The phaseolin (phas) promoter drives the copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues when a nucleosome is positioned over its three phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis leaves was accomplished by ectopic expression of the transcription factor PvALF (Phaseolus vulgaris ABI3-like factor), and application of abscisic acid (ABA). PvALF belongs to a family of seed-specific transcriptional activators that includes the maize viviparious1 (VP1) and the Arabidopsis abscisic acid-insensitive3 (ABI3) proteins. The major goal of the study is to gain insight to the regulation of seed-specific gene expression in three different aspects. First, since ABI3 (homolog of PvALF) is involved in ABA-mediated expression of several seed-specific protein genes in Arabidopsis, understanding its transcriptional regulation will provide insight to the mechanism by which PvALF expression is controlled. To achieve this, ABI3 promoter deletion analysis using either $-glucuronidase (gus) or green fluorescent protein (gfp) reporter gene fusions have identified various regulatory regions within the ABI3 promoter including two upstream activating sequences and a minimal seed specific expression region. In addition, a 405 bp 5' UTR was shown to play a negative role in ABI3 expression, possibly through post-transcriptional mechanisms. Second, placement of PvALF expression under control of an estradiol-inducible promoter permitted chronological ChIP analysis of changes in histone modifications, notably increased acetylation of H3-K9, as phas chromatin is remodeled (potentiated). A different array of changes (trimethylation of H3-K4) is associated with ABA-mediated activation. In contrast, H3-K14 acetylation decreased upon phas potentiation and increased on activation. Whereas decreases in histone H3 and H4 levels were detected during PvALF-mediated remodeling, slight increases occurred following ABA-mediated activation, suggesting the restoration of histone-phas interactions or the redeposition of histones in the phas chromatin. The observed histone modifications thus provide insight to the factors involved in euchromatinization and activation of a plant gene. Finally, ectopically expressed ABI5 and PvALF renders the activation of phas ABA-independent, suggesting ABI5 acts downstream of ABA during phas activation.

Seed maturation is an important process that is evolutionarily advantageous, allowing for seed dispersal and germination under favourable growth conditions. The B3-domain transcription factor FUSCA3 (FUS3) is a master regulator of seed maturation and controls developmental phase transitions through hormonal regulation in Arabidopsis thaliana. The aim of this study was to determine the post-translational regulation of FUS3 during embryonic and vegetative development. Here, FUS3 was found to interact with the E3 ubiquitin ligase ABI3-INTERACTING PROTEIN2 (AIP2) in yeast two-hybrid, in vitro, and in planta assays. Analysis of transcriptional and translational reporters also showed overlapping spatial and temporal expression patterns of AIP2 and FUS3. Furthermore, in vitro FUS3 degradation was delayed in aip2-1 mutant and increased FUS3-GFP levels were observed during mid-embryogenesis in aip2-1. Finally, double transgenic plants overexpressing AIP2 and FUS3 showed reduced FUS3 levels and reversion of the gain-of-function FUS3 phenotypes back to WT. Together, these results indicate that AIP2 is a negative regulator of FUS3.

<p>The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The <i>napA</i> gene encodes for a <i>Brassica napus</i> 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The <i>napA</i> promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to <i>napA</i> through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster.</p><p>We also show that the RY/G cluster is responsible for repression of <i>napA</i> expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the <i>napA</i> RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the <i>napA</i> RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.</p>

The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The napA gene encodes for a Brassica napus 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The napA promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to napA through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster. We also show that the RY/G cluster is responsible for repression of napA expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the napA RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the napA RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.

La tolérance à la dessiccation (TD) est définie comme l'aptitude à survivre à l'état sec et à reprendre un métabolisme normal après le retour à des conditions hydriques favorables. Contrairement aux graines orthodoxes, qui acquièrent la TD au cours de leur maturation, les graines récalcitrantes ne survivent pas à la dessiccation. L'analyse comparative du développement de ces deux types de graines constitue donc un modèle intéressant pour mettre en évidence des mécanismes spécifiquement impliqués dans la TD. Par des approches protéomique et transcriptomique, ce travail a permis de caractériser le développement de graines récalcitrantes de Castanospermum australe et de le comparer à celui de graines orthodoxes de Medicago truncatula, espèce de la même sous-famille des Fabacées. Nos résultats montrent que certaines protéines de type LEA (Late Embryogenesis Abundant) sont absentes ou faiblement accumulées dans les graines matures de C. australe comparé à celles de M. truncatula. Le profil des LEA ressemble à celui du mutant Mtabi3, déficient pour le régulateur majeur de la maturation des graines orthodoxes ABI3 (ABscissic acid Insensitive 3). L'analyse transcriptomique révèle une forte répression de CaABI3 et de ses gènes cibles en fin de développement chez les graines de C. australe, alors qu'ils restent fortement exprimés tout au long de la maturation des graines de M. truncatula. Deux gènes codant pour ABI3 ont été clonés chez C. australe : CaABI3 et CaABI3-like. Par sur-expression ectopique dans des racines de M. truncatula, seul CaABI3-like est en mesure d'activer les mêmes cibles que MtABI3. De plus, CaABI3 ne complémente pas le mutant abi3-5 d'Arabidopsis thaliana. Cette analyse renforce l'importance d'ABI3 pour expliquer la sensibilité à la TD des graines récalcitrantes. Elle révèle par ailleurs que la graine mature de C. australe présente certaines caractéristiques d'une graine prête à germer tout en exprimant de nombreux gènes de réponse au stress.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 3 uppsatser.

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