Analytical Methods

Sulfur occurs in plants in different chemical forms (250), and nearly all of them have been tested as indicators for sulfur nutritional status. The parameters analyzed by laboratory methods for the purpose of diagnostics can be divided into three general classes: biological, chemical, and composed parameters.

Biological parameters are the sulfate and glutathione content. Many authors proposed the sulfate- S content as the most suitable diagnostic criterion for the sulfur supply of plants (241,242,251-255). They justify their opinion by referring to the role of sulfate as the major transport and storage form of sulfur in plants (256,257). Other authors, however, attribute this function also to glutathione (55,258,259). Based on this concept, Zhao et al. (260) investigated the glutathione content as a diagnostic parameter for sulfur deficiency.

Although indeed directly depending on the sulfur supply of the plant (64,103), neither of the compounds is a very reliable indicator for the sulfur status because their concentrations are governed by many other parameters, such as the actual physiological activity, the supply of other mineral nutrients, and the influence of biotic and abiotic factors (5,63,256,261). Biotic stress, for instance, increased the glutathione content by 24% (63). Amino acid synthesis is influenced by the deficiency of any nutrient and thus may indirectly cause an increase in sulfate or glutathione in the tissue. An example for this action is the increase in sulfate following nitrogen deficiency (103,262,263). Significant amounts of sulfate may also be physically immobilized in vacuoles (see Section).

In plant species synthesizing glucosinolates, sulfate concentrations can also be increased by the release of sulfate during the enzymatic cleavage of these compounds after sampling (103). As enzymatically released sulfate can amount to the total physiological level required, this type of postsampling interference can be a significant source of error, yielding up to 10% higher sulfate concentrations (63,103). It is probably also the reason for some extraordinarily high critical values for sulfate concentrations reported for brassica species (220,264). The preference for sulfate analysis as a diagnostic criterion may also come from its easier analytical determination compared to any other sulfur compound or to the total sulfur concentration (265).

Hydrogen iodide (HI)-reducible S, acid-soluble sulfur, and total sulfur are chemical parameters used to describe the sulfur status of plants. None of them is related to a single physiological sulfurcontaining compound. The HI-reducible sulfur or acid-soluble sulfur estimate approximately the same amount of the total sulfur in plant tissue (~50%). The acid-soluble sulfur is the sulfur extracted from plant tissue by a mixture of acetic, phosphoric, and hydrochloric acids according to Sinclair (167), who described this extractant originally for the determination of sulfate. Schnug (103) found in tissue samples from more than 500 field-grown oilseed rape and cereal plants that the acidsoluble sulfur content (y) is very closely correlated with the total sulfur content (x). The slope of the correlations is identical, but the intercept is specific for species with or without S-containing secondary metabolites:

oilseed rape: y = 0.58x - 1.25; r = 0.946

cereals: y = 0.58x - 0.39; r = 0.915

As the total sulfur content in Sinclair's (167) solution is easy to analyze by ICP, this extraction method seems to be a promising substitute for wet digestion with concentrated acids or using x-ray fluorescence spectroscopy for total sulfur determination (53,103,266-268).

The total sulfur content is most frequently used for the evaluation of the sulfur nutritional status (see Section 7.5.3). Precision and accuracy of the analytical method employed for the determination of the total sulfur content are crucial. In proficiency tests, X-ray fluorescence spectroscopy proved to be fast and precise (269,270). Critical values for total sulfur differ in relation to the growth stage (242,261), but this problem is also true for all the other parameters and can be overcome only by a strict dedication of critical values to defined plant organs and development stages (103). If this procedure is followed strictly, the total sulfur content of plants has the advantage of being less influenced by short-term physiological changes that easily affect fractions such as sulfate or glutathione.

Composed parameters are the nitrogen/sulfur (N:S) ratio, the percentage of sulfate-S from the total sulfur concentration, and the sulfate/malate ratio. The concept of the N/S ratio is based on the fact that plants require sulfur and nitrogen in proportional quantities for the biosynthesis of amino acids (271-273). Therefore, deviations from the typical N/S ratio were proposed as an indicator for sulfur deficiency (239,274-281). Calculated on the basis of the composition of amino acids in oilseed rape leaf protein, the optimum N/S ratio for this crop should theoretically be 12:1 (103,282), but empirically maximum yields were achieved at N/S ratios of 6:1 to 8:1 (216,242,253,283). Distinct relationships between N/S ratio and yield occur only in the range of extreme N/S ratios. Such N/S ratios may be produced in pot trials but do not occur under field conditions (see Figure 7.16).

There is no doubt that balanced nutrient ratios in plant tissues are essential for crop productivity, quality, and plant health, but the strongest argument against using the N/S ratio to assess the nutritional status is that it can result from totally different N and sulfur concentrations in the plant tissue. Surplus of one element may therefore falsely be interpreted as a deficiency of the other (284). The suitability of N/S ratios as a diagnostic criterion also implies a constancy (273,285-288), which is at least not true for species with a significant secondary metabolism of S-containing compounds such as Brassica and Allium species (289,290). Additionally, it requires the determination of two elements and thus is more laborious and costly.

The percentage of sulfate-S of the total sulfur content has been proposed as a diagnostic criterion (240-242,251-255). Except for laboratories operating x-ray fluorescence spectroscopy, which allows the simultaneous determination of sulfate-S and total sulfur (291,292), this determination doubles the analytical efforts without particular benefit. The sulfate/malate ratio is another example of a composed parameter (293). Though both parameters can be analyzed by ion chromatography in one run, the basic objection made with regard to sulfate (see above), namely its high variability, also applies to malate.