Diana M. R. Harvey

Quantitative ion localization within Suaeda maritima leaf mesophyll cells

Grown under saline conditions, Suaeda maritima accumulates Na+ and Cl- into its leaves, where individual mesophyll cells behave differently in their compartmentation of these ions. Measurements of ion concentrations within selected subcellular compartments show that freeze-substitution with dry sectioning is a valuable preparative technique for analytical electron microscopy of highly vacuolate plant material. Using this approach, absolute estimates were made of Na+, K+ and Cl- concentrations in the cytoplasm, cell walls, chloroplasts and vacuoles of leaf mesophyll cells.

Evidence for the cytoplasmic localization of betaine in leaf cells of Suaeda maritima

An attempt has been made to localize glycinebetaine in shoots of Suaeda maritima L. Dum. using a technique based on the formation of an iodoplatinate precipitate. Deposits were largely restricted to the cytoplasm of salt-grown plants and were analysed by transmission analytical electron microscopy. The results are considered to support the hypothesis that glycinebetaine acts as a cytoplasmic osmoticum to balance high vacuolar salt levels in certain halophytes.

The use of freeze-substitution in the preparation of plant tissue for ion localization studies

A method, utilizing freeze substitution, is described for the preparation of plant tissue for analytical electron microscopy. The fine structure of the cytoplasm was adequately preserved after freezing leaf tissue in 2‐methylbutane at −170°C. Furthermore, following substitution in ether, losses of sodium and potassium from the tissue were less than 4% of the original ion content and the loss of chloride was less than 1%. The merits of the procedure as a means of tissue preparation for ion localization studies are discussed.

The effects of salinity on ion concentrations within the root cells of Zea mays L.

Zea mays is a salt-sensitive crop species which in saline (≦100 mol m-3 NaCl) conditions suffers considerable growth reduction correlated with elevated Na+ and Cl- concentration within the leaves. To increase understanding of the regulation of ion uptake and transport by the roots in saline conditions, ion concentrations within individual root cortical cells were determined by X-ray microanalysis. There was variation in Na+, K+ and Cl- distributions among individual cells, which could not be correlated with their spatial position in the roots. Generally, however, in response to saline growth conditions (100 mol m3 NaCl) Na+ and Cl- were mostly localized in the vacuoles, although their concentrations were also sometimes increased in the cytoplasm and cell walls. The concentration of K+ in the cytoplasm was usually maintained at a level (mean 79 mol m-3) compatible with the biochemical functions ascribed to this ion.

Optimization and investigation of the use of 2,2-dimethoxypropane as a dehydration agent for plant tissues in transmission electron microscopy.

After obtaining initially inconsistent results using 2,2-dimethoxypropane (DMP) to dehydrate pea root and stem segments for transmission electron microscopy, it was found that consistent total dehydration of 3-mm pea root and 1- and 3-mm pea stem lengths was only obtained after 2 15 min exposure to DMP, i.e., much longer than originally recommended (L. L. Muller and T. J. Jacks, 1975, J. Histochem. Cytochem., 23, 107). Satisfactory dehydration of a variety of plant tissues was achieved after 2 15 min dehydration in DMP, and no differences in cell ultrastructure were observed between ethanol and DMP-dehydrated samples following three different fixation procedures (glutaraldehyde/OsO4, acrolein/OsO4, and KMnO4). The value of DMP in the retention of cell contents during dehydration was assessed in procedures for the localization of (1) rat liver peroxisomal soluble catalase using 3,3-diaminobenzidine and (2) Na+ K+ and Cl by precipitation techniques in Suaeda maritima leaf segments.

The validity of the lead precipitation technique for the localization of ATPase activity in plant cells.

SummaryThe claim that osmium-containing deposits which lack lead are frequently and incorrectly interpreted as enzymatic reaction products in lead precipitation techniques for ATPase localization in plants is without foundation. Proper controls clearly demonstrate the enzymatic origin of membrane-located deposits and the presence of lead is confirmed by analytical electron microscopy.

Determination of the sodium, potassium and chloride ion concentrations in the chloroplasts of the halophyteSuaeda maritima by non-aqueous cell fractionation

SummaryLeaf material from the halophyteSuaeda maritima L. Dum. grown under both saline and non-saline conditions was fractionated under non-aqueous conditions in order to determine the ion content of various subcellular compartments. Fractions containing cell walls, nuclei and chloroplasts were successfully prepared and contents of DNA, chlorophyll, protein and Na+, K+, and Cl− determined. The cell wall fraction was not apparently heavily contaminated by the other fractions and had a low ion content although the nuclear fraction was contaminated by other organelles. The ion contents of chloroplasts were determined and the results discussed in relation to earlier microscopical data.

The preparation of calibration standards for sodium, potassium and chlorine analyses by analytical electron microscopy

Attempts were made to prepare sodium and potassium thin section standards for analytical electron microscopy by introducing, in serial concentrations, a complex between the appropriate salt and macrocyclic polyether ([15] crown‐5 for sodium and [18] crown‐6 for potassium) into epoxy resin. Of the tested salts, sodium tetraphenylborate, tetrafluoroborate and cyanide failed to produce homogeneous standards, and those standards containing sodium or potassium iodide or bromide were analytically inhomogeneous at concentrations greater than 20 meq 1−1. Only sodium thiocyanate and potassium cyanide standards were analytically homogeneous at concentrations up to 600 meq 1−1. A chlorine standard, which was analytically homogeneous at concentrations up to 1000 meq 1−1, was prepared by incorporating 1,2,4‐trichlorobenzene into an epoxy resin.

Improvement of quantitation of biological X-ray microanalysis

Absolute measurements of elemental concentrations within thin biological samples are often made by reference to a series of standards which resemble the samples in chemical and physical properties and the linear relationship between (p‐b)/c and concentration. This principle requires that the chemical and physical properties of the matrix remain constant throughout a series of standards with different elemental contents and throughout different regions of the samples. Some of the changes undergone by specimens during X‐ray microanalysis, e.g. loss of elements or organic mass loss, are also influenced by the composition of the matrix. A simple empirical modification to the linear (p‐b)/c versus concentration relationship is presented to account for some of these effects and therefore improve quantitation of analyses.

Precipitation Procedures For Sodium, Potassium And Chloride Localization In Leaf Cells Of The Halophyte Suaeda Maritima

Attempts have been made to localize sodium and potassium with antimonate and cobaltinitrite reagents in Suaeda leaf segments by rapid aqueous and freeze‐substitution precipitation procedures and to localize chloride by freeze‐substitution precipitation with silver ions. Substantial sodium losses occurred from the tissue during both types of preparation although potassium and chloride losses were considerably reduced by the use of freeze‐substitution when compared with the rapid aqueous procedures. The losses during freeze‐substitution resulted from the required use of ethanol as substitution solvent (rather than acetone or diethyl ether, in which the reagents were much less soluble). Analytical electron microscopy showed that silver and cobaltinitrite specifically precipitated chloride and potassium respectively, but antimonate did not specifically precipitate sodium. The vacuoles were important sites of silver and antimonate deposits, although the chloroplasts were the major sites of cobaltinitrite localization. The value of these techniques in ion localization studies is discussed.