Bot. Mag. Tokyo 82: 424-428 (November 25, 1969)

Effect of Benzimidazole and Nicotinamide

on the Fine Structure of Elodea

by Yoshio YOSHIDA, * * E. R. WAYGOOD

Adenine Dinucleotide

Chloroplasts*

and P. K. ISAAC

Received July 14, 1969

Abstract

An electron microscope study of the effect of benzimidazole and nicotinamide adenine dinucleotide on the senescence of Elodea chloroplasts is described. It was found that benzimidazole results in maintaining at least, or even in enhancing tendency of the lamellar structure of the chloroplast, whereas nicotinamide adenine dinucleotide remarkably accelerates the degeneration normally occurring during senescence. It is suggested that this result may account for the increased rate of

photosynthesis found in benzimidazole treated leaves.

Introduction

In previous papers Yoshida"2,3,4> reported that the senescence of chloroplasts was closely correlated with the function of the nucleus, and that the addition of nicoti-namide adenine dinucleotide (NAD) accelerated the process of senescence. It has been known for some time that benzimidazole delays the onset of chlorosis in detached leaves.5,6,7,8' Investigations in this laboratory with leaves of Elodea and also wheat have confirmed the accelerating effect of NAD on their senescence, a

phenomenon which was overcome by the presence of benzimidazole in the medium.9' A brief review of the benzimidazole effect in wheat leaves has been summarized by Waygood.10' The purpose of this paper was to study the changes in the fine structure of chloroplasts in Elodea leaves when either floated on water or treated with NAD and benzimidazole in an attempt to provide a basis on which it might b e possible to correlate any morphological changes with the metabolic patterns of chloroplasts undergoing these treatments.

Material and Methods

Elodea gigantea was grown in an aquarium in Hoagland's nutrient solution diluted 10-fold. A 19 hr photoperiod was provided by two 20 watt sylvania Gro-

Lux fluorescent lamps 20 cm above the surface of the water. The water was aerated. Leaves from healthy growing tips were excised, rinsed in distilled water and immersed in the test solution in petri dishes. The solutions used were distilled

Department of Botany, University of Manitoba, Winnipeg, Manitoba, Canada. * This work was supported by Grants-in-Aid from the Canada Department of Agriculture

(EMR-14) and the National Research Council of Canada (PRBT-10). ** Permanent Address: Department of Biology , Faculty of Science, Niigata University

Niigata, Japan.

November, 1969 Effect of Benzimidazole and NAD on Chloroplasts 425

water and 10-3 M solutions of NAD, and benzimidazole, and both simultaneously. The petri dishes were placed in a growth chamber at 21 ° under continuous illumi-nation at 420 f t-c of mixed fluorescent and incandesent light for 5 days.

Material was fixed for electronmicroscopy with cold buffered glutaraldehyde fixative11 at approximately 0° for about 48 hr, then postfixed with cold buffered

permanganate fixative for 1 hr, and followed by dehydration through a graded acetone series (30%, 50%, 70%, 90% 2 x 15 min. each and absolute acetone 30 min. two changes) at room temperature. Finally they were impregnated and embedded in selectron-methacrylate resin mixture.12' Ultrathin sections were cut with a LKB ultramicrotome and examined in a Philips EM-75 B electron microscope.

Results

Leaves suspended in distilled water showed a gradual daily progressive alter-ation to a pale greenish yellow chlorosic condition. The chloroplasts shrank and

gradually lost their starch content. Treatment with NAD accelerated chlorosis and after 4-5 days the leaves were almost completely yellow, the chloroplasts were more shrunken and there was a greater loss of starch content. On the other hand, when leaves were treated with NAD and benzimidazole simultaneously, the effect of NAD was very much reduced. The leaves showed little deterioration and their green colour was maintained. Leaves treated with benzimidazole showed scarcely any alteration in their appearance during treatment; the chloroplasts were of a similar size to chloroplasts of immediately detached leaves, and their green colour and starch content were also similar.

Electron microscope observation. Since the fine structure of the chloroplast has been studied extensively and described by other authors,13' a general description of the fine structure of the untreated Elodea chloroplast is omitted here. The normal lamellar structure with well developed grana is shown in Fig. 1.

The effect of keeping the detached leaf in continuous light for 5 days in dis-tilled water is shown in Fig. 2, where the senescent chloroplast is seen to be shorter than the normal, a little swollen, and vacuolated. The grana stacks are still largely unaltered, but the stroma lamellae are beginning to show signs of disruption. These senescent trends are markedly enhanced by NAD treatment (Fig. 3) after which they become almost circular in section and the stroma lamellae are reduced in number and disorganized. Although the grana are still clearly distinguishable, the number of lamellae contained in each granum is reduced to approximately half the normal number and the spacing between them is increased.

When the detached leaves are treated with both NAD and benzimidazole simul-taneously (Fig. 4) the tendency for the chloroplasts to decay and for the stroma lamellae to become disorganized is largely suppressed. It is clearly noticeable that benzimidazole overcomes the effect of NAD on the senescence of chloroplasts.

Suspension of the detached leaves in a solution of benzimidazole results in senescence being arrested. This is reflected in the form and structure of the chloro-plasts (Fig. 5), which show slight reduction in size, but maintain the fine structure intact, or even suggest an enhancing tendency of the lamellar system. In addition,

peculiar electron dense globules with transparent centers can often be found in the stroma region of these chloroplasts. However, they occur only rarely in the other treatments or control but mainly in benzimidazole treated chloroplasts. This

426 YOSHIDA, Y., et al. Vol. 82

gr.

1. 2. 3.

4.

5.

Fig. 1-5. Electron micrographs of chlorop.l.asts. grana, str : stroma lamellae, v : vacuolated space, glb : globules, m : mitochondria.

Chloroplasts from untreated El odea leaves. Chloroplasts from leaves immersed in water for 5 days. Note vacuolation. Chloroplasts from leaves immersed in NAD solution for 5 days showing ac- celerated deterioration. Chloroplasts from leaves immersed in a solution of NAD and benzimidazole for 5 days showing the suppression of NAD effect.

Chloroplasts from leaves immersed in a solution of benzimidazole for 5 days showing the prevention of senescence and even enhancing tendeney of the lamellar system.

November, 1969 Effect of Benzimidazole and NAD on Chloroplasts 427

suggests that they are not artifact in fixation technique, but may be an accumula-

tion of real metabolite increased by benzimidazole effect.

Discussion

Godavari and Waygood9' already showed that the NAD accelerates the chlorosis of detached leaves, but its effect is arrested by treatment with benzimidazole. The changes of chlorophyll level and of conformation must be a reflection of a general function of chloroplasts. Therefore it is of interest that such phenomena really correlate with an actual change of fine structure of chloroplasts.

In chloroplast senescence, the first changes appear in the disorganization of the stroma lamellae together with a reduction in their number. NAD causes typical deterioration of chloroplast structure. Especially, it is interesting that benzimidazole acts not only to maintain, but even to enhance, the organization of grana stacks and stroma lamellae system as compared with the control after detachment. It is well known that the grana are the main loci of chlorophyll localization and the

photochemical reaction. From these facts, one may speculate that the fine structure of chloroplasts may correspond to the physiological activity of leaves after detach-ment. This point is emphasized by the latest observation of Pathak17' and Waygood10' that benzimidazole results in the effective maintenance of photophosphorylation of detached wheat leaves at a high level, and that even in a yellow leaves after 4 days water floating, benzimidazole accelerates the production of enough ATP to carry on photosynthesis. Mishra13' also has demonstrated that benzimidazole in-creased ATP in chloroplasts and promoted the synthesis of NADP which disappears rapidly from chloroplasts of wheat leaves floated on water. Chloroplasts of senescing Elodea leaves in distilled water are almost always characterized by the appearance of large areas of low electron density (Fig. 2). In wheat leaves, senescence is accompanied by a depletion of its reserve carbohydrate but this is accompanied also by an increased synthesis of free fructose and glucose.19> A similar situation may exist in Elodea leaves too, (Yoshida, unpublished data). Hence it is of some interest to speculate that they might be reservoirs of such free monosaccharides, since no such vacuoles are noticed in the NAD treated material, which may be due to the acceleration of carbohydrate breakdown caused by the addition of NAD. Person et al.5' demonstrated that benzimidazole prevented the respiratory increase that occurs after leaf detachment, and Buchel et a120' and Beechey21' reported that benzimidazole shows an uncoupler effect, similar to 2, 4-dinitrophenol, on the oxidative phosphorylation in isolated mitochondria of rat heart, and it inhibits the ATP-dependent NAD reduction coupled with succinate in electron-transport of mitochondria. It may be suggested, therefore, that one of the effect of benzimidazole is conceivable in view of the supressing NAD. Furthermore, some recognizable globules were observed in the chloroplasts treated

with bezimidazole. Ikeda and Ueda22' reported that the occurrence of characteristic

granules were noted in senescent chloroplasts of Elodea leaf cells which were cultured in tap water in dark for 5 days and fixed with osmium. They concluded that these granules may be transferred from carotenoid pigment liberated from the disintegrating lamellar system after dark culture. According to Weier23', the round, dark bodies common after osmium fixation are not encountered in potassium per-manganate, but conversely, the irregularly darkened bodies named "star bodies" were present in most plastids but were scarce or absent from the fall collections.

428 YOSHIDA, Y., et al. Vol. 82

In our present work, no such granules as in Ikeda and Ueda's report were obvious

in senescent chloroplasts in permanganate fixation. Conversely, peculiar globules

were pronounced in benzimidazole treated chloroplasts. It may be possible that these globules responding to parmanganate are allied to the "star bodies" of Weier23.

Although a nature of these materials has not been conclusively resolved, it is inferred that they are deposite of a metabolite in the chloroplasts caused by the effect of

benzimidazole, since they were very little encountered in other treatments.

While the mechanism of action of benzimidazole yet remains to be elucidated, it is reasonable to assume from these diverse effects on the photosynthetic apparatus that their site of action in leaves resides largely in the chloroplasts, culminating in

an integrated lamellar structure. Whether their effect is direct or indirect is not known, nevertheless the results shown in this paper provide some morphological support on the biochemical point of view on the effect of benzimidazole on the

chloroplast activity'o"7,24>

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