Since childhood I have been intrigued by the marvelous color variety of flowering plants: why and how do so many colors occur? Why so few blue flowers and so many yellow? How do the flowers produce their colors and of what substances are they made? I had many questions but very few answers. [At this point I must say I'm not a trained botanist or even a decent gardener. (My wife has that attribute, thankfully.) I'm simply a retired chemist with botanical questions.]

After earning a living in a commercial chemical direction that had nothing to do with gardening, botany or any of their aspects, I retired.  However, I was still interested in flower colors so I began a few studies of yellow wild flowers because of their ubiquity and ease of obtaining them. The flower I have studied the most has been a Missouri region variety (Rudbeckia missouriensis) of the Black-eyed Susan, Rudbeckia hirta.  R. missouriensis is a smaller variety of its larger North American cousin, R. hirta, and has blossoms perhaps one-half the size of the better known plant. Both are members of the worldwide Daisy (asteraceae) family with its 1,000 genera and 20,000 species. Some other family flowers and plants are the sunflower, coneflower, dandelion, coreopsis, yarrow, goldenrod, thistle, tansy, etc., etc. [A botanical description of Rudbeckia missouriensis is at the end of this article.]

Fig. 1    R. missouriensis	Fig. 2      R. missouriensis

Extracting somewhat pure, unchanged color pigments from wild flowers is challenging, especially in a very small home laboratory not equipped for organic synthesis. Once extracted, examining spectra to attempt any identification is also challenging, frustrating (especially without pigment comparisons) and time consuming. So, I recently took time away from the project to do other things. One of these was to use my microscope to have a go at visually searching for the yellow pigments in the petals themselves. (Actually, I thought this might be more entertaining rather than really useful.)

I don't have a microtome but, by employing the ingenuous double razor blade method reported by Walter Dioni (Micscape February 2004) I produced reasonably useful slices. To maintain some cell osmotic pressure, the material was cut under water (Dioni method) then mounted in weak saline solution under cover glasses; I made no attempt at permanent mounts. All the observations were essentially photographic and thus relatively brief.

STEM
I looked first at transverse slices of R. missouriensis stems that had dried-on-the-stalk. (Here in Missouri we are in a summer-long drought.) Figures 3 and 4, below, reveal a multi-sided, deep faceted dry stem with its central phloem core more than half gone and the small vascular bundles surrounding the stem are closed. Figures 5 and 6 of a healthy stem, on the other hand, show that both the fluid bearing central phloem cells and the vascular bundles are intact and functioning. The greenish areas along the stem boundary, fig. 6, contain many chloroplasts in which chlorophyll is present. Two closer views of the chlorophyll cell regions are seen in figures 7 and 8, both being composites of more than one photo. Probably the needles seen in the cells in figure 9  are calcium oxalate (a metabolic by-product) along with tiny bundles of chlorophyll-containing cells.
 

Fig. 3  Dry Stem,  Rheinberg,  40X	Fig. 4   Dry Stem,  100X

Fig. 5  Fresh Stem,  40X	Fig. 6  Rheinberg filter,  100X

Fig. 7   Composite, 100X	Fig. 8   Composite,  400X	Fig. 9   Calcium Oxalate raphides,  400X

A longitudinal slice (LS) of the dry stem, figures 10 and 11, also reveals the loss of the central phloem cells. Bubbles in Fig. 10 are from water in the slide prep.

PERIANTH
A longitudinal slice through the perianth, the flower base section composed of the sepals (calyx) and the petals (corolla), was made to look for pigmented cells, especially where the petals attached. As might be expected, Fig. 12 and 13 at the bottom edge featured cells filled with opaque red-orange pigment.  Cells higher up the slice, Fig. 14, contained areas of chlorophyll.

PETAL SURFACE
The surface of a few cut petals was examined in oblique light; orderly rows of nipple-like (papillae) elongated surface cells filled with yellow pigment were seen. Figures 15 and 16 show the rows; figures 17 and 18 capture the papillae up close, 17 being very nearly a side view while 18 is almost straight on. Papillae, which are small thickenings of the cuticle and may be hollow or solid, often give a velvet appearance to a petal. (The cuticle is a non-cellular outer protective layer.)

PETALS
Finally, transverse sections of the petals themselves were put under the 'scope.  Flower petals are basically leaves that have become transformed into the colorful but relatively fragile "glory of the plant". Many petals of flowers show their leaf likeness by possessing a few or many vein-like creases running through their structures. Figure 19, below, is a TS of the central portion of a R. missouriensis petal. A crease or dip in the section is evident. Figures 20 and 21 at 100X reveal small pockets of red pigment along their bottom edges. Figure 22 at 400X focuses on the bottom portion of the petal crease and shows both chlorophyll cellular pockets as well as a rich scattering of red pigment cells. Figures 23 and 24 continue the focus on the bottom edge and the interior cellular structures with pigment packets very much in evidence.

These areas of intense red-orange pigments most likely are carotenoid/xanthophyll compounds but may also be flavonoids. R. missouriensis contains both chemical groups though the carotenoids/xanthophylls seem to be present in larger amounts (my initial work appears to indicate this). In Nature, there are a number of yellow flowers that appear to be colored by some combination of carotenoid/flavinoid compounds. The carotenoids are oil soluble and are found in plastids within chromoplasts of the petals. (Interestingly, carotenoids often crystallize within chromoplasts.) Flavinoids are water soluble and are located in vacuole cavities. Anthrocyanins of the flavanoid family are present in many flowers and are the major contributors to red, purple and blue flowers. They too have a role in producing yellow in flowers but that role is as yet somewhat murky.  And, the physical nature of the petal surface may likely have an effect on its color.

Observations of the papillae (below) were interesting. In one, figure 28, most of the liquid formerly in the tips of the elongated cells has disappeared leaving what appears to be a crystalline pigment mass. The transparent nature of the cuticle tissue of the papillae is quite distinct as well. Conversely, in figure 29 the entire papillae are filled and a clear liquid in motion was seen churning  around the tip ends.  And, while there may be some crystalline pigments, most of the dark yellow mass appeared to be in solution.  Figure 30 is a photo composite of the transverse slice, top to bottom. Because of compositing, the photo is somewhat darker than the individual pictures. However the petal structure difference between top and bottom is clear. (The papillae themselves are quite small: their average length is 0.077 mm and diameter is 0.027 mm.)

The papillae were looked at under highest magnification, figures 31 to 33, and more evidence of pigment clumping and/or crystallization was found in figure 32. In figure 31 the channel through which fluid flow was taking place is seen on the left side of the papilla and small amounts of pigment can be detected within the flow. Also the cuticle tip of the papilla is somewhat thicker than its walls and this was characteristic of most of the papillae observed. Almost all of the right papilla in figure 33 is empty of pigment though a mass of red-yellow pigment (?) sits turgidly at the bottom, seeming to mingle with chlorophyll containing cells.

At this point, I'm still studying my data and pictures to determine if I've really learned anything about the yellow pigment source(s) of Missouri's Black-eyed Susan, Rudbeckia missouriensis. Maybe yes, maybe no.

Oh, I almost forgot: I have a little puzzle for Micscape readers. In looking over the petal surfaces at 400X, I discovered two tiny critters, or I think they're critters, nestling between the papillae of the upper surface. See figures 34 and 35. Both measure approximately 0.01 mm in width and 0.04 mm in length. Any idea what these unknown (to me, anyway) critters, bugs, cocoons, etc., might be??? Editor's note added October 2006: Readers kindly emailed Richard to remark on the nature of these organisms; they are 'Alternaria, a common fungal spore that affects plants' and are described in the October 2006 issue.

I am interested to hear from Micscape readers and all comments are welcome.

I use a Nikon Eclipse 200 trinocular microscope equipped with an Qioptiq digital coupler and a Nikon CP 4500 camera.
All photos were processed in Adobe Photoshop® 7.0 and some are of differing size because I was looking for visual data and not necessarily the best picture.

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