Energy-dispersive x-Ray Analysis of Phosphorus, Potassium, Magnesium, and Calcium in Globoid Crystals in Protein Bodies from Different Regions of Cucurbita maxima Embryos

Abstract

The seeds of Cucwubit maximas contain protein bodies with electrondense globoid crystals. Because of their density globoid crystals are ideal material for energy-dispersive x-ray (EDX) analysis studies of elemental composition. Fixation trials were carried out to test globoid crystal extraction during glutaraldehyde fixation, water washing, and ethanol dehydration. Glutaraldehyde fixation without subsequent washing or dehydration alone produced no significant changes in elemental composition of cotyledon globold crystals. If glutaraldebyde fixation was followed by water washes or ethanol debydration there was some loss of the major globoid crystal elements but the relative percentages of the elements P, K, Ca, and Mg remained relatively unchanged. In this paper results of a study of the P, K, Mg, and Ca content of globold crystals in different tissues of squash embryos are presented. The globoid crystals in the radicle were found to be the least dense in the embryo. Globoid crystals from all embryo regions contained P, K, and Mg. In the various embryo regions P and Mg maintained relatively constant proportions of the globoid crystal composition while K and Ca varied. Of particular significance is the distribution of Ca which is generaly an immobile element. Calcium was found in highest amounts in the globoid crystals of the radicle and stem regions while globoid crystals in much of the cotyledon contained little, if any, Ca. The Ca storage thus seems to be spatially arranged in a manner that would aid early growth of the root-shoot axis.In order for us to understand the nutritional state GLOBOID CRYSTAL COMPOSITION tained from a single fruit grown at CSIRO, Adelaide, South Australia.Two seeds of similar size from the Stoke's seed supply (seeds I and 2) and two seeds from a fruit grown at CSIRO (seeds 3 and 4) were selected for EDX2 analysis. The seed coats were removed and tissue was sampled from the radicle, stem, cotyledon base, cotyledon center, cotyledon side, and cotyledon tip of each seed. Since the embryo axis of squash is very short, the stem sample was selected as the portion of the axis to which the cotyledons were attached. The cotyledon base sample was that portion of the cotyledon immediately adjacent to the cotyledonary node. A 3-mm2 region at the center of the cotyledon was used as the cotyledon center sample. The cotyledon side sample came from a region (2 x 5 mm) at the edge of the cotyledon, midway between the cotyledon base and the cotyledon tip. For the cotyledon tip sample a 2-mm2 region of tissue was taken from the edge of the cotyledon in the region furthest removed from the cotyledonary node.Tissues from the cotyledon and stem regions were sliced 0.5 to 1 mm thick prior to fixation. The samples were prepared for EDX analysis according to the method of Lott (11). Tissues from the dry seeds were fixed in 5% glutaraldehyde in distilled H20, dehydrated, infiltrated with Spurr's low viscosity epoxy resin, and polymerized. Sections, 150 to 170 nm thick, were mounted on copper grids ha...

Calcium Distribution in Globoid Crystals of Cucurbita Cotyledon Protein Bodies

“…These findings are consistent with earlier studies (7,10 Since only large globoid crystals were analyzed in the Lott et al (7) study it seems unlikely that globoid crystals from epidermal cells or most provascular cells would have been chosen because of their small size. It seems probable that the occasional globoid crystal with high Ca content reported by Lott et al.…”

“…It seems probable that the occasional globoid crystal with high Ca content reported by Lott et al. (7) Some of the available EDX analysis studies of globoid crystal composition in cotyledons have noted the presence of Ca-containing and Ca-lacking globoid crystals. Such differences have been reported to occur in cotyledons of almond (5), macadamia (4), and jojoba (1).…”

“…(7). Determining whether or not traces of Ca are present in a sample can be difficult because the major K. peak for Ca centered at 3.69 kev is overlapped by the minor Ke peak of K, centered at 3.59 kev.…”

“…No OS04 was used in these studies since it can bring about extraction of cations and even phosphorus from globoid crystals (6). Glutaraldehyde fixation alone does not seem to alter globoid crystal composition drastically in C. maxima (7) and thus this procedure was used in the studies reported here.…”

“…EDX analysis of 150 to 190 nm sections was carried out as described previously (7). 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.…”