The Sporulation Rhythm ofNeurospora crassa:

Example of a Circadian Clock Rhythm

(Peter Ruoff, Stavanger University)

 

Mycel and spore formation in the asexual life cycle of Neurospora crassa

Neurospora crassa has a sexual and an asexual life cycle. It is during the asexual cycle the sporulation rhythm can be observed. The picture below indicates the different stages during the asexual cycle.

[N. crassas 
asexual life cycle]



Let's begin with the spores: they begin to germinate and form a mycel at high nutrient conditions and light. The mycel develops further by forming aerial hyphae which finally bear again conidia.The pictures below illustrate the various stages in the development of Neurospora.



 

Conidia seen in microscope with 400x magnification

 

Hyphae and germinating conidia (1000x magnification)

 

Mycel (100x magnification)

 

DAPI-treated mycel: showing blue DNA-fluorescence of nuclei

 

The sporulation rhythm of Neurospora crassa

If a piece of mycel is transferred to a low nutrient medium and continuous darkness (DD-condition; DD: from German, Dauer-Dunkel, constant darkness) the circadian oscillator starts.

The oscillations can be seen as regular-repeating zones of sporulation when the mycel is placed on agar-medium under DD conditions: during the growth sporulation occurs every 22 h (when using the wild-type bd-mutant).

Rhythmic sporulation of N. crassa observed in a 20 cm petri-dish. A piece of mycel was placed in the middle of the dish which contained agar with minimal medium. After several days in darkness the rhythmic formation of conidia was observed. The color of the conidia is orange due to carotenoids.

 


Study of the sporulation rhythm in growthtubes

Normally the rhythm is studied by using petri-plates or so-called "racetubes". Racetubes are glass tubes that contain the agar medium.

 [schematic picture of 
racetube experiment]  


Racetubes have the advantage that the mycel can grow over a larger distance and that the period of the sporulation can be easily determined as the distance between conidiation peaks when the growth velocity is known. A schematic sideview of a racetube is shown in the picture above (left). It shows part of the tube where a piece of mycel has been placed on agar. While the tubes are kept in continuous darkness, rhythmic bands begins to form. Neurospora does not respond to red light and work that should be performed under dark conditions can be done under a red safety light (right picture above).

The photograph to the right shows racetubes seen from above. The growing mycel has formed rhythmic bands of aerial hyphae with spores. The white cotton plugs avoid contamination of the sterile agar medium by other microorganisms while air can still enter the tubes. As ourselves, Neurospora needs oxygen to live!

In this particular experiment the extracellular pH in the agar was varied from pH 4 (top racetube) to pH 9 (bottom) with intervals of 0.5 units. It is clearly seen that the growth becomes slower at higher pH. The growth rate is determined by marking the growing front of the mycel every 24 hours. In most instances the growing velocity is on average constant. By determining the growth rate it is then possible to measure the period of the rhythm. This is simply done by measuring the distance between the centers of neighboring sporulation areas and dividing this distance by the growth velocity.

Although the growing velocity is depended on pH (and other environmental factors, as temperature, nutrition supply, etc.), the period is generally (and also in this example) found to be unaltered, i.e., around 21.5 hours. This constancy of the period even under different environmental influences (temperature, pH, nutrition) is due to one or several compensation mechanisms within the cells, which keep the period constant. It is now generally believed that circadian rhythms act as time-measuring devices: physiological clocks.

 

 

A video of the sporulation rhythm

 

We have extracted two QuickTime® video clips (one 1.9 Mb, the other 3.6 Mb) from a movie taken by Prof. Van Gooch, University of Minnesota. It shows the rhythmic sporulation under a red savety light (N. crassa is blind for red light). Note that in the viedeo the racetubes are seen from above.

In order to see these movies, you will need a QuickTime player. If you don't have it (or are unsure about it), go to www.apple.com to download the latest version of the free player.


click here for the 1.9 Mb video

or

here for the 3.6 Mb clip


[picture from sporulation 
movie]

 

References


Jason C. Thoen and Van Gooch: Time Lapse Video Showing an Internal Circadian Clock in Mold (Neurospora) Growth.

Rowland H. Davis: Neurospora. Contributions of a Model Organism. Oxford University Press, Oxford, 2000. ISBN 0-19-512236-4.

David D. Perkins, Alan Radford and Matthew S. Sachs: The Neurospora Compendium. Chromosomal Loci. Academic Press, San Diego, 2001. ISBN 0-12-550751-8.

Rowland H. Davis and David. D. Perkins: Neurospora: a model of model microbes. Nature Reviews | Genetics, 2002, 3 , 7-13.



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