Protein Body Composition in Cuburbita maxima Cotyledons as Determined by Energy Dispersive X-Ray Analysis

Abstract

Energy dispersive x-ray analysis was used to study the composition of certain protein body components in Cucurbita maxima cotyledons. The globoid crystal was rich in phosphorus, potassium, and magnesium. This elemental composition provides further evidence that the globoid crystal in squash cotyledon protein bodies is composed of phytin, a myoinositol hexaphosphoric acid salt of potassium and magnesium. Calcium, a common component of phytin in many species, was absent or present in only trace amounts in the globoid crystals of squash. Results In Cucurbita maxima the structure of the protein bodies in cotyledon tissues of dormant and germinating seeds has been investigated with a variety of electron microscopic techniques (8)(9)(10). Cotyledon mesophyll cell protein bodies were found to have at least four components which are the proteinaceous matrix, the protein crystalloid, the soft globoid, and the globoid crystal. Due to the electron density of the globoid crystal, it was proposed that this region is rich in phytin (8).The energy dispersive x-ray analysis system makes possible an elemental analysis of chosen tissue regions (3). All elements of atomic number 11 (sodium) and higher in the periodic To check on the possibility that the fixation and dehydration procedure used above could have extracted materials from the globoid crystals, cotyledon tissue from dry seeds was sliced 1 mm thick, quick frozen in liquid nitrogen, ground in a mortar and pestle, and freeze-dried at low temperature using the method of Darley and Lott (2). The freeze-dried powder was dusted onto Formvar-coated grids prior to analysis. The electron-dense globoids could be readily identified.Analysis was conducted with a Philips 300 transmission electron microscope fitted with a Model 606 x-ray spectrometer, a Model 707A energy dispersive analysis of x-rays system and an EDIT system for EDAX International Inc. Samples at maximum distance from the grid bars were chosen for analySiS. RESULTSFixed and sectioned cotyledon tissue from seeds grown in several different places in North America was analyzed with EDX. The EDX analysis showed that magnesium, phosphorus, and potassium were the only major elements present in the globoid crystals from inside protein bodies of seeds grown at Rocky Ford, Colorado (Fig. 1); Oregon (Fig. 5); Summerland, British Columbia (Fig. 6); and Hamilton, Ontario (Fig. 7). The copper peaks present in Figures 3, 4, and 6 are due to x-rays originating from the copper support grids.EDX analysis of a section of a protein crystalloid surrounded by the proteinaceous matrix region revealed the presence of phosphorus, sulfur, and traces of chlorine (Figs. 2 and 3 X-RAY ANALYSIS OF SQUASH PROTEIN BODIESledons revealed the presence of phosphorus, magnesium, and potassium (Figs. 8 and 9) in approximately the same amounts and ratios as was found in globoid crystals in fixed and sectioned materials (Figs. 1, 5, 6 and 7). In one small-sized globoid crystal a trace of calcium was detectable (Fig. 9). DISCUSSION Phosphorus...

Calcium Distribution in Globoid Crystals of Cucurbita Cotyledon Protein Bodies

“…With the exception of tiny globoid crystals in very small provascular regions and in epidermal cells, the spot size used was equal to or smaller than the size of the globoid crystal under investigation. Past work with C. maxima (3) showed that some proteinaceous material in the region being analyzed will not contribute greatly, if at all, to the P, K, Ca, and Mg peaks.…”

“…Tissues from individual seeds were kept separate so that seed-to-seed variation could be studied. Tissue samples were glutaraldehyde fixed and embedded according to the method of Lott (3). No OS04 was used in these studies since it can bring about extraction of cations and even phosphorus from globoid crystals (6).…”

“…The protein reserves are thought to be located primarily in the proteinaceous matrix and protein crystalloid regions. The mineral reserves usually occur in the electron-dense globoid crystals and are generally considered to be largely phytin, a cation (K, Mg, Ca) salt of inositol hexaphosphoric acid (3,4,(11)(12)(13).Recently, Lott et al (7) published results of an energy-dispersive x-ray analysis study of globoid crystal composition in 10 different regions of Cucurbita maxima embryos. Although P, K, and Mg were commonly found in globoid crystals in all squash embryo regions, the Ca distribution showed definite differences between embryo regions.…”

“…The protein reserves are thought to be located primarily in the proteinaceous matrix and protein crystalloid regions. The mineral reserves usually occur in the electron-dense globoid crystals and are generally considered to be largely phytin, a cation (K, Mg, Ca) salt of inositol hexaphosphoric acid (3,4,(11)(12)(13).…”

Localization of Phosphorus and Cation Reserves in Lilium longiflorum Pollen

“…Characteristically, the EDX spectra of seed globoids show large P and cation (most notably Mg, Ca, K) signals relative to those signals generated from surrounding cytosolic regions (13)(14)(15)(16)(17)(18). The elemental composition of globoids is indicative of the presence of phytin which is composed of salts of myo-inositol hexakisphosphate (14,15).…”