February 29th, 2012 | Category: Papers | Leave a comment

Nessun dorma

Here is a detour. In this instance a trip into plant metabolism in order to mark the online publication of our latest paper in New Phytologist earlier this month. Whilst there isn’t any synthetic biology here there is biology, and an illustration of how genetics and the environment combine to influence plant behaviour.

The Great British Summer

To say that the summer of 2007 in the UK was poor would be an understatement, June was one of the wettest months on record, and in the midst of all this wet was a field of barley just outside Cambridge (Fig. 1). The barley plants were part of a field trial co-ordinated between the National Institute of Agricultural Botany and the John Innes Centre. They were, in fact, a motley collection of mutants mutants impaired in their ability to create starch (their storage reserve) in the seed endosperm. Growing alongside these low-starch mutants were different parental control lines.

Fig. 1. Phil Howell in better growing times.

Fig. 1. Barley plants and Phil Howell growing happily together in drier times.

Whilst many of the enzymes (and their respective genes) that are responsible for generating starch polymers are known, little is known about the mechanisms which govern their assembly into semi-crystalline starch granules. We were to use these plants, or more specifically the grain collected from them, to learn about the genetic control of starch structure.

During analysis however, it became apparent that the starch mutants had behaved differently in the wet conditions than their fatter, starch-rich parental lines. The low starch mutants had not been sleeping. This was an observation which led us in an unexpected direction.

None shall sleep

Seed dormancy is of crucial importance to seed fitness in two important respects. Firstly dormancy allows seeds to travel through time, waiting for the moment when conditions are right to germinate, giving the plant the best chance of success.

Secondly (and sadly lacking a link to David Attenborough), seed dormancy is required in order to prevent premature germination on the plant, i.e. before the completion of seed maturity. It is this second behaviour, the repression of germination during grain development, that had altered in the low-starch mutants, and it had manifest itself as the phenomenon known as preharvest sprouting (PHS).

Fig. 2. Bad grain.

Fig. 2. Badly behaved grain.

PHS is the partial or complete on-ear germination of grains prior to harvest. An example of a preharvest sprouted grain (found by Nuffield-sponsored sixth form student Alice Craggs, who helped to quantify PHS in our grains) can be seen in Fig. 2. To quantify the extent of PHS across our collection we employed various techniques from simply counting and categorising grains, to the determination of germination induced enzyme activity and the wonderful Hagberg falling number assay.

The results were clear: 10 out of 12 of our low-starch mutant lines demonstrated more PHS than their respective parental control line.

So what causes PHS? PHS is a complicated phenomenon influenced not only by many genetic components but also by environmental queues [1]. It is known however that PHS is inversely correlated with dormancy [2]; in other words, grains in a deep dormancy are less likely to sprout when the weather is poor than light sleepers. With this in mind we sought to investigate whether or not changes had occurred to the dormancy status of the low-starch lines.

For these experiments a select number of lines were grown under glass, away from the travails of the weather. Seeds were removed at various points during development and assayed immediately for their germination responses to certain conditions. For example, altering the germination temperature or the developmental stage at which the grains are harvested alters the rate at which the grains germinate.

These tests were designed to determine if dormancy status was indeed different between the low-starch lines and their parental controls. As with the PHS data the results were clear: low-starch mutants were more likely to germinate from earlier in grain development than their parental lines, they germinated faster, and they were less sensitive to elevated temperature (a factor which normally delays germination). These results all indicated a reduction in seed dormancy in the low-starch grains consistent with the increase in PHS in field grown plants. But why was dormancy reduced?

Cross-talk

Two further important findings followed.

Fig. 3. Wheat caryopsis from the Seed Biology Place

Fig. 3. Wheat caryopsis (image from seedbiology.de, click to embiggen).

Firstly, the interaction between grain tissues had altered. It is important to know that in barley (amongst many other species), seed dormancy is imposed upon the embryo by the surrounding structures, rather than being self-imposed (termed ‘non-deep physiological dormancy‘). Removing the embryo from surrounding structures (Fig. 3) in such species allows germination. Removal of the hull in barley for example greatly increases germination rates. What was of interest to us was that in the parental lines, removal of the hull but with retention of the endosperm did not restore germination rates to equivalent rates of isolated embryos – the endosperm/pericarp tissue therefore also inhibits germination. This endosperm-mediated suppression of germination is lost as the grain dries and starchy endosperm dies. Crucially, in the low-starch mutants, this endosperm/pericarp inhibition of germination was completely lost: the developing endosperm/pericarp no longer represses germination. The embryo of these grains behaved as if the endosperm was already dead.

The second important finding surrounds one of the key players in seed dormancy, the phytohormone abscisic acid (ABA). In wheat and barley dormancy correlates both with the amount of ABA in the tissue and with the sensitivity of the embryo to ABA [3]. Assisted by another Nuffield Scholar, Rachel Mumford, we set out to determine if either of these changes were likely. We extracted and quantified ABA content from both embryo and endosperm tissue  but were unable to find elevated levels of ABA consistent with the dormancy changes observed. On the other hand, application of exogenous ABA revealed that low-starch lines were much less sensitive to ABA than controls. The signal appears to be mediated via ABA-sensitivity, not ABA content.

What does this mean?

One of the most common knock-on effects of a reduction in starch synthesis is an accumulation of sugars, in particular sucrose. Metabolite analysis of these low starch lines confirmed that this was the case and sucrose may turn out to be the key player in this story. At this stage however, the story, becomes rather murky. Sugar signalling networks are often interlinked with hormonal signalling networks, and sugar concentrations are can also affect expression of factors that mediate ABA-induced gene expression [4].  It will be interesting to learn however whether this point of contact between primary metabolism and phytohormone signalling, and between endosperm and embryo, provides a mechanism by which the endosperm can suppress embryo germination whilst it is actively laying down reserves.

Our results reveal a causal link between the rate of starch accumulation and susceptibility to PHS in barley. Previously it has been shown that ABA signals can modify carbon metabolism in developing grains. In following-up our initial observations of an increased occurrence of PHS in the low-starch mutants we have discovered that the opposite is also true; that starch synthesis in the endosperm generates signals that influence ABA sensitivity and the imposition of dormancy in the developing embryo.

Refs.

The full paper is Howard, T.P., Fahy, B., Craggs, A., Mumford, R., Howell, P., Leigh, F., Greenland, A., and Smith. A.M.  Barley mutants with low rates of endosperm starch synthesis have low grain dormancy and high susceptibility to pre-harvest sprouting. New Phytologist 2012 DOI: 10.1111/j.1469-8137.2011.04040.x.

[1] Gale MD, Flintham JE, Devos KM. 2002. Cereal comparative genetics and preharvest sprouting. Euphytica 126(1): 21-25.

Flintham J, Adlam R, Bassoi M, Holdsworth M, Gale M 2002 Mapping genes for resistance to sprouting damage in wheat. Euphytica 126:39-45

[2] Gerjets T, Scholefield D, Foulkes MJ, Lenton JR, Holdsworth MJ. 2010. An analysis of dormancy, ABA responsiveness, after-ripening and pre-harvest sprouting in hexaploid wheat (Triticum aestivum L.) caryopses. Journal of Experimental Botany 61: 597–607.

[3] Walker-Simmons M. 1987. ABA levels and sensitivity in developing wheat embryos of sprouting resistant and susceptible cultivars. Plant Physiology 84: 61–66.

[4] Ramon M, Rolland F, Sheen J. 2008. Sugar sensing and signaling. The Arabidopsis Book 6: e0117. doi:10.1199/tab.0117

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