Proanthocyanidins in Cereals and Brassicaceae: A Cross-Species Approach on their Roles for Seed-Coat Biophysical Properties, Dormancy and Germination

Seeds are at the beginning and end of the life cycle of all higher plants. Many wild species show a high degree of dormancy that prevents seeds from germinating immediately after shedding, and delays germination until more favourable conditions for plant growth are encountered. Domestication of crop plants from such wild species has often resulted in significant loss of dormancy as prehistoric farmers selected in favour of seeds with rapid germination. However, some degree of dormancy is an important quality trait in many crops, as it prevents seeds from germinating prematurely while still attached to the mother plant. This is a serious problem in cereals such as bread wheat, because wet weather near harvest time can cause seeds to germinate within the ear ("pre-harvest sprouting"). This causes release of enzymes that partially degrade of the starch (flour) and thereby impacts negatively on bread-making quality. Sprout-induced seeds produce so much enzyme that a small proportion of such grains within a crop can result in the entire harvest being suitable only as animal feed, with consequent economic loss to the farmer.

Thus, too little dormancy can result in pre-harvest sprouting, while too much dormancy may lead to non-uniform germination in the field. This is important for high-quality seeds also of broadleaf crop species from the cabbage family (Brassicaceae). The speed and uniformity of germination of crop seeds after sowing is an equally important seed quality trait and together with dormancy directly affects crop production. Clearly, an understanding of the processes that determine the level of dormancy and the speed of germination are essential to enable us to design and breed new varieties of crop plants which perform well even in stressful environments (under climate change). One clear contributory factor in many species is the seed coat, damage to which or removal can result in complete loss of dormancy and in faster germination. This appears to be associated with the presence of reddish-brown tannins in the seed coat; the importance of these seed coat tannins is clear from plants that have lost the ability to make the compounds, resulting in pale-coloured seeds with lower levels of dormancy and faster germination. For example, white-grained wheat is much more prone to pre-harvest sprouting than a red-grained variety and is therefore difficult to grow successfully in the wet UK climate. White-grained wheat has several advantages over red-grained types, including a higher yield of white flour and the production of "white wholemeal" with the taste of white bread but the fibre and nutrient properties of normal red wholemeal.

There are several ways in which the tannins in the seed coat could affect dormancy and germination: they may increase the physical strength of the coat to prevent germination, they may affect the permeability of the coat to water, hormones or oxygen, which are required for germination, or precursors or metabolites of the tannins might directly suppress seed germination. These different hypotheses have not previously been directly tested, but our collaborative team has developed the materials and the methods through which we can examine each in turn. We plan to look at two species, cress and wheat, as models for eudicot (broadleaf) and monocot (cereal) species, respectively. We have developed varieties of both species that are impaired in the late steps in tannin production in the seed coat, so that we can examine the effects on tannins for seed coat properties, on dormancy and the speed of germination. At the same time we have pioneered methods for measuring the strength, extensibility and permeability of the isolated seed coats. We will relate these properties to the interaction with environmental factors such as temperature, to provide a comprehensive understanding of the roles of tannins in coat-imposed dormancy and germination speed of seeds.

The aim of this project is to determine the mechanisms by which condensed tannins, or proanthocyanidins (PAs), present in the seed coat of many plant species determine coat-imposed seed dormancy as well as the speed and uniformity of seed germination. PAs are polymerised flavan-3-ols that are synthesized from flavonoid precursors in the integument, a maternal tissue which on maturation and cell death becomes the testa, or true seed coat. There are a number of hypotheses to explain the mechanisms through which PAs promote seed dormancy and inhibit germination. These include possible roles in determining the biophysical properties of the testa, including its mechanical strength/resistance and permeability to oxygen or plant hormones, and also the possibility that mobile PA precursors or metabolites may directly suppress embryo germination upon imbibition.

In previous projects we have developed materials and techniques that will allow us to directly test these various hypotheses in an eudicot and a monocot species. These include mutant and trangenic lines of cress (Lepidum sativum) and wheat (Triticum aestivum) with complete or partial lesions in the later steps of PA biosynthesis and also methods to directly measure puncture force, extensibility and permeability of the testa and other outer seed layers of our target species. These methods require a suitably large seed size which is met by the species under study and we have verified the feasibility of our techniques. In addition, we propose to investigate the processes in seed maturation during which PAs assembled in the vacuoles of integument cells become associated with the cell wall, and how this results in changes to its biophysical properties. We further propose to investigate how the PA-related biophysical properties are altered at different ambient temperatures during seed imbibition and how this affects embryo growth, coat dormancy and germination speed and uniformity.

Our research will not only generate new and deep scientific insight and fundamental advancements of academic interest, and will be published in internationally recognized peer-reviewed journals, it also has direct relevance to applied scientists and organizations including the crop breeding and seed industries. Broadleaf crop seeds (eudicots including Brassica crops, vegetables, spicy sprouts (cress), sugarbeet) and cereal grains (monocots including wheat and barley) are the beginning and the end of all important food supply chains. High-quality seeds are fundamental for the global seed industry with ca. $40 billion annual turnover. Germination and dormancy are key traits for crop plants, as they determine establishment in the field and the quality of the harvested product. Germination and dormancy are fundamental for adaptation mechanisms to changing environments, e.g. early stages in plant life-history and plant reproduction are especially vulnerable to abiotic stresses such as temperature extremes (climate change) and threaten food security. Pre-harvest sprouting (PHS) of cereals crops is a major problem in many parts of the world, with wet weather before harvest resulting in premature germination of grain and severe losses (ca. $1 billion annually) in crop quality and economic value.

The group of Prof. Gerhard Leubner (GL) has not only a strong track record in basic research, but also in applied projects in cooperation with seed industry, while Andy Phillips's (AP) laboratory has good relations with wheat breeders and has been involved in several projects with a strong commercial focus. Our results in this project are relevant for moving beyond to crops and for transfer of knowledge and techniques to seed industry, e.g. for coat-dormancy and seed technology (priming/pelleting/coating). Our current projects with seed industry include sugarbeet seed technology and Dr Tina Steinbrecher (TS) as a biomechanical engineer leads a project on the biomechanics of wheat grain milling. A collaboration application in the UK Agri-Tech Catalyst programme on vegetable seed priming is together with Germains Seed Technology (Norfolk, UK). This interaction with the seed industry will lead to fostering global economic performance and especially to enhanced economic competiveness of the UK seed industry.

The central scientific objective of our project is to provide fundamentally novel and comprehensive insight into the molecular mechanisms how proanthocyanidins (PAs) in the seed coats of a eudicot (cress) and a monocot (wheat) model match local demands (such as temperature) for seed germination timing. Dissemination pathways for this include applied seed conferences. GL was chairman/lead speaker at the tri-annual 29th ISTA (International Seed Testing Association) Congress 2010 in Cologne, Germany. ISTA sets seed testing rules and includes government bodies and institutes as members. AP and AH both presented work at the most recent International Symposium on PHS in Red Deer, Canada. GL is also the curator of The Seed Biology Place website (www.seedbiology.eu) which is internationally among the top-10 visited websites (ca. 700 visits/month) disseminating information about seed biology for research and education purposes. Seeds are fascinating and therefore have the potential to get the public interested in plants. To address the wider public we will actively contribute with talks, posters, and seed exhibitions/displays to outreach events at RHUL, RRes and UK botanical gardens. Seed biology will be fostered as a new focus at RHUL and in addition to teaching our outreach activities aim on attracting new students. The research assistants in our collaboration project will gain experience in interaction and communication across disciplines. Training and expertise in different techniques, including next-generation-sequencing and biophysical technologies, are needed in the seed technology industry, and will be delivered by this project.