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1.
C R Biol ; 345(4): 61-110, 2023 Feb 16.
Article in English | MEDLINE | ID: mdl-36847120

ABSTRACT

Elucidating the mechanisms that control seed development, metabolism, and physiology is a fundamental issue in biology. Michel Caboche had long been a catalyst for seed biology research in France up until his untimely passing away last year. To honour his memory, we have updated a review written under his coordination in 2010 entitled "Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research". This review encompassed different molecular aspects of seed development, reserve accumulation, dormancy and germination, that are studied in the lab created by M. Caboche. We have extended the scope of this review to highlight original experimental approaches implemented in the field over the past decade such as omics approaches aimed at investigating the control of gene expression, protein modifications, primary and specialized metabolites at the tissue or even cellular level, as well as seed biodiversity and the impact of the environment on seed quality.


L'élucidation des mécanismes qui contrôlent le développement, le métabolisme et la physiologie des graines est une question fondamentale en biologie. Michel Caboche a longtemps été un catalyseur de la recherche en biologie des graines en France jusqu'à son décès prématuré l'année dernière. Pour honorer sa mémoire, nous avons mis à jour une revue écrite sous sa coordination en 2010 intitulée « Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research ¼. Cette revue englobait différents aspects moléculaires du développement des graines, de l'accumulation des réserves, de la dormance et de la germination, qui sont étudiés dans le laboratoire créé par M. Caboche. Nous avons étendu la portée de cette revue pour mettre en évidence des approches expérimentales originales mises en œuvre dans le domaine au cours de la dernière décennie, telles que les approches omiques visant à étudier le contrôle de l'expression des gènes, les modifications des protéines, les métabolites primaires et spécialisés au niveau des tissus ou même des cellules, tout en tenant compte de la biodiversité des graines et de l'impact de l'environnement sur leur qualité.


Subject(s)
Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Seeds/genetics , Molecular Biology , Biology , France , Germination/genetics , Plant Dormancy/genetics , Gene Expression Regulation, Plant
2.
Cells ; 10(10)2021 09 26.
Article in English | MEDLINE | ID: mdl-34685527

ABSTRACT

The conjugation of sterols with a Glc moiety is catalyzed by sterol glucosyltransferases (SGTs). A portion of the resulting steryl glucosides (SG) are then esterified with a long-chain fatty acid to form acyl-SG (ASG). SG and ASG are prevalent components of plant cellular membranes and influence their organization and functional properties. Mutant analysis had previously inferred that two Arabidopsis SGTs, UGT80A2 and UGT80B1/TT15, could have specialized roles in the production of SG in seeds, despite an overlap in their enzymatic activity. Here, we establish new roles for both enzymes in the accumulation of polysaccharides in seed coat epidermal cells (SCEs). The rhamnogalacturonan-I (RG-I) content of the inner layer of seed mucilage was higher in ugt80A2, whereas RG-I accumulation was lower in mutants of UGT80B1, with double mutant phenotypes indicating that UGT80A2 acts independently from UGT80B1. In contrast, an additive phenotype was observed in double mutants for increased galactoglucomannan (GGM) content. Double mutants also exhibited increased polymer density within the inner mucilage layer. In contrast, cell wall defects were only observed in mutants defective for UGT80B1, while more mucilage cellulose was only observed when UGT80A2 was mutated. The generation of a range of phenotypic effects, simultaneously within a single cell type, demonstrates that the adjustment of the SG and ASG composition of cellular membranes by UGT80A2 and UGT80B1 tailors polysaccharide accumulation in Arabidopsis seeds.


Subject(s)
Epidermal Cells/metabolism , Glucosyltransferases/metabolism , Mannans/metabolism , Polysaccharides/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Cell Wall/metabolism , Cellulose/metabolism , Glucosyltransferases/genetics , Phenotype
3.
Plant Cell Physiol ; 57(4): 660-74, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26637538

ABSTRACT

Mature seeds are an ultimate physiological status that enables plants to endure extreme conditions such as high and low temperature, freezing and desiccation. Seed longevity, the period over which seed remains viable, is an important trait not only for plant adaptation to changing environments, but also, for example, for agriculture and conservation of biodiversity. Reduction of seed longevity is often associated with oxidation of cellular macromolecules such as nucleic acids, proteins and lipids. Seeds possess two main strategies to combat these stressful conditions: protection and repair. The protective mechanism includes the formation of glassy cytoplasm to reduce cellular metabolic activities and the production of antioxidants that prevent accumulation of oxidized macromolecules during seed storage. The repair system removes damage accumulated in DNA, RNA and proteins upon seed imbibition through enzymes such as DNA glycosylase and methionine sulfoxide reductase. In addition to longevity, dormancy is also an important adaptive trait that contributes to seed lifespan. Studies in Arabidopsis have shown that the seed-specific transcription factor ABSCISIC ACID-INSENSITIVE3 (ABI3) plays a central role in ABA-mediated seed dormancy and longevity. Seed longevity largely relies on the viability of embryos. Nevertheless, characterization of mutants with altered seed coat structure and constituents has demonstrated that although the maternally derived cell layers surrounding the embryos are dead, they have a significant impact on longevity.


Subject(s)
Plant Dormancy/physiology , Seeds/physiology , DNA Repair , Oxidative Stress , Polyphenols/metabolism , RNA, Plant/physiology , Seeds/cytology , Signal Transduction , Waxes
4.
Plant Cell Physiol ; 55(9): 1646-59, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25008975

ABSTRACT

Among secondary metabolites, flavonoids are particularly important for the plant life cycle and could be beneficial for human health. The study of Arabidopsis thaliana transparent testa mutants showed that seed flavonoids are important for environmental adaptation, reactive oxygen species homeostasis, dormancy and longevity. Compared with Arabidopsis and maize (Zea mays L.), far less research has been conducted on rice (Oryza sativa L.) particularly for cultivars with non-pigmented seeds. In this study, we describe the localization, nature and relative abundance of flavonoids in mature and germinated non-pigmented Nipponbare seeds using a combination of confocal microscopy, mass spectrometry and gene expression analysis. The mature seed exclusively accumulates flavones mostly in the embryo and to a lesser extent in the pericarp/testa. Due to the variety of flavone conjugation patterns, 21 different flavones were identified, including sulfated flavones never mentioned before in cereals. Schaftoside (apigenin-6-C-glucoside-8-C-arabinoside) and its two isomers represent nearly 50% of all rice seed flavones and are the only flavonoids accumulated in the pericarp/testa seed compartment. These 21 conjugated flavones showed a very stable profile during rice seed germination sensu stricto, while expression of key flavone synthesis genes strongly increases before the completion of germination. We discuss the potential roles of these rice seed flavones in a seed biology context.


Subject(s)
Flavones/metabolism , Gene Expression Regulation, Plant , Oryza/metabolism , Seeds/metabolism , Chromatography, Liquid , Flavones/chemistry , Flavones/isolation & purification , Germination , Oligonucleotide Array Sequence Analysis , Oryza/chemistry , Oryza/genetics , Oryza/ultrastructure , RNA, Plant/genetics , Seeds/chemistry , Seeds/genetics , Seeds/ultrastructure , Tandem Mass Spectrometry , Water/physiology
5.
J Exp Bot ; 65(3): 885-93, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24532452

ABSTRACT

NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation.


Subject(s)
Anion Transport Proteins/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant , Nitrates/metabolism , Proanthocyanidins/metabolism , Signal Transduction , Alleles , Anion Transport Proteins/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Color , Flavonoids/analysis , Flavonoids/metabolism , Gene Expression , Genetic Complementation Test , Homozygote , Laccase/genetics , Laccase/metabolism , Mutation , Nitrates/analysis , Oxidation-Reduction , Phenotype , Seeds/genetics , Seeds/metabolism
6.
Plant Methods ; 8(1): 16, 2012 May 09.
Article in English | MEDLINE | ID: mdl-22571391

ABSTRACT

BACKGROUND: Seed transmission constitutes a major component of the parasitic cycle for several fungal pathogens. However, very little is known concerning fungal or plant genetic factors that impact seed transmission and mechanisms underlying this key biological trait have yet to be clarified. Such lack of available data could be probably explained by the absence of suitable model pathosystem to study plant-fungus interactions during the plant reproductive phase. RESULTS: Here we report on setting up a new pathosystem that could facilitate the study of fungal seed transmission. Reproductive organs of Arabidopsis thaliana were inoculated with Alternaria brassicicola conidia. Parameters (floral vs fruit route, seed collection date, plant and silique developmental stages) that could influence the seed transmission efficiency were tested to define optimal seed infection conditions. Microscopic observations revealed that the fungus penetrates siliques through cellular junctions, replum and stomata, and into seed coats either directly or through cracks. The ability of the osmosensitive fungal mutant nik1Δ3 to transmit to A. thaliana seeds was analyzed. A significant decrease in seed transmission rate was observed compared to the wild-type parental strain, confirming that a functional osmoregulation pathway is required for efficient seed transmission of the fungus. Similarly, to test the role of flavonoids in seed coat protection against pathogens, a transparent testa Arabidopsis mutant (tt4-1) not producing any flavonoid was used as host plant. Unexpectedly, tt4-1 seeds were infected to a significantly lower extent than wild-type seeds, possibly due to over-accumulation of other antimicrobial metabolites. CONCLUSIONS: The Arabidopsis thaliana-Alternaria brassicicola pathosystem, that have been widely used to study plant-pathogen interactions during the vegetative phase, also proved to constitute a suitable model pathosystem for detailed analysis of plant-pathogen interactions during the reproductive phase. We demonstrated that it provides an excellent system for investigating the impact of different fungal or plant mutations on the seed transmission process and therefore paves the way towards future high-throughput screening of both Arabidopsis and fungal mutant.

7.
Plant J ; 61(6): 971-81, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20409271

ABSTRACT

Seeds play a fundamental role in colonization of the environment by spermatophytes, and seeds harvested from crops are the main food source for human beings. Knowledge of seed biology is therefore important for both fundamental and applied issues. This review on seed biology illustrates the important progress made in the field of Arabidopsis seed research over the last decade. Access to 'omics' tools, including the inventory of genes deduced from sequencing of the Arabidopsis genome, has speeded up the analysis of biological functions operating in seeds. This review covers the following processes: seed and seed coat development, seed reserve accumulation, seed dormancy and seed germination. We present new insights in these various fields and describe ongoing biotechnology approaches to improve seed characteristics in crops.


Subject(s)
Arabidopsis/genetics , Genomics , Seeds/growth & development , Arabidopsis/growth & development , Arabidopsis/metabolism , Biotechnology/trends , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genome, Plant , Germination , Plant Growth Regulators/metabolism , Seed Storage Proteins/metabolism , Seeds/genetics , Seeds/metabolism
8.
Plant Cell Rep ; 28(4): 601-17, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19153740

ABSTRACT

As part of an ongoing research program dedicated to the understanding of proanthocyanidin (PA) accumulation in Brassica napus seed coat, transgenic rapeseed plants carrying a 2.3-kb fragment of the Arabidopsis thaliana BAN promoter (ProAtBAN) fused to the uidA reporter gene (GUS) were generated. Analysis of these plants revealed that ProAtBAN was activated in B. napus seed coat, following a spatio-temporal pattern that was very similar to the PA deposition profile in rapeseed and also to the one previously described in Arabidopsis. ProAtBAN activity occurred as soon as the early stages of embryogenesis and was restricted to the cells where PAs were shown to accumulate. Therefore, the Arabidopsis BAN promoter can be used to trigger gene expression in B. napus seed coat for both genetic engineering and functional validation of candidate genes. In addition, these data strongly suggest that the transcriptional regulatory network of the BAN gene is conserved between Arabidopsis and rapeseed. This is consistent with the fact that similarity searches of the public rapeseed sequence databases allowed recovering the rapeseed homologs for several BAN regulators, namely TT1, TT2, TT8, TT16 and TTG1, which have been previously described in Arabidopsis.


Subject(s)
Brassica napus/metabolism , Proanthocyanidins/metabolism , Promoter Regions, Genetic , Seeds/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Brassica napus/genetics , DNA, Plant/genetics , Gene Expression Regulation, Plant , Molecular Sequence Data , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Seeds/genetics , Sequence Alignment , Sequence Analysis, DNA
9.
C R Biol ; 331(10): 796-805, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18926494

ABSTRACT

The seed constitutes the main vector of plant propagation and it is a critical development stage with many specificities. Seed longevity is a major challenge for the conservation of plant biodiversity and for crop success. Seeds possess a wide range of systems (protection, detoxification, repair) allowing them to survive in the dry state and to preserve a high germination ability. Therefore, the seed system provides an appropriate model to study longevity and aging.


Subject(s)
Germination/physiology , Seeds/physiology , Arabidopsis/embryology , Arabidopsis/genetics , Cell Survival , Cyanides/metabolism , DNA Repair , Desiccation , Flavonoids/physiology , Plant Proteins/biosynthesis , Plant Proteins/physiology , Preservation, Biological , Reactive Oxygen Species/metabolism , Seeds/cytology , Vitamin E/physiology , gamma-Aminobutyric Acid/physiology
10.
Plant J ; 55(6): 940-53, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18532978

ABSTRACT

SUMMARY: In Arabidopsis thaliana, several MYB and basic helix-loop-helix (BHLH) proteins form ternary complexes with TTG1 (WD-Repeats) and regulate the transcription of genes involved in anthocyanin and proanthocyanidin (PA) biosynthesis. Similar MYB-BHLH-WDR (MBW) complexes control epidermal patterning and cell fates. A family of small MYB proteins (R3-MYB) has been shown to play an important role in the regulation of epidermal cell fates, acting as inhibitors of the MBW complexes. However, so far none of these small MYB proteins have been demonstrated to regulate flavonoid biosynthesis. The genetic and molecular analyses presented here demonstrated that Arabidopsis MYBL2, which encodes a R3-MYB-related protein, is involved in the regulation of flavonoid biosynthesis. The loss of MYBL2 activity in the seedlings of two independent T-DNA insertion mutants led to a dramatic increase in the accumulation of anthocyanin. In addition, overexpression of MYBL2 in seeds inhibited the biosynthesis of PAs. These changes in flavonoid content correlate well with the increased level of mRNA of several structural and regulatory anthocyanin biosynthesis genes. Interestingly, transient expression analyses in A. thaliana cells suggested that MYBL2 interacts with MBW complexes in planta and directly modulates the expression of flavonoid target genes. These results are fully consistent with the molecular interaction of MYBL2 with BHLH proteins observed in yeast. Finally, MYBL2 expression studies, including its inhibition by light-induced stress, allowed us to hypothesise a physiological role for MYBL2. Taken together, these results bring new insights into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation of its developmental and environmental regulation.


Subject(s)
Anthocyanins/biosynthesis , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Plant , Genes, Plant , Light , Mutagenesis, Insertional , Mutation , Promoter Regions, Genetic , RNA, Plant/genetics , Reverse Transcriptase Polymerase Chain Reaction , Seeds/genetics , Seeds/metabolism , Transcription Factors/genetics , Transcription, Genetic , Transfection
11.
Plant Cell ; 19(6): 2023-38, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17601828

ABSTRACT

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.


Subject(s)
Antiporters/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Flavonoids/metabolism , Proanthocyanidins/metabolism , Seeds/metabolism , Transcription Factors/metabolism , Vacuoles/metabolism , Anthocyanins/chemistry , Anthocyanins/metabolism , Anthocyanins/pharmacology , Arabidopsis/drug effects , Cytoplasmic Vesicles/drug effects , Flavonoids/biosynthesis , Flavonoids/chemistry , Glucosides/chemistry , Glucosides/metabolism , Glucosides/pharmacology , Mutation/genetics , Proanthocyanidins/biosynthesis , Proanthocyanidins/chemistry , Promoter Regions, Genetic/genetics , Protein Transport/drug effects , Seeds/chemistry , Seeds/cytology , Seeds/drug effects , Substrate Specificity/drug effects , Vacuoles/drug effects , Yeasts/drug effects
12.
Trends Plant Sci ; 12(1): 29-36, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17161643

ABSTRACT

Flavonoids protect plants against various biotic and abiotic stresses, and their occurrence in human diet participates in preventing degenerative diseases. Many of the biological roles of flavonoids are attributed to their potential cytotoxicity and antioxidant abilities. Flavonoid oxidation contributes to these chemical and biological properties and can lead to the formation of brown pigments in plant tissues as well as plant-derived foods and beverages. Flavonoid oxidation in planta is mainly catalyzed by polyphenol oxidases (catechol oxidases and laccases) and peroxidases. These activities are induced during seed and plant development, and by environmental stresses such as pathogen attacks. Their complex mode of action is regulated at several levels, involving transcriptional to post-translational mechanisms together with the differential subcellular compartmentalization of enzymes and substrates.


Subject(s)
Flavonoids/metabolism , Plants/metabolism , Flavonoids/biosynthesis , Flavonoids/chemistry , Gene Expression Regulation, Plant , Maillard Reaction , Oxidation-Reduction , Pigmentation , Plants/enzymology , Plants/genetics
13.
Annu Rev Plant Biol ; 57: 405-30, 2006.
Article in English | MEDLINE | ID: mdl-16669768

ABSTRACT

Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. This review presents a current view of the genetic and biochemical control of flavonoid metabolism during seed development. It focuses mainly on proanthocyanidin accumulation in Arabidopsis, with comparisons to other related metabolic and regulatory pathways. These intricate networks and their fine-tuned regulation, once they are determined, should contribute to a better understanding of seed coat development and the control of PA and flavonol metabolism. In addition, flavonoids provide an interesting model to study various biological processes and metabolic and regulatory networks.


Subject(s)
Flavonoids/genetics , Flavonoids/metabolism , Seeds/metabolism , Gene Expression Regulation, Plant , Subcellular Fractions/metabolism
14.
Planta ; 224(1): 96-107, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16395586

ABSTRACT

Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.


Subject(s)
Arabidopsis/metabolism , Flavonoids/biosynthesis , Seeds/metabolism , Arabidopsis/embryology , Arabidopsis/genetics , Flavonoids/chemistry , Flavonols/chemistry , Kinetics , Mutation , Proanthocyanidins/chemistry
15.
Plant Cell ; 17(11): 2966-80, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16243908

ABSTRACT

The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV-visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/growth & development , Flavonoids/metabolism , Laccase/metabolism , Oxidoreductases/metabolism , Seeds/enzymology , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/isolation & purification , Base Sequence , Catechol Oxidase/genetics , Catechol Oxidase/isolation & purification , Catechol Oxidase/metabolism , DNA, Complementary/analysis , DNA, Complementary/genetics , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Plant/physiology , Laccase/genetics , Laccase/isolation & purification , Molecular Sequence Data , Mutation/genetics , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/isolation & purification , Polymers/metabolism , Promoter Regions, Genetic/genetics , Seeds/genetics
16.
Plant Physiol ; 134(4): 1598-613, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15047896

ABSTRACT

To investigate the role of stored and neosynthesized mRNAs in seed germination, we examined the effect of alpha-amanitin, a transcriptional inhibitor targeting RNA polymerase II, on the germination of nondormant Arabidopsis seeds. We used transparent testa mutants, of which seed coat is highly permeable, to better ascertain that the drug can reach the embryo during seed imbibition. Even with the most permeable mutant (tt2-1), germination (radicle protrusion) occurred in the absence of transcription, while subsequent seedling growth was blocked. In contrast, germination was abolished in the presence of the translational inhibitor cycloheximide. Taken together, the results highlight the role of stored proteins and mRNAs for germination in Arabidopsis and show that in this species the potential for germination is largely programmed during the seed maturation process. The alpha-amanitin-resistant germination exhibited characteristic features. First, this germination was strongly slowed down, indicating that de novo transcription normally allows the synthesis of factor(s) activating the germination rate. Second, the sensitivity of germination to gibberellic acid was reduced 15-fold, confirming the role of this phytohormone in germination. Third, de novo synthesis of enzymes involved in reserve mobilization and resumption of metabolic activity was repressed, thus accounting for the failure in seedling establishment. Fourth, germinating seeds can recapitulate at least part of the seed maturation program, being capable of using mRNAs stored during development. Thus, commitment to germination and plant growth requires transcription of genes allowing the imbibed seed to discriminate between mRNAs to be utilized in germination and those to be destroyed.


Subject(s)
Amanitins/pharmacology , Arabidopsis/genetics , Germination/genetics , Proteome/analysis , RNA, Messenger/genetics , Seeds/genetics , Arabidopsis/drug effects , Arabidopsis/growth & development , Cycloheximide/pharmacology , Germination/drug effects , Germination/physiology , Gibberellins/pharmacology , Mutation , Nucleic Acid Synthesis Inhibitors/pharmacology , Plant Growth Regulators/pharmacology , Protein Synthesis Inhibitors/pharmacology , Proteome/genetics , RNA Polymerase II/drug effects , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Messenger/metabolism , Seeds/drug effects , Seeds/metabolism , Uridine Monophosphate/metabolism
17.
Plant Cell ; 15(11): 2514-31, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14555692

ABSTRACT

Anthocyanidin reductase encoded by the BANYULS (BAN) gene is the core enzyme in proanthocyanidin (PA) biosynthesis. Here, we analyzed the developmental mechanisms that regulate the spatiotemporal expression of BAN in the developing Arabidopsis seed coat. PA-accumulating cells were localized histochemically in the inner integument (seed body and micropyle) and pigment strand (chalaza). BAN promoter activity was detected specifically in these cells. Gain-of-function experiments showed that an 86-bp promoter fragment functioned as an enhancer specific for PA-accumulating cells. Mutations in regulatory genes of PA biosynthesis abolished BAN promoter activity (transparent testa2 [tt2], tt8, and transparent testa glabra1 [ttg1]), modified its spatial pattern (tt1 and tt16), or had no influence (ttg2), thus revealing complex regulatory interactions at several developmental levels. Genetic ablation of PA-accumulating cells targeted by the BAN promoter fused to BARNASE led to the formation of normal plants that produced viable yellow seeds. Importantly, these seeds had no obvious defects in endosperm and embryo development.


Subject(s)
Arabidopsis/genetics , Proanthocyanidins/biosynthesis , Seeds/genetics , Tannins/biosynthesis , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Base Sequence , Cell Differentiation/genetics , Cell Differentiation/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , MADS Domain Proteins/genetics , MADS Domain Proteins/metabolism , Molecular Sequence Data , Mutation , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Proanthocyanidins/metabolism , Promoter Regions, Genetic/genetics , Seeds/growth & development , Seeds/metabolism , Sequence Homology, Nucleic Acid
18.
Plant Cell ; 14(10): 2463-79, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12368498

ABSTRACT

Screening for seed pigmentation phenotypes in Arabidopsis led to the isolation of three allelic yellow-seeded mutants, which defined the novel TRANSPARENT TESTA16 (TT16) locus. Cloning of TT16 was performed by T-DNA tagging and confirmed by genetic complementation and sequencing of two mutant alleles. TT16 encodes the ARABIDOPSIS BSISTER (ABS) MADS domain protein. ABS belongs to the recently identified "B-sister" (B(S)) clade, which contains genes of unknown function that are expressed mainly in female organs. Phylogenetic analyses using a maximum parsimony approach confirmed that TT16/ABS and related proteins form a monophyletic group. TT16/ABS was expressed mainly in the ovule, as are the other members of the B(S) clade. TT16/ABS is necessary for BANYULS expression and proanthocyanidin accumulation in the endothelium of the seed coat, with the exception of the chalazal-micropylar area. In addition, mutant phenotype and ectopic expression analyses suggested that TT16/ABS also is involved in the specification of endothelial cells. Nevertheless, TT16/ABS apparently is not required for proper ovule function. We report the functional characterization of a member of the B(S) MADS box gene subfamily, demonstrating its involvement in endothelial cell specification as well as in the increasingly complex genetic control of flavonoid biosynthesis in the Arabidopsis seed coat.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , MADS Domain Proteins/genetics , Pigments, Biological/biosynthesis , Plant Epidermis/genetics , Proanthocyanidins , Seeds/genetics , Amino Acid Sequence , Anthocyanins/biosynthesis , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Base Sequence , Cell Size/genetics , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Flavonoids/biosynthesis , Gene Expression Regulation, Plant , MADS Domain Proteins/metabolism , Molecular Sequence Data , Mutation , Phenotype , Phylogeny , Pigmentation/genetics , Plant Epidermis/cytology , Plant Epidermis/metabolism , Seeds/cytology , Seeds/growth & development , Sequence Analysis, DNA , Sequence Homology, Amino Acid
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