Agriculture and Sustainability Archives - SeeSD

DELAY OF GERMINATION 1-LIKE 4 acts as an inducer of seed reserve accumulation

admin — Mon, 29 Jul 2019 07:00:00 +0000

By Khadidiatou Sall, Bas J. W. Dekkers, Mariko Nonogaki, Yoshihiko Katsuragawa, Ryosuke Koyari, David Hendrix, Leo A. J. Willems, Leónie Bentsink, Hiroyuki Nonogaki

[Published on Onlinelibrary.wiley.com]

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Summary

More than 70% of global food supply depends on seeds. The major seed reserves, such as proteins, lipids, and polysaccharides, are produced during seed maturation. Here, we report that DELAY OF GERMINATION 1-LIKE 4 (DOGL4) is a major inducer of reserve accumulation during seed maturation. The DOGL family proteins are plant-specific proteins of largely unknown biochemical function. DOGL4 shares only limited homology in amino acid sequence with DOG1, a major regulator of seed dormancy. DOGL4 was identified as one of the outstanding abscisic acid (ABA)-induced genes in our RNA sequencing analysis, whereas DOG1 was not induced by ABA. Induction of DOGL4 caused the expression of 70 seed maturation-specific genes, even in germinating seeds, including the major seed reserves ALBUMIN, CRUCIFERIN and OLEOSIN. Although DOG1 affects the expression of many seed maturation genes, the major seed reserve genes induced by DOGL4 are not altered by the dog1 mutation. Furthermore, the reduced dormancy and longevity phenotypes observed in the dog1 seeds were not observed in the dogl4 mutants, suggesting that these two genes have limited functional overlap. Taken together, these results suggest that DOGL4 is a central factor mediating reserve accumulation in seeds, and that the two DOG1 family proteins have diverged over the course of evolution into independent regulators of seed maturation, but retain some overlapping function.

Introduction

Seed development can be divided into two major phases: embryogenesis and maturation. Events during embryogenesis include histodifferentiation, which establishes the foundation of the major tissues, organs and a body plan of plants (Bewley et al., 2013). In the maturation phase, the differentiated embryo accumulates seed reserves, such as proteins, lipids and polysaccharides (Bewley et al., 2013).

The seed maturation program is regulated mainly by abscisic acid (ABA). 9-cis-Epoxycarotenoid dioxygenase (NCED) is a rate-limiting enzyme for ABA biosynthesis (Nambara and Marion-Poll, 2005). NCED9 is expressed in the testa, the seed coat, and the embryo of developing Arabidopsis seeds, whereas NCED6 is expressed in the endosperm (Lefebvre et al., 2006). NCED5 is expressed in the embryo and the endosperm (Frey et al., 2012). All of these genes contribute to ABA production in developing seeds, which is essential for normal seed maturation (Meurs et al., 1992).

Another event during seed maturation that is induced by ABA is seed dormancy, which is an intrinsic pause in germination in otherwise favorable germination conditions (Bentsink and Koornneef, 2008; Bewley et al., 2013; Nonogaki, 2014). DELAY OF GERMINATION 1 (DOG1) is a central regulator of seed dormancy (Bentsink et al., 2006), which encodes for a plant-specific protein that enhances ABA signaling through its binding to protein phosphatase 2C (PP2C) ABA HYPERSENSITIVE GERMINATION1 (AHG1) and AHG3 (Née et al., 2018; Nishimura et al., 2018). There are five DOG1-LIKE (DOGL) genes – DOGL1, DOGL2, DOGL3, DOGL4 and DOGL5 – in the Arabidopsis genome (Nishimura et al., 2018). DOGL3 and DOGL5 (DOGL5.2, alternative spliced form similar to DOG1) also bind to PP2C (Nishimura et al., 2018). Overexpression of DOGL3 causes ABA hypersensitivity in seed germination, like DOG1, whereas DOGL5 overexpression does not result in increased ABA sensitivity in seeds (Nishimura et al., 2018). No phenotypes were observed for the dogl1, dogl2 and dogl3 single mutants (Bentsink et al., 2006). The dogl4 mutants exhibit moderately enhanced seed dormancy (Zhu et al., 2018), suggesting instead that DOGL4 is a negative regulator of seed dormancy, unlike DOG1. The dog1 single knock-out mutant seeds exhibit a non-dormant phenotype (Bentsink et al., 2006). Taken together, DOG1 and DOGLs do not seem to share a great deal of redundancy in terms of seed dormancy imposition.

An additional function of DOG1 outside seed dormancy has been demonstrated through genetic and molecular approaches. DOG1 has some synergistic effects with ABA INSENSITIVE 3 (ABI3), a major factor in ABA signaling. The dog1-1 mutation enhances weak phenotypes of the abi3-1 mutants and causes downregulation of the seed maturation genes (Dekkers et al., 2016). These results suggest that DOG1 has additional biological roles in the seed maturation program to dormancy. The involvement of DOGLs in the seed maturation program, other than seed dormancy, is not known.

In this study, we identified DOGL4 as a DOG1 family gene that is induced by ABA and found that DOGL4 induction was robust enough to cause the expression of a number of seed maturation-specific genes, even in germinating seeds. The biological role of DOGL4 will be discussed in the context of the evolution of diverged functions of the DOG1 family proteins in seed maturation and dormancy mechanisms.

Transcriptomics of nine-cis-epoxycarotenoid dioxygenase 6 induction in imbibed seeds reveals feedback mechanisms and long non-coding RNAs

admin — Mon, 11 Sep 2017 07:00:00 +0000

By Khadidiatou Sall, David Hendrix, Taira Sekine, Yoshihiko Katsuragawa, Ryosuke Koyari and Hiroyuki Nonogaki

[Published online by Cambridge University Press]

Abstract

Induction of nine-cis-epoxycarotenoid dioxygenase 6 (NCED6), an abscisic acid (ABA) biosynthesis gene, alone is sufficient to suspend germination in testa-ruptured seeds, which are at the final step of germination. Molecular consequences of NCED6 induction in imbibed seeds were investigated by RNA sequencing. The analysis identified many unknown and uncharacterized genes that were up-regulated by NCED6 induction, in addition to the major regulators of ABA signalling. Interestingly, other NCEDs were up-regulated by NCED6 induction, suggesting that the major rate-limiting enzymes in the ABA biosynthesis pathway are subject to positive-feedback regulation. ZEAXANTHIN EPOXIDASE and ABSCISIC ALDEHYDE OXIDASE3, which function upstream and downstream of NCED, were also up-regulated in seeds by NCED6 induction, which suggests that the distinct layers of positive feedback loops are coordinately operating in the NCED6-induced seeds. SOMNUS (SOM), which was also up-regulated by NCED6 induction, was the major mediator of enhanced ABA signalling in NCED6-induced seeds. SOM exerted negative effects on GA biosynthesis, which also contributes to a high ABA–GA ratio and reinforces the suppressive state of germination. Besides these coding genes, long intergenic non-coding RNAs (lincRNAs) were also up-regulated upon NCED6 induction (termed N6LINCRs). Conditional expression of N6LINCR1 altered gene expression profiles in seeds. Twenty-six genes were up-regulated and 66 genes were down-regulated by the induction of N6LINCR1. These results suggest that some of N6LINCRs have a regulatory role in gene expression in seeds, which potentially contributes to the regulation of germination by ABA.

The Analysis of Hormone-dependent Transcription Factors and lncRNAs in Seeds by RNA Sequencing

admin — Fri, 11 Aug 2017 18:16:00 +0000

By Khadidiatou Sall for the completion of her PhD thesis at Oregon State University

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Abstract : Seed dormancy is defined as the inability of viable seeds to germinate under conditionsotherwise favorable for germination. Dormancy provides a strategy for seeds to germinateat an appropriate time. Abscisic acid (ABA) is a major hormone involved in the regulationof seed dormancy. To elucidate the molecular mechanisms of dormancy, the twoexperimental systems, Gene Switch (GS), a chemically induced system and positivefeedback (PFB) system, a spontaneous system that does not require a chemical ligand,were created. Both systems were engineered to induce nine-cis-epoxycarotenoiddioxygenase (NCED), a rate-limiting enzyme of ABA biosynthesis. The GS systemincreased ABA levels in seeds more than 20 fold while PFB increases ABA more than 73fold, with both preventing imbibed seeds from germinating. To understand dormancymechanisms, the molecular consequences of NCED6 induction in the GS system wereinvestigated by RNA sequencing (RNA-seq). Many genes involved in ABA biosynthesisand signaling were identified. Other NCEDs, such as NCED5 and NCED9 and other ABAbiosynthesis genes, such as ZEAXANTHIN EPOXIDASE and ABSCISIC ALDEHYDEOXIDASE3, which function upstream and downstream of NCED, were upregulated inseeds by NCED6 induction. These results suggest that the distinct layers of PFB loops arecoordinately operating in the NCED6-induced seeds. In addition to known genes, manyuncharacterized genes including DELAY OF GERMINATION1 (DOG1)-LIKE-4 (DOGL4)and five long intergenic noncoding RNAs (lincRNAs) termed N6LINCRs were identifiedand characterized. DOGL4 shares only limited homology in amino acid sequence toDOG1, a major regulator of seed dormancy. Induction of DOGL4 alone in imbibed seedscaused expression of 70 seed maturation-specific genes, including those coding for themajor seed reserves, such as albumin, cruciferin, and oleosin, as well as sugar and lipidtransporters that are not affected by dog1-1 mutation. These results suggest that DOGL4 isa master regulator of reserve accumulation in seeds and has a distinct role from that ofDOG1. N6LINCR1, one of the lincRNAs identified by RNA-seq was preferentiallyexpressed from one strand and showed a clear response to ABA and gibberellin (GA) inseeds. RNA-seq analysis showed that induction of N6LINCR1 upregulated 26 genes anddownregulated 66 genes, suggesting that this lincRNA has a regulatory role in geneexpression in seeds, which potentially contributes to ABA regulation of germination.Besides their importance in basic research, the GS and PFB systems created in this thesisresearch provide efficient technologies, which can prevent preharvest sprouting (PHS),precocious germination in cereal crops. Moreover, DOGL4, a major seed maturationregulator identified by this study, also offers a great potential to increase protein and lipidcontents in grain crops and contribute to agricultural production and food security.

Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds

admin — Wed, 12 Feb 2014 07:00:00 +0000

By Mariko Nonogaki, Khadidiatou Sall, Eiji Nambara, Hiroyuki Nonogaki [Published on Onlinelibrary.wiley.com]

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Summary

Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA-stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE-binding factor) expression in Arabidopsis Columbia-0 seeds, which caused 9- to 73-fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non-dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre-harvest sprouting during crop production, and therefore contributes to translational biology.

Introduction

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.

Results

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).