Green Islands – mining cytokinins

A little while ago I wrote about the phenomenon of  “green islands” caused by ants keeping insect herbivores away from trees.   If, however, you work on leaf miners, the term green islands means something else entirely.  Instead of referring to a feature of the landscape, it refers to a feature of the leaf, which unless you are Toby*, is definitely not a landscape-level phenomenon 😊

While some insects, aphids for example, induce senescence to improve the quality of their host plant and some plants induce senescence and early leaf-fall in those leaves that have been colonised by gall aphids in order to reduce their infestation load (Williams & Whitham, 1986), there are other insects that try desperately to prevent senescence so as to prolong their feeding life on what would otherwise be a dead leaf.

Green island leaf mine of the moth, Stigmella atricapatella – Many thanks to Mike Shurmer for the photographs.

The phenomenon of the green islands in autumn leaves associated with leaf mining Lepidoptera has been known about for some time (Hering, 1951), but although the adaptive value of this was easy to see, the causal mechanism remained unknown for some time. Similarly, plant pathologists had also noticed that one of the symptoms of powdery mildew infections is the appearance of a green ring around the necrotic spot caused by the fungus (von Tubeuf, 1897); if not a green island, a green atoll 😊

Green island or green atoll? Powdery mildew on wheat https://slideplayer.com/slide/9073461/27/images/14/Green+island+on+wheat+infected+with+wheat+powdery+mildew.jpg

That fungi produced secretions containing plant growth substances such as the auxin (plant hormones) indole acetic acid has been known since the 1930s (Thimann, 1935) and it was later hypothesised that the levels present in the surrounding leaf tissue were associated with the resistance or lack thereof, to the fungal agent (e.g. Shaw & Hawkins, 1958). A further class of plant growth substances, initially termed kinins because of their similarity to kinetin (a cell growth promoting plant hormone, but later renamed cytokinins** (Skoog et al., 1965)) were discovered by Folke Skoog and co-workers (Miller et al., 1956) and linked to the production of green islands by plant pathogens (reviewed by Skoog & Armstrong, 1970).

“What about the leaf miners?” I hear you ask. You will be pleased to know that entomologists were not too far behind. Lisabeth Engelbrecht working on Nepticulid leaf miners on birch (Betula pendula) and Aspen (Populus tremula) set up a study (Engelbrecht, 1968) to test her hypothesises that the green islands were caused as a result of insect saliva or by the larvae physically cutting the leaf veins that would otherwise have delivered the chemical signal responsible for beginning leaf senescence. She discovered that the green islands contained large concentrations of cytokinin  (Engelbrecht, 1968) and working with other colleagues discovered that the labial glands of leaf mining larvae also contained cytokinin, but was unsure as to whether the cytokinin originated from the larvae or were formed in the leaf in response to chemicals in the saliva or frass of the larvae (Engelbrech et al., 1969), although if you read the paper it is quite clear that she is convinced that the source of the cytokinin is from the larvae and not the plant.

After all this excitement about insect produced cytokinin and green islands things seemed to go a bit dead.  I found a couple of passing references to the possibility that leaf mining Lepidopteran larvae use cytokinin to produce a green island to extend larval life after leaf abscission (Miller, 1973; Faeth, 1985) and an opinion piece discussing the possible adaptive role of using green islands to prolong larval life after leaf fall (Kahn & Cornell, 1983), but, surprisingly, nothing experimental to test this hypothesis. Oddly, I did find a paper testing the idea that early leaf abscission was an induced defence against leaf miners, where green islands were mentioned in the introduction but not mentioned again (Stiling & Simberloff, 1989).

Don’t get me wrong, plant pathologists and entomologists working on insect galls were still writing about the role of cytokinin (e.g. Murphy et al., 1997: Mapes & Davies, 2001), but leaf miner green island research seemed to have died.  Suddenly, however, in the mid-2000s the French ‘discovered’ leaf miners and David Giron and colleagues, showed how the leaf miner Phyllonorycer blancardella manipulates the nutritional quality of their host leaves by increasing the levels of cytokinin in the surrounding leaf tissue (Giron et al., 2007).

‘Green island’ formed by Phyllonorycter blancardella (From Giron et al., 2007).

 

As we know from aphids, where insects play, bacterial symbionts are never far away, and sure enough it wasn’t long before it was shown that Wolbachia ‘infections’ were helping the leaf miners produce their ‘green islands’. Wilfried Kaiser and colleagues treated leaf miner larvae with antibiotics to remove the symbiont and found that the ‘cured’ larvae, although still able to feed and form leaf mines, were unable to produce ‘green islands’ and the levels of cytokinin were much lower than that found in the ‘green islands’ formed by untreated leaf miners (Kaiser et al., 2010).

Influence of Wolbachia on green island formation. To the left, infected leaf miners (Phyllonorycter blancardella) happily surrounded by nutritious plant tissue; to the right, ‘cured’ by antibiotics, the leaf miner soon runs out of food (Kaiser et al., 2010)

The same group have also documented the mechanism by which the leaf miners and their symbionts work together (Body et al., 2013) and also, using molecular phylogenies and ecological trait data, shown that the existence of the ‘green island’ phenotype and Wolbachia infections are associated with the evolutionary diversification of the Gracillarid leaf miners (Gutzwiller et al., 2015).

You might expect that these findings would have stimulated renewed interest in the ‘green island’ phenomenon, but you would be wrong.  Despite the fact that at the time of writing this article (September 10^th 2019) Kaiser et al. (2010) had, according to the Web of Science, been cited 105 times, only three papers dealing with this phenomenon have been published, the most recent appearing in early 2018 (Zhang et al., 2018) and, incidentally, by the same group that published the Kasier et al. (2010) study. It would appear that as with ‘green islands’, the study of the phenomenon is also very localised.

References

Allen, P.J. (1942) Changes in the metabolism of wheat leaves induced by infection with powdery mildew. American Journal of Botany, 42, 425-435.

Body, M., Kaiser, W., Dubreuil, G., Casas, J. & Giron, D. (2013) Leaf-miners co-opt microorganisms to enhance their nutritional environment. Journal of Chemical Ecology, 39, 969-977.

Engelbrecht, L. (1968) Cytokinin in den ,,grunen Inseln” des Herbstlauibes. Flora oder Allgemeine botanische Zeitung. Abt. , Physiologie und Biochemie, 159, S, 208-214.

Englebrecht , L., Orban, U. & Heese, W. (1969) Leaf-miner caterpillars and cytokinins in the “green islands” of autumn leaves. Nature, 223, 319-321.

Faeth, S.H. (1985) Host leaf selection by leaf miners: interactions among three trophic levels. Ecology, 66, 870-875.

Gutzwillner, F., Dedeine, F., Kaiser, W., Giron, D., & Lopez-Vaamonde, C. (2015) Correlation between the green-island phenotype and Wolbachia infections during the evolutionary diversification of Gracillariidae leaf-mining moths. Ecology & Evolution, 5, 4049-4062.

Hering, E.M. (1951) Biology of the Leaf Miners, Dr W Junk, The Hague, Netherlands

Herrick, G.W. (1922) The Maple Case-Bearer Paraclemensia Acerifoliella Fitch. Journal of Economic Entomology, 15, 282-288.

Kahn, D.M. & Cornell, H.V. (1983) Early leaf abscission and folivores: comments and considerations. American Naturalist, 122, 428-432.

Kaiser, W., Huguet, E., Casas, J., Commin, C. & Giron, D. (2010)  Plant green-island phenotype induced by leaf-miners is mediated by bacterial symbionts. Proceedings of the Royal Society B, 277, 2311-2319.

Mapes, C.C. & Davies, P.J. (2001) Cytokinins in the ball gall of Solidago altissima and in the gall forming larvae of Eurosta solidaginis. New Phytologist, 151, 203-212.

Miller, C. O., Skoog, F., Okumura, F. S., Von Saltza, M. H., & Strong, F. M. (1956). Isolation, structure and synthesis of Kinetin, a substance promoting cell division. Journal of the American Chemical Society, 78, 1375–1380.

Miller, P.F. (1973) The biology of some Phyllonorycter species (Lepidoptera: Gracillariidae) mining leaves of oak and beech. Journal of Natural History, 7, 391-409.

Murphy, A.M., Pryce-Jones, E., Johnstone, K. & Ashby, A.M. (1997) Comparison of cytokinin production in vitro by Pyrenopeziza brassicae with other plant pathogens. Physiological & Molecular Plant Pathology, 50, 53-65.

Shaw, M. & Hawkins, A.R. (1958) the physiology of host-parasite relations V. A preliminary examination of the level of free endogenous Indoleacetic acid in rusted and mildewed cereal leaves and their ability to decarboxylate exogenously supplied radioactive indoleacetic acid. Canadian Journal of Botany, 34, 389-405.

Skoog, F. & Armstrong, D.J. (1970) Cytokinins. Annual Review of Plant Physiology, 21, 359-384.

Skoog, F., Strong, F.M. & Miller, C.O. (1965) Cytokinins. Science, 148, 532-533.

Stiling, P.D. & Simberloff, D. (1989) Leaf abscission – induced defense against pests or response to damage ? Oikos, 55, 43-49.

Thimann, K.V. (1935) On the plant growth hormone produced by Rhizopus suinus. Journal of Biological Chemistry, 109, 279-291.

Von Tubeuf, K.F. (1897) Diseases of Plants, Longmans, Green & Co, London.

Walters, D.R., McRoberts, N. & Fitt, B.D.L. (2008) Are green islands red herrings? Significance of green islands in plant interactions with pathogens and pests. Biological Reviews, 83, 79-102.

Williams, A.G. & Whitham, T.G. (1986) Premature leaf abscission: an induced plant defense against aphids. Ecology, 67, 1619-1627.

Zhang,  H., Dubreuil, G., Faivre, N., Dobrev, P., Kaiser, W., Huguet, E., Vankova, R. & Giron, D.  (2018) Modulation of plant cytokinin levels in the Wolbachia‐free leaf‐mining species Phyllonorycter mespilella. Entomologia experimentalis et applicata, 166, 428-438.

 

*Toby Alone (La Vie Suspendue) by Timothée de Fombelle, is a fantastic novel, which I only fairly recently discovered, but can heartily recommend.

** Cytokinins are a class of plant growth substances that promote cell division, or cytokinesis, in plant roots and shoots. They are involved primarily in cell growth and differentiation, but also affect apical dominance, axillary bud growth, and leaf senescence. Wikipedia