European mistletoe under the microscope

[Microscopy_is_fun]

It's winter time, and botany doesn't offer too many things to look at during this time of the year. The European Mistletoe (Viscum album) is available all year long, but becomes visible especially in winter time, when the green bushes growing on leaf-less trees can be seen from far away (Fig.1).


Fig.1: Mistletoes in the crown of trees

Most of the mistletoes grow high up in the trees and are therefore not accessible for the curious microscopist. But a few can be found low enough (Fig.2), and the green leaves as well as the white berries are clearly visible. The sticky berries are food for various birds, and that's how the mistletoe spreads: The birds drop some of the seeds onto the bark of the tree, and a certain percentage of these seeds will germinate and stretch their haustoria into the bark and wood of the host plant.


Fig.2: Close-up of mistletoe

The haustoria reach quite deep into the wood of the host (Fig.3), where they hook up with the xylem of the host plant. In that way, the mistletoe gets the necessary water to keep its metabolism running. The tree does not benefit from the mistletoe, and if there are too many parasitic mistletoes living on the tree, the host plant might eventually die.


Fig.3: Longitudinal section through host wood and mistletoe

The haustoria need to pass several barriers before they can hook up their xylem with that of he tree. The biochemical processes behind these mechanisms are not fully understood. But the microscopic images of a cross-section (Fig.4, 50µm thick section, stained with Etzold FCA, viewed in transmission with 5x objective) reveal some of the fights that must be going on during these processes: We see the parenchyma of the non-lignified haustoria (HT in Fig.4) and the tracheids TR in the center of the haustoria. The lignified wood of the host (HW, older wood in orange-red, fresh wood in purple) tries to encapsulate the foreign, parasitic tissue of the mistletoe. The micrograph is taken from a 50µm thick cross-section stained in Etzold FCA and imaged with Leica HC PL Fluotar 5x. The image is a z-stack based on 5 individual images.


Fig.4: Micrograph of cross-section through haustoria

Fig. 5 shows a longitudinal section through a sample, also stained with Etzold FCA. It can be seen how the haustoria (blue) and their tracheids (purple) successfuly connect with the host wood and give the mistletoe access to the water system of the host plant.


Fig.5: Micrograph of longitudinal section through haustoria

P.S.: I am neither botanist nor English native speaker, therefore feel free to correct me if something is not right in my text.

[Microscopy_is_fun]

I somehow like the graphical look of the micrograph of the cross-section, therefore I post the picture once more, in this case without annotations.


Fig.6: Cross-section without annotations

[Microscopy_is_fun]

The stem of the mistletoe is quite easy to cut, since it is neither too hard nor too soft for slicing with a sledge microtome. I made a set of slices and stained them with various stains. I also had a look at an unstained and freshly cut sample under fluorescence excitation.

The slices were cut with a home-made sledge microtome from a fresh, two year old stem of a mistletoe. The thickness of the slices was around 40µm. All images were taken as panorama of 4 sub-images using a Leica HC PL Fluotar 5x lens. The panorama was stitched using Lightroom, followed by post-processing using Photoshop.

Fig. 7 indicates some of the anatomical features which we can find in a cross-section (stained with Etzold FCA). The lignified sclerenchyma (SK) is visible in bright red, as well as the xylem (XY) which is embedded in the parenchyma of the stem. I assume that the phloem (PH) is located between sclerenchyma and xylem.

Fig. 7: Anatomical features in the stem of a mistletoe.

The fluorescence image was taken from a slice which was embedded in glycerol, excitation wavelength was blue (Leica filter I3).The red fluorescence of the chloroplasts in the parenchyma is well visible, in contrast to the cellulose cell walls in the parenchyma, which are not visible here. The cells in the xylem and sclerenchyma can also be identified easily due to their green fluorescence.


Fig.8: Fluorescence image of mistletoe stem (cross-section)

Additionally, I stained samples with Etzold FCA, safranin-astrablue and Wacker stain (acridine red-acriflavine-astrablue). After slicing and before staining, the slices were fixed with FAA for about 30min, and afterwards rinsed with aqua dest to remove the FAA. Staining took place for around 8min (Etzold FCA and safranin-astrablue). In the case of Wacker stain, a three-step staining was applied (10min acridine red, 15sec acriflavine and 2 min astrablue). Between the three steps the slices were rinsed with water.

Finally, all slices were dehydrated in 99,95 isopropanol and mounted in Euparal.

Interestingly, the Etzold FCA stain gave pretty much the same result as the safranin-astrablue stain. The Wacker samples turn more into greenish colors. Obviously, the yellow acriflavine and the astrablue stain the same tissues in the slices, resulting in large greenish areas.



Fig.9: Stem stained with Etzold FCA


Fig.10: Stem stained with safranin-astrablue


Fig.11: Stem stained with Wacker (acridine red-acriflavin-astrablue).

[imkap]

This is great, I'm fascinated

[Hobbyst46]

Great images. Well done !

[MichaelG.]

What an excellent study !!

Thanks for sharing it.

MichaelG.

[patta]

Great, I've always wondered what was happening there! Images are worth thousand words

If maybe you could cut a thinner section and higher magnification of the parasite-host interface?
Easy to ask!

[Microscopy_is_fun]

@imkap, Hobbyst46, MichaelG: Thanks for your kind feedback! Great to see that the pictures find interest.

@patta : you are completely right, the sections of the wood-haustoria interfaces are too thick. The reason for this is the limited stiffness of my DIY-microtome, which is not designed for heavy-duty wood-cutting. I have a stiffer (and substantially heavier) microtome in our second home. Once I get there (in spring) I will try to obtain thinner sections. The samples are prepared and sitting in FAA already.

[Sabatini]

Great work

[Microscopy_is_fun]

Once I get there (in spring) I will try to obtain thinner sections.

Here are two images of a thinner mistletoe section (20µm). One image taken with 5x magnification, panorama of 12 images. The second image is a z-stack (5 images) taken with 20x objective.

The sample was stained with acridine red, acriflavín and astra blue. The lignified host plant is in red, the mistletoe in green/blue.

[perrywespa]

Beautiful images and great explanation! Thanks.

[woyjwjl]

Great popular science teaching!

[Microscopy_is_fun]

Great popular science teaching!

Beautiful images and great explanation! Thanks.

Thanks for your feedback!

[KurtM]

Great to see that the pictures find interest.

Indeed they are - this is just the sort of thing this forum could use much more of in my opinion.

Bravo! Lovely images and great discussion, well done. Botany isn't my area, and that is precisely why such a post as this is of great value to me. Thank you, I have learned something, and I hope to see more.

[Microscopy_is_fun]

Botany isn't my area, and that is precisely why such a post as this is of great value to me.

Hi Kurt,
thanks for the encouraging words! The nice thing about botany: Even in the smallest frontyard garden there are enough opportunities for many years of microscopic projects. And as bonus, some of the pictures are fun to look at.

[KurtM]

...as bonus, some of the pictures are fun to look at.

In my best Charlie Chan impersonation, contradiction please: ALL of the pictures are fun to look at!