Studies on the insoluble states of anthocyanin in rose petals (1974-79)
Hitoshi Yasuda

Jour. Fac. Sci. Shinshu Univ. 9: 63-69 (1974)
I. The insoluble state of anthocyanin and its relationship to petal color, together with a new instance of this relationship.

Abstract

The upper epidermal cells of certain cultivars of black roses, e.g. Charles Mallerin, Josephine Bruce, Bonne Nuit and so on, occasionally hold in the cell vacuoles massive structures colored purple or purplish-red.

It was observed histochemically that these massive structures showed a color change from either purplish-red or from purple to red with dilute hydrochloric acid and to bluish purple with a weak solution of sodium hydroxide. From these results the massive structure found in the red rose petals can be regarded as the insoluble state of anthocyanin.

In the surface view of those petals having these massive structures in their upper epidermal cells, the numerous black spots, which are considered to be shadows of epidermal cells, are visible even in petals whose height/width ratio of the upper epidermal cells are within the range of the red petal ones, It can therefore be said that the insoluble state of anthocyanin present in the red rose petals may play an important role in the blackening effect on the petal color.

Introduction

It is generally known that anthocyanins are present in solution in the cell sap. However, it is considered to be a rare occurrence when anthocyanin appears in insoluble states in plant cells or in tissues. According to BLANK'S review), the insoluble states of anthocyanin can be classified by the following three types:

(A) a crystallized type in cell plasma (e.g Allium) and in sap (e.g., juice of the blood orange).

(B) a stored type in the cell wall (e.g. Sphagnum, Marchantia, Preissia etc.).

(C) an "anthocyanophore" type in the cell vacuole (e.g. Erythrea, Fuchsia, Iris, Dianthus, Delphinium, Pulmonaria and so on).

Very recently, the present author reported that the cell vacuole of the bluing petals of the red rose, in some cases, includes anthocyanin as a component of the blue spherule, the basis of which may principally consist of a tannin-like substance). This example could be considered as another type of the insoluble state of anthocyanin.

As stated above, several observations have been made on the insoluble states of anthocyanins, but there has been relatively little information concerning their cytological and physiological investigations.

As a beginning step in detailed investigations on the insoluble state of anthocyanin, especially of the anthocyanophore type, the present paper is concerned with its significance relative to the petal color, thus providing a new example found in some petals of the so called "black rose".

References

  1. BLANK, F. (1947) Bot. Rev. 13 : 241
  2. WEBER, F. (1936) Protoplasma 22 : 100
  3. YASUDA, H. (1970) Bot. Mag. Tokyo 83 233
  4. ______ (1965) Jour. Fac. Lib. Art. Sci. Shinshu Univ. No. 15 : 15
  5. ______ (1965) Bot. Mag. Tokyo 89 : 357
  6. ______ (1964) Jour. Fac. Lib. Art. Sci, Shinshu Univ. No. 14 31
  7. ______ (1965) ibid. No. 15 : 23
  8. LIPPMAA, T. (1926) Beih. Bot. Zentralbl. 43 : 127
  9. YASUDA, H. (1965) Bot. Mag. Tokyo 80 357
  10. ZIESLIN, N. and HALEVY, A. H. (1969) Jour. Amer. Soc. Hort. Sci. 94 : 729

Cytologia 41: 487-492 (1976)
II. Histochemical observation on its basal substance.

In the previous paper (Yasuda 1974), it was reported that the blackish petals of certain rose cultivars (e. g. Charles Mallerin, Bonne Nuit and Josephine Bruce) occasionally include an insoluble state of anthocyanin in the central vacuoles of the upper epidermal cells, and it was also pointed out that this state contributes toward the elucidation for the presence of blackish tone to the petal color of these cultivars. In the same paper, it was described how this insoluble state of anthocyanin showed a closely similar appearance to the anthocyanophore which had been named by Weber (1934, 1936), especially to that found in Erythraea reported by Lippmaa (1926).

If this insoluble state of anthocyanin is analogous to the anthocyanophore, it should then be something of a massive structure composed of basal substances associated with anthocyanin. Hence, it will be necessary for the moment to attack the question of whether or not such massive structure is really related to the state of anthocyanin. In order to answer this question, the present observations were made and some histochemical tests on the basal substance of the state of anthocyanin were undertaken.

Summary

The present paper dealt with some histochemical observation on the basal substance of the insoluble state of anthocyanin appearing in the upper epidermal cells of petals, using a black rose cultivar, Charles Mallerin.

The results obtained are summarized below.

  1. The satisfactory fixation and decoloration of the insoluble state of anthocyanin were accomplished by Muller's solution, 10% neutral buffered formalin and Scott's solution, leaving a massive structure.
  2. The insoluble state of anthocyanin treated with the solutions above mentioned was stained orange with safranin, and red with ruthenium red very strongly.
  3. These stainings are strongly suggestive that the pectic substance is involved in the basal substance of the insoluble state of anthocyanin.
  4. It may be considered that the pectic substance of this massive structure is not calcium salt of pectic acid but protopectin.
  5. The insoluble state of anthocyanin in the present material can be interpreted as a special structure of the pectic substance, protopectin, associated anthocyanin.

Literature cited


Cytologia 44: 687-692 (1979)
III. The observation of the developmental process of the massive structure.

Yasuda (1974) revealed an example of the insoluble states of anthocyanin in the upper epidermal cells of the petals of some black rose cultivars, e. g. Charles Mallerin, Josephine Bruce, Bonne Nuit and others. The histochemical observations of the insoluble state of anthocyanin in rose petals (Yasuda 1976), led to an assumption that anthocyanin may be associated with the wall constituent material; the basic substance of which is probably protopectin.

The other significant problem induced from this novel intracellular structure, would be the clarification of how this structure is developed in the epidermal cells.

At present besides the present material, other examples of the insoluble state of anthocyanin in the plant cells or tissues have been extensively reported by several authors (Weber 1936, Blank 1946, Yasuda 1970, 1974). However, literatures available to elucidate the occurrence of these insoluble states of anthocyanin in plant cells are insufficient.

In the present investigation the author reports a base-line on the sequence of the developmental process on the massive structure which develops by associating with anthocyanin and makes the pigment insoluble.

Summary

The development of the massive structure which appeared as a protuberance of the internal cell wall and is composed of the insoluble state of anthocyanin, was morphologically observed using a black rose cultivar, Charles Mallerin.

The massive structure initiated in the various appearances, for example, thread-like, T-shaped, L-shaped, auched, meteoric and so on, in the portion near the tip of the upper epidermal cells of the petals in the early age of half-opened stage of flowering.

With advance of the flowering age, the young massive structure undergoes a marked increase both in length and in thickness.

In further progress of flowering age the rod-like, sometimes branched, feature of the structure appeared and grew longer and thicker occupying the large portion of the central vacuole of the epidermal cells.

The relationship between the massive structure and the tonoplast was discussed.

Literature cited