Regulation of Seed Germination: The Involvement of Multiple Forces Exerted via Gibberellic Acid Signaling

Abstract

Seed germination is a key step in the life cycle of all seed plants, and it is of immense significance in agriculture. Germination is controlled by multiple environmental cues such as light and moisture, and endogenous factors, especially phytohormones. Gibberellic acid (GA) has long been identified as a critical hormone in the regulation of seed germination by countering the inhibition imposed by abscisic acid (ABA) (Holdsworth et al., 2008Holdsworth M.J. Bentsink L. Soppe W.J. Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination.New Phytol. 2008; 179: 33-54Crossref PubMed Scopus (651) Google Scholar, Lee et al., 2010Lee K.P. Piskurewicz U. Tureckova V. Strnad M. Lopez-Molina L. A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds.Proc. Natl. Acad. Sci. USA. 2010; 107: 19108-19113Crossref PubMed Scopus (143) Google Scholar). The underlying molecular mechanism involves multiple facets, including DELLA proteins such as RGA-LIKE2 (RGL2; Lee et al., 2002Lee S. Cheng H. King K.E. Wang W. He Y. Hussain A. Lo J. Harberd N.P. Peng J. Gibberellin regulates Arabidopsis seed germination via RGL2, a GAI/RGA-like gene whose expression is up-regulated following imbibition.Genes Dev. 2002; 16: 646-658Crossref PubMed Scopus (479) Google Scholar, Ravindran et al., 2017Ravindran P. Verma V. Stamm P. Kumar P.P. A novel RGL2–DOF6 complex contributes to primary seed dormancy in Arabidopsis thaliana by regulating a GATA transcription factor.Mol. Plant. 2017; 10: 1307-1320Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) along with other key signaling intermediates and specific biophysical forces as has recently been demonstrated (Sánchez-Montesino et al., 2018Sánchez-Montesino R. Bouza-Morcillo L. Marquez J. Ghita M. Duran-Nebreda S. Gómez L. Holdsworth M.J. Bassel G. Oñate-Sánchez L. A regulatory module controlling GA-mediated endosperm cell expansion is critical for seed germination in Arabidopsis.Mol. Plant. 2018; https://doi.org/10.1016/j.molp.2018.10.009PubMed Scopus (43) Google Scholar). The increase in GA levels upon imbibition is essential for the rupture of testa and the endosperm, and subsequent emergence of the radicle. The complexity of GA signaling and the associated molecular genetic networks working in a coordinated manner to influence biophysical parameters that ultimately result in seed germination is beginning to emerge. The role of GA in regulating germination received greater attention once the signaling intermediates were identified. The role of DELLA proteins, especially RGL2 as a key transcriptional repressor of GA signaling to regulate seed germination, has been well established (Lee et al., 2002Lee S. Cheng H. King K.E. Wang W. He Y. Hussain A. Lo J. Harberd N.P. Peng J. Gibberellin regulates Arabidopsis seed germination via RGL2, a GAI/RGA-like gene whose expression is up-regulated following imbibition.Genes Dev. 2002; 16: 646-658Crossref PubMed Scopus (479) Google Scholar). The classic model for DELLA action is that of negative regulation where DELLA sequesters transcription factors in the absence of GA. With an increase in GA, e.g., from the aleurone layer in imbibed seeds, DELLA proteins will be polyubiquitinated and degraded via the proteasome, thereby releasing the transcription factor(s) from inhibition. In addition, recent studies have begun revealing a positive mode of action for DELLA proteins where they function as transcriptional activators. In an effort to study the downstream targets of RGL2 in Arabidopsis thaliana, GATA12 (encoding a GATA-type zinc finger transcription factor) was identified as a gene that is positively regulated by RGL2 (Ravindran et al., 2017Ravindran P. Verma V. Stamm P. Kumar P.P. A novel RGL2–DOF6 complex contributes to primary seed dormancy in Arabidopsis thaliana by regulating a GATA transcription factor.Mol. Plant. 2017; 10: 1307-1320Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Furthermore, it was shown that GATA12 expression is negatively regulated by GA in a DELLA-dependent manner, and its transcript levels are depleted by seed dormancy-breaking treatments such as cold stratification and after-ripening. GATA12 promoter has several GAMYB- and DOF-associated motifs that are known to be GA and RGL2 responsive, respectively (Stamm et al., 2012Stamm P. Ravindran P. Mohanty B. Tan E.L. Yu H. Kumar P.P. Insights into the molecular mechanism of RGL2-mediated inhibition of seed germination in Arabidopsis thaliana.BMC Plant Biol. 2012; 12: 179Crossref PubMed Scopus (38) Google Scholar, Ravindran et al., 2017Ravindran P. Verma V. Stamm P. Kumar P.P. A novel RGL2–DOF6 complex contributes to primary seed dormancy in Arabidopsis thaliana by regulating a GATA transcription factor.Mol. Plant. 2017; 10: 1307-1320Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). DNA BINDING1 ZINC FINGER6 (DOF6) is a plant-specific transcription factor that inhibits germination in freshly harvested seeds (Rueda-Romero et al., 2012Rueda-Romero P. Barrero-Sicilia C. Gómez-Cadenas A. Carbonero P. Oñate-Sánchez L. Arabidopsis thaliana DOF6 negatively affects germination in non-after-ripened seeds and interacts with TCP14.J. Exp. Bot. 2012; 63: 1937-1949Crossref PubMed Scopus (99) Google Scholar). Its overexpression induced upon seed imbibition triggers delayed germination and ABA-hypersensitive phenotypes. Ravindran et al., 2017Ravindran P. Verma V. Stamm P. Kumar P.P. A novel RGL2–DOF6 complex contributes to primary seed dormancy in Arabidopsis thaliana by regulating a GATA transcription factor.Mol. Plant. 2017; 10: 1307-1320Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar showed that RGL2 interacts with DOF6, and this novel complex binds to the DOF motifs in the promoter of GATA12 to enhance its expression and enforce primary dormancy. Freshly harvested (unstratified) seeds of GATA12 suppression lines have reduced dormancy compared with the wild-type, while ectopic expression lines show enhanced seed dormancy. As discussed above, RGL2 lacks a DNA-binding domain and requires other transcription factors to induce expression of target genes. It is obvious that GATA12 is not the only target of RGL2–DOF6 complex, and it is conceivable that future investigations will reveal other important downstream targets that are critical components of the molecular network to enforce primary seed dormancy (Figure 1). Moreover, DOF motifs were also found to be enriched in the downstream targets of another DELLA protein, GA INSENSITIVE (GAI). GAI interacts with DOF AFFECTING GERMINATION1 (DAG1) and represses the expression of the GA biosynthetic gene, AtGA3ox1, in seeds (Boccaccini et al., 2014Boccaccini A. Santopolo S. Capauto D. Lorrai R. Minutello E. Serino G. Costantino P. Vittorioso P. The DOF protein DAG1 and the DELLA protein GAI cooperate in negatively regulating the AtGA3ox1 gene.Mol. Plant. 2014; 7: 1486-1489Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These recent studies highlight the importance of DOF proteins in mediating DELLA activity in the regulation of seed dormancy and germination. The importance of mechanical force adjustments by expansion of endosperm cells surrounding the elongating embryo prior to radicle emergence as critical for successful germination has been identified in Arabidopsis thaliana (Bassel et al., 2014Bassel G.W. Stamm P. Mosca G. Barbier de Reuille P. Gibbs D.J. Winter R. Janka A. Holdsworth M.J. Smith R.S. Mechanical constraints imposed by 3D cellular geometry and arrangement modulate growth patterns in the Arabidopsis embryo.Proc. Natl. Acad. Sci. USA. 2014; 111: 8685-8690Crossref PubMed Scopus (130) Google Scholar, Sánchez-Montesino et al., 2018Sánchez-Montesino R. Bouza-Morcillo L. Marquez J. Ghita M. Duran-Nebreda S. Gómez L. Holdsworth M.J. Bassel G. Oñate-Sánchez L. A regulatory module controlling GA-mediated endosperm cell expansion is critical for seed germination in Arabidopsis.Mol. Plant. 2018; https://doi.org/10.1016/j.molp.2018.10.009PubMed Scopus (43) Google Scholar, Stamm et al., 2017Stamm P. Topham A.T. Mukhtar N.K. Jackson M.D. Tome D.F. Beynon J.L. Bassel G.W. The transcription factor ATHB5 affects GA-mediated plasticity in hypocotyl cell growth during seed germination.Plant Physiol. 2017; 173: 907-917Crossref PubMed Scopus (29) Google Scholar). Using cell surface area measurements by confocal imaging and 3D geometry reconstruction, Sánchez-Montesino et al., 2018Sánchez-Montesino R. Bouza-Morcillo L. Marquez J. Ghita M. Duran-Nebreda S. Gómez L. Holdsworth M.J. Bassel G. Oñate-Sánchez L. A regulatory module controlling GA-mediated endosperm cell expansion is critical for seed germination in Arabidopsis.Mol. Plant. 2018; https://doi.org/10.1016/j.molp.2018.10.009PubMed Scopus (43) Google Scholar identified that the region of the endosperm surrounding the lower hypocotyl and the radicle exhibit the highest expansion rates in imbibed seeds. In these zones of the germinating Arabidopsis seeds, they identified that the rate of endosperm cell expansion is positively correlated with expansion rates of the embryo axis. Although embryo growth during germination is mostly provided by cell elongation along the embryonic axis (Sliwinska et al., 2009Sliwinska E. Bassel G.W. Bewley J.D. Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl.J. Exp. Bot. 2009; 60: 3587-3594Crossref PubMed Scopus (114) Google Scholar), GA also mediates activation of cell division in the embryonic root apical meristem (RAM). Under unfavorable conditions, DELLAs restrict cell-cycle progression in the RAM by sequestering TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR14 (TCP14) and TCP15 (Figure 1, Resentini et al., 2015Resentini F. Felipo-Benavent A. Colombo L. Blazquez M.A. Alabadi D. Masiero S. TCP14 and TCP15 mediate the promotion of seed germination by gibberellins in Arabidopsis thaliana.Mol. Plant. 2015; 8: 482-485Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These results highlight the importance of biophysical forces in regulating seed germination. Subsequently, NAC transcription factors NAC25 and NAC1-like (NAC1L) were identified as activators of EXPANSIN2 (EXPA2) expression that regulates the GA-mediated endosperm cell expansion during seed germination (Sánchez-Montesino et al., 2018Sánchez-Montesino R. Bouza-Morcillo L. Marquez J. Ghita M. Duran-Nebreda S. Gómez L. Holdsworth M.J. Bassel G. Oñate-Sánchez L. A regulatory module controlling GA-mediated endosperm cell expansion is critical for seed germination in Arabidopsis.Mol. Plant. 2018; https://doi.org/10.1016/j.molp.2018.10.009PubMed Scopus (43) Google Scholar). The DELLA protein RGL2 sequesters NAC25 upon interaction and represses the activation of EXPA2 (Sánchez-Montesino et al., 2018Sánchez-Montesino R. Bouza-Morcillo L. Marquez J. Ghita M. Duran-Nebreda S. Gómez L. Holdsworth M.J. Bassel G. Oñate-Sánchez L. A regulatory module controlling GA-mediated endosperm cell expansion is critical for seed germination in Arabidopsis.Mol. Plant. 2018; https://doi.org/10.1016/j.molp.2018.10.009PubMed Scopus (43) Google Scholar) under a low GA environment, which is an excellent example of negative regulation of target gene expression by DELLA protein (Figure 1). In support of this observation, NAC-binding cis elements were frequently encountered in the promoters of genes that were upregulated by GA and downregulated by DELLAs. For example, a group of genes encoding cell wall remodeling enzymes (CWREs) have a high proportion of NAC-binding cis elements. This partially explains how GA regulates the coordinated expression of CWREs, including EXPA2, through NAC transcription factors. Similarly, DELLAs sequester two HD-ZIP transcription factors, namely, ATML1 and PDF2, and thereby block GA signaling in the epidermis under low GA conditions. However, upon imbibition, ATML1/PDF2 are released from DELLAs and become free to activate EXPA8 and epidermis-specific GDSL lipase gene (LIP1) expression, thereby initiating elongation of epidermal cells to allow seeds to germinate (Rombolá-Caldentey et al., 2014Rombolá-Caldentey B. Rueda-Romero P. Iglesias-Fernández R. Carbonero P. Oñate-Sánchez L. Arabidopsis DELLA and two HD-ZIP transcription factors regulate GA signaling in the epidermis through the L1 box cis-element.Plant Cell. 2014; 26: 2905-2919Crossref PubMed Scopus (49) Google Scholar). A better picture of how GA responses are regulated downstream of the central regulators of GA signaling, namely DELLA proteins, is beginning to emerge. Arabidopsis seed dormancy and germination serves as an excellent experimental system for such studies. DELLAs lack a DNA-binding domain and thus transcriptionally regulate target genes by forming the complexes with specific transcription factors (Boccaccini et al., 2014Boccaccini A. Santopolo S. Capauto D. Lorrai R. Minutello E. Serino G. Costantino P. Vittorioso P. The DOF protein DAG1 and the DELLA protein GAI cooperate in negatively regulating the AtGA3ox1 gene.Mol. Plant. 2014; 7: 1486-1489Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, Rombolá-Caldentey et al., 2014Rombolá-Caldentey B. Rueda-Romero P. Iglesias-Fernández R. Carbonero P. Oñate-Sánchez L. Arabidopsis DELLA and two HD-ZIP transcription factors regulate GA signaling in the epidermis through the L1 box cis-element.Plant Cell. 2014; 26: 2905-2919Crossref PubMed Scopus (49) Google Scholar, Yoshida et al., 2014Yoshida H. Hirano K. Sato T. Mitsuda N. Nomoto M. Maeo K. Koketsu E. Mitani R. Kawamura M. Ishiguro S. DELLA protein functions as a transcriptional activator through the DNA binding of the indeterminate domain family proteins.Proc. Natl. Acad. Sci. USA. 2014; 111: 7861-7866Crossref PubMed Scopus (158) Google Scholar, Ravindran et al., 2017Ravindran P. Verma V. Stamm P. Kumar P.P. A novel RGL2–DOF6 complex contributes to primary seed dormancy in Arabidopsis thaliana by regulating a GATA transcription factor.Mol. Plant. 2017; 10: 1307-1320Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Different molecular mechanisms drive GA responses in seeds depending on the cell layer and developmental phase. In addition, RGL2 is a contributing factor to promote ABA synthesis in the endosperm, and thereby it can help to repress embryo growth and maintain seed dormancy (Lee et al., 2010Lee K.P. Piskurewicz U. Tureckova V. Strnad M. Lopez-Molina L. A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds.Proc. Natl. Acad. Sci. USA. 2010; 107: 19108-19113Crossref PubMed Scopus (143) Google Scholar). A bidirectional communication between embryo and endosperm seems essential to balance the biomechanical forces that control germination. The signal from the embryo that regulates endosperm cell expansion has not yet been identified. With the knowledge that DELLA proteins can function as both transcriptional activators and repressors, it is important to identify additional DELLA-interacting proteins and their target genes to fully unravel the mechanism of dormancy release and germination. With better understanding of this, manipulation of GA action at the right developmental stages and appropriate seed tissues will become an attractive avenue to engineer agronomically superior seeds.