISTRY, MOLECULAR BIOLOGY AND FUNCTIONS.
(Hamilton, RJ., Ed.), Oily Press, Dundee, 91-129, 1995.
[10] Morice, IM. and Shorland, FB. Composition of the surface waxes of apple fruits and changes during storage.
JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, 24: 1331–1339, 1973.
[11] Belding, RD., Blankenship, SM., Young, E. and Leidy, RB. Composition and variability of epicuticular waxes
in apple cultivars. JOURNAL OF THE AMERICAN SOCIETY FOR HORTICULTURAL SCIENCE, 123: 348-256,
1998.
[12] Baker, EA. Chemistry and morphology of plant epicuticular waxes. In: THE PLANT CUTICLE. (Cutler, DF.,
Alvin, KL. and Price, CE., Eds.), Academic Press, London, 139–165, 1982.
[13] Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I. and Wilhelmi, H. Classification and
terminology of plant epicuticular waxes. BOTANICAL JOURNAL OF THE LINNEAN SOCIETY, 126: 237–260,
1998.
[14] Jetter, R. and Riederer, M. Composition of cuticular waxes on Osmunda regalis fronds. JOURNAL OF
CHEMICAL ECOLOGY, 26: 331-399, 2000.
[15] Skene, DS. The fine structure of apple, pear, and plum fruit surfaces, their changes during ripening, and
their response to polishing. ANNALS OF BOTANY, 27: 581-582, 1963.
[16] Glenn, GM., Rom, CR., Rasmussen HP. and Poovaiah, BW. Influence of cuticular structure on the
appearance of artificially waxed ‘Delicious’ apple fruit. SCIENTIA HORTICULTURAE, 42: 289-297, 1990.
[17] Roy, S., Watada, AE., Conway, WS., Erbe, EF. and Wergin, WP. Low-temperature scanning electron
microscopy of frozen hydrated apple tissues and surface organisms. HORTSCIENCE, 29: 305–309, 1994.
[18] Curry, EA. Ultrastructure of epicuticular wax aggregates during fruit development in apple (Malus domestica
Borkh.) JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY, 80: 668-676, 2005.
[19] Veraverbeke, EA. Van Bruaene, N. Van Oostveldt, P. and Nicolaï, BM. Non-destructive analysis of the wax
layer of apple (Malus domestica Borkh.) by means of confocal laser scanning microscopy. PLANTA, 213: 525533, 2001.
[20] Veraverbeke, EA., Lammertyn, J., Saevels, S. and Nicolaï, BM. Changes in chemical wax composition of
three different apple (Malus domestica Borkh.) cultivars during storage. POSTHARVEST BIOLOGY AND
TECHNOLOGY, 23: 197-208, 2001.
[21] Rinallo, C. and Mori, B. Damage in apple (Malus domestica Borkh) fruit exposed to different levels of rain
acidity. JOURNAL OF HORTICULTURAL SCIENCE, 71: 17–23, 1996.
[22] Roy, S., Conway, WS., Buta, GJ., Watada, AE., Sams, CE., Erbe, DF. and Wergin, WP. Heat treatment
affects epicuticular wax structure and postharvest calcium uptake in ‘Golden Delicious’ apples. HORTSCIENCE,
29: 1056-1058, 1996.
[23] Lurie, S., Fallik, E. and Klein, JD. The effect of heat treatment on apple epicuticular wax and calcium uptake.
POSTHARVEST BIOLOGY AND TECHNOLOGY, 8: 271–277, 1996.
[24] Kasperbauer, MJ. and Wilkinson, RE. Mulch surface color affects accumulation of epicuticular wax on
developing leaves. PHOTOCHEMICAL AND PHOTOBIOLOGICAL SCIENCES, 2: 861-866, 1995.
[25] Solovchenko, A. and Merzlyak, M. Optical properties and contribution of cuticle to UV protection in plants:
Experiments with apple fruit. PHOTOCHEMICAL AND PHOTOBIOLOGICAL SCIENCES, 2: 861-866, 2003.
[26] Pierson, CF., Ceponis, MJ. and McColloch, LP. Market diseases of apples, pears and quinces. USDA,
AGRICULTURE HANDBOOK NO. 376, 2-69, 1971.
[27] Porritt, SW., Meheriuk, M. and Lidster, P. Postharvest disorders of apples and pears. AGRICULTURE
CANADA PUBLICATION 1737E, 11-53, 1982.