Introduction
Seed germination is subjected to very precise regulation, the complexity of which originates both in the action of various external factors (temperature, oxygen, light, water potential of the medium) and in characteristics within the seeds themselves (Bewley and Black, Reference Bewley and Black1994; Bewley, Reference Bewley1997). Germination of a seed lot results from the functioning of the genome, but is also largely dependent on numerous factors which intervene throughout the seed life, from its development on the parent plant up until sowing, including storage conditions (Priestley, Reference Priestley1986; Bewley and Black, Reference Bewley and Black1994). A seed population is therefore heterogeneous, which leads to a lack of uniform performance, especially when environmental conditions at sowing are not optimal. Seed quality includes genetic purity, health quality and seed germination and vigour (McDonald, Reference McDonald1998).
Successful stand establishment requires high-quality seeds, i.e. seeds that: (1) germinate completely; (2) germinate quickly and simultaneously; (3) produce normal and vigorous seedlings; and (4) have germination which shows little sensitivity to external factors, enabling them to germinate in a wide range of agro-climatic conditions (Corbineau and Côme, Reference Corbineau and Côme2006). To these criteria, storability of the seeds can be added. For seed companies a major challenge toward improving crop yield is a better control of seed vigour. Therefore methods of evaluation of seed quality, providing accurate prediction of seed performance under field conditions, must be developed. Such methods are also required for assessing the effectiveness of technologies used to enhance seed performance, such as priming or seed health treatments. The objective of this short review is to demonstrate that a better understanding of the cellular, molecular and biochemical mechanisms involved in the acquisition of seed vigour during seed development, in the germination process and in seed deterioration during ageing, could yield new markers of seed quality, besides the classical physiological tests.
Overview of acquisition, loss and improvement of seed vigour
Seed dehydration is the normal terminal event in the development of ‘orthodox’ seeds (Vertucci and Farrant, Reference Vertucci, Farrant, Kigel and Galili1995) that ensures the switch from a developmental mode to a germination one (Kermode, Reference Kermode1997). The involvement of the maturation-drying phase in the acquisition of seed vigour is subject to discussion. Some authors have claimed that seeds attain maximum germinability at the end of seed-filling phase and therefore age (TeKrony and Egli, Reference TeKrony, Egli, Ellis, Black, Murdoch and Hong1997), others found that storability, which can be considered as an indicator of vigour, improves during maturation drying (Sanhewe and Ellis, Reference Sanhewe and Ellis1996; Kermode, Reference Kermode1997; Bailly et al., Reference Bailly, Audigier, Ladonne, Wagner, Coste, Corbineau and Côme2001). Numerous cellular and biochemical events appear to be associated with seed maturation and, consequently, acquisition of desiccation tolerance; these include modification of ultrastructural characteristics such as cellular vacuolization, synthesis of late embryogenesis abundant (LEA) proteins or heat-shock proteins (HSP), accumulation of the raffinose family oligosaccharides and activation of antioxidant defence systems (Vertucci and Farrant, Reference Vertucci, Farrant, Kigel and Galili1995; Kermode, Reference Kermode1997; Bailly, Reference Bailly2004; Buitink and Leprince, Reference Buitink and Leprince2008; Leprince and Buitink, Reference Leprince and Buitink2010).
Loss of seed vigour, and subsequently of seed viability, is highly dependent on temperature and seed moisture content (Priestley, Reference Priestley1986). Free radicals and lipid peroxidation are widely considered to be major contributors to seed deterioration, including loss of membrane integrity, reduced energy metabolism, impairment of RNA and protein synthesis, and DNA degradation (Priestley, Reference Priestley1986; Hendry, Reference Hendry1993; Bailly et al., Reference Bailly, Benamar, Corbineau and Côme1996; McDonald, Reference McDonald1999; Bailly, Reference Bailly2004). Proteomic analyses revealed that the loss in seed vigour resulted in changes in proteins in the dry seeds and in the inability of the low vigour seeds to display a normal proteome during germination (Job et al., Reference Job, Rajjou, Lovigny, Belghazi and Job2005; Oracz et al., Reference Oracz, El-Maarouf-Bouteau, Farrant, Cooper, Belgazhi, Job, Job, Corbineau and Bailly2007; Bailly et al., Reference Bailly, El-Maarouf-Bouteau and Corbineau2008; Rajjou and Debeaujon, Reference Rajjou and Debeaujon2008; Rajjou et al., Reference Rajjou, Lovigny, Groot, Belghazi, Job and Job2008). Antioxidant metabolites and enzymatic detoxifying mechanisms (catalase, glutathione reductase, superoxide dismutase) limit lipid oxidation and oxidative damage to proteins and nucleic acids (McDonald, Reference McDonald1999; Bailly, Reference Bailly2004; Bailly et al., Reference Bailly, El-Maarouf-Bouteau and Corbineau2008; Rajjou and Debeaujon, Reference Rajjou and Debeaujon2008). Besides these mechanisms, repair of protein damage through the activity of l-isoaspartyl methyltransferase might also play an important role in seed longevity and vigour (Ogé et al., Reference Ogé, Bourdais, Bove, Collet, Godin, Granier, Boutin, Job, Jullien and Grappin2008).
Seed germination has been characterized by a triphasic curve of water uptake, with a rapid imbibition (phase I) followed by a plateau phase when moisture content exceeds 40–50% [fresh weight (FW) basis] (phase II), and a further increase in water uptake associated with radicle elongation (phase III) (Bewley and Black, Reference Bewley and Black1994; Bewley, Reference Bewley1997). Phase I results in a gradual resumption of cellular metabolism (respiratory activity, RNA and protein synthesis, initiation of reserve degradation, etc.); phase II is associated with various events, including an accumulation of osmotic solutes, activation of H+ ATPase in the plasmalemma and weakening of the surrounding tissues; whereas phase III corresponds to radicle growth. Recent proteomic and transcriptomic analysis of seeds has led to the identification of specific proteins which might contribute to seed vigour (Gallardo et al., Reference Gallardo, Job, Groot, Puype, Demol, Vandekerckhove and Job2001; Rajjou et al., Reference Rajjou, Gallardo, Debeaujon, Vandekerckhove, Job and Job2004; Job et al., Reference Job, Rajjou, Lovigny, Belghazi and Job2005; Catusse et al., Reference Catusse, Job and Job2008a, Reference Catusse, Strub, Job, Van Dorsselaer and Jobb). Using sugar-beet seed as a model, Catusse et al. (Reference Catusse, Strub, Job, Van Dorsselaer and Job2008b) have been able to reconstruct the metabolic pathways involved in germination and vigour, and to demonstrate that the germination vigour is built up during seed maturation on the parent plant.
Seed priming is widely used for enhancing seed performance by improving the rate of germination and decreasing the seed sensitivity to external factors. This technique is based on the progress of germination, and the objective of priming is to allow a controlled water uptake by the seeds up to the end of phase II, before the radicle protrudes from the seed coat (Corbineau and Côme, Reference Corbineau and Côme2006). Priming improves germination in a wider range of temperatures and in hypoxia, and also restores the germination of aged seeds (Bailly et al., Reference Bailly, Benamar, Corbineau and Côme1998). Modulating vigour by ageing and priming allows seed batches of various vigour to be obtained, the proteomic and transcriptomic analysis of which may result in the identification of new markers of quality (Gallardo et al., Reference Gallardo, Job, Groot, Puype, Demol, Vandekerckhove and Job2001; Rajjou et al., Reference Rajjou, Huguet, Robin, Belghazi, Job and Job2006; Catusse et al., Reference Catusse, Job and Job2008a, Reference Catusse, Strub, Job, Van Dorsselaer and Jobb).
Which germination tests for the assessment of seed vigour?
Official germination testing is performed using rules produced by the International Seed Testing Association (ISTA) or the Association of Official Seed Analysts (AOSA), prescribing substrate (paper, sand), moisture (quantity of water to moist the germination substrate, quality of water), light, temperature (constant or alternating temperatures) and dormancy-breaking techniques when required. These tests are usually carried out under near-optimal conditions for germination (ISTA, 2006), and therefore give information about the viability of the seed population, but do not predict its field performance when the sowing conditions are generally not optimal.
Measurements of seedling dry weight (soybean, maize) or shoot elongation (barley, wheat, oat, maize, ryegrass and fescue grass) have also been developed to evaluate growth potential. The topographical tetrazolium test (TTZ) allows seed viability to be determined, measuring the red staining of living tissues after reducing the colourless tetrazolium salt to a red, non-diffusible dye, formazan, catalysed by dehydrogenase activity (Moore, Reference Moore and Heydecker1973). This test is very suitable when seed germination is difficult or in the presence of dormancy.
Besides the ISTA tests, various germination tests are used in order to predict the behaviour of a seed lot at sowing. They are based on the kinetic criteria of germination under suboptimal conditions (too low or too high temperatures, low water potential of the medium, hypoxia, high salinity, etc.). Germination rates, expressed as mean germination time (MGT) or the time to attain 50% germination, are often used to evaluate seed vigour. Physiological tests based on the sensitivity/tolerance of seeds to stress during germination are also often performed to evaluate seed vigour or to discriminate among different batches. In the cold test, for example, seeds are imbibed for 4–7 d at low temperature (usually 10°C) and then germinated at 20–25°C for the assessment of normal seedlings. This is widely used for maize and vegetable sweet corn (Van Waes, Reference Van Waes1995; McDonald, Reference McDonald1999; Nijenstein and Kruse, Reference Nijenstein and Kruse2000); similar tests can be used for soybean.
The responsiveness of seeds to ageing is considered to be a vigour test and is used for numerous species. The accelerated ageing test consists of placing seeds at high relative humidity (75–100%) and warm temperature (35–45°C) (Delouche and Baskin, Reference Delouche and Baskin1973; TeKrony, Reference TeKrony1993; TeKrony and Egli, Reference TeKrony, Egli, Ellis, Black, Murdoch and Hong1997). The controlled deterioration test differs from accelerated ageing by imbibing seeds to a precise water content (generally between 19 and 24% FW) before the warm temperature treatment (Powell and Matthews, Reference Powell and Matthews1981; Powell, Reference Powell and van de Venter1995; TeKrony and Egli, Reference TeKrony, Egli, Ellis, Black, Murdoch and Hong1997). It provides information about the tolerance of seeds to stress and on their storability potential (Priestley, Reference Priestley1986).
Which biological, biochemical and cellular markers for seed vigour?
Understanding the mechanisms involved in the acquisition of quality during seed development, loss of quality during seed ageing and in the germination process, may allow the identification of three main groups of markers for the assessment of seed vigour: (1) indicators of cell deterioration [membrane damage, accumulation of reactive oxygen species (ROS), protein carbonylation, RNA oxidation, etc.]; (2) indicators of the protective/defence system effectiveness (soluble sugars, specific proteins, antioxidant contents, antioxidant defence enzymes, such as catalase, etc.); and (3) indicators of the germination process (respiratory activity, cell-cycle induction, hydrolytic activity, etc.).
Indicators of membrane properties
Leakage of low molecular weight solutes (sugars, amino acids, ions, etc.) occurs during seed imbibition and is associated with the phase transition of membranes from the dry gel state to a liquid crystalline state. The degree of leakage correlates with the time taken to complete this phase transition and increases with seed deterioration. It is then used as an indicator of the effectiveness of membranes as barriers to solute diffusion, and consequently of seed vigour (Powell, Reference Powell1986; Hampton, Reference Hampton and van de Venter1995). Low seed quality is generally associated with high leakage during the imbibition phase. However, this test cannot be used for all crops, in particular because of the presence of high sugar contents (sweet corn), a semipermeable membrane (McDonald, Reference McDonald1999) or phenolic compounds in the seed coat. Such a test is recommended by ISTA for large-seeded legumes (Hampton and TeKrony, Reference Hampton and TeKrony1995).
In higher plants, ethylene is mainly synthesized through the pathway from S-adenosyl methionine (SAM) to 1-aminocyclopropane 1-carboxylic acid (ACC), the latter being converted to ethylene through ACC oxidase activity (Yang and Hoffmann, Reference Yang and Hoffmann1984). ACC-dependent ethylene production is a good indicator of membrane deterioration, since the in vivo activity of ACC oxidase depends on membrane integrity or properties (Odawara et al., Reference Odawara, Watanabe and Imaseki1977; Porter et al., Reference Porter, Borlakoglu and John1986). Decrease in ACC conversion to ethylene is one of the early indicators of cell membrane damage during dehydration of recalcitrant Araucaria angustifolia embryos (Salmen Espindola et al., Reference Salmen Espindola, Noin, Corbineau and Côme1994) and developing pea seeds (Corbineau et al., Reference Corbineau, Picard, Fougereux, Ladonne and Côme2000) or during cold temperature exposure of chilling-sensitive tissues (Chen and Patterson, Reference Chen and Patterson1985; Corbineau et al., Reference Corbineau, Engelman and Côme1990, Reference Corbineau, Bogatek, Picard, Côme, Sanchez-Diaz, Irigoyen, Aguirreolea and Pithan1999). A close relationship between ACC-dependent ethylene production and seed vigour has been reported in various species (Samimy and Taylor, Reference Samimy and Taylor1983; Gorecki et al., Reference Gorecki, Ashino, Satoh and Esashi1991; Khan, Reference Khan1994; Chojnowski et al., Reference Chojnowski, Corbineau and Côme1997). A curvilinear relationship of germination parameters to ethylene production seems to be fitted by second-degree polynomial and logarithmic equations, as suggested by Khan (Reference Khan1994) and a sub-linear relationship exists between ethylene production during the first 20 h of imbibition and the vigour of carrot seeds primed for different durations, as assessed by germination rate (T50) at 20°C (Corbineau, unpublished data).
Indicators of the efficacy of protective systems
In terms of seed germinability, carbohydrate metabolism, specific proteins such as LEA proteins and HSPs, as well as the capacity of seed tissues to protect cellular structures against activated forms of oxygen, are of particular interest (Horbowicz and Obendorf, Reference Horbowicz and Obendorf1994; Vertucci and Farrant, Reference Vertucci, Farrant, Kigel and Galili1995; Buitink and Leprince, Reference Buitink and Leprince2008; Leprince and Buitink, Reference Leprince and Buitink2010). The possibility of repairing injury upon rehydration may also be very important in seed quality.
Oligosaccharides, mainly those of the raffinose family of oligosaccharides (RFO) which accumulate during seed maturation on the parent plant, are considered to contribute to the stabilization of intracellular glasses by increasing cytoplasmic viscosity and the glass-to-liquid transition temperature, which are likely to slow down ageing-related modifications (Bernal-Lugo and Leopold, Reference Bernal-Lugo and Leopold1995; Sun, Reference Sun1997). However, Buitink et al. (Reference Buitink, Hemminga and Hoekstra2000) demonstrated that oligosaccharides are not involved in the stabilization of the glassy state in Impatiens and bell pepper seeds that had been osmoprimed. Also, accumulation of soluble sugars during seed maturation depends on the rate of seed dehydration (Blackman et al., Reference Blackman, Obendorf and Leopold1992; Black et al., Reference Black, Corbineau, Gee, Guy and Côme1999; Corbineau et al., Reference Corbineau, Picard, Fougereux, Ladonne and Côme2000) and might be used as indicator of drying conditions. In pea, a clear relationship exists between electrolyte leakage and RFO/sucrose ratio in the cotyledons (Corbineau et al., Reference Corbineau, Picard, Fougereux, Ladonne and Côme2000). Moreover, numerous data (Bernal-Lugo and Leopold, Reference Bernal-Lugo and Leopold1992, Reference Bernal-Lugo and Leopold1995; Horbowicz and Obendorf, Reference Horbowicz and Obendorf1994; Lin and Huang, Reference Lin and Huang1994; Sun and Leopold, Reference Sun and Leopold1994; Steadman et al., Reference Steadman, Pritchard and Dey1996; Sun, Reference Sun1997; Sinniah et al., Reference Sinniah, Ellis and John1998) have suggested that seed sugar content, particularly the ratio of oligosaccharides to sucrose, might be used as an indicator of seed storability. Besides sugars, proteins might play an important role in the formation of intracellular glass during dehydration (Sun and Leopold, Reference Sun and Leopold1994; Sinniah et al., Reference Sinniah, Ellis and John1998; Wolkers et al., Reference Wolkers, McCready, Brandt, Lindsey and Hoekstra2001; Boudet et al., Reference Boudet, Buitink, Hoekstra, Rogniaux, Larre, Satour and Leprince2006; Tunnacliffe and Wise, Reference Tunnacliffe and Wise2007). In Brassica campestris, Sinniah et al. (Reference Sinniah, Ellis and John1998) demonstrated a relationship between potential longevity and both the oligosaccharide/total sugar ratio and the 58 kDa heat-stable protein content. A strong correlation also exists between longevity in Medicago truncatula and the level of sucrose and stachyose, as well as some LEA polypeptides (Chatelain, Reference Chatelain2011).
The ability of seeds to withstand desiccation during maturation and their tolerance to ageing, i.e. acquisition and loss of seed quality during seed life, might be related, at least partly, to their ability to scavenge ROS to avoid deleterious events (Vertucci and Farrant, Reference Vertucci, Farrant, Kigel and Galili1995). The main detoxifying systems involve enzymes, mainly superoxide dismutase (SOD), catalase (CAT) and enzymes of the ascorbate–glutathione cycle, and antioxidant compounds such as reduced glutathione and ascorbate, and α-tocopherol (Scandalios, Reference Scandalios1997; Bailly, Reference Bailly2004). The efficacy of ROS scavenging through antioxidant enzyme activities has been shown to increase during seed development in bean (Bailly et al., Reference Bailly, Audigier, Ladonne, Wagner, Coste, Corbineau and Côme2001) and sunflower (Bailly et al., Reference Bailly, Leymarie, Lehner, Rousseau, Côme and Corbineau2004), and CAT activity is linearly correlated with seed moisture content in sunflower (Bailly et al., Reference Bailly, Leymarie, Lehner, Rousseau, Côme and Corbineau2004). In sunflower seeds, reinvigoration of aged seeds by osmopriming is associated with the restoration of the cell detoxifying defence mechanisms, resulting from the recovery of SOD, CAT and glutathione reductase (GR), and a relationship exists between the germination rate, assessed by T50 at 15°C, and antioxidant enzyme activity (Bailly et al., Reference Bailly, Benamar, Corbineau, Côme, Ellis, Black, Murdoch and Hong1997, Reference Bailly, Benamar, Corbineau and Côme1998). There is also a linear relationship between T50 at 10°C and CAT activity in seeds of Brassica napus aged for different durations (Corbineau, unpublished data). However, it is difficult to establish a clear relationship between antioxidant system activity at harvest and seed quality in wheat (Lehner et al., Reference Lehner, Bailly, Flechel, Poels, Côme and Corbineau2006), sugar-beet and carrot seeds (Corbineau, unpublished data).
Events occurring during the germination process
The rate of oxygen uptake during seed imbibition, before radicle protrusion, is often correlated with seed vigour, as assessed by subsequent rates of germination and seedling growth in pea, Zea mays, Capsicum annuum and Brassica seeds (Woodstock and Grabe, Reference Woodstock and Grabe1967; Carver and Matthews, Reference Carver and Matthews1975, Halpin-Ingham and Sundstrom, Reference Halpin-Ingham and Sundstrom1992; Bettey and Finch-Savage, Reference Bettey and Finch-Savage1996), but is not a good indicator of the germination of other species (Côme and Corbineau, Reference Côme, Corbineau and Taylorson1989). In Brassica, activities of glucose 6-phosphate dehydrogenase and pyrophosphate:fructose 6-phosphate 1-phosphotransferase were correlated with T50 (Bettey and Finch-Savage, Reference Bettey and Finch-Savage1996). Imbalances in metabolism in aged or low-quality seeds may also result in accumulation of acetaldehyde and ethanol (Priestley, Reference Priestley1986). In canola seed, both compounds appeared to be promising for assessment of seed vigour (Buckley and Buckley, Reference Buckley and Buckley2009), and 24-h assays for seed deterioration have been developed by Agriculture and Agri-Food Canada.
Identification of biochemical and molecular events associated with germination has emphasized the importance of cell repair (mitochondria, DNA, proteins, etc.), protein synthesis from stored mRNA, initial mobilization of reserves and activation of the cell cycle (Bewley, Reference Bewley1997; Boubriak et al., Reference Boubriak, Kargiolaki, Lyne and Osborne1997; Ogé et al., Reference Ogé, Bourdais, Bove, Collet, Godin, Granier, Boutin, Job, Jullien and Grappin2008; Rajjou and Debeaujon, Reference Rajjou and Debeaujon2008). The beneficial effects of priming are associated with various metabolic, biochemical, cellular and molecular events, including synthesis of proteins and RNA, degradation of LEA and heat-stable proteins, mobilization of reserves (decrease in oligosaccharides, increase in soluble 11-S globulin B-subunit, etc.), and induction of cell-cycle processes (β-tubulin accumulation, percentage of 4C DNA nuclei). In sugar-beet, pepper and tomato seeds, a positive correlation was found between the induction of DNA replication, measured by the increase in 4C nuclei or the 4C/2C ratio, and the efficacy of the priming treatment (Lanteri et al., Reference Lanteri, Saracco, Kraak and Bino1994; Özbingöl et al., Reference Özbingöl, Corbineau, Groot, Bino and Côme1999; Sliwinska et al., Reference Sliwinska, Jing, Job, Job, Bergervoet, Bino and Groot1999). However, in tomato, there were some limits to this relationship, in particular when priming was performed at too low or too high a temperature (Özbingöl et al., Reference Özbingöl, Corbineau, Groot, Bino and Côme1999). There is also a sub-linear relationship between sucrose and oligosaccharide contents, and sucrose/oligosaccharide ratio and the T50 at 10°C in carrot seeds (Corbineau and Côme, Reference Corbineau and Côme2006). A linear relationship was found between the extent of 11S globulin B-subunit and the advancement of priming in sugar-beet seeds (Job et al., Reference Job, Kersulec, Ravasio, Chareyre, Pépin and Job1997), whereas accumulation of endo-β-mannanase is also a good indicator of the priming effect in tomato (Still et al., Reference Still, Dahal and Bradford1997). However, for the same effectiveness of priming, the accumulation of soluble B-subunit per seed depended on the priming technique used (Capron et al., Reference Capron, Corbineau, Dacher, Job, Côme and Job2000). In addition, Bailly et al. (Reference Bailly, Benamar, Corbineau, Côme, Ellis, Black, Murdoch and Hong1997) demonstrated that catalase activity is sub-linearly correlated with the germination rate of primed sunflower seeds.
Conclusion
A better understanding of the biochemical, cellular and molecular mechanisms involved in the acquisition of seed vigour, in the germination process and in seed deterioration would allow various markers to be proposed for assessing seed quality. Some of these markers seem to be promising for evaluating seed maturity, seed performance in suboptimal conditions or/and priming efficiency. However, a single marker is often not enough for a good evaluation of seed quality and, very often, a marker cannot be applied to different species or even to different batches of the same species. For seed companies, the determination of the marker must also be fast and easy to perform in routine.
Seed quality depends on the conditions of seed development and seed storage, and it can be modulated through treatments such as priming. However, it is important to distinguish between markers of advancement of germination (i.e. priming) and those of seed vigour at harvest. Seed quality studies would also benefit from non-invasive techniques for assessing seed viability and vigour. For example, X-ray radiography, a non-destructive method, providing information on mechanically damaged, empty and filled seeds in a batch (Halmer, Reference Halmer, Black and Bewley2000); chlorophyll fluorescence (Jalink et al., Reference Jalink, van der Schoor, Frandas, van Pijlen and Bino1998); and infrared thermography, which can be used to predict seed viability in various species (Kranner et al., Reference Kranner, Kastberger, Hartbauer and Pritchard2010). Biophysical techniques, such as differential scanning calorimetry, can also be used to detect changes in thermal properties of lipids, which occur during loss of seed vigour or after priming treatment (Walters et al., Reference Walters, Landré, Hill, Corbineau and Bailly2005).
Acknowledgements
I am grateful to all seed companies (Advanta, BASF, Benary, Clause, Gauthier Semences, Limagrain, Syngenta, Tézier, Vilmorin and others), and research and/or professional Institutions (CNRS, FNAMS, GEVES, GNIS, INRA, SNES) with which I have collaborated on seed quality within the frame of national or European contracts. I also deeply thank Professor D. Côme, head of the laboratory until 1997, and first President of the International Society for Seed Science, who developed this applied research at the interface with basic research.
