Effect of root heating on the tolerance of barley leaf cells and ultrastructure of chloroplasts and mitochondria

Abstract

In Nature, unfavorable factors affect frequently not the whole plant but its separate organs (or parts), which however influence functioning of its other organs (parts), which did not experience stress. For example, even short-term heating plant roots could improve leaf cell tolerance to elevated temperature [1‐3] due to various physiological and biochemical changes in the leaf cells [4‐6]. Such data indicate a capability of signal transduction from one plant organs (parts) to others reporting about temperature changes, although the mechanisms of this phenomenon are not completely deciphered and are still the object of discussion. To understand them, it is of importance to establish not only functional (physiological, biochemical, and molecular) but also possible structural changes arising in the plant cells under the local action of temperature. However, only few related studies [7, 8] are known, which do not permit of presenting a general pattern. In this connection, the objective of this work was to examine structural changes in chloroplasts and mitochondria occurring during the development of improved heat tolerance of leaf cells after the action of high hardening temperature on plant roots. Experiments were performed on barley ( Hordeum vulgare L., cv. Otra) seedlings grown in filter paper rolls on the Knop nutrient medium (pH 6.2‐6.4) in the chamber of the phytotron at 25 ° C, a relative humidity of 60‐70%, an illuminance about 10 klx, and a photoperiod of 14 h. The root system of 7-day-old seedlings was heated at 38 ° C for 1, 4, or 24 h in a specially constructed chamber [2] permitting the maintenance of a desired temperature only around the root system; seedling shoots were kept at 25 ° C. Exposure duration and temperature were chosen on the basis of previous studies [9]. The first leaves were used for the assessment of their heat tolerance and the analysis of the organelle ultrastructure. Leaf cell heat tolerance was assessed from a temperature (LT 50 ) killing 50% of palisade cells of excised leaf disks (0.3 cm 2 in area) after their 5-min heating in the water thermostat. A desired temperature in the thermostat was maintained with an accuracy of ± 0.1 K. Cell viability was assessed from the coagulation of the cytoplasm and chloroplast destruction using a light Mikmed-2 microscope ( × 40 objective, LOMO, St. Petersburg). To examine the ultrastructure of chloroplasts and mitochondria, leaf disks were fixed at 0‐4 ° C with 3% glutaraldehyde prepared in phosphate buffer (pH 7.4) and then postfixed in 2% OsO 4 . The material dehydrated in the series of ethanol concentrations and acetone was stained with uranyl acetate and embedded in the mixture of Epoxy resins. The sections of the leaf palisade parenchyma were prepared using an LKB-4800 ultramicrotome, contrasted with lead citrate, and examined with a JEM-100 electron microscope at magnification of 6000‐25000. The tables and figures present mean values from 2‐ 3 independent experiments performed in 5‐10 replicates and their standard errors. The values significant at P ≤ 0.05 are discussed.