Quantitative electron probe microanalysis of biological thin sections: the use of stem for measurement of local mass thickness.

A method for performing quantitative electron probe microanalysis on ultrathin (less than 30 microgram/cm2) biological samples is described and evaluated. The technique is based on a measurement of the characteristic peak count rate and the degree of beam attenuation as the primary electron beam passes through the sample. Using this method it is possible to measure the concentration of a given element such as sodium in sections ranging in mass-thickness from several microgram/cm2 up to 30 microgram/cm2 with an accuracy of better than 10%. For sections having a mass thickness of approximately 11 microgram/cm2 the minimum detectable concentration for sodium was found to be 20 mmolar or 4 X 10(-2) wt.%. The interaction of the electron beam with the sample is also discussed with emphasis on characterizing the variation in sample mass with radiation dose.

Lateral Transport of Ions into the Xylem of Corn Roots: II. Evaluation of a Stelar Pump.

When an excised corn (Zea mays) root pretreated with chloride was exposed for 10 minutes to pulse labeling with (30)Cl and then transferred to unlabeled chloride, the activity in the xylem exudate reached a maximum about 4 minutes after pulse labeling was discontinued and then declined sharply. The rate at which labeled chloride was transported across the root into the xylem and basipetally therein was on the order of 75 to 250 centimeters per hour. Consequently, symplasmic movement of chloride in corn roots is fast and may not be rate-limiting in transfer from the root surface to the xylem. Experiments on pulse labeling with (22)Na gave similar results. A large fraction of the absorbed (22)Na was not translocated into the exudate but was tightly sequestered in a cell compartment, probably the vacuole.Electron probe analysis was used to reveal the pattern of potassium distribution in cross sections taken 10 to 11 millimeters from the tip. The cytoplasm and vacuoles of the xylem parenchyma cells accumulated potassium to a much greater extent than cortical and other stelar cells. Ultrastructural studies showed that the cytoplasm of the xylem parenchyma cells contains numerous membrane systems. It was concluded that the xylem parenchyma cells secrete ions from the symplasm into the conducting vessels, and it was suggested that this secretion is driven across the plasmalemma by a carrier-mediated transport.

Electron probe analysis of freeze-substituted, epoxy resin embedded tissue for ion transport studies in plants.

A new technique is described to prepare plant material for electron probe analysis. Root segments 1 mm in length were frozen at-170°, freeze-substituted with anhydrous ether at-30° and infiltrated with Spurr's low-viscosity epoxy resin embedding medium at low temperatures. Sections 1 and 2µ thick were cut anhydrously using hexylene glycol in the ultramicrotome trough, mounted on the polished surface of a Be disc and vacuum coated with 150-200 Å aluminum.The new technique allows retention of water-soluble ions at the original sites in the tissue and is superior to cryostat sectioning in spatial resolution of electron probe analysis and in the preservation of cellular structures.The lateral transport of K(+) into the xylem of corn roots has been successfully studied by electron probe analysis of freeze-substituted, epoxy resin embedded material.

Isolation of active mitochondria from tomato fruit.

An improved method for isolating mitochondria from tomato fruit (Lycopersicon esculentum Mill.) is described. The fruit is chilled, and the tissue of the fruit wall cut by hand into very thin slices with a razor blade while immersed in a buffer containing 0.4 m sucrose, 2 mm MgCl(2), 8 mm EDTA, 4 mm cysteine, 10 mm KCl, 0.5 mg per ml bovine serum albumin 50 mm tris-HCl, pH 7.6. The pH is monitored and kept within the range of 7.0 to 7.2 by dropwise addition of 1 n KOH during cutting. The tissue is strained through 8 layers of cheesecloth and centrifuged at 2000 x g for 15 minutes. The supernatant is then centrifuged at 11,000 x g for 20 minutes, and the sediment is washed once with a medium containing 0.4 m sucrose, 10 mm KCl, 1 mm MgCl(2), 10 mm tris-HCl, 10 mm KH(2)PO(4) and bovine serum albumin (0.5 mg per ml), pH 7.2. Electron microscope studies show that this method gives homogeneous, relatively intact mitochondria; they have a higher respiratory control ratio than those reported by other workers. The method was also tested successfully on fruits of cantaloupe and ;Honey Dew' melon.

Auxin and gibberellin effects on cell growth and starch during abscission in cotton.

An increase in starch content of cells in the abscission zone of the cotton explant appeared correlated with an increase in number of cells. A large increase in the number of cells in the abscission zone, concomitant with an increase in starch content, followed treatment with gibberellin as compared to auxin. In the final stages of abscission starch was hydrolyzed in the cells of the separation layer. Some starch remained after the petiole abscised.A positive phloroglucinol-hydrochloric acid reaction in the cells of the petiole distal to the line of separation indicated the presence, not of lignin, but of soluble sugars and uronic acids. This reaction was especially intense following gibberellic acid treatment.It was concluded that gibberellin in accelerating abscission leads to (1) an increase in cell number and starch content in the abscission zone, (2) the hydrolysis of starch in the separation layer just before abscission, and (3) the breakdown of polysaccharides and the release of soluble sugars and uronic acids. Auxin, an abscission retardant, either delays or prevents these events.