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Cellular Rupture and Release of Protoplasm and Protein Bodies from Bean and Pea Cotyledons During Imbibition

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Cellular Rupture and Release of Protoplasm and Protein Bodies from Bean and Pea Cotyledons During Imbibition Food Structure Volume 6 Number 2 Article 4 1987 Cellular Rupture and Release of Protoplasm and Protein Bodies From Bean and Pea Cotyledons During Imbibition Stephen C. Spaeth Joe S. Hughes Follow this and additional works at: https://digitalcommons.usu.edu/foodmicrostructure Part of the Food Science Commons Recommended Citation Spaeth, Stephen C. and Hughes, Joe S. (1987) "Cellular Rupture and Release of Protoplasm and Protein Bodies From Bean and Pea Cotyledons During Imbibition," Food Structure: Vol. 6 : No. 2 , Article 4. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol6/iss2/4 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Food Structure by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. FOOD MICROSTRUCTURE, Vol. 6 (1987 ) , pp. 127-134 Scanning Mi croscopy International, Chicago (AMF O'Hare), IL 60666 USA CELLULAR RUPTURE AND RELEASE OF PROTOPLASM AND PROTEIN BODIES FROH BEAN AND PEA COTYLEDONS DURING IMBIBITION Stephen C, Spaeth* and Joe S. Hughes** Grain Legume Genetics and Physiology Research Unit Agricult.ural Research Servi;e 0 0 8 Depa r~~e~t· o ;p;oror:dme;ctie nfceAgarnidc~l~uar: Nu t ;idtion** Washington State Univers i ty Pullman, Wa shington, 99164 Introductio n Imbibition is a critical phase in germina­ Legwne seeds and excised cotyledons which tion and processing of legume seeds because cell­ are soaked i n water or sown in wet soil release ular disruption during imbibition may influence intracellular substances into the surrounding seedling vigor and processing quality Cellular water (Schroth and Cook, 1964; Duke and Kakefuda, disruption of cotyledonary surfaces of beans 1981, Duke et al., 1983, Spaeth, 1987), Substan­ (Phaseolus vulgaris L.) and peas (Pisum sacivum tial quantities of nutrients can be lost from L.) and the cellular contents released during seeds during soaking, e.g., 60% of K content in imbibition were examined with scanning electron 24 h (Simon, 1984). In crop production, losses microscopy. Two types of c e llular disruption of intracell ular materials are associated with we r e observed during imbibition: ruptures and poor seedl ing vigor and stand (Larson, 1968, fractures. Individual cells and small groups of Powell and Matthews, 1978) because cells are cells on t he surfnces of cotyledons ruptured ruptured and storage reserves are not nvailable after immersion in water. Ruptured cells had for growth of seedlings Loss of intracellular flaps of cell walls which remained attached to substances is also associated with increased intact portions of cell walls. Fractured cells pathogen growth (Sch roth and Cook, 1964) because spl it in half, and remnant portions of cell walls nutrient sources become available to soil-borne were completely separated from each other. pathogens. Disrupted cells on the interior surfaces of The loss of int racellular substances has blister cavities were of the fractured type. been attributed to large scale disruption of Materials released from cotyledonary tissues manifested as transverse cracks (Morris tissues consisted of both dense aggregates of et. al., 1970), displacement of tissue strips protein bodies and a dispersed phase of proto­ (Duke and Kakefuda, 1981), and blistering of plasm. In some cases, protoplasm and protein embryonic axes (Dunn et al., 1980) and cotyle­ bodies on cotyledonary surfaces were found donary surfaces (Spaeth, 1987). Multicellular adjacent to single cell ruptures and, in others, blist:ers on surfaces of bean cotyledons ranged the sites of losses were not found. The presence from less than 0. 4 to more than 0. 8 mm in of protoplasm and protein bodies and absence of diameter (Spaeth, 1987). One mechanism for sites for their release indicate an additions 1 release of intracellular materials from blisters mechanism other than fracture or rupture may is pressure-driven extrusion (Spaeth , 1987). contribute co losses of intracellular substances Partially hydrated protoplasm, protein bodies during imbibition. and, in some cases , starch grains (granules) exhibit characteristics of a viscous fluid which is extrud e d as streams of material t h rough irreg­ ular orifices in blisters. Solute leakage from cotyledonary tissue is Initial paper rec eived April 6, 1987 not always accompanied by visible tissue disrup­ Manuscript rec eived October 16, 1987 tion. While cotyledons of one bean cultivar Direc t inquiries to S.C . Spaeth produced surface blisters, a second bean cultivar Telephone number: 509 335-9521 and two pea cult i vars extruded small diameter streams of intracellular material but did not form visible blisters (Spaeth, 1987). Seeds with intact seed coats may leak substantial amounts of intracellular substances, yet not exhibit visible Key wo rds: beans, Phaseolus vulgaris (L), peas, tissue disrup tion (Simon and Raja Harun, 1972, Pisum sacivum (L), cellular rupture, protoplasm Duke and Kake f uda, 1981, Simon, 1984). The extrusion , nutrient loss, solute leakage, soaking existence of cellular ruptures has been inferred injury, imbibitional stress, imbibitional injury, from the appearance of intracellul ar substances, protein bodies. e. g. ot·ganelle specific enzymatic marke·cs (Duke 127 S. C. Spaeth and J. S. Hughes and Kakefuda, 1981), which are too large for dehydrated in a graded ethanol series (30 to diffusion through intact membranes. Evans Blue 100%) (Hughes and Swanson, 1985). Fixed samples stain has been used to demonstrate plasma were dried in carbon dioxide with a critical membrane rupture during imbibition (Duke and point drier (Bomar SPC·l500) 1 , sputter coated Kakefuda, 1981; Schoetcle and Leopold, 1984). with gold (Hummer·Technics) 1 • All samples were Detailed microscopic studies of cell rupture viewed and photographed at: 20 kV with an ETEC U ·1 would help to elucidate mechanisms of solute Scanning Electron Hicroscope 1 • leakage and its role in reduced legume seed viability. The first objective of this research was to examine cellular disruption on a scale smaller In addition to the large scale cracking and than transverse cracks, crescent cracks, and multicellular blisters previously reported, blisters. The second and third objectives were epidermal cells of legume cotyledons were to identify various types of cellular disruption, ruptured during imbibition. Areas of Apollo bean and to identify some materials lost from cotyle· cotyledons which were not blistered exhibited dons during imbibition Scanning electron ruptured cells and the contents from the ruptured microscopy (SEM) was used co examine excised cells on adjacent unblistered tissue (Fig 1). cotyledons a nd protoplasm lost during imbibition. Only a few of the total number of cells on the surface were ruptured (Fig. l). Holes consisted Materials and Methods of individual or small groups of ruptured cells. Flaps of cell walls, which were formed by the Seeds of two bean cult:ivars (Phaseolus rupture process, were attached to remaining walls vulgaris L. cv Apollo, a snap bean, and cv Royal of ruptured cells. Cell contents released from Red Mexican, a dry bean) and two pea cultivars cells did not contain recognizable constituents (Plsum sativum L. cv OSU 605, a wrinkled pea, and (Fig. 1). Cell contents from ruptures did not cv Carfield 81, a round pea) were obtained from form extrusion streams as did cell contents from local commercial or research sources. Seeds were blisters (Spaeth, 1987). equilibrated to constant water content over a The scale of individual cell rupture was saturated solution of potassium carbonate. Water too small to be seen without SEM, therefore, it content of seeds was determined by oven drying at was not possible to use light microscopy to 10s•c for 48 h. Initial water contents of bean directly confirm that individual cells or small and pea cotyledons were 11 and 10% of seed dry groups of cells ruptured during soaking To weight, respectively. Coats of dry seeds were carefully removed with a scalpel. Individual cotyledons were £.i&..........l. Ruptured cells (R) with wall flaps (F) immersed in 6 ml of distilled, deionized water at and cell contents (C) on t h e surface of Apollo 24•c. Cotyl edons and extrusion streams from snap·bean cotyledons soaked prior to fixation cotyledons were observed with a dissecting micro · Bar - 10 pm. scope during imbibition. Cotyledons for SEH were prepared in several .E.i&...,___,f. Surface cells of Apollo bean cotyledons different ways. To prepare unfixed samples for which had single drops of water applied to SEH, some cotyledons were simply excised from dry cotyledonary surfaces. Low magnification (2a) seeds. Other cotyledons were excised, soaked in shows dry (untreated) tissue (D), perimeter of water for 30 min, blotted to remove excess water, the treated area (P), ruptured cells (R), unrup· and then allowed to dry at ambient temperature tured cells (U), cell contents (C), smooth and humidity. In one set of Apollo bean cotyle· surface (S) with fissures (f). Bar - 100 ~m. dons, a droplet of water was applied to abaxial Same seed at higher magnification (2b) shows surfaces. After the water was absorbed into the ruptured cells (R), cell contents (C), and cotyledon or evaporated, the sample was reequili· fissures (F) in the epidermal surface covered brated at ambient relative humidity. Another set with a film of cell contents. Bar - 10 pm. of bean cotyledons was fractured dry. Fracture surfaces were washed free of protoplasm a nd .Eig,_,_l. Ruptured cells (R) in OSU 605 pea cotyle­ cellular inclusions as described by Wo lf and dons which were soaked but not fixed. Wall flaps Baker (1980), Dry tissues of all unfixed samples (F) were attached to undisturbed portions of were sputter coated with gold (Hummer·Technics) 1 • ruptured cell walls. Cotyledon surface was free To prepare fixed samples for SEM, cotyle· of cellular contents. Bar- 10 pm. dons were immersed in 6 ml distilled, deionized water unt:il blisters extrusion ~· Fracture surfaces of transverse cracks appeared (20 min).
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