Seed dormancy is defined as a failure of germination under conditions that are otherwise favorable for the emergence of an embryo from a seed (Bewley et al., 2013). The key genes that play a central role in induction of seed dormancy have been identified through genetic and molecular approaches (Bentsink et al., 2006; Liu et al., 2007; Graeber et al., 2013). Some of these are not directly associated with hormonal regulation (Nakabayashi et al., 2012), while others function directly through hormone balance in seeds (Kushiro et al., 2004; Lefebvre et al., 2006).
The major plant hormone involved in the induction and maintenance of seed dormancy is ABA (Seo et al., 2009; Bewley et al., 2013), which induces transcription factors and other regulatory proteins involved in suppression of germination (Piskurewicz et al., 2008; Lee et al., 2010). Abscisic acid is also associated with the seed maturation program including the induction of storage proteins and other proteins associated with desiccation tolerance (Galau et al., 1987). The levels of ABA in seeds are determined by its biosynthesis, deactivation and transport (Seo and Koshiba, 2011). Biosynthesis of ABA in seeds is mainly regulated by the rate-limiting enzyme 9-cis-epoxycarotenoid dioxygenase (NCED) (Lefebvre et al., 2006; Martinez-Andujar et al., 2011), while 8′-hydroxylase plays an important role in ABA deactivation (Kushiro et al., 2004; Millar et al., 2006; Barrero et al., 2009).
It has been demonstrated that the induction of NCED6, using the plant gene switch system (PGSS), a chemically induced gene expression system, was sufficient to suppress the germination of imbibed seeds of wild-type (WT) Arabidopsis thaliana, ecotype Columbia-0 (Col), after 3-days’ pre-chilling (dormancy release) treatment (Martinez-Andujar et al., 2011). The suppression of germination by NCED6 induction was cancelled by fluridone, an inhibitor of carotenoid (hence ABA) biosynthesis, suggesting that the dormancy phenotypes in the PGSS NCED6 seeds were dependent on de novo ABA biosynthesis. In fact, ABA levels in the NCED6-induced seeds were more than 20-fold higher than the ABA levels in uninduced seeds (Martinez-Andujar et al., 2011). These results showed that NCED expression could serve as a sole determinant of seed dormancy under certain conditions.
While NCED induction in imbibed seeds suppresses germination, seed dormancy in natural systems is induced in developing seeds during maturation and results obtained from imbibed seeds do not necessarily confirm the role of NCED as a single determinant of seed dormancy induction. Conditional expression of DOG1, one of the best-characterized seed dormancy-specific genes, which is expressed during the seed maturation stage (Bentsink et al., 2006), does not suppress seed germination when it is induced in imbibed seeds (Nakabayashi et al., 2012). Thus, induction of dormancy-associated genes only in imbibed seeds may not represent their roles in the natural system. It is necessary to create an efficient experimental system that enables enhanced NCED expression in developing seeds for analysis of the biochemical and molecular mechanisms of seed development and dormancy regulated by ABA.
Chemical induction (Piskurewicz et al., 2008; Martinez-Andujar et al., 2011) of NCED in developing seeds can be tested by painting siliques with a ligand. However, genes might not be induced efficiently in seeds due to impermeability of the testa to a ligand. In addition, application of a ligand to siliques affects the maternal tissues, such as the replum, valves and funicular tissues, which could alter seed development and dormancy. Drenching the soil or media surrounding the roots of the maternal plants could deliver a chemical ligand to developing seeds via the vascular system. However, in this case also, the maternal plants can be affected by ectopic gene induction. The major objective of this study was to develop an efficient experimental system to enhance NCED expression in developing seeds in a specific manner that does not depend on chemical induction but still allows us to experimentally examine whether NCED expression alone or a single metabolic change during seed maturation can serve as a determinant of seed dormancy in mature seeds.
To this end, we expressed NCED with an ABA-regulated promoter which is activated during the seed maturation stage. This system is expected to cause positive feedback through ABA biosynthesis and signaling (details in Results). Although we planned all experiments in Arabidopsis, we were interested in creating the experimental system using genes from cereal crops, because if the dormancy controlling system is established successfully it would not only provide an efficient experimental system for basic research but also establish a basic technology for preventing pre-harvest sprouting (PHS), a serious problem in crop production, which could contribute to food security in our society. A NCED was isolated from Sorghum bicolor and used with an ABA-regulated promoter from wheat (Triticum aestivum), which is activated in developing seeds during the maturation stage. The role of NCED expression during seed maturation as a determinant of seed dormancy, its relation to ABA biosynthesis in mature seeds during imbibition and positive feedback mechanisms in the natural seed dormancy system will be discussed in this paper. The utility of the experimental system for seed dormancy research and its potential for PHS prevention in crop species will also be discussed.
Isolation of sorghum NCED
Sorghum NCED sequences were searched through the Sorghum GDB database (http://www.plantgdb.org/SbGDB/) using the cDNA sequence of A. thaliana NCED6 (AtNCED6, NM_113327.2), which identified Sb01g013520.1 (and Sb02g003230.1 later). The Sb01g013520.1 gene was used for further analyses and experiments. The deduced amino acid sequence of Sb01g013520.1 contained the domains conserved throughout the known NCEDs in both monocots and dicots (Figure S1 in Supporting Information). Sb01g013520.1 was termed SbNCED. Since the SbNCED gene does not contain introns, the coding sequence was isolated from sorghum genomic DNA and used for functional analyses.
The SbNCED sequence was also aligned with the sequences of AtNCED5, AtNCED6 and AtNCED9 that are expressed in Arabidopsis seeds (Figure S2). For AtNCED9, the protein sequence starting with the second methionine (position 51, MASTT…) was used for alignment because it has been suggested that the first 150 bp of the annotated coding sequence (NM_106486.2) might include the 3′ region of the NCED9 promoter sequence, which adds 50 extra amino acids (position 1, MTIIT…) to the N-terminus (Frey et al., 2012). The AtNCED3 sequence was also included in the alignment because SbNCED was annotated as a putative ortholog of AtNCED3 in the Sorghum GDB database. SbNCED exhibited high similarities to both AtNCED3 and the seed-expressed NCEDs (Figure S2). SbNCED was used to create amplification of the ABA biosynthesis and signaling pathways (see next section).