Desiccation Tolerance and Sensitivity in Medicago Truncatula and Inga Vera Seeds
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Desiccation tolerance and sensitivity in Medicago truncatula and Inga vera seeds José Marcio Rocha Faria Promotoren: Prof. Dr. A.M.C. Emons Hoogleraar bij de leerstoelgroep Plantencelbiologie, Wageningen Universiteit Prof. Dr. L.H.W. van der Plas Hoogleraar bij de leerstoelgroep Plantenfysiologie, Wageningen Universiteit Co-promotoren: Dr. H.W.M. Hilhorst Universitair hoofddocent bij de leerstoelgroep Plantenfysiologie, Wageningen Universiteit Dr. A.A.M. van Lammeren Universitair hoofddocent bij de leerstoelgroep Plantencelbiologie, Wageningen Universiteit Promotiecommissie: Prof. Dr. ir. M. Koornneef (Wageningen Universiteit/MPI, Keulen, Duitsland) Prof. Dr. L.A.C.J. Voesenek (Universiteit Utrecht) Prof. Dr. O. Leprince (Institut National d'Horticulture, Angers, France) Dr. W. Finch-Savage (Warwick HRI, University of Warwick, Wellesbourne, Warwick, UK) Dit onderzoek is uitgevoerd binnen de onderzoekschool Experimentele Plantenwetenschappen (EPW) José Marcio Rocha Faria Desiccation tolerance and sensitivity in Medicago truncatula and Inga vera seeds Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit Prof. Dr. M. Kropff in het openbaar te verdedigen op maandag 1 mei 2006 des namiddags te vier uur in de Aula Faria, J.M.R. (2006). Desiccation tolerance and sensitivity in Medicago truncatula and Inga vera seeds. PhD thesis, Wageningen University, Wageningen, The Netherlands. With summaries in English, Dutch and Portuguese. ISBN: 90-8504-417-0 I dedicate this thesis to the three women of my life: Lucília, my mother; Regiane, my wife; and Alissa, my daughter. God bless you! Contents Chapter 1. General introduction 1 Chapter 2. Desiccation sensitivity and cell cycle aspects in seeds of Inga vera 17 subsp. affinis Chapter 3. Changes in DNA and microtubules during loss and re-establishment of 43 desiccation tolerance in germinating Medicago truncatula seeds Chapter 4. Changes in gene expression during loss and re-establishment of 69 desiccation tolerance in germinated Medicago truncatula seeds Chapter 5. Improvement of storability of Inga vera subsp. affinis embryos 87 Chapter 6. General discussion 109 Summary 117 Samenvatting 121 Resumo 125 Acknowledgements 129 Curriculum vitae 133 Chapter 1 General introduction Introduction Desiccation tolerance is one of the most outstanding features found in the plant kingdom. Seeds that possess such an attribute, the so-called orthodox seeds, can be dried and stored for many years without significant loss of viability. Seeds that lack this characteristic, the so- called recalcitrant seeds, represent a big challenge for those who need to keep them in seed banks for germplasm conservation purposes. While many studies have been devoted to reveal the secrets of desiccation tolerance, less effort has been put into clarifying the recalcitrance phenomenon. In order to gain insight into this theme, this thesis deals with physiological, cytological and molecular aspects of desiccation sensitivity in seeds of Inga vera (a recalcitrant-seeded species) and in germinating seeds of Medicago truncatula (an orthodox-seeded species). Seed storage behavior Seeds are the basis of human feeding and the main way of plant propagation. For those reasons farmers have acquired the habit of storing seeds for planting in the subsequent years, since remote ages. To date, storage of crop seeds is an important step in the agricultural productive chain. In recent years, awareness of the loss of plant diversity1 has captured worldwide attention and germplasm conservation has become necessary as a means of maintaining species diversity to prevent genetic erosion (Marzalina and Krishnapillay, 1999). In such context, stored seeds can be used for restoration of degraded ecosystems. Plants produce seeds that respond differently to desiccation and storage and, based on such distinct behavior, Roberts (1973) classified the seeds as orthodox and recalcitrant. Orthodox seeds acquire desiccation tolerance during their development and undergo substantial drying in the final developmental phase (Fig. 1). After shedding they may be dried even further by artificial means and their viability may be extended in a predictable 1 During the 1990s, the loss of natural forests was in average 16.1 million hectares per year, of which 15.2 million occurred in the tropics (FAO, 2000). Chapter 1 way by storage at low temperature and low relative humidity (Roberts, 1973; Roberts and Ellis, 1989; Berjak and Pammenter, 1997). Most crop species, such as wheat, rice, bean, soybean, maize and barley, produce orthodox seeds. Dispersion/harvest Imbibition Start of DT loss Re-establishment of DT tolerance Desiccation Histodiffe- Maturation Desiccation Storage Germination and rentiation seedling growth Figure 1. Acquisition and loss of desiccation tolerance (DT) in orthodox seeds. DT is acquired during development enabling the seeds to withstand maturation drying. After dispersion or harvest, DT is maintained during storage and even for some time after imbibition (beginning of germination). With the progress of germination DT starts to decline until it is totally lost upon radicle protrusion or with further seedling growth. Even after DT is totally lost, it can still be re-established (dotted line) by applying mild stresses. Adapted from McKersie (1996). In contrast with the orthodox types, recalcitrant seeds do not undergo maturation drying and are shed at relatively high moisture contents (0.8-4.0 g H2O/g dry matter). They are desiccation-sensitive both before and after shedding and do not survive if desiccated to water potentials below -15 MPa. Recalcitrant seeds have a very limited post-harvest lifespan in the hydrated condition (Berjak and Pammenter, 1997, 2001). They remain metabolically active upon shedding and, consequently, germinate readily after abscission (Berjak et al., 1989). Moreover, many recalcitrant seeds, particularly those of tropical origin, are also chilling sensitive, and, thus, cannot be stored at temperatures below approximately 15oC (Pammenter and Berjak, 1999; Dussert et al., 1999). Recalcitrant seeds are mainly produced by tree species. In terms of habitat, most recalcitrant species (89%) grow in wet-forest, riverine, flooded, or coastal environments. Most species (79%) are native to the tropics (Farnsworth, 2000). The best known examples are the economically important species cacao (Theobroma cacao), rubber-tree (Hevea brasiliensis), avocado (Persea americana), mango (Mangifera indica) and coconut (Cocus nucifera). Although not abundant as in the tropics, 2 General introduction recalcitrance is also found in the Temperate Zone in some important tree species belonging to the genera Castanea, Quercus, Aesculus and Acer (Connor and Bonner, 2001). A third category of seeds, called intermediate, was introduced by Ellis et al. (1990) to group seeds that show intermediate post-harvest behavior, i.e. they are relatively desiccation tolerant, but do not withstand removal of water to levels as low as orthodox seeds. They survive desiccation to about -150 MPa, and can generally be stored for periods of intermediate length. Such seeds are often chilling sensitive, even in the dehydrated state. Coffee - Coffea spp (Ellis et al., 1990) and neem - Azadirachta indica (Sacandé, 2000) are examples of species with intermediate seeds. It is thus clear that while it is relatively easy to store orthodox and intermediate seeds, it is still a challenge to store recalcitrant seeds for longer periods. To progress towards a protocol for storage of this category of seeds, one of the first steps is to gain insight into the causes of their desiccation sensitivity. This can be done by studying both the recalcitrant seeds themselves and germinating/germinated orthodox seeds, since the latter are a useful tool for understanding desiccation sensitivity (Farrant et al., 1997; Sun, 1999). Desiccation tolerance and sensitivity Desiccation tolerance (DT) in plants can be considered as the ability to rehydrate successfully after the removal of 80-90% of protoplasmic water, reaching moisture contents below 0.3 g H2O/g dry matter (Oliver et al., 2000; Hoekstra et al., 2001). There are three criteria which a plant or plant structure must meet in order to survive such severe loss of protoplasmic water: 1) limitation of the damage suffered by the tissues during desiccation to a repairable level, 2) maintenance of its physiological integrity in the dry state, 3) mobilization of repair mechanisms upon rehydration aiming to revert the damages caused by desiccation and/or rehydration (Bewley, 1979). While few plants are capable of withstanding full desiccation, orthodox seeds undergo this event as a programmed and final stage in their development (Kermode, 1997). Desiccation tolerance enables seed survival during storage or environmental stress and ensures better dissemination of the species (Leprince et al., 1993) A number of processes and mechanisms have been implicated in the acquisition of DT in orthodox seeds, including a) the accumulation of putatively protective molecules, such as the late-embryogenesis-abundant proteins (LEAs), sucrose and certain oligosaccharides, b) reduction of the degree of vacuolisation, c) switching off of metabolism, d) presence of active antioxidant systems, and e) presence and operation of repair mechanisms during rehydration. The absence or ineffective operation of one or more of these processes or mechanisms could determine the desiccation sensitivity in recalcitrant seeds (Pammenter