Fluctuation of Arabidopsis seed dormancy with relative humidity and temperature during dry storage

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Hormonal regulation of dormancy and germination

Two phytohormones, ABA (abscisic acid) and GA (gibberellins), are the main hormones that are involved in regulating seed dormancy, although roles for additional hormones such as ethylene, brassinosteroid (BR), auxin and strigolactone have also been described (Kucera et al., 2005). ABA acts as a positive regulator of dormancy and negatively regulates germination while GA and ethylene has the opposite effect.

Abscisic acid

The plant hormone ABA is ubiquitous among higher plants where it participates in the control of many crucial aspects of plant growth and development (Zeevaart and Creelman, 1988; Cutler et al., 2010). In seeds, ABA regulates several processes essential for seed viability and germination, in concert with other as yet unidentified developmental factors. These processes include the accumulation of protein and lipid reserves, the induction of dormancy, and the acquisition of tolerance to desiccation. During seed development, ABA levels peak during mid-maturation and this coincides with the induction of dormancy levels (Kermode, 2005). ABA which is synthesized during seed development can be of dual origin, the embryo and/or the maternal tissues (Kucera et al., 2005). The ABA biosynthesis pathway is well known and the germination phenotypes of a large number of mutants of its biosynthetic pathway have been characterized (Nambara and Marion-Poll, 2003) (Figure 5). Cellular ABA content is regulated by the balance between its biosynthesis and catabolism (Nambara and Marion-Poll, 2003). Nine-cis-epoxycarotenoid dioxygenases (NCEDs) catalyse the cleavage of C40 9-cis-epoxycarotenoids, 9-cis-neoxanthin and 9-cis-violaxanthin, which is a primary regulatory step in ABA biosynthesis (Schwartz et al., 1997). Zeaxanthin epoxidase (ZEP), which is a xanthophyll cycle enzyme, is also postulated to have a regulatory function in this process (Marin et al., 1996). Members of the CYP707A family of P450 mono-oxygenases encode ABA 8’-hydroxylase, which is involved in a committed step in the ABA 8’-hydroxylation pathway, and they play a regulatory role in the control of ABA content (Kushiro et al., 2004; Saito et al., 2004; Millar et al., 2006). All plant species examined to date encode NCED and CYP707A as multigene families, and differential combinations of these members contribute to tissue- and environment specific regulation (Lefebvre et al., 2006; Seo et al., 2006). The ABA content in dry Arabidopsis seeds at harvest is not related to depth of dormancy (Ali-Rachedi et al., 2004) but expression of dormancy at high temperature is associated with maintenance of a high level of ABA content during the first 24-48 h of incubation at 25°C, while ABA decreases in seeds incubated at temperatures which allow germination (Ali-Rachedi et al., 2004). This ABA decrease depends largely on CYP707A2 activity (Ali-Rachedi et al., 2004; Kushiro et al., 2004). High ABA content and sensitivity are often associated in dormant seeds (Yoshioka et al., 1998; Corbineau and Côme, 2003; Argyris et al., 2011; Benech-Arnold et al., 2006; Leymarie et al., 2008).
Figure 5. ABA metabolic pathway (Adapted from Nambara et al., 2010). Each box indicates an enzyme, grey boxes indicate enzymes with a regulatory function in seeds. ZEP, zeaxanthin epoxidase; NSY, neoxanthin synthase; NCED,9-cis-epoxycarotenoid dioxygenase, CYP707 A, ABA 8’-hydroxylase;PA, phaseic acid; DPA, dihydrophaseic acid.


GA play an important role in diverse growth and developmental processes throughout the whole life cycle of plants, including seed germination, stem elongation, leaf expansion and flower development (Richards et al., 2001). The biochemical pathway for GA biosynthesis is well characterized and the majority of genes encoding enzymes in this pathway has been identified (Sun, 2010). The activation and deactivation of GA have been extensively reviewed by Yamaguchi (2008). Briefly, terpene synthase and P450 enzymes are involved in early stages of GA biosynthesis, which leads to the production of GA12 (Figure 6). GA12 is then converted to a bioactive form, GA4 by GA20-oxidase (GA20ox) and GA3-oxidase (GA3ox). Key enzymes in the GA deactivation pathway are encoded by the GA2ox genes which use C-19 and C-20-GAs. The DELLA family of GRAS proteins, which are characterized by two leucine rich areas flanking a VHIID motif, are negative regulators of plant growth and have been shown to have a role in seed germination (Lee et al., 2002; Tyler et al., 2004; Cao et al., 2006; Penfield et al., 2006). In Arabidopsis there are five DELLA proteins, RGA, GAI, RGA LIKE1 (RGL1), RGL2 and RGL3. DELLA proteins are nuclear transcriptional regulators, which repress GA signaling by causing transcriptional reprogramming (Sun, 2010). The GA signal targets DELLAs for degradation via ubiquitination and the 26S proteasome pathway, releasing the plant from the DELLA-mediated growth constraint (Silverstone et al., 2001). This targeting of DELLAs for degradation occurs when GA binds to its soluble receptor, GA INSENSITIVE DWARF1 (GID1). The GID1-GA complex then causes the binding to DELLA and consequently degradation (Hirano et al., 2007). Seed dormancy is alleviated through modulation of GA synthesis (Ogawa et al., 2003). In Arabidopsis, a de novo biosynthesis of GAs is required during imbibition (Karssen et al., 1989; Nambara et al., 1991).
Figure 6. Principal pathway of GA metabolism in plants (Sakamoto et al., 2004).

ABA and GA balance

The integration of ABA and GA signaling can occur as shown previously through the regulation of one hormone content by the other (Seo et al., 2006, 2009). In the ga1-3 mutant, ABA biosynthetic genes are activated and ABA deactivation genes suppressed, suggesting that GA regulates ABA metabolism (Oh et al., 2006). Additionally, RGL2 is responsible for promoting ABA synthesis when GA levels are reduced. The increase in ABA levels in turn forms a positive feedback loop by promoting RGL2 expression (Piskurewicz et al., 2008). Following transfer of imbibed seeds to 4°C and then 20°C, a decrease in ABA content occurs before germination (Ali-Rachedi et al., 2004). This decrease in ABA levels is also apparent upon imbibition at warm temperatures (Chiwocha et al., 2005), suggesting that the promotion of germination by cold stratification does not require a reduction in ABA levels. The balance between ABA and gibberellin levels and sensitivity is a major regulator of seed dormancy (Finch-Savage and Leubner-Metzger, 2006).


Ethylene is a gaseous plant hormone that regulates numerous developmental processes in higher plants, from seed germination to senescence and is important for plant responses to biotic and abiotic stresses (Mattoo and Suttle, 1991; Abeles et al., 1992). Ethylene is implicated in the promotion of germination of non-dormant seeds of many species (Corbineau et al., 1990; Esashi, 1991; Kepczynski and Kepczynska, 1997; Matilla, 2000). Ethylene biosynthesis pathway in germinating seeds is the same as the one described in other plant organs, 1-aminocyclopropane-1-carboxylic acid (ACC) is oxidized by ACC oxidase (ACO) to form ethylene. Responses to ethylene in Arabidopsis are mediated by a family of five receptors called Ethylene Response1 (ETR1), ETR2, Ethylene Insensitive 4 (EIN4), Ethylene Response Sensor 1 (ERS1), and ERS2 (Chang et al., 1993; Hua and Meyerowitz, 1998; Hua et al., 1998; Sakai et al., 1998). CTR1 acts as a negative regulator of the pathway that, in turn, inhibits downstream components of the pathway, preventing ethylene responses (Kieber et al., 1993).

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Transcriptional regulation of gene expression

The growth, development, and function of an organism is a reflection of gene expression. Control of gene expression is exerted by multiple steps such as transcription, RNA splicing, mRNA export, mRNA degradation, translation, and posttranslational events (Figure 7). In the next chapters, the mechanisms of transcription, post-transcriptional and translation events are discussed in details.
Figure 7. An overview of the flow of information from DNA to protein in a eukaryote.

Transcriptional regulation in plants

Gene regulation is essential to life from the simplest virus to the most complex mammal. The transcription of DNA to make messenger RNA is highly controlled by the cell. In order for a higher organism to function, genes must be turned on and off in coordinated groups in response to a variety of situations. For a plant this may be “abiotic” stress such as drought, or heat, “biotic” stress such as insects, viral or bacterial infection. The job of coordinating the function of groups of genes falls to proteins called transcription factors.
Transcription factors are proteins that regulate gene expression. Each gene is preceded by a promoter region that includes a binding site for the RNA polymerase and a variety of other features,
including the “TATA box” (a short segment of repeating thymidine and adenine residues) and one or
more enhancer sites, which serve as a binding location for transcription factors (Roeder, 1996). For a messenger RNA to be created, an initiation complex must assemble in the promoter region. This complex consists of over 40 proteins, including the RNA polymerase, TATA binding protein, and one
or more transcription factors. Transcription factors work by binding to DNA at the enhancer site and/or to other proteins in the initiation complex. Through these interactions, transcription factors are able to control whether RNA polymerase moves forward along the DNA to produce a message.
Transcription factors play important and diverse roles in gene expression, including chromatin remodeling and recruitment/stabilization of the Pol II transcription-initiation complex (Karam et al., 1998). Major classes of transcription factors are activators and repressors (Lefstin and Yamamoto, 1998).
Plant transcription factors contain a variety of structural motifs that allow for binding to specific DNA sequences. These two components of transcription are normally described as cis-acting and trans-acting factors. Cis-acting elements are defined like non-coding DNA sequence in the vicinity of the structural portion of a gene that are required for gene expression. Trans-acting factors were usually considered to be proteins that bind to the cis-acting sequences to control gene expression. The functional analysis of interactions between transcription factors and other proteins is very important for elucidating the role of these transcriptional regulators in different signaling cascades. In table 1, we present an overview of cis-element for the major families of transcription factors in plant.
The development of transcriptomics in seed science has led to considerable progress in the understanding of seed development and germination and its regulation by environmental factors. These studies revealed thousands of differentially expressed genes during these developmental phase transition and provided detailed maps of regulatory processes identified key transcription factors. They include seed maturation regulators such as ABSCISIC INSENSITIVE 3 (ABI3), genes involved in hormonal regulation or specific genes of dormancy such as DELAY OF GERMINATION 1 (DOG1) which encodes a protein of unknown function (Bentsink et al., 2006). Studies related to the expression of the Arabidopsis genome following imbibition of mature seeds indicate that there are very large changes in genome expression that are associated with the transition from dormancy to germination (Holdsworth et al., 2008). Using ABA and GA biosynthesis and signalling mutants, it has been demonstrated that these two hormones play essential but antagonistic roles in dormancy and germination. The balance between the levels of these two hormones and their respective signalling pathways regulate both induction and maintenance of dormancy, and promotion of germination in relation to the history of the maternal environment (Finkelstein et al., 2008). New developments in – omics approaches consist of generating gene regulatory networks that reveal developmental modules and key regulators related to seed germination (Bassel et al., 2011; Figure 8) and longevity (Verdier et al., 2013).

Table of contents :

I. State of the art
I.1 Seed dormancy and germination
I.1.1 Arabidopsis seed development and structure
I.1.2 Seed germination
I.1.3 External factors regulating germination
Water content
I.1.4 Seed dormancy
Factors of dormancy
After ripening
I.1.5 Hormonal regulation of dormancy and germination
Abscisic acid
ABA and GA balance
I.2 Transcriptional regulation of gene expression
I.2.1 Transcriptional regulation in plants
I.3 Post-transcriptional regulation of gene expression
I.3.1 Regulation of mRNA translation in plants
Regulation of translation initiation
Regulation of eIF2 activity
Regulation of eIF4E and eIFisoE activity
5′ to 3′ Exoribonucleases
3’-5’ Exoribonucleases
Specialized decay pathways
I.4. Objectives of the thesis
II. Fluctuation of Arabidopsis seed dormancy with relative humidity and temperature during dry storage
II.1 Introduction
II.2 Material and methods
II.3 Results
II.4 Discussion
III. Germination potential of dormant and non-dormant Arabidopsis seeds is driven by distinct recruitment of mRNAs to polysomes 
III.1 Introduction
III.2 Results
III.3 Discussion
III.4 Materiels and methodes
IV. 5′ to 3′ mRNA decay contributes to the regulation of Arabidopsis seed germination by dormancy
IV.1 Introduction
IV.2 Results
IV.3 Discussion
IV.4 Methods


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