Ultrastructural changes in chloroplasts of Quercus rotundifolia Lam. in response to evernic acid

Annals of Botany 57, 407^413, 1986

407

Ultrastructural Changes in Chloroplasts of Quercus rotundifolia Lam. in Response to Evernic Acid. C. ASCASO and S. RAPSCH. Instituto de Edafología y Biología Vegetal, Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain Accepted: 23 September 1985

ABSTRACT The amount of total chlorophyll, chlorophylls a and b as well as the ratio of a to b decreased in chloroplasts isolatedfrom Quercus rotundifolia leaves, kept for 17 d in a solution of 35-5/ÍM evernic acid in 1 mM Na HCO3, when compared with the chloroplasts of control leaves (kept in NaHCO3). The chloroplasts in the spongy parenchyma were smaller and the amount of starch and plastoglobuli lower. The number of grana per chloroplast section, the number of thylakoids per grana and the height of grana stacks were also less in the chloroplasts of leaves treated with evernic acid. Quantitative ultrastructural differences were determined by rneans of electrón microscopy and image analysis techniques. Key words: Quercus rotundifolia Lam., chloroplasts, ultrastructure, lichens, evernic acid. INTRODUCTION

Lichens produce a large number of secondary aliphatic and phenolic metabolites, many of which are extracellular producís found on the surface of hyphae in the form of crystals (Hale, 1973, 1976; Hawksworth, 1976). Since this is a peculiarity of lichen symbiosis, lichen substances nave been the object of numerous studies to establish their physiological role and evolutionary significance. Through the research of Schatz et al. (1954), Schatz (1963) and Vicente (1975), it is known that the lichen substances deposited on thallus hyphae have a chelating effect through which the thallus receives inorganic cations from the substrate where they live. It is also known that the hyphae of lichens on a tree substrate penétrate the tissue as far as the xylem vessels (Ascaso, González and Vicente, 1980; Orus and Ascaso, 1982) Little is known about the effect of lichen thalli attached to higher plants. They can cause trees and bushes to defolíate (Hale, 1983), though it is not known whether this is due to lichen substances. In the course of investigating their possible influence on defoliation, phorophyte shoots have been incubated in substances isolated from lichen thalli. In this manner, Orus, Estevez and Vicente (1981) found that lichen substances, isolated from Evernia prunastri (L.) Ach. thalli, interfered with the physiological processes of Quercus rotundifolia Lam. foliage. For example, the inhibition of photolytic activity through the chelation of the chloroplast Mn2+. Similarly, Ascaso, Orus and Estevez (1983) observed that the thylakoids swelled and disintegrated while structures associated with thylakoid-free stroma regions appeared which were similar to the stromacentres described by Gunning, Steer and Cochrane (1968). Inoue, Noguchi and Hubo (1983) confirmed the findings of Orus et al. (1981) by incubating chloroplasts, isolated from Q. mongolia and spinach leaves, in usnic acid. 0305-7364/86/030407 + 07 $03.00/0

© 1986 Annals of Botany Company

408

Ascaso and Rapsch—Effect

of Evernic Acid on Chhroplast Ultrastructure

In the present study, the main lichen substance of the E. prunastri thallus, evernic acid, was used to treat Q, rotundifolia shoots. These species are commonly found growing in association. The ultrastructural changes in chloroplasts were studied morphometrically by using image analysis techniques. Chlorophyll content of leaves was also determined. MATERIALS AND METHODS

Quercus rotundifolia seedlings were grown from acorns in trays of soil in the laboratory in natural photoperiods at room temperature. When the seedlings were one year oíd, shoots were detached and incubated in a solution of 35-5 /¿M evernic acid (Sigma) in 1 niM NaHCO3, pH 6-5. Other shoots of the same age were incubated in NaHCO3 solution, but without evernic acid. The concentration of evernic acid was based on the work of Cifuentes (1985). Temperature, humidity and photoperiod were those in the laboratory; not specifically controlled. Chlorophyll determinations Chloroplasts were isolated by the method of Whatley and Arnon (1963). Chlorophyll was determined in 80 per cent acetone extracts of leaves using Arnon's (1949) equations to calcúlate Chlorophyll a, b and total chlorophyll concentration. Electron microscopy After treatment, leaf svgments (about 1 mm2) were fixed in 3-25 per cent glutaraldehyde in O-1 M Sorensen buffer, pH 7-1, for 3 h, washed in buffer, post-fixed in 1 per cent osmium tetroxide and dehydrated ir a graded series of ethanol. Samples were embedded in Spurr's médium (Spurr, 1969), polymerized at 70 °C for 24 h, and sectioned with an Ultracut-E Reichert Ultratome. The sections were stained with lead citrate for 6 min at 20 °C (Reynolds, 1963). Ultrathin sections were examined with a Philips EM 300 electrón microscope and image analysis was done with a Mop-Videoplan (Kontron) semi-automatic image analyzer. Statistical analysis was done using Student's /-test. RESULTS

After 17 d, the leaves of shoots treated with evernic acid were more chlorotic and senescent than those in the NaHCO3 control group. The total amount of chlorophyll and chlorophylls a and b were lower in the leaves treated with evernic acid (Table 1). No differences in general leaf structure were found by examination in the transmission electrón microscope. However there is a difference in the appearance of palisade parenchyma cells after evernic acid treatment (Fig. 1 A). The chloroplasts are no longer TABLE 1. Chlorophyll content of chloroplasts from Q. rotundifolia leaves treated with evernic acid Total chlorophyll Control Evernic acid

(/íg g"1 f. Wt)

Chlorophyll a (/íg g-1 f. wt)

Chlorophyll b (/*g g-1 f. wt)

Chl a/Chl b

119-33 + 4-31 91 -47 ±3-00

84-01+2-37 59-70 + 3-01

35-30 + 3-01 31-75±2-30

2-38 1-88

Means + s.d. Replícate of five samples.

Ascaso and Rapsch—Effect

of Evernic Acid on Chloroplast Ultrastructure

409

FIG. 1. Electron micrographs of cells from leaves of Quercus rotundifolia. A, B, Treated with evernic acid for 17 d. A, Palisade parenchyma; B, spongy parenchyma. C,D, untreated control leaves; C, chloroplast from spongy parenchyma; D, detail of chloroplast from spongy parenchyma. Abbreviations: ch, chloroplast; g, grana; m, mitochondria; pg, plastoglobuli; s, starch grain; t, thylakoid; v, vacuole; cw, cell wall.

410

Ascaso and Rapsch—Effect

of Evernic Acid on Chloroplast Ultrastructure

in their typical peripheral position and the large central vacuoles are broken and in disarray. In the cells of the spongy parenchyma too (Fig. 1B), the chloroplasts are not cióse to the plasmalemma and a large number of vesicles have occupied the cell interior. The organization of the plasmalemma, the cytoplasm with chloroplasts and mitochondria, and the large central vacuole surrounded by tonoplast is partly disrupted. The appearance and organization of xylem vessels is not apparently changed by evernic acid treatment. Since the cells of the spongy parenchyma are closest to the vascular bundle, we particularly examined chloroplasts in this mesophyll tissue. The thylakoid lamellae of these chloroplasts were in good order compared with those studied in fresh Q. rotundifolia leaves (Ascaso and Rapsch, 1985). The thylakoids were well structured with more or less prominent grana stacks, but without the widening of the thylakoid lumen, which was observed in freshly collected leaves. These chloroplasts had several grains of starch in their stroma while plastoglobuli were rare and large, the largest being the most electron-transparent (Fig. 1C). Figure 1D shows an enlarged chloroplast región with grana. The thylakoids appear in negative contrast; this often happens in chloroplasts of Quercus leaves because the material between the thylakoid membranes reacts with osmium tetroxide to créate a very electron-dense product. After incubating in evernic acid, a small proportion of chloroplasts (5-10 per cent) look like those in Fig. 2A. Most of the chloroplast membranes are broken and, in consequence, the chloroplasts become very irregular in shape. This may be due to the mechanical tensión produced in the vacated stroma spaces previously occupied by plastoglobuli. The other chloroplasts, with a less damaged surrounding membrane, have a remarkably dilated thylakoid lumen (Fig. 2B). The sectional área of these chloroplasts was small compared with those in the controls. Chloroplasts in the spongy parenchyma from evernic acid treated leaves are shown in Fig. 2C (compare untreated in Fig. 1 D). Here, the thylakoid lumen and stroma react with osmium tetroxide, creating an electron-dense product. Moreover, there are structures that appear to be stromacentres. These units are hexagonal with a width of 10-11 nm. The sectional áreas and perimeters of chloroplasts were reduced significantly (P < 0-01) in evernic acid treated leaves. The numbers of starch grains and plastoglobuli per /¿m2 of chloroplast were also reduced. The grains occupied only half as much of the chloroplast área (2-4 per cent) as those in control leaves (4-8 per cent) and the number of grana in each chloroplast section was smaller (Table 2). The smaller size of chloroplasts in treated leaves was associated with fewer grana per /