• Aucun résultat trouvé

Environmental control of seed germination in Arabidopsis thaliana: the role of GA and ABA signaling pathways

N/A
N/A
Protected

Academic year: 2022

Partager "Environmental control of seed germination in Arabidopsis thaliana: the role of GA and ABA signaling pathways"

Copied!
201
0
0

Texte intégral

(1)

Thesis

Reference

Environmental control of seed germination in Arabidopsis thaliana:

the role of GA and ABA signaling pathways

PISKUREWICZ, Urszula Maria

Abstract

Seed germination is a drastic developmental transition taking the plant from a highly protected, desiccated and quiescent form of life (dry seed) into a more fragile, vegetative seedling. Seed germination is tightly controlled by the environment, which determines the relative levels of two phytohormones: GA (gibberellins) and ABA (abscisic acid).

Consequently, GA and ABA are key regulators of Arabidopsis seed germination. GA stimulates germination and its synthesis upon seed imbibition is necessary for germination to take place. On the other hand, environmental conditions unfavorable for seed germination trigger ABA synthesis, which in turn arrests germination. Until now, metabolic and signaling pathways of these two hormones were studied separately. The purpose of my thesis was to re-examine the role of GA and ABA on the control of seed germination in order to understand how relative levels of these two hormones are coordinated within the seed.

PISKUREWICZ, Urszula Maria. Environmental control of seed germination in

Arabidopsis thaliana: the role of GA and ABA signaling pathways. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4246

URN : urn:nbn:ch:unige-122358

DOI : 10.13097/archive-ouverte/unige:12235

Available at:

http://archive-ouverte.unige.ch/unige:12235

Disclaimer: layout of this document may differ from the published version.

(2)

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Départment de botanique Professeur Luis Lopez-Molina et de biologie végetale

Environmental Control of Seed Germination in Arabidopsis thaliana:

the Role of GA and ABA signaling pathways

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Urszula Maria Piskurewicz de

Pologne

Thèse No 4246

GENÈVE

Atelier d’impression ReproMail 2010

(3)
(4)

Table of contents

Table of contents ... 1

Summary ... 7

Résumé ... 9

Abbreviations ... 11

Introduction ... 12

Dry seed properties ... 12

Dry seed anatomy ... 12

Embryo growth arrest in developing seeds is orchestrated by ABA ... 13

Embryonic growth is arrested upon completion of the first phase of embryogenesis ... 13

The second phase of embryogenesis involves accumulation of food reserves and desiccation tolerance proteins ... 14

Food reserves accumulation ... 14

Desiccation tolerance acquisition ... 15

Seed dormancy is likely established during seed maturation ... 15

Dry seed content ... 17

Control of non-dormant seed germination ... 17

Highlights of seed germination ... 17

Control of seed germination by the seed coat ... 19

A role of endosperm as a nutritive tissue ... 19

The endosperm undergoes a process of weakening ... 20

(5)

ABA- and GA-dependent physiological and gene expression processes occur in endosperm

cells ... 20

Control of seed germination by GA ... 21

GA biosynthesis ... 22

The synthesis of ent-kaurene, the first committed step in GA synthesis ... 22

Cytochrome P450 oxygenases convert ent-kaurene to GA12 ... 23

Oxydation by GA20- and GA3-oxidases leads to production of bioactive GA ... 24

Subcellular localization of GA biosynthetic genes ... 25

Localization of GA biosynthetic genes during seed germination ... 26

GA catabolism ... 27

2β-hydroxylation is the main process of GA inactivation ... 27

Other GA inactivation mechanisms ... 27

GA homeostasis and feedback mechanism ... 28

GA perception and signal transduction ... 28

The GA receptor is encoded by GID1 ... 28

Characterization of DELLA factors ... 29

GA-mediated degradation of DELLA factors ... 30

A Model for GA-dependent DELLA protein degradation ... 30

Regulation of DELLA factors activity ... 31

DELLA protein phosphorylation ... 31

O-GlcNAc- modification by SPY ... 32

Inactivation by sequestration into a complex ... 33

Potential DELLA target genes ... 33

Global gene expression analysis ... 33

Attempts to find direct DELLA targets ... 34

DELLAs might sequester transcription factors to modulate gene expression ... 35

Control of seed germination by ABA ... 36

ABA biosynthesis ... 36

Plastid- localized ABA biosynthesis pathway ... 36

The cytoplasm- localized ABA biosynthetic pathway ... 38

ABA catabolism ... 38

ABA inactivation by hydroxylation ... 38

ABA inactivation by conjugation with glucose ... 39

The ABA signal transduction pathway ... 39

A role of PP2C protein phosphatases in ABA signal transduction ... 40

A role of bZIP transcription factors in ABA signal transduction ... 41

Other transcription factors involved in ABA signal transduction ... 42

Other ABA-Insensitive loci ... 43

A role of SnRK2 protein kinases in ABA signal transduction ... 43

(6)

ABA receptors ... 45

G proteins as a putative ABA receptor ... 45

Magnesium-protoporphyrin IX chelatase as a putative ABA receptor ... 46

Additional approaches to identify ABA receptors ... 46

A model for ABA perception and signal transduction ... 47

Regulation of GA and ABA levels during seed germination ... 49

Control of seed germination by light ... 50

Light perception by phytochromes induces changes in endogenous GA and ABA levels 50

Control of dormant seed germination ... 52

Dormancy is imposed during the seed maturation phase ... 52

A key role of de novo ABA synthesis on dormancy maintenance ... 53

The seed coat is necessary to maintain dormancy ... 54

A role of testa in dormancy maintanance ... 54

A role of endosperm on dormancy maintenance ... 55

Genetic determinants of dormancy ... 55

DOG1, the first dormancy-promoting gene isolated after QTL analysis ... 55

RDO, the role of histone ubiquitination in dormancy maintenance ... 56

Dormancy breaking factors ... 57

After-ripening ... 57

Exposure to nitrate breaks seed dormancy ... 57

Objectives and results of this study ... 58

Results ... 62

The Gibberellic Acid Signaling Repressor RGL2 Inhibits Arabidopsis Seed Germination by Stimulating Abscisic Acid Synthesis and ABI5 Activity .... 62

The GA-signaling repressor RGL3 represses testa rupture in response to changes in GA and ABA levels ... 80

Far-red light inhibits germination through DELLA-dependent stimulation of

ABA synthesis and ABI3 activity ... 84

(7)

A seed coat bedding assay shows that RGL2-dependent release of ABA by the

endosperm controls embryonic dormancy in Arabidopsis ... 98

Discussion ... 117

Environmental control of seed germination involves changes in GA and ABA levels ... 117

The role of DELLA factors to repress testa rupture ... 118

The role of DELLA factors to sustain high endogenous ABA levels under low GA conditions ... 120

DELLA factors promote high ABA accumulation in imbibed seeds ... 120

DELLAs may sustain high ABA levels by stimulating XERICO expression ... 122

How does ABA inhibit endosperm rupture? ... 124

Regulation of DELLA factors expression ... 125

ABA promotes RGL2 mRNA accumulation ... 125

The role of DELLA factors in promoting seed dormancy ... 126

ABA levels follow a similar profile in dormant and ga1 seeds upon imbibition ... 126

Potential mechanisms breaking dormancy ... 128

Perspectives for future studies ... 129

How do DELLA factors inhibit testa rupture and promote ABA synthesis during seed germination? ... 129

How is DELLA factor activity regulated? ... 129

How is dormancy broken? ... 131

References ... 132

(8)

Acknowledgments ... 146

Appendices ... 147

Supplementary figures for: The Gibberellic Acid Signaling Repressor RGL2 Inhibits Arabidopsis Seed Germination by Stimulating Abscisic Acid Synthesis and ABI5 Activity ... 147

Supplementary figures for: Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity ... 163

Supplementary figures for: A seed coat bedding assay shows that RGL2- dependent release of ABA by the endosperm controls embryonic dormancy in Arabidopsis ... 175

Material and Methods ... 181

Abstract ... 181

Introduction ... 181

Materials ... 182

Total RNA Isolation from Arabidopsis Seed Material ... 182

Genomic DNA Isolation from Arabidopsis Seed Material ... 183

Standard Protein Isolation for Immunoblot Analysis ... 183

Acetone Precipitation of Proteins for Immunoblot Analysis ... 183

Optimization of Protein Transfer to Membrane for Immuno-Detection ... 183

In Vivo Labeling of Proteins ... 184

Immunopurification of Protein for Radio-Detection or Mass Spectrometry Analysis184

Methods ... 184

(9)

Total RNA Isolation from Arabidopsis Seed Material ... 184

Preparing Total RNA for Quantitative RT-PCR or Microarray-Analysis ... 185

Genomic DNA Isolation from Arabidopsis Seed Material ... 186

Standard Protein Isolation for Immunoblot Analysis ... 187

Acetone Precipitation of Proteins for Immunoblot Analysis ... 187

SDS-Polyacrylamide Gel Assembly ... 188

Optimization of Protein Transfer to Membrane for Immuno-Detection ... 188

Transfer by diffusion: ... 189

In Vivo Labeling of Proteins ... 189

Immunopurification of Proteins for Radio-Detection or Mass Spectrometry Analysis189

Notes ... 190

References ... 192

Figures: ... 193

(10)

Summary

Seed germination is a drastic developmental transition taking the plant from a highly protected, desiccated and quiescent form of life (dry seed) into a more fragile, vegetative seedling.

Seed germination is tightly controlled by the environment, which determines the relative levels of two phytohormones: GA (gibberellins) and ABA (abscisic acid). Consequently, GA and ABA are key regulators of Arabidopsis seed germination. GA stimulates germination and its synthesis upon seed imbibition is necessary for germination to take place. On the other hand, environmental conditions unfavorable for seed germination trigger ABA synthesis, which in turn arrests germination. ABA and GA levels are negatively correlated during seed germination: seeds that germinate have high GA and low ABA levels, and seeds that are arrested have low GA and high ABA levels. Until now, metabolic and signaling pathways of these two hormones were studied separately. However, available evidence suggests that GA-and ABA-dependent control of seed germination does not operate independently and cross-talk between these two phytohormones is expected.

The purpose of my thesis was to re-examine the role of GA and ABA on the control of non- dormant seed germination in order to understand how relative levels of these two hormones are coordinated within the seed. We have shown that when GA synthesis is inhibited, DELLA factors are necessary to: 1) block testa rupture, 2) stimulate de novo ABA synthesis. Elevation of endogenous ABA levels, resulting from de novo synthesis of ABA, not only promotes accumulation and activity of ABA response factors, ABI5 and ABI3, to block endosperm rupture but also stimulates RGL2 mRNA accumulation creating a positive feedback loop that ensures that RGL2 levels remain high. We showed that the role of GAI and RGA to inhibit testa rupture and elevate ABA levels becomes important under canopy conditions, where a stabilized PIL5, a bHLH transcription factor, stimulates their accumulation. We showed that ABI5’s activity to repress endosperm rupture is positively regulated by phosphorylation via a SnRK2-like type kinase (PKABA1). We showed that DELLA-dependent elevation in endogenous ABA levels might involve XERICO, a zinc-finger protein that contains a RING-H2 motif. Finally, we have applied our new model for the control of non-dormant seed germination to describe the phenomenon of seed dormancy. We showed that dormancy is a state where GA-dependent down-regulation of

(11)

RGL2 expression is not taking place leading to constitutively high ABA levels upon imbibition of a dormant seed, thus ensuring that germination remains repressed.

(12)

Résumé

La germination de la graine permet la transition d'un organisme quiescent, desséché et hautement protégé (la graine sèche) en une plantule autotrophe, plus fragile et exposée. Cette transition est finement contrôlée par l'environnement, qui régule notamment le niveau de deux phytohormones : les gibbérellines (GA) et l'acide abscissique (ABA). GA et ABA sont deux régulateurs majeurs de la germination des graines d'Arabidopsis. En effet, les gibbérellines stimulent la germination et leur synthèse après imbibition des graines est absolument requise afin de permettre cette transition développementale. Au contraire, des conditions environnementales défavorables à l'établissement d'une nouvelle plantule induisent la synthèse d'ABA, qui est responsable de l'arrêt de la germination dans ces conditions. Les niveaux de GA et d'ABA sont inversement corrélés durant la germination : les graines qui germent présentent des niveaux élevés de GA et de faibles quantités d'ABA, alors que des graines arrêtées ont des niveaux élevés d'ABA et de faibles quantités de GA. Les études sur les voies métaboliques et de signalisation de ces deux hormones ont toujours été réalisées séparément jusqu'à maintenant. Cependant, des données suggèrent que GA et ABA ne contrôlent pas la germination de manière indépendante mais qu'il existe une interaction entre ces deux voies pour finement réguler cette transition développementale critique.

L'objectif de ma thèse était de réexaminer les fonctions respectives de l'ABA et des gibbérellines dans le contrôle de la germination de graines d'Arabidopsis non dormantes, dans le but de mieux comprendre comment sont coordonnées ces deux voies hormonales au sein de la graine. J'ai montré que, lorsque la synthèse de GA est inhibée, les facteurs de type DELLA sont nécessaires pour : 1/

bloquer la rupture des téguments, et 2/ stimuler la synthèse de novo d'ABA. L'élévation des niveaux d'ABA endogène, résultant d'une synthèse de novo, induit non seulement l'accumulation et l'activité des facteurs de réponse à l'ABA (ABI3 et ABI5) pour réprimer la rupture de l'albumen, mais stimule également l'accumulation des transcrits RGL2, initiant ainsi une boucle de rétroaction positive qui assure le maintien de niveaux élevés de RGL2. J'ai également montré que l'activité de répression de la rupture de l'albumen par ABI5 est induite par la phosphorylation de ce facteur par une protéine kinase de type SnRK2. J'ai aussi montré que l'élévation des niveaux d'ABA médiée par les facteurs de type DELLA pourrait impliquer XERICO, une protéine à doigts de zinc contenant un motif RING-H2. Enfin nous avons appliqué notre nouveau modèle pour le contrôle de la germination de graines non dormantes au cas des graines dormantes. J'ai pu montrer que la

(13)

dormance est un état dans lequel les gibbérellines sont incapables d'induire la diminution des niveaux de RGL2, maintenant ainsi de manière constitutive des niveaux élevés d'ABA après imbibition, ce qui assure une répression efficace de la germination.

(14)

Abbreviations

ABA: abscisic acid

ABF: ABRE binding factor ABH: ABA Hypersensitive ABI: ABA Insensitive

ABRE: ABA Responsive Element BG: beta-glucosidase

bHLH: basic Helix-Loop-Helix CHLH: Mg-chelatase H subunit DOG1: Delay Of Germination DPA: dihydrophaseic acid

EUI: Elongated uppermost internode FUS: Fusca

GA: Gibberellins GAI: GA-Insensitive

GAMT: GA methyl transferase GFP: Green Fluorescent Protein GID: GA Insensitive

GUS: Glucuronidase

HUB1: Histone Monoubiquitination1 LEA: Late Embryogenesis Abundant LEC: Leafy cotyledon

NIL: Near Isogenic Lines OST: Open stomata PA: phaseic acid

PIF: Phytochrome Interacting Factor PIL: Phytochrome Interacting Factor- Like

PP2C: Protein Serine/Threonine phosphatase class C

QTL: Quantitative Trait Loci RDO: Reduced Dormancy RGA: Repressor of ga1-3 RGL: RGA- Like

SLR1: Slender rice SLY: Sleepy

SnRK: Sucrose non-fermenting 1- Related Kinase

(15)

Introduction

Most plants are sessile organisms, and therefore they cannot avoid harsh environmental conditions. Thus, the place where a seed germinates determines where the plant spends its whole life. Given these constraints, it is perhaps unsurprising that land plants have evolved the capacity to adjust their developmental programs to better cope with environmental changes. Seed germination is no exception and is tightly controlled by the surrounding environment. This work addresses the question how environmental cues are integrated within seeds to influence their ability to germinate. Arabidopsis thaliana is a model organism for studying plant biological processes genetically and we have used Arabidopsis seeds to study control of germination. As an introduction, I will first describe the main properties of dry seeds that allow them to survive years of harsh environmental conditions. Then, I will describe the onset of processes that leads to the transition from a dry seed to a seedling under favorable environmental conditions. Finally, I will describe metabolic and signaling pathways of two key plant hormones controlling seed germination.

Dry seed properties

Dry seed anatomy

Dry seeds are the end point of embryogenesis. They consist of an embryo that is surrounded by a seed coat (see Figure 1). The Arabidopsis seed coat consists of a single-cell layer of endosperm tissue that surrounds the embryo and outer layer of dead tissue called testa.

Figure1. The anatomy of an Arabidopsis seed. Source:

(16)

Embryogenesis begins wit and sperm cell nuclei into a zygote that further develops into a plant embryo, and 2) the fusion of the two central cell nuclei with a second sperm cell nucleus, thus forming a primary (Goldberg et al., 1994). This triploid tissue divides rapidly and becomes the food source for embryo development and later on for germination (Berger, 1999). As the embryo grows within the seed, the endosperm is progressively absorbed by the embryo so that upon completion of embryogenesis the mature Arabidopsis seed contains only one cell layer of endosperm (Berger, 1999). The testa develops from the tissue originally surrounding the ovule (the integuments), and is therefore of maternal origin. The endosperm is physiologically active, in contrast to the testa whose cells died during the late phase of embryogenesis (Haughn and Chaudhury, 2005). The seed coat protects the embryo from mechanical injury and a harsh environment.

Embryo growth arrest in developing seeds is orchestrated by ABA

As a dry seed, the plant is able to pause its life cycle and withstand unfavorable conditions for extended periods of time. The important properties of a dry seed that allow this selective advantage are acquired during two major phases of seed formation: in the first phase the embryo development takes place and in the second one seed maturation processes occur.

Transcription factors such as LEC1, LEC2, FUS3 and ABI3 are essential for orchestrating those processes (To et al., 2006) and the phytohormone abscisic acid (ABA) is important for regulating their expression and activity (Finkelstein et al., 2002). FUS3, LEC2 and ABI3 are three homologous B3 transcription factors (Giraudat et al., 1992; Luerßen et al., 1998; Stone et al., 2001) and LEC1 encodes a protein related to the mammalian HAP3 subunit of CCAAT-binding factor, suggesting that LEC1 is also a regulator of transcription (Lotan et al., 1998).

Embryonic growth is arrested upon completion of the first phase of embryogenesis

The first phase of embryogenesis is morphogenesis, which starts with the formation of a single-cell zygote and ends in the heart stage when all embryo structures have been formed (Mayer et al., 1991). It is followed by a growth phase during which the growing embryo

(17)

eventually fills the seed sac (Goldberg et al., 1994). During the embryonic growth phase the volume of the Arabidopsis embryo increases about tenfold, while that of the endosperm is reduced. At the end of the embryonic growth phase, cell division in the embryo arrests. This growth arrest correlates with increased concentration of ABA in the seed. It was shown that ABA can induce the expression of a cyclin-dependent kinase inhibitor (ICK1), which may account for the cell cycle arrest atthe G1/S transition (Wang et al., 1998). Increased levels of ABA at this stage of seed development are important for the regulation of expression and activity of LEC1, FUS3 and ABI3 for preventingpremature germination at the end of the cell division phaseof embryogenesis (Raz et al., 2001). Indeed, when wild type embryos from the heart stage until the early seed maturation stage were excised from the seed and plated on water-agar plates, embryos showed no growth capacity and remained the same size (Raz et al., 2001). However, embryos of fus3, lec1, lec2 and abi3 excised at the same stage as wild type, continued growth and developed into seedlings, suggesting that FUS3, LEC1, LEC2 and ABI3 are four transcription factors necessary to exert embryonic growth arrest upon completion of the first phase of embryogenesis (Raz et al., 2001).

The second phase of embryogenesis involves accumulation of food reserves and desiccation tolerance proteins

The second phase of embryogenesis (the so called “maturation phase”) involves food reserves accumulation and acquisition of dormancy and desiccation tolerance (Goldberg et al., 1994). Similar to the first phase of seed development, processes occurring in the second phase are coordinated by LEC2, FUS3 and ABI3, while ABA keeps orchestrating their expression and activity (Finkelstein et al., 2002).

Food reserves accumulation

Maturing seeds accumulate lipids and storage proteins in the embryo as well as in the endosperm cells (Baud et al., 2002). Two major Arabidopsis seed storage proteins (SSP) are 2S and 12S, and they can represent up to one third of the seed’s dry weight (Pang et al., 1988;

(18)

Guerche et al., 1990; Baud et al., 2002). SSP genes are highly expressed and tightly regulated both spatially and temporally. FUS3, LEC2 and ABI3 were shown to control At2S3 storage protein accumulation (Kroj et al., 2003). It was suggested that FUS3 and LEC2 promote At2S3 expression by directly binding to its promoter sequences, while ABI3 acts more indirectly, for example playing a role of a cofactor in an activation complex (Kroj et al., 2003).

Desiccation tolerance acquisition

During late maturation phase, specific proteins referred to as Late Embryogenesis Abundant (LEA) proteins accumulate in the embryo (Delseny et al., 2001). Based on their accumulation pattern and biochemical properties, LEA proteins were proposed to be involved in the seed desiccation tolerance. AtEm1 and AtEm6 are two proteins that belong to the LEA group and are believed to protect the Arabidopsis embryo during the phase of desiccation by replacing water, thus providing alternative hydrogen bonding opportunities (Bray, 1993). A common feature of the Em genes in all plant species studied so far is that their expression is modulated by the plant hormone ABA (Williamson et al., 1985). Indeed, Em genes can be induced in immature seeds or young seedlings by exogenously applied ABA (Soderman et al., 2000). Similarly, the expression of those genes is reduced in ABA-deficient and ABA-insensitive mutants (Finkelstein and Lynch, 2000; Lopez-Molina L, 2000; Soderman et al., 2000; Carles et al., 2002). Expression analysis of Em genes showed that their expression depends on the accumulation of LEC1, FUS3, ABI3, ABI4 and ABI5 (Parcy et al., 1994; Parcy et al., 1997; Lopez-Molina L, 2000; Soderman et al., 2000; Carles et al., 2002). ABI5 was shown to directly bind to promoter sequences of AtEm genes (Carles et al., 2002; Lopez-Molina et al., 2002). This suggests that ABA promotes Em accumulation by stimulating the expression and activity of ABA-induced transcription factors, which in turn promote Em expression.

Seed dormancy is likely established during seed maturation

Finally, seed dormancy is established during the maturation phase. Dormancy is a property of a mature, viable dry seed that prevents its germination even under favorable environmental conditions, unless the state of dormancy is alleviated. Dormancy may represent an

(19)

adaptive trait that decreases competition between individuals of the same species and/or prevents germination out of the season.

In nature, numerous factors may contribute to break seed dormancy, thus allowing seeds to germinate. Among them, after-ripening (dry storage of seeds for extended period of time) and stratification (water imbibition of seeds in cold and darkness) are wide spread environmental factors breaking dormancy. Different Arabidopsis accessions show various levels of seed dormancy. The commonly used Landsberg erecta or Columbia ecotypes show low levels of dormancy and their seeds germinate after a very short period of after-ripening (2-4 weeks).

Ecotypes such as Cape Verde Island (Cvi) or C24 show high levels of dormancy and their seeds will not germinate unless stratified or after-ripened for extended period of time (several weeks of dry storage for Cvi).

The mechanism by which dormancy is first established and later alleviated is not known however, ABA is suggested to play a crucial role on dormancy imposition. Indeed, several mutants with altered ABA biosynthesis or signaling pathways produce seeds that are viviparous i.e. their embryo growth within a seed is not arrested and they germinate precociously on the mother plant (McCarty et al., 1989; Nakashima et al., 2009). However, as described above, ABA plays a crucial role during seed development, thus ABA deficient mutants might produce seeds that lack many properties of the “normal”, mature wild type seed. Similarly, the four ABA- induced transcription factors mentioned above (ABI3, FUSCA3, LEC1 and LEC2) were proposed to play an important role in dormancy imposition mostly due to the fact that mutations in each of those genes lead to non dormant seed production. However, abi3, fus3, lec1 and lec2 mutant seeds lack the important properties of a mature dry seed. Indeed, they are not desiccation tolerant, they have decreased expression of seed storage proteins and lec1 and lec2 mutants have defects in embryonic cotyledon identity (Parcy et al., 1994; Kroj et al., 2003). Thus, it is difficult to conclude what is the role of ABI3, FUS3, LEC1 and LEC2 in maintaining dormant state of a seed once the fully mature seed is produced. Indeed, the differences in LEC1, FUS3 and ABI3 expression during seed development of various strong or moderate dormancy seeds were not found (Baumbusch et al., 2004).

(20)

Dry seed content

In summary, at maturity a wild type dry seed will have accumulated: storage compounds that will later fuel the process of germination, osmoprotective proteins AtEm1 and AtEm6 and transcription factors that at later stages of maturation promoted their accumulation ( for example ABI3 and ABI5). Dry seeds still contain high levels of ABA that play an important role in orchestrating seed maturation processes. Dry seeds have very low water content (5-10% of weight) and, when freshly harvested from the mother plant, seeds are dormant. In such dry and highly protected form, dry seeds can survive harsh environmental conditions for many years awaiting proper conditions to initiate germination.

In the next chapter I will describe factors that control germination of a seed that has lost its dormancy during after-ripening. As mentioned above, the ecotypes commonly used to study seed germination show moderate levels of dormancy and their seeds germinate after 3-4 weeks of after-ripening. In the last chapter of Introduction I will further describe seed dormancy.

Control of non-dormant seed germination

Highlights of seed germination

Under favorable germination conditions, i.e. proper temperature and light conditions, imbibition by water triggers non-dormant seed germination. The first visible germination event is rupture of the testa layer which takes place approximately 24 hours after seed imbibition (see Figure 2). Thereafter, about 36 hours after imbibition, the elongation of the embryonic axis pierces the layer of the endosperm. Endosperm rupture is a common criteria to define seed germination (Kucera B, 2005). Next, the embryonic axis further elongates, cotyledons soon expand and start greening so that 48 hours after seed imbibition the autotrophic phase of the plant life cycle is established.

(21)

Figure 2 Highlights of wild type seed germination under normal germination conditions

At the molecular level, dry seed imbibition triggers rapid down-regulation of ABA levels within the first 12 hours, degradation of osmoprotective proteins (AtEm1, AtEm6) and mobilization of food storage compounds. Thus, the plant embryo abandons its highly protected form and enters a more fragile stage. For successful seedling establishment, proper environment conditions are crucial. Temperature, water availability and light quality are sensed by a seed and trigger internal signals that determine its developmental fate. Two phytohormones: abscisic acid (ABA) and gibberellins (GA) are central players contributing to seed germination. GA synthesis starts upon seed imbibition (Ogawa et al., 2003) and GA is essential for the early germination events of testa and endosperm rupture to occur. Indeed, ga1-3 mutants (unable to synthesize GA) are unable to rupture testa and endosperm unless exogenous GA is provided (Koornneef M., 1980). Similarly, under the canopy, when GA synthesis in imbibed seeds is repressed, seeds fail to rupture testa and endosperm (Oh et al., 2007).

ABA plays a role during embryogenesis to promote seed maturation. In the vegetative phase of the plant life cycle, ABA acts as a stress hormone. In particular, ABA is synthesized upon osmotic stress to promote stomata closure (Merlot et al., 2002; Mustilli et al., 2002).

During germination a sudden osmotic stress will trigger ABA synthesis, which in turn will: 1) arrest germination (including endosperm rupture, axis elongation, cotyledon expansion and greening)(Finkelstein et al., 1998; Finkelstein and Lynch, 2000; Lopez-Molina L, 2000;

Kinoshita et al., 2010), 2) promote osmotolerance by stimulation of AtEm1 and AtEm6 expression (Finkelstein and Lynch, 2000; Lopez-Molina L, 2000; Carles et al., 2002). Although ABA is very efficient in inhibiting endosperm rupture and embryonic axis elongation, it does not prevent rupture of the testa (Muller et al., 2006; Piskurewicz et al., 2008).

Because of these key properties of GA and ABA, it has been commonly assumed that the relative ratio of those two antagonistic phytohormones determines the seed potential to germinate

Testarupture Endospermrupture Full radical tip elongation

Cotyledon expansion and greening

(22)

(Oh et al., 2007).

Control of seed germination by the seed coat

As mentioned above, the ga1 mutant cannot germinate. However, when the seed coat (i.e.

testa and endosperm) is removed, ga1 embryos develop into young seedlings even in the absence of exogenous GA application. This observation led to the conclusion that GA is required to overcome the germination constraint imposed by the seed coat (Debeaujon and Koornneef, 2000). However, due to the fact that the seed coat is composed of two distinct tissue layers (testa and endosperm) it is still unclear which of them plays a more prominent and active role. Because the testa is composed of cells that are dead, it might indicate that only living endosperm cells are actively inhibiting seed germination by a yet unidentified mechanism. Consistent with this view, removal of the testa layer from imbibed, dormant wild type seeds (that cannot germinate, similar to ga1-3 seeds) was not sufficient to trigger their germination as long as the endosperm layer was covering the embryo (Bethke et al., 2007). This experiment was not performed on ga1-3 seeds, however it strongly suggests that the endosperm layer plays a predominant inhibitory role to maintain seed dormancy (see below) and control germination.

A role of endosperm as a nutritive tissue

The importance of the endosperm layer was mostly studied in the context of its role as a nutritive tissue (Penfield et al., 2004; Penfield et al., 2005). Indeed, numerous protein storage vacuoles (PSV) and oleosomes occupy most of the mature endosperm cell volume (Bethke et al., 2007). Within the first 12 hours upon seed imbibition, the amount of PSVs per cell transiently increases, but thereafter a progressive reduction in the number of PSVs per cell occurs. It has been observed that these small PSVs merge to form a single large vacuole (a process called vacuolation). Various germination stimulants increase endosperm vacuolation even in endosperm cells that are separated from the embryo (Bethke et al., 2007). Vacuolation of the endosperm cells correlates with the ability of the seed to germinate and it has become a convenient marker of endosperm activity during seed germination.

(23)

The endosperm undergoes a process of weakening

Less in known about a putative role of the endosperm layer as a constraint to radicle protrusion. It was proposed that prior to radicle protrusion, the endosperm undergoes the process called weakening, during which cell wall modifying enzymes (mostly hydrolases) trigger loosening of endosperm cell adhesions. Indeed, upon imbibition, Arabidopsis endosperm cells near the radicle tip became less angular at the time of germination (Bethke et al., 2007; Belin C, 2010) suggesting that thinning and weakening of the cell walls was occurring and cells are assuming a more rounded shape since they are no longer attached to each other. After several days of seed imbibition endosperm cells (mostly those surrounding the radicle tip) become spherical, with only a thin band of cell wall that remains (Bethke et al., 2007; Belin C, 2010).

However, in Arabidopsis, enzymes involved in endosperm cell wall modifications are not known. It was proposed that in the Solanaceae family such enzymes are endo-β-mannanases and β-1,3-glucanases ( reviewed in (Leubner-Metzger, 2003; Yuan JS., 2007). Indeed, β-1,3- glucanase is induced after testa rupture and just prior to endosperm rupture in Nicotiana tabacum seeds. β-1,3-glucanase is localized in the micropylar endosperm where the radicle will emerge and its expression is strongly repressed by ABA treatment (Leubner-Metzger et al., 1995).

ABA- and GA-dependent physiological and gene expression processes occur in endosperm cells

The inhibitory effect of ABA on endosperm weakening was further evidenced by direct measurement of endosperm weakening by the puncture force method in Lepidium sativum, a close relative of Arabidopsis (Muller et al., 2006). Those experiments directly showed that endosperm weakening was inhibited by ABA and promoted by GA and that it was an organ- autonomous process, i.e. endosperm tissue separated from the embryo was able to loosen its cells adhesions and respond to exogenously added ABA and GA. These results confirm that the endosperm is a biologically active tissue, and show that it is able to synthesize factors needed for its weakening (Muller et al., 2006). It was also shown that endosperm weakening required induction by an early signal from the embryo (Muller et al., 2006). Consistent with endosperm being an active tissue, transcriptome analysis of Arabidopsis endosperms revealed that it highly

(24)

expresses many classes of genes, for example those involved in storage reserve mobilization, protein degradation and amino acid transport (Penfield et al., 2006). Moreover, ABA treatment led to changes in expression of many cell wall-related enzymes in the endosperm. Importantly, the endosperm expressed all ABA and GA biosynthetic and catabolic enzymes, as well as all known signaling components of these pathways suggesting that it might play an active role in GA- and ABA-dependent control of Arabidopsis seed germination (Penfield et al., 2006).

Control of seed germination by GA

Gibberellins were first isolated from the fungus Gibberella fujikuroi in 1926 by the Japanese scientist Eiichi Kurosawa, who was investigating the causative agent of the “foolish seedling” disease of rice. Diseased rice seedlings exhibited excessive shoot elongation and when they reached maturity, their grains were either absent or poorly developed. Kurosawa demonstrated that diseased rice plants were infected with a fungal pathogen, G. fujikuroi, which secreted a factor that increased the rate of shoot elongation. This fungal factor was crystallized and named gibberellin. Gibberellins were isolated also from healthy (i.e. not infected by fungi) plants and were shown to be not only fungal metabolites but also endogenous regulators of many aspects of growth and development of plants. In Arabidopsis, in addition to its role to promote seed germination, GA promotes leaf and root growth, floral induction under short-day conditions, inflorescence stem elongation, anther and petal development, and fruit and seed development.

Gibberellins (GAs) are a group of tetracyclic diterpenes. The GAs can be divided into two groups: C20-GAs, which contain 20 carbon atoms, and C19-GAs, which have lost the C-20 and carry a γ-lactone ring (see Figure 3). Variations in the types of gibberellins result from differences in the substituent groups (for example hydroxyl group) or in the sequence in which groups are added to the GA skeleton. Among 136 gibberellins that have been identified so far only few are biologically active (for example GA1, GA3, GA4 and GA7) (Figure 3). The number of a GA is based on the chronological order of its discovery and not on its position in the biosynthetic pathway. The detailed analysis of metabolic genes expression, combined with the analysis of changes in the contents of precursors, active and deactivated gibberellins suggested

(25)

that GA4 is the main endogenous active gibberellin in germinating seeds of Arabidopsis thaliana (Ogawa et al., 2003). However, it cannot be excluded that other bioactive forms of gibberellins might contribute to germination control. Thus, throughout my thesis, GA is a generic term describing all the gibberellins that are necessary, and therefore active, to promote seed germination. Most of the non-active gibberellins are precursors or catabolites of bioactive GA.

Figure 3. Major bioactive forms of GAs. Source: Tai-ping Sun (2008) Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis. September 24, 2008. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists. http://www.aspb.org/publications/arabidopsis/

GA biosynthesis

Biosynthesis of GA can be divided into three stages:

1) synthesis of ent-kaurene from geranyl geranyl diphosphate (GGDP) in plastids, 2) conversion of ent-kaurene to GA12 via cytochrome P450 monooxygenases associated with the endoplasmic reticulum

3) synthesis of GA intermediates and synthesis of active GA in cytoplasm.

The synthesis of ent-kaurene, the first committed step in GA synthesis

GGDP is a common precursor for GA, carotenoids and chlorophylls synthesis. Because ABA is formed by cleavage of carotenoids (see below), GGDP is also a precursor for ABA

(26)

synthesis. GGDP is converted to ent-kaurene, the first committed intermediate in the GA biosynthesis pathway by a two-step cyclization reaction: the first one is catalysed by ent-copalyl diphosphate synthase (CPS) and the second one by ent-kaurene synthase (KS) (Figure 4). In Arabidopsis CPS is encoded by a single gene GA1 (Kamiya, 1994) and KS by a single gene GA2 (Yamaguchi S., 1998) (see Table1). These two terpene synthases, catalyzing the two consecutive cyclization reactions, may form a complex (West C., 1982). ga1 and ga2 null mutants are not able to germinate due to the lack of GA synthesis. However, after seed coat removal, embryos of ga1 and ga2 develop into severe dwarf and male-sterile plants. All those defects can be reversed by exogenously applied GA (Koornneef M., 1980). GA1 mRNA levels are low throughout plant development (Silverstone A., 1997b) and they rise in rapidly growing tissues. The expression pattern of GA2 is similar to GA1 with overall higher mRNA levels during plant development (Yamaguchi S., 1998) suggesting that levels of active GA are mostly controlled by expression of GA1. Over-expression of CPS or KS in transgenic Arabidopsis plants did not affect levels of bioactive GA or the plant’s phenotype (Fleet C., 2003) suggesting that other genes encode rate limiting enzymes acting at later stages of GA biosynthetic pathway.

Figure 4. Plastid and ER localized GA biosynthetic pathway. Source: Tai-ping Sun (2008) Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis. September 24, 2008. The Arabidopsis Book. Rockville, MD:

American Society of Plant Biologists. http://www.aspb.org/publications/arabidopsis/

Cytochrome P450 oxygenases convert ent-kaurene to GA12

In the second step of GA synthesis, ent-kaurene is oxidized which is followed by ring contraction. These processes are catalyzed by ent-kauren oxidase (KO) and ent-kaurenoic acid

(27)

oxidase (KAO) and result in the production of GA12 (Figure 4). KO is encoded by a single gene in Arabidopsis: GA3 (Helliwell et al., 1998) and KAO is encoded by two genes: KAO1 and KAO2 (Helliwell, 2001b) (Table1).The ga3 null mutant is non germinating, dwarf and male- sterile plant, and exogenous GA application reverts those defects (Koornneef M., 1980).

Similarly, wild type seeds treated with paclobutrazol (PAC), an inhibitior of KO activity, are not able to germinate due to GA synthesis inhibition. Over-expression of KO and KAO in transgenic Arabidopsis does not lead to changes in bioactive GA levels nor to visible changes in plant phenotype (Swain et al., 2005).

Oxydation by GA20- and GA3-oxidases leads to production of bioactive GA

In the third step of GA synthesis, GA12 can be converted to GA53 by 13-hydroxylation and then these two forms undergo a series of oxidation events catalyzed by GA 20-oxidases (GA20ox) and GA 3-oxidases (GA3ox) to produce bioactive GA 4, GA1 and GA3 (Figure 5).

Figure 5. Cytoplasm localized GA biosynthetic and catabolic pathways. Source: Tai-ping Sun (2008) Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis. September 24, 2008. The Arabidopsis Book.

Rockville, MD: American Society of Plant Biologists. http://www.aspb.org/publications/arabidopsis/

(28)

GA20-oxydases are encoded by a family of five genes: GA20ox1-5 (Phillips A., 1995;

Rieu et al., 2008) (Table 1). GA20ox1, GA20ox2 and GA20ox3 are the most highly expressed GA20ox genes during vegetative and early reproductive stages, as well as during seed germination and in dry seeds (Rieu et al., 2008). Phenotypic analysis of a ga20ox1ga20ox2 double mutant revealed that these two GA 20-oxidases display overlapping roles throughout plant development. However, seed germination in this double mutant was not affected suggesting that other GA 20-oxidases contribute to or can substitute in GA synthesis during seed germination (Rieu et al., 2008). Over-expression of GA20ox leads to higher bioactive GA levels and to early flowering and increased stem growth phenotypes suggesting that they are rate limiting enzymes.

GA3-oxydases are encoded by a four gene family: GA30x1-4 (Chiang et al., 1995;

Yamaguchi et al., 1998; Mitchum et al., 2006)(Table1). GA3ox1 and GA3ox2 are the most highly expressed GA3ox genes during vegetative and reproductive stages as well as during seed germination (Mitchum et al., 2006). Only the ga3ox1 ga3ox2 double mutant combination shows a non-germinating phenotype, suggesting that these two GA 3-oxidases act redundantly during germination (Mitchum et al., 2006).

Subcellular localization of GA biosynthetic genes

CPS and KS are localized in the stroma of plastids (Kamiya, 1994; Helliwell, 2001a)(see Figure 6). Transient expression of KO and KAO fused to green fluorescent protein (GFP) in tobacco leaves localized KO to the outer surface of the plastid envelope while KAO localization was associated with the endoplasmic reticulum (ER) (Helliwell, 2001a). The localization of these two enzymes out of the plastid suggests that ent-kauren is exported from the plastid for oxidation processes performed by KO and KAO. The product of those processes, GA12, is further processed in the cytoplasm where the GA20- and GA 3-oxidases are localized.

(29)

Figure 6. Subcellular localization of GA biosynthetic and catabolic pathways. Source: Tai-ping Sun (2008) Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis. September 24, 2008. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists. http://www.aspb.org/publications/arabidopsis/

Localization of GA biosynthetic genes during seed germination

CPS promoter activity during germination was studied in transgenic Arabidopsis plants expressing promoter CPS:CPS-GUS fusion. CPS promoter activity was localized in the shoot apex and the provasculature of cotyledons and in the embryo axis (Silverstone A., 1997b;

Yamaguchi S., 2001)(Figure 7). The observation that transcripts of KO were detected in the cortex and endodermis of the embryo axis (Yamaguchi S., 2001) suggested that ent-kauren is transported from the embryo provasculature to these tissues during GA synthesis in germinating seeds (Figure 7). Transcripts of GA3ox1 and GA3ox2 were localized in the cortex and endodermis of an embryo suggesting that in these tissues bioactive GA is produced during germination (Yamaguchi S., 2001) (Figure 7).

Figure 7. Physical separation of early and later steps in GA biosynthesis in germinating embryos: X-gluc staining of the promoter CPC-CPS-GUS line and in situ hybridization analysis of KO, GA3ox1and GA3ox2 mRNAs. Source:

Tai-ping Sun (2008) Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis. September 24, 2008. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists.

http://www.aspb.org/publications/arabidopsis/

(30)

GA catabolism

2β-hydroxylation is the main process of GA inactivation

Levels of bioactive GA are determined by the rate of its synthesis and degradation.

Bioactive GA and its intermediates are inactivated in the process of 2β-hydroxylation catalyzed by GA 2-oxidases (GA2ox). GA2-oxidases are encoded by an eight member gene family (Thomas et al., 1999) (Schomburg et al., 2003). Each gene shows a tissue specific expression pattern. Over-expression of GA2ox7 or GA2ox8 genes leads to lower GA levels and to a dwarf plant phenotype, typical for mutants with GA deficiency (Schomburg et al., 2003).

Other GA inactivation mechanisms

There are two other mechanisms of GA inactivation: epoxidation (Zhu et al., 2006) and methylation (Varbanova et al., 2007). 16α,17-epoxidation of non-13-hydroxylated GA is catalyzed by cytochrome P450 monooxygenase CYP714D1 that is encoded by the EUI gene in rice (Zhu et al., 2006). Mutation in this gene leads to an extremely elongated uppermost internode in rice with high overaccumulation of bioactive GA in this organ. When EUI is over- expressed, bioactive GA levels decrease, suggesting that EUI deactivates GA (Zhu et al., 2006).

However, functional ortholog(s) of this gene are not known yet in Arabidopsis.

Methylation of bioactive GA was proposed to be an alternative GA inactivation mechanism. The Arabidopsis GA methyltransferases, encoded by GAMT1 and GAMT2, are mainly expressed in siliques and in developing seeds. Whole siliques (including seeds) of single null mutants accumulated higher levels of various gibberellins when compared to WT, with double null mutant accumulating the highest levels of gibberellins, especially of GA1. However, methylated forms of GA were not found in WT siliques (Varbanova et al., 2007). The authors suggested that methylation by GAMT1 and GAMT2 might lead to fast GA inactivation and subsequent degradation during seed maturation (Varbanova et al., 2007).

(31)

GA homeostasis and feedback mechanism

GA biosynthesis and catabolism are under tight control (see below). One of the mechanisms controlling levels of bioactive GA is a negative feedback loop mechanism exerted by GA itself, leading to changes in its biosynthetic and catabolic gene expression. Indeed, application of exogenous GA or mutations leading to higher GA signaling result in down- regulation of GA biosynthetic genes GA20ox1, GA20ox2, GA20ox3 and GA3ox1 (Chiang et al., 1995; Yamaguchi et al., 1998; Thomas et al., 1999; Rieu et al., 2008), while GA catabolic genes GA2ox1 and GA2ox2 are up-regulated (Thomas et al., 1999). Conversely, inhibition of GA biosynthesis or decreased GA signaling leads to up-regulation of GA biosynthetic genes and down-regulation of GA catabolic genes mentioned above. Expression of early GA biosynthetic genes such as GA1, GA2 and GA3 is not affected by feedback mechanism (Helliwell et al., 1998) suggesting that the content of bioactive GA is regulated mostly by the expression of GA20ox1, GA20ox2, GA20ox3, GA3ox1, GA2ox1 and GA2ox2.

GA perception and signal transduction

The GA receptor is encoded by GID1

The GA receptor was first identified in rice by studying the GA-Insensitive Dwarf mutant gid1 (Ueguchi-Tanaka et al., 2005). The identification of the GA receptor was facilitated by the fact that it is encoded by only one gene in rice. In contrast, Arabidopsis contains three GID1 orthologs (GID1a, GID1b, GID1c) that exhibit partially redundant functions (Griffiths et al., 2006; Nakajima et al., 2006). Indeed, none of the single gid1 mutants in Arabidopsis displays an obvious phenotype, the double mutant combinations show partial GA deficiency phenotypes and only the gid1a gid1b gid1c triple mutant is completely non-responsive to GA (Griffiths et al., 2006). Rice and Arabidopsis GID1s were shown to be soluble GA receptors that show a high similarity to hormone-sensitive lipases (HSL) and can bind bioactive GA with high affinity but not inactive GA derivatives. GID1-GFP is localized to both the cytoplasm and nucleus in transgenic plants (Ueguchi-Tanaka et al., 2005). Both the gid1 mutant in rice and the gid1a gid1b gid1c triple mutant in Arabidopsis are GA-non responsive severe dwarfs with defects in

(32)

flowering and the Arabidopsis triple mutant cannot germinate unless the seed coat is removed (Griffiths et al., 2006; Iuchi et al., 2007).

In a current model, GA promotes seed germination, leaf and root growth, floral induction, inflorescence stem elongation, anther and petal development, fruit and seed development because it promotes degradation of regulatory proteins (DELLA factors, described below) that act as repressors of these physiological processes. Consistent with this model, ga1 and gid1a gid1b gid1c mutants accumulate high levels of these repressors.

Characterization of DELLA factors

DELLA factors belong to the plant-specific GRAS family of regulatory proteins that exhibit sequence similarities to each other in their C-termini but vary in their N-terminal sequences (reviewed in (Bolle, 2004). One sub-branch of the GRAS family consists of genes that contain at their N-terminus highly conserved DELLA and VHYNP motifs and a variable region rich in Ser and Thr. These genes are called DELLA genes after their N-terminal domain. There are five DELLA genes in the Arabidopsis genome (RGA, GAI, RGL1, RGL2, RGL3) and one DELLA factor in rice encoded by SLR. All of them function as negative regulators of GA signaling. DELLAs are proposed to be transcriptional regulators, although they do not have a clearly identified DNA binding domain. They are proposed to bind to transcription factors and therefore modulate transcription events (see below). Their repression activities partially overlap, but there are also some distinct characteristics of each DELLA repressor. GAI and RGA play a predominant role in repressing vegetative growth and in the ga1-3/gai/rga mutant vegetative growth is almost completely recovered (Dill, 2001). However, mutations in GAI and RGA do not improve germination or flower development of the ga1-3 mutant, suggesting that other DELLAs control these processes. In particular, although all DELLA genes are expressed upon seed imbibition, only mutation in RGL2 allows ga1-3 seeds to germinate (Lee et al., 2002). Additional mutations in DELLA genes improve the germination potential of the ga1/rgl2 double mutant, suggesting that other DELLA factors contribute to the repression of germination when GA synthesis is prevented (Cao et al., 2005). Similarly, RGA, RGL1 and RGL2 play a role during floral development (Tyler et al., 2004). GAI and RGA mRNA is ubiquitously expressed in all tissues while that of RGL1, RGL2 and RGL3 is mostly expressed in germinating seeds and/or

(33)

flowers and siliques (Tyler et al., 2004) .

GA-mediated degradation of DELLA factors

As mentioned above, characterization of the rice gid1 mutant led to the discovery of the GA receptor. Analysis of another GA-insensitive dwarf rice mutant, gid2, suggested the mechanism by which the GA signal is transmitted. GID2 encodes a protein that contains an F- box motif at its N-terminus. Arabidopsis contains an ortholog of GID2 called SLY1 (Steber CM, 1998). Such proteins are often a part of SCF (SKP1, CULLIN1, F-box)-type E3 ubiquitin ligase complexes that trigger polyubiquitination in specific proteins. Both gid2 and sly1 mutants are GA-unresponsive dwarfs with reduced fertility. Both of them accumulate high levels of DELLA proteins that are not degraded by exogenously applied GA (Sasaki et al., 2003; Dill et al., 2004).

Thus, it is proposed that GID2 (in rice) and SLY1 (in Arabidopsis) specifically recruit DELLA proteins for polyubiquitination and subsequent degradation by the 26S proteasome (Dill et al., 2004; Fu et al., 2004). In addition, sly1 shows very strong seed dormancy that is alleviated after an extended period of after-ripening (Steber CM, 1998; Ariizumi, 2007). SLY1 is expressed in all tissues, while its homolog SNEEZY (SNE) is mostly expressed in flowers (Strader et al., 2004).

A Model for GA-dependent DELLA protein degradation

The discovery of a GA receptor and an F-box containing protein as positive regulators of GA signaling, together with a role of DELLA factors as negative regulators, allowed a model to be proposed for GA signal transduction. In the present model (see Figure 8), bioactive GA binds in the pocket site of the GID1 receptor triggering its conformational change so that its N-terminal part forms a lid to which the DELLA factor binds via its DELLA/ VHYNP domain (Murase et al., 2008; Shimada et al., 2008). Binding of the DELLA protein to the GA-GID1 complex causes a conformational change of the DELLA factor that in turn allows its binding to an F-box protein SLY1/GID2. Interaction between the GA-GID1-DELLA complex and SLY1 triggers DELLA protein ubiquitination and degradation through the 26S proteasome machinery (Bolle, 2004; Dill et al., 2004). Thus, GA promotes expression of its target genes by releasing the repression which is imposed by DELLA factors by triggering their degradation.

(34)

Figure 8. A model of GA-regulated GID1–DELLA protein interactions. Source: K Murase et al. Nature 456, 459- 463 (2008); doi:10.1038/nature07519

Regulation of DELLA factors activity

As mentioned above, sly1 mutants in Arabidopsis persistently accumulate high levels of DELLA factors. When freshly harvested from the mother plant, sly1 seeds cannot germinate, but after extended period of after-ripening, they germinate despite persistently high RGL2 accumulation (Ariizumi, 2007). This suggests that the activity of RGL2 to repress germination in sly1 can be lost. There are two potential mechanisms for DELLA protein activity regulation described so far: phosphorylation and O-GlcNAc-modification.

DELLA protein phosphorylation

DELLA proteins were reported to exist in phosphorylated form (Sasaki et al., 2003; Fu et al., 2004). It was shown that the F-box protein, SLY1, in Arabidopsis or GID2 in rice interact with phosphorylated forms of GAI or SLR, respectively (Fu et al., 2004; Gomi et al., 2004).

These results suggested that DELLAs are phosphorylated prior to their degradation. However, experiments performed in rice callus cells showed that both forms of SLR1, phosphorylated and non-phosphorylated, could be degraded by GA treatment (Itoh et al., 2005). Moreover, GA- induced RGL2 degradation was blocked by Ser/Thr phosphatase inhibitors but not by Ser/Thr kinase inhibitors suggesting that phosphatase activity might be prerequisite for RGL2 degradation (Hussain et al., 2005). Furthermore, substitutions of the conserved serine and threonine residues that mimicked the status of constitutive phosphorylation of RGL2 rendered

(35)

this protein more resistant to GA-induced degradation (Hussain et al., 2005). Thus, it is still unclear what is the biological relevance of DELLA protein phosphorylation.

O-GlcNAc- modification by SPY

spy (spindly) null mutants exhibit phenotypes that resemble wild-type plants repeatedly treated with exogenous GA (Jacobsen and Olszewski, 1993) and to varying degrees this mutation suppresses all phenotypes of GA deficiency (Jacobsen and Olszewski, 1993). Indeed, spy alleles were found in screens for a suppressor of the dominant gai-1 mutation, i.e. mutation in the DELLA motif that renders the GAI protein resistant to GA-mediated degradation (Wilson and Somerville, 1995; Peng et al., 1997). Moreover, the spy mutation partially rescues the dwarf phenotype of rga-Δ17 (a mutation where the DELLA motif is missing in RGA, rendering this protein resistant to GA mediated degradation) without altering RGA protein accumulation or changing its nuclear localization. This might suggest that RGA partially loses its repressive activity when the SPY gene is mutated (Silverstone et al., 2007) and that SPY is a general positive regulator of DELLA activity.

SPY (SPINDLY) is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) (Jacobsen and Olszewski, 1993; Jacobsen SE, 1996). OGTs add a GlcNAc monosaccharide to Ser/Thr residues of nuclear and cytosolic proteins. Many of the proteins modified by OGTs are phosphoproteins and, in some cases, phosphorylation and O-GlcNAc modification can occur at the same site (Slawson and Hart, 2003). DELLA factors contain a putative GlcNAc site mainly in the poly Ser/Thr region suggesting that SPY activity might interfere with the nearby protein phosphorylation site. Indeed, reduced SPY expression (by RNAi) in the rice gid2 mutant leads to elevated phosphorylation of SLR1, without changing the total SLR1 protein levels (Shimada et al., 2006). Down-regulation of rice SPY by RNAi in gid2 partially rescues the dwarf phenotype of this mutant. This might suggest that SPY increases SLR1 activity by preventing its phosphorylation due to the competition for the sites of O-GlcNAcylation and phosphorylation. It also suggests that when SLR1 is phosphorylated, it is less active.

(36)

Inactivation by sequestration into a complex

Arabidopsis sly1 mutants accumulate higher DELLA protein amounts than ga1-3 or triple gid1a gid1b gid1c mutants yet their phenotype is less severe, i.e. they are less dwarf and they can germinate after an extended period of after-ripening. Surprisingly, GID1 over-expression rescued the sly1 dwarf phenotype and low fertility without decreasing the levels of the DELLA protein (RGA)(Ariizumi et al., 2008). The severity of the sly1 phenotype growth rescue by GID1 over- expression depended on the level of GID1 accumulation, GA synthesis and the presence of a functional DELLA motif (Ariizumi et al., 2008). Similar results were obtained in the rice gid2 mutant (Ueguchi-Tanaka et al., 2008). These results suggested that DELLA proteins can lose their repressive activity when they are sequestered to the GA-GID1-DELLA complex. This mechanism could account for a SLY1/GID2 –independent GA signaling which does not necessitate DELLA degradation.

Potential DELLA target genes

One of the most interesting and open questions is how DELLA factors exert their repression activity on seed germination, plant vegetative growth and flowering.

Global gene expression analysis

Microarray experiments were performed in search for DELLA target genes (Ogawa et al., 2003; Cao et al., 2006; Zentella et al., 2007). Ogawa et al. searched for GA-regulated genes during seed germination comparing transcriptomes of ga1-3 mutant seeds germinating in the absence (no germination) or presence (germination) of exogenous GA4 and comparing them to transcriptome of wild type seeds. This study identified 230 genes that were up-regulated in response to GA and 127 genes that were down-regulated. Among GA-upregulated genes were genes involved in cell elongation like aquaporins (a membrane proteins that facilitate water uptake into the symplast), expansins and a set of genes whose functions are associated with cell- wall loosening activities (like xyloglucan endotransglycosylase/hydrolase (XTH)) and might be involved in weakening of tissues surrounding the embryo and/or in embryo growth (Ogawa et

(37)

al., 2003). The other group of GA-upregulated genes were those involved in cell cycle (Ogawa et al., 2003). Cao et al. compared global gene expression patterns during germination of the ga1-3 mutant to that of the ga1-3 gait6 rga-t2 rgl1-1 rgl2-1 mutant and compared them to the pattern of a wild type plant. They found that about half of the total of GA-regulated genes were regulated in a DELLA dependant manner. Their analysis identified a group of cell-wall loosening enzymes being DELLA-regulated, consistent with previous reports (Ogawa et al., 2003; Cao et al., 2006). Both reports showed that a significant number of genes related to other phytohormones were altered in response to GA and in a DELLA-dependent manner. Those genes include ten ABA-related genes, seven auxin related and five ethylene related genes (Ogawa et al., 2003; Cao et al., 2006). Another prominent group of GA-induced and DELLA-dependent genes is composed of transcription factors belonging to the MYB family (seven genes), zinc- finger family (four genes), bHLH family (four genes) (Ogawa et al., 2003; Cao et al., 2006).

However, these reports did not show whether DELLA factors directly bind to any of their potential targets. Indeed, all reported targets could be located further downstream in the GA response pathway.

A global gene expression approach in search for DELLA targets has obvious limitations.

Changes in transcriptome expression might reflect the physiological state of a seed, for instance its commitment to germinate, and not be directly due to the presence or absence of a DELLA factor.

Attempts to find direct DELLA targets

Zentella et al. searched for direct targets of RGA by analyzing early transcriptome changes in response to induction of RGA accumulation (Zentella et al., 2007). This approach resulted in 14 putative direct targets of RGA, and the existence of RGA protein-DNA interactions was confirmed for eight of them by ChIP experiments (Zentella et al., 2007). Among those eight genes were GID1a, GID1b and a group of putative transcription factors. Moderate enrichments of promoter sequences of those eight genes in ChIP experiments suggested that RGA might associate with its target promoters via interaction with additional DNA binding proteins. Enrichment in GID1a and GID1b sequences was consistent with reports showing that GA treatment or mutation in DELLA factors lead to decreased levels in GA signaling

(38)

components such as GA receptor (Griffiths et al., 2006). Moderate but consistent enrichment was also observed for promoter sequences of the XERICO gene (Zentella et al., 2007). Expression of this gene was induced by DELLA and repressed by GA. XERICO is predicted to be a RING–H2 motif containing zinc-finger protein (Ko et al., 2006). XERICO over-expression leads to drought tolerance and to an increase in endogenous ABA levels (Ko et al., 2006) and the xerico mutant displays slight ABA insensitivity during seed germination, most likely due to their lower ABA levels in dry and imbibed seeds (Zentella et al., 2007). These observations suggested that the DELLA protein, RGA, can promote ABA synthesis by stimulating expression of XERICO (Zentella et al., 2007). The role of the interaction with five other putative direct targets of RGA such as the transcription factors MYB, bHLH137, WRKY27 and the transcriptional regulators SCL3 and LBD40 was not investigated.

Taken together, DELLA factors seem to function as: 1) regulators of GA homeostasis by regulating the components of GA biosynthesis and perception; 2) transcriptional regulators promoting the accumulation of negative components of GA signaling components or inhibiting the accumulation of positive components; 3) integrators of GA signaling with other hormone biosynthetic and signaling pathways (for example ABA).

DELLAs might sequester transcription factors to modulate gene expression

Recently, an alternative role for DELLAs’ function was proposed: they were shown to interact with bHLH transcription factors, PIF3 and PIF4, in order to prevent hypocotyl elongation during photomorphogenesis of Arabidopsis seedlings (de Lucas et al., 2008; Feng et al., 2008). PIF3 and PIF4 are bHLH transcription factors that promote hypocotyl cell elongation in dark-grown seedlings by inducing expression of their target genes. In the proposed model, DELLAs act as transcriptional regulators that sequester the transcription factors, PIF3 and PIF4, to the complex thus preventing their binding to their target genes. In turn, decreased expression of PIF’s target genes leads to inhibition of hypocotyl elongation under light conditions ((de Lucas et al., 2008; Feng et al., 2008)).

(39)

Control of seed germination by ABA

Abscisic acid (ABA) regulates many aspects of plant growth and development including embryo maturation, seed dormancy, germination, cell division and elongation, as well as responses to environmental stresses like drought, salinity and pathogen attack by modulating stomatal aperture, plant architecture and tolerance to osmotic stresses.

ABA biosynthesis

Abscisic acid (ABA) is a lipophilic 15-C weak acid that is formed by cleavage of carotenoids that contain 40 atoms of carbon. Because carotenoids synthesis takes place in plastids, the first three steps in ABA synthesis are localized in this organelle.

Plastid- localized ABA biosynthesis pathway

In a simplified view, the plastid- localized ABA biosynthesis pathway can be divided into three steps (reviewed in (Nambara and Marion-Poll, 2005):

1) Conversion of β-carotene to zeaxanthin and then to violaxanthin, the latter process being catalysed by zeaxanthin epoxidase (ZEP). ZEP is encoded by ABA1 in Arabidopsis (E Marin, 1996) (Figure 9, Table 2).

2) Violaxanthin isomerisation yielding 9-cis-Violaxanthin and 9-cis-Neoxanthin (Figure 9).

3) Oxidative cleavage of 9-cis-Violaxanthin and 9-cis-Neoxanthin yielding 15-C xanthoxin.

This cleavage reaction is considered the first committed step in ABA production. It is performed by nine-cis-epoxycarotenoid dioxygenase (NCED) enzymes (Schwartz et al., 1997). NCED genes belong to a multigene family, and in Arabidopsis five of them (NCED2, 3, 5, 6, and 9) are involved in ABA synthesis (Iuchi et al., 2001) (Table2).

Expression analysis of NCED genes showed the highest relative expression level of NCED6 and NCED9 during seed development (Lefebvre et al., 2006). Moreover, NCED6 was mostly expressed in the seed coat, while NCED9 was expressed highly in the embryo and to a lesser extent in the seed coat (Lefebvre et al., 2006). NCED proteins are

Références

Documents relatifs

Furthermore, histological analysis identified in mature seeds an electron-dense endospermic cuticular film covering endosperm cells on their external side (Fig 2). The

Results: We show that DAG1 expression is controlled at the epigenetic level through the H3K27me3 mark during the seed-to-seedling transition, and that DAG1 directly represses also

Interestingly, our results revealed that expression of RGA, but not of GAI, is significantly affected in dag2 mutant seeds exposed to R light, suggesting that DAG2 may

After 5 days in the dark, stratified seeds of the mutant lines germinated completely as wild-type seeds (Figure 2D); on the contrary, in the absence of stratification, wild-type,

The treatment of dormant seeds with ethylene (D/ET) promotes seed germination, and abscisic acid (ABA) treatment reduces non-dormant (ND/ABA) seed germination in sunflowers

Key words: Allelopathy, bibliometrics, bioassay, germination, inhibition of germination, permanent effects, recovery of germination, stimulation of germination, weed

The review of the literature data about crystallographic as well as some thermodynamic properties of the unary and binary phases relevant to the present study is based

Interestingly, reserves remobilization seems to start very early in palm seeds (at stage I) and, thus, is part of germination stricto sensu, whereas in most species (such