Histochemical assay of proanthocyanidin heterogeneity in cell cultures

Plant Science, 52 (1987) 99--104 Elsevier Scientific Publishers Ireland Ltd.

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H I S T O C H E M I C A L A S S A Y O F P R O A N T H O C Y A N I D I N H E T E R O G E N E I T Y IN C E L L CULTURES*

HELEN A. STAFFORD**, HOPE H. LESTER and ROSELLEN M. WEIDER

Biology Department, Reed College, Portland, OR 97202 (U.S.A.) (Received February 23rd, 1987) (Revision received May 1lth, 1987) (Accepted May l l t h , 1987) The distribution patterns and intracellular localization of proanthocyanidins (condensed tannins) were studied microscopically in cell suspension cultures derived from leaves of Ginkgo biloba L., Pseudotsuga menziesii Franco (Douglas fir),and Ribes sanguineum Pursh. (Flowering current) after staining with the nitroso reagent. The specificity and variations of the reaction are discussed.

Key words: proanthocyanidins; condensed tannins; cell cultures; nitroso stain

Introduction

Secondary metabolism in cell cultures has g e n e r a l l y been s t u d i e d b y m e a n s of biochemical procedures using extracts of cells. Such assays give average values of products per culture. Histochemical techniques, on the other hand, can show the variation in content per cell as well as the distribution patterns between cells and the relationship b e t w e e n morphological and biochemical heterogeneity can be compared. We previously reported the accumulation of P A s {condensed tannins) and r e l a t e d compounds, based on a test tube colorimetric assay, in extracts from cell suspension cultures (CSC) derived from leaves of Ginkgo

biloba, Pseudotsuga menziesii and Ribes sanguineurn [1,2]. In order to determine which cells in these cultures produced these secondary products and their intracellular localization, we have now added the nitroso *Supported by National Science Foundation grant DMB 82118301. **To whom all correspondence should be sent. Abbreviations: CSC, cell suspension cultures; PAs, proanthocyanidins.

reagent to culture cells and studied staining patterns microscopically [3--6]. The cultures already mentioned are of interest since their P A s consist of varying ratios of procyanidins {with o-diphenolic B-rings) and prodelphinidins {with triphenolic B-rings) [1,2]. Materials and methods

Cell cultures derived from leaves were maintained as described in previous papers [1,2]. The cultures of Pseudotsuga, Ginkgo and Ribes were subcultured approximately every 3 weeks for 8, 4 and 2 years, respectively. The nitroso stain procedure was basically that of Reeves [3]. One drop each of the following solutions was added in sequence (or 3 drops of a mixture) to a sample of cells: (a) 10% (w/v) sodium nitrite, (b) 20% {w/v) urea, {c) 10% (v/v) acetic acid. After 3--5 min of gentle stirring with a glass rod, 3 drops of 2 N N a O H were added. Descriptions in the text were based on microscopic examination of smears of fresh cultures as well as on photomicrographs of these cells taken with a MC 63 photomicrographic camera mounted on Zeiss 9901 microscope with Kodak Ektachrome Tungsten Film (ASA 50). A blue

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filter (1 m m in thickness) over the lamp source was useful in removing background reflections. Standards of higher oligomers of PAs were obtained by the elution in 70% (v/v) methanol of the basal streak area [2] after 2d i m e n s i o n a l p a p e r c h r o m a t o g r a p h y of extracts from CSC of P. menziesii as a source of procyanidins, and needles of Metasequoia glyptostroboides H. Hu and Cheng as a source high in prodelphinidins. Another p r o d e l p h i n i d i n p r e p a r a t i o n was k i n d l y supplied by L.J. Porter, Petone, New Zealand. Other standards were obtained as previously described [1,2]. Results and discussion

The nitroso histochemical stain Although it is not specific for PAs, and the mechanism of its various color reactions with different phenolic compounds has not been r i g o r o u s l y s t u d i e d w i t h m o d e r n methods, the nitroso reaction followed by NaOH was a useful histochemical stain for our cultures because PAs were the only major phenolic compounds present. The

reaction has sometimes been used uncritically and has mistakenly been considered to be a specific test for so-called 'catechol tannins' (presumably meaning compounds with odiphenolic B-rings or procyanidins) by Reeves [3,4], who also confused these oligomers with chlorogenic acid or its alkali induced polymers. The nitroso reaction has been reported to involve the formation of nitrosophenols in the presence of nitrous acid. The nitrosonium ion is added to an available position that is either ortho or para to the hydroxyl group of a benzene ring to produce a nitroso compound that is in tautomeric equilibrium with a quinonemonoxime. Upon the addition of NaOH, colored quinoid salts are formed, with the specific color determined by the number and position of the hydroxyls of the phenolic compound involved [5]. Caffeic acid, however, an ortho-diphenol with a free acid group, produces a bright red color prior to the addition of NaOH. The quinic acid ester, chlorogenic acid, gives a slight orange or tan coloration until alkali is added, after which the typical bright red color was observed, reported to have an absorption peak at 520

Table I.

Comparison of percent of stained cells and color variations with the nitroso mixture before and after addition of NaOH with total P A content and ratio of procyanidins to prodelphinidins. Color reactions with NaOH alone were negligible in all cases. CSC

% nitroso positive a

Color variations

Pre-NaOH

Total PA c content Post-NaOH b

PC:PD d

pg/mg

dry wt. Pseudotsuga

90

Mainly brown, few orangish brown

Ginkgo

40--50

Wide range of orange and brownish red colors Mainly brown, few orar~gish brown

Ribes

5--10

Mainly brownish red and bright red, few orangish red Mainly brownish red, some bright red Mainly brown, some brownish red

575

1:0

565

1:1

87

1:5

aIncludes all ranges of colors: orange, red and brown of varying intensities. The colors of cells in a series of microscopic fields were recorded and totaled as stained or unstained. Total cells counted were about 2800, 2200 and 800 for Pseudotsuga, Ginkgo and Ribes, respectively.

ball showed a definite intensification of color upon addition of NaOH. CRegular test tube assay for PAs with b u t a n o l - - H C l reagent (data from Ref. 2~. dRatios of procyanidins (PC) to prodelphinidins (PD) estimated on residues after regular PA assay (data from Raf. 2).

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nm [7]. Controls are needed to distinguish between the effects of N a O H on the postulated nitrosophenols and on the original phenolic compounds. Ortho diphenolic flavanoids such as dihydroquercetin, catechin, epicatechin and oligomeric procyanidins all gave a red color with the nitroso reagents plus NaOH, compared with a yellow color with N a O H alone (at about 50--100 ~g/100 ~1 on a spot plate). Monomeric flavanoids with a triphenolic Bring such as epigallocatechin, gallocatechin and dihydromyricetin gave yellow to gold colors with the nitroso mixture plus NaOH, bu the yellow was mainly due to N a O H alone. Oligomeric prodelphinidins, on the other hand, gave an intensified brown color with the nitroso mixture and NaOH.

The nitroso stain in cell culture smears When the nitroso mixture plus N a O H was added to smears of cell cultures, a range of orange, red, reddish brown and brown colors was obtained (Table I). In general, reddish colors were browner with Ribes and redder with Pseudotsugc~ This would be expected because of the prevalence of prodelphinidins compared with procyanidins in the Ribes cultures. Since P A s and their flavan-3-ols were the only major phenolic compounds in our culture extracts, we feel that all the visible staining was due to these compounds. NaOH alone caused little staining of the cells, in contrast to its effect on standard phenolic compounds discussed earlier. Pre-NaOH effects, however, were observed (Table I). This was especially true with Ginkgo smears, where a significant number of orange and red colors were detected microscopically in some cells prior to the addition of the NaOH. However, there was no chromatographic evidence for significant quantities of caffeic acid which is known to produce pre-NaOH red colors. Cells in CSC of Pseudotsuga gave mainly a brown color. Such brown products prior to the addition of NaOH have been reported previously [3,7]. The cause of this pre-NaOH coloration is

unclear, especially as it was much more evident in cultures than when the nitroso stain was used on free hand sections of tissues.

Vacuolar localization A major use of the nitroso stain was in determining the intracellular localization of the products stained with the least damage to the cellular constituents. The products stained with the nitroso reagents plus N a O H were clearly localized either in one or more vesicles of varying sizes in presumably y o u n g cells near the ends of chains of cells, or in a large central vacuole of mature cells. The apical, youngest cell of a chain was generally unstained. This intracellular localization is consistent with other microscopic and electron microscopic (EM} studies t h a t indicated biosynthesis in vesicles, budded off from the e n d o p l a s m i c reticulum, t h a t subsequently coalesce to form the large central vacuole [8,9]. Staining with the vital stain, neutral red, indicated a similar array of red colored small vesicles or large central vacuoles. While the colors of the nitroso stain were diffuse, with no indication of an aggregated state, there is EM evidence of aggregation of P A s and even an association with membranes inside the vacuole, including the tonoplast [8]. Distribution pattern of the nitroso stain The approximate percent of cells in our cultures giving a positive nitroso reaction is shown in Table I. This value included the entire range of orangish reds, reds and browns. No purely yellow colors were observed in cell smears. (Very few dead cells were present as indicated by a lack of cytoplasmic staining with E v a n s Blue). In general, a b o u t 90% of the cells of Pseudotsuga CSC showed positive colors, while only about 50% of the Ginkgo cells and only 10% of Ribes cells were stained. Unfortunately, while the number of stained vs. unstained cells can be easily determined, any spectrophotometric quantification per

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Fig. 1. Photomicrographsto illustrate the distribution pattern of nitroso positivecellsin CSC of Pseudotsuga(A), Ginkgo (B) andRibes(C). × 170. cell would be very difficult because of the color variations and the non-homogeneity of the vacuolar staining. The variation in intensity was sometimes great. Some of this variation per cell could be due to a developmental sequence of cells of different ages, clearly seen in some of the chains of cells. This type of information about the d i s t r i b u t i o n of P A s a m o n g cells or concentration per cell given by the nitroso stain is quite different from the test tube spectrophotometric analysis with the butanol --HC1 reagent [1,2], also summarized in Table I, which gives an average value for all cells in

a culture. Since both cultures contained similar average amounts of PAs, but differ in the percent of nitroso positive cells (50% for Ginkgo and 90% for Pseudotsuga), the PA content per cell m u s t be higher for Ginkgo than for stability of the PA phenotype Pseudotsuga cells. PA containing cells appeared to have divided; this has also been reported in other cultures and in intact tissues [10,11]. Two basic distribution patterns of the expression of the PA phenotype were observed. In one, the PA phenotype tended to be retained in daughter cells of either clumps or chains of

103 cells in our cultures, while in the other, its expression was much more random in daughter cells {Fig. 1). Pseudotsuga cultures were c h a r a c t e r i z e d by a w i d e s p r e a d expression of the PA phenotype in daughter cells, although in varying amounts per cell. Ribes cultures were similar in that they contained relatively long chains and large clusters of cells that were either stained or unstained, but the distribution of the stained cells was very limited within the culture in comparison with that of Pseudotsugo~ On the other hand, stained and unstained cells in Ginkgo .cultures were much more randomly arranged within a chain of cells or clump that presumably were derived from one cell line, indicating that the phenotype for PA accumulation was not always expressed in the daughter cells upon cells division. These two basic differences in phenotypic expression in daughter cells may have their counterparts in PA distribution within intact plants. For instance, presumed PAs were found in all endodermal cells [12], while only occasional isolated cells with PAs were observed in xylem parenchyma cells of cotton seedling roots [13]. We observed similar widespread positive PA phenotypes in some tissues while only scattered PA positive phenotypes were seen in others when we used the nitroso stain on free hand sections of Pseudotsuga needles. For instance, the vacuoles of most cells in the palisade mesophyll layer on the adaxial side were heavily stained a bright red, while only isolated red-stained cells in the spongy mesophyll or parenchyma within the veins were observed.

Relationship of the distribution pattern of PAs to morphological heterogeneity Cultures from the three plants studied varied considerably in the size and shape of the individual cells and in the degree of aggregation of ceils (Fig. 1). We have consistently selected for both the greenest and smallest clumps. Pseudotsuga CSC consisted of aggregates {less than 3 rain in

size) made up of a core of relatively isodiametric cells in size {25 /~m) with chains of more elongated cells (about 21 × 53 grn) extending from the outer surface, and isolated chains of 5--7 elongated cells that m a y have broken off from the larger aggregates. These morphological patterns were very similar to those reported by Durzan et al. in white spruce [14]. In comparison, the cells in Ginkgo cultures were more isodiametric and homogeneous in size (about 40 × 44 gin). The aggregates (less than 5 rnm in diameter) were more tightly packed and arranged in more orderly rows than the cultures of either of the other two species and fewer isolated chains of cells were observed. The cultures originally contained aggregates larger than 5 rain in diameter; the P A content was less on a dry weight basis, but no vascularization was observed. Ribes C S C were the most variable in both size and shape (diameters ranged from 15--61 ~m and lengths from 15--147 ~m). Both aggregates (less than 0.5 mm in diameter) and long chains of cells were observed; the wall interconnections appeared to be very loose. The PA phenotype did not appear to be related to cell size or shape in any of the cultures since PAs were found in a wide range of cells forms.

Origin of the heterogeneity in the positive PA phenotype in cell cultures The genetic variability in our cultures is unknown; unfortunately, we cannot as yet grow clones from single cells. However, in the case of Pseudotsuga cultures, the positive PA phenotype was expressed in all but the youngest cells, although variable in amount per cell judging from the intensity of the nitroso stain, and was inherited in the sense that it was transmitted to daughter cells [15]. The problem of variability and stabilization of the production of secondary products in cultures has been discussed recently by Ellis [16,17]. In the particular cases studied, the differences were phenotypic rather than genotypic, since single cell clones reproduced

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the original distribution pattern of variation in capacity for secondary metabolite production. The PA positive phenotype in daughter cells of Ginkgo, in which stained cells appear to give rise to colorless cells and vice versa (see Fig. 1), is an example of instability somewhat like that reported by Dougall in carrot cell cultures, in which a similar variable production of anthocyanins per cell was observed in clones derived from either high or low accumulating cells [18]. Regulation of these phenotypic patterns of secondary products in cultures remains an intriguing problem. In summary, there does not appear to be any c o n s i s t e n t r e l a t i o n s h i p b e t w e e n morphological and biochemical (detected as PAs) heterogeneity in our cultures. Despite a lack of specificity and difficulties in effective quantification on a cell basis, the nitroso stain is a quick and useful histochemical technique for showing the intracellular localization and the distribution pattern of nitroso positive cells, especially in cell cultures that, based on other biochemical analyses, contain only PAs and their related compounds.

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